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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
07 January 2015 to 09 February 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Study performed accoring to OECD Guideline 471 and the GLP principles.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2015
Report Date:
2016

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
- Name of test material (as cited in study report): Spearmint Essential Oil, ex Mentha spicata
- Physical state: Clear colourless to pale yellow liquid
- Analytical purity: confidental
- Impurities (identity and concentrations): confidental
- Purity test date: confidental
- Lot/batch No.: confidental
- Expiration date of the lot/batch: confidental
- Storage condition of test material: at room temperature

Method

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 specified
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital – 5,6-Benzoflavone induced S9 from Sprague Dawley rat liver
Test concentrations with justification for top dose:
Preliminary toxicity test: 50, 158, 500, 1580, 5000 µg/plate

Exp 1 (plate incorporation)
TA1535 - S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 +S9 3200, 1600, 800, 400, 200 and 100 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 ±S9 S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1537 ±S9 200, 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate
WP2 uvrA –S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
WP2 uvrA +S9 5000, 2500, 1250, 625 and 313 µg/plate

Exp. 2 (pre incubation)
WP2 uvrA –S9 5000, 2500, 1250, 625, 313, 156 and 78.1 µg/plate
WP2 uvrA + S9 5000, 2500, 1250, 625, 313 and 156 µg/plate
TA1535 + S9 3200, 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA1535 − S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA98 ± S9 1600, 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA100 + S9 1600, 800, 400, 200, 100 and 50.0 µg/plate
TA100 − S9 800, 400, 200, 100, 50.0 and 25.0 µg/plate
TA1537 ± S9 400, 200, 100, 50.0, 25.0 and 12.5 µg/plate

Exp. 3 (pre incubation)
TA1535 −S9 25.0, 12.5, 6.25, 3.13, 1.56 and 0.781 µg/plate
TA1535 +S9 100, 50.0, 25.0, 12.5, 6.25 and 3.13 µg/plate

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: compatible with the survival of the bacteria and the S9 metabolic activity
Controls
Untreated negative controls:
yes
Remarks:
sterile water
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
Experiment I: Plate incorporation, Experiment II and III: preincubation
DURATION
- Preincubation period: 30 min
- Exposure duration: 72 h at 37 C

NUMBER OF REPLICATIONS: Three replicates

Solubility was determined in a preliminary test.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies
with increasing dose levels.
Statistics:
regression analysis, t test

Results and discussion

Test results
Key result
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
- Precipitation: not detected

RANGE-FINDING/SCREENING STUDIES: the results of the preliminary toxicity test revealed cytotoxicity in some concentration levels, mentioned as thinning of the background lawn. Details can be found below, in section 'Any other information on results including tables'.

COMPARISON WITH HISTORICAL CONTROL DATA: some exceptions, but were considered acceptable
Sterility: confirmed based on absent clonies on additional agar plates.

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Table 1: PRELIMINARY TOXICITY TEST WITHOUT METABOLIC ACTIVATION

Dose level (µg/plate)

TA-1535

Rev/pl.

TA-1537

Rev/pl.

TA-98

Rev/pl.

TA-100

Rev/pl.

WP2uvrA

Rev/pl.

untreated

21

20

37

169

26

0.00

24

23

31

118

24

50.0

39

16*

35

121

24

158

25*

13*

36

108

28

500

59*

12*

26*

104

24

1580

65*

12*

40*

76*

18*

5000

50*

M

34*

M

13*

*: thinning of the background lawn
M: microcolony formation

Table 2: PRELIMINARY TOXICITY TEST WITH METABOLIC ACTIVATION

Dose level (µg/plate)

TA-1535

Rev/pl.

TA-1537

Rev/pl.

TA-98

Rev/pl.

TA-100

Rev/pl.

WP2uvrA

Rev/pl.

untreated

19

21

35

169

34

0.00

22

27

32

132

31

50.0

23

15*

28

119

20

158

22

23*

46

133

27

500

28

16*

31*

123

30

1580

31*

12*

23*

75*

22

5000

13*

M*

18*

13*

17*

*: thinning of the background lawn
M: microcolony formation

**Tables of the main mutagenicity experiments 1, 2 and 3 can be found in the attached document (Tables_Results_Spearmint Spicata) below, section 'Attached background material'.

Applicant's summary and conclusion

Conclusions:
negative with and without S9. Spearmint Spicata did not induce reverse mutations in S. typhimurium or E.coli, in the presence and absence of metabolic activation, under the conditions of this test.
Executive summary:

In a bacterial reverse mutation assay, Spearmint Spicata was tested in order to examine its potential to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli. The following strains were used: TA1535, TA1537, TA98, TA100 and WP2uvrA. Experiments were conducted both in the absence and presence of metabolic activation (liver S9 fraction, induced with phenobarbitone and betanaphthoflavone). A preliminary test was performed in order to determine cytotoxicity and define appropriate concentration levels for the main experiments. Both the plate incorporation and pre incubation methods were used. The study was performed according to the OECD Guideline 471.

No precipitation was seen at all concentrations tested, while some toxicity was detected. Concentrations levels were adapted based on these reults. No significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation or exposure method. The positive controls induced marked increases in the No of revertant colonies, which show the validity of this result.

It was concluded that Spearmint Spicata does not show any mutagenic activity under the conditions of this test. Therefore, the test substance does not need to be classified under the conditions of this test according to the criteria outlined in Annex I of 1272/2008/EC (CLP).