Juniper, Juniperus mexicana, ext., isomerized, acetylated

Genetic toxicity: in vitro

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:

12-01-2015 to 04-06-2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study performed under GLP. Minor deviation (in the range-finder: WP2uvrA strain) that was evaluated and does not impact the reliability of the study.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2015

Report date:

2015

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Minor deviation: In the dose range finding test in tester strain WP2uvrA in the absence of S9-mix, the precipitation of the test item on the plates was not evaluated. Due to infection at the highest dose level of 5000 µg/plate. No impact on the study

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

Minor deviation; see above

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)

Deviations:

yes

Remarks:

Minor deviation; see above

Test guideline 4

Qualifier:

according to guideline

Guideline:

JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals

Version / remarks:

Guideline stipulated by the Japanese Ministry of Health, Labour and Welfare, Ministry of Economy, Trade and Industry and Ministry of the Environment (revised March 31st, 2011)

Deviations:

yes

Remarks:

Minor deviation; see above

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: March 2013 ; signature: May 2013

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Juniper, Juniperus mexicana, ext., isomerized, acetylated

EC Number:

293-297-3

EC Name:

Juniper, Juniperus mexicana, ext., isomerized, acetylated

Cas Number:

91053-33-7

Molecular formula:

Not applicable (a generic molecular formula cannot be provided for this specific UVCB substance)

IUPAC Name:

(1S,2R,5S,7S)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-ene; 1-[(1R,2R,5S,7R)-2,6,6,8-tetramethyltricyclo[5.3.1.0¹,⁵]undec-8-en-9-yl]ethan-1-one

Test material form:

liquid

Details on test material:

- Physical state: Liquid.
- Storage condition of test material: At room temperature protected from light.
- Other: Pale yellow

Method

Target gene:

histidine or tryptophan locus

Species / strain

Species / strain 1

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system: Rat liver S9
- source of S9: Purchased from recognised supplier (dates within full study report)
- method of preparation of S9 mix: Documented in the full study report. Stored at -196ºC
- concentration or volume of S9 mix and S9 in the final culture medium: Dose-range finding test and First Mutagenicity Assay: 5% S9 ; Second Mutagenicity Assay: 10% S9
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability): A Certificate of S9 QC and Production Certificate including Activity was not presented in the full study report, however would likely be available to the GLP laboratory. The report did indicate that the S9-batch had been previously “characterised with the mutagens Benzo-(a)-pyrene and 2-aminoanthracene, which require metabolic activation, in tester strain TA100 at concentrations of 5 μg/plate and 2.5 μg/plate, respectively” ; information typically available in a Quality Certificate for S9. Prior to being utilised within the GLP study. Additionally, during the study concurrent positive control substances all produced marked increases in the number of revertant colonies and the activity of the S9 fraction was found to be satisfactory.

Test concentrations with justification for top dose:

Dose range finding study (plate-incorporation method): 0, 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate
Experiment 1 (plate-incorporation method): 0, 52, 164, 512, 1600, 5000 µg/plate
Experiment 2 (plate-incorporation method): At least five test item dose levels were selected in Experiment 2 in order to achieve both a minimum of three to four non-toxic doses and the toxic/guideline limit of the test item. The dose levels were selected based on the results of Experiment 1.
All strains (absence and presence of S9-mix): 0, 492, 878, 1568, 2800, 5000 µg/plate.
Experiment 3 (plate-incorporation method): WP2uvrA strain only (presence of S9-mix): 0, 492, 878, 1568, 2800, 5000 µg/plate ; tested to verify the observation in Experiment and/or biological relevance

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: A solubility test was performed. The test item could not be dissolved in water or dimethyl sulfoxide at up to 50 mg/mL. The test item was soluble in ethanol. Preparation of test solutions started with solutions of 50 mg/mL applying a mixing step on the vortex, resulting in a clear solution. The lower test concentrations were prepared by subsequent dilutions in ethanol. Test item concentrations were used within 2.5 hours of preparation.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191

Remarks:

Without metabolic activation S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

With metabolic activation S9

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: minimum of two (2)

METHOD OF TREATMENT/ EXPOSURE:
- Test item added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: Not applicable.
- Exposure duration/duration of treatment: Top agar in top agar tubes was melted by heating to 45°C. The following solutions were successively added to 3 ml molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/mL) of one of the tester strains, 0.1 ml of a dilution of the test item in ethanol and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. The plates were inverted and incubated in the dark at 37.0 ± 1.0 °C for 48 ± 4 h. After this period revertant colonies (histidine independent (His+) for Salmonella typhimurium bacteria and tryptophan independent (Trp+) for Escherichia coli were counted. The revertant colonies were counted automatically with a Colony Counter. Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test item precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope for evidence of thinning (cytotoxicity).

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method, e.g.: background growth inhibition; relative total growth (RTG)
- Any supplementary information relevant to cytotoxicity: Plates with sufficient test article precipitate to interfere with automated colony counting were counted manually and evidence of test item precipitate on the plates was recorded. The condition of the bacterial background lawn was evaluated, both macroscopically and microscopically by using a dissecting microscope.

METHODS FOR MEASUREMENTS OF GENOTOXICIY
The revertant colonies were counted automatically with a Colony Counter.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.
A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is greater than three (3) times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

- OTHER: Dose range finding test on TA100 and WP2urvA with and without 5% (v/v) S9-mix; First mutation assay on TA1535, TA1537 and TA98 with and without 5% (v/v) S9-mix. To obtain more information about the possible mutagenicity of the test item, a second mutation experiment was performed on all strains, in the absence of S9-mix and in the presence of 10% (v/v) S9-mix. Based on the results of the first mutation assay, the test item was tested up to the dose level of 5000 μg/plate in strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Based on the results of the earlier assays. To verify the results obtained with tester strain WP2uvrA, an additional experiment was performed. In the third mutation experiment, test item was tested at a concentration range of 492 to 5000 µg/plate in the presence of 10% (v/v) S9-mix in tester strain WP2uvrA.

Rationale for test conditions:

In accordance with the OECD TG 471 guidelines.

Evaluation criteria:

See 'Any other information on materials and methods' for details on evaluation of the assay and positive criteria.

Statistics:

No formal hypothesis testing was done. See 'Any other information on materials and methods' for details on the acceptability and evaluation criteria of the assay.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

In tester strain TA100, fluctuation in the number of revertant colonies below the laboratory historical control data range was observed at the mid doses of 492 µg/plate and 878 µg/plate. This reduction was not considered to be caused by toxicity.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

True negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No data.
- Data on osmolality: No data.
- Possibility of evaporation from medium: Not reported.
- Water solubility: Not soluble in water, ethanol vehicle utilised.
- Precipitation and time of the determination: The test item precipitated on the plates as oily droplets at the top dose level of 5000 μg/plate.
- Definition of acceptable cells for analysis: Not applicable.
- Other confounding effects: None reported.

RANGE-FINDING/SCREENING STUDIES (if applicable):
Test item was tested in the tester strains TA100 and WP2uvrA with concentrations of 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 μg/plate in the absence and presence of S9-mix. Based on the results of the dose range finding test, the following dose range was selected for the mutation assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 52, 164, 512, 1600 and 5000 μg/plate.

STUDY RESULTS
- Concurrent vehicle negative and positive control data: See tables. All concurrent vehicle negative controls were acceptable, and all concurrent positive controls produced expected positive responses.

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible: See tables.
- Statistical analysis; p-value if any: Not applicable.
- Any other criteria: Based on the results of the earlier assays. To verify the results obtained with tester strain WP2uvrA, an additional experiment was performed. In the third mutation experiment, test item was tested at a concentration range of 492 to 5000 µg/plate in the presence of 10% (v/v) S9-mix in tester strain WP2uvrA. During Experiment 1: in tester strain WP2uvrA in the presence of S9-mix, the test item induced an up to 2.2-fold increase in the number of revertant colonies compared to the solvent control at dose levels of 492 and 878 μg/plate (second mutation experiment). However, this increase was only observed at the two lowest dose levels tested and were not seen in two independently repeated experiments. Furthermore, the increase was up to 2.2-fold, which is below the acceptance criteria for a positive result of 3-fold. Therefore, this increase is considered to be not biologically relevant and the test item is considered to be not mutagenic. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two- or three-fold, increase in the number of revertants in two independently repeated experiments.

Ames test:
- Signs of toxicity: See tables.
- Individual plate counts: See tables.
- Mean number of revertant colonies per plate and standard deviation See tables.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data: The current Positive HCD dataset is presented in the full study report.
- Negative (solvent/vehicle) historical control data: The current background spontaneous revertant counts in concurrent untreated controls and/or or vehicle controls ; historic negative controls are presented in the full study report.

Any other information on results incl. tables

Table 1 : Dose range finding test: Mutagenic response of test item in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with one strain of Salmonella typhimurium and one Escherichia coli strain
	TA100		WP2uvrA
Without S9-mix
Positive control	903	± 34	1644	± 258
Solvent control	89	± 10	20	± 2
1.7	86	± 15	31	± 6
5.4	85	± 6	23	± 3
17	114	± 5	21	± 6
52	97	± 8	28	± 2
164	101	± 3	34	± 9
512	70	± 3	23	± 3
1600	70	± 12	23	± 6 n
5000	81	± 7 n 2 NP	30	± 8 n i 2
With S9-mix #1
Positive control	1338	± 205	298	± 35
Solvent control	100	± 14	30	± 10
1.7	109	± 16	30	± 7
5.4	94	± 14	25	± 6
17	80	± 6	26	± 8
52	84	± 3	31	± 7
164	77	± 15	36	± 13
512	72	± 8	34	± 12
1600	70	± 11	27	± 6
5000	75	± 12 n 2 NP	33	± 4 n 2 NP

#1: Plate incorporation assay (5% S9)

#2: Oily droplets of the test item were observed

NP: No precipitate

i: Plate infected

n: Normal bacterial background lawn

 

Table 2 Experiment 1: Mutagenic response of test item in the Salmonella typhimurium reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium
	TA1535		TA1537		TA98
Without S9-mix
Positive control	909	± 73	687	± 40	1237	± 114
Solvent control	15	± 4	9	± 4	26	± 2
52	14	± 6	8	± 4	15	± 5
164	16	± 3	6	± 3	16	± 4
512	13	± 3	3	± 2	15	± 4
1600	17	± 5	7	± 3	17	± 6
5000	20	± 2 n 2 NP	7	± 3 n 2 NP	29	± 6 n 2 NP
With S9-mix #1
Positive control	288	± 10	622	± 104	1335	± 129
Solvent control	16	± 7	8	± 0	30	± 7
52	18	± 6	10	± 3	22	± 2
164	15	± 4	8	± 4	23	± 5
512	17	± 5	6	± 2	19	± 5
1600	18	± 4	5	± 2	21	± 5
5000	23	± 1 n 2 NP	12	± 7 n 2 NP	23	± 10 n 2 NP

#1: Plate incorporation assay (5% S9)

#2: Oily droplets of the test item were observed

NP: No precipitate

n: Normal bacterial background lawn

 

Table 3 Experiment 2: Mutagenic response of test item in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with different strains of Salmonella typhimurium and one Escherichia coli strain
	TA1535		TA1537		TA98		TA100		WP2uvrA
Without S9-mix
Positive control	870	± 26	482	± 42	1008	± 20	852	± 35	1050	± 51
Solvent control	18	± 2	8	± 3	14	± 2	91	± 8	36	± 4
492	16	± 6	5	± 2	23	± 6	79	± 13	30	± 5
878	21	± 5	7	± 0	17	± 11	98	± 5	29	± 5
1568	24	± 7	8	± 4	19	± 6	88	± 11	34	± 5
2800	17	± 3	10	± 10	22	± 7	90	± 12	30	± 2
5000	19	± 4 n 2 NP	7	± 5 n 2 NP	19	± 6 n 2 NP	96	± 6 n 2 NP	32	± 7 n 2 NP
With S9-mix #1
Positive control	262	± 32	526	± 33	653	± 60	1670	± 165	313	± 17
Solvent control	25	± 1	11	± 3	35	± 9	99	± 13	39	± 9
492	24	± 5	6	± 2	29	± 5	39	± 6	86	± 9
878	23	± 3	9	± 4	34	± 11	30	± 4	73	± 5
1568	22	± 10	8	± 4	29	± 4	80	± 11	37	± 5
2800	27	± 10	9	± 8	32	± 9	74	± 6	27	± 9
5000	21	± 6 n 2 NP	7	± 2 n 2 NP	28	± 5 n 2 NP	79	± 24 n 2 NP	46	± 11 n 2 NP

#1: Plate incorporation assay (10% S9)

#2: Oily droplets of the test item were observed

NP: No precipitate

n: Normal bacterial background lawn

Table 4 Experiment 3: Mutagenic response of test item in the Escherichia coli reverse mutation assay

Dose (µg/plate)	Mean number of revertant colonies/3 replicate plates (± S.D.) with the Escherichia coli strain WP2uvrA
With S9-mix #1
Positive control	402	± 17
Solvent control	37	± 14
492	22	± 4
878	27	± 5
1560	26	± 8
2800	17	± 7
5000	21	± 6 n 2 NP

#1: Plate incorporation assay (10% S9)

#2: Oily droplets of the test item were observed

NP: No precipitate

n: Normal bacterial background lawn

Applicant's summary and conclusion

Conclusions:

Interpretation of results :
negative
Under the conditions of this study, the test item was considered to be non-mutagenic in the presence and absence of S9 activation. Negative and strain specific positive control values were within laboratory historical control data.

Executive summary:

The study was performed to the requirements of OECD Guideline 471, EU Method B13/14, US EPA OCSPP 870.5100 and Japanese guidelines for bacterial mutagenicity testing under GLP, to evaluate the potential mutagenicity of the test item in a bacterial reverse mutation assay using S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in both the presence and absence of S-9 mix. Within the dose range finding test, the test item was tested up to concentrations of 5000 μg/plate in the absence and presence of S9-mix in the strains TA100 and WP2uvrA. The test item precipitated on the plates as oily droplets at the top dose level of 5000 μg/plate. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was not observed including up to the top dose of 5000 μg/plate in both tester strains. Based on the results of the dose range finding test, the test item was tested in the first mutation assay at a concentration range of 52 to 5000 μg/plate in the absence and presence of 5% (v/v) S9-mix in tester strains TA1535, TA1537 and TA98. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed. In a follow-up experiment of the assay with additional parameters, the test item was tested at a concentration range of 492 to 5000 μg/plate in the absence and presence of 10% (v/v) S9-mix in tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA. Cytotoxicity, as evidenced by a decrease of the bacterial background lawn, was not observed at the top dose of 5000 μg/plate in both tester strains. To verify the results obtained with tester strain WP2uvrA, an additional experiment was performed. In the third mutation experiment, the test item was tested at a concentration range of 492 to 5000 µg/plate in the presence of 10% (v/v) S9-mix in tester strain WP2uvrA. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants were observed. In tester strain WP2uvrA in the presence of S9-mix, the test item induced an up to 2.2-fold increase in the number of revertant colonies compared to the solvent control at dose levels of 492 and 878 µg/plate (second mutation experiment). However, this increase was only observed at the two lowest dose levels tested and were not seen in two independently repeated experiments. Furthermore, the increase was up to 2.2-fold, which is below the acceptance criteria for a positive result of 3-fold. All other bacterial strains showed negative responses over the entire dose range, i.e. no dose-related, two- or three-fold, increase in the number of revertants in two independently repeated experiments. Negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate, and that the metabolic activation system functioned properly. There were no biologically relevant increases in the frequency of revertant colonies recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation (S9‑mix). It was concluded that, under the conditions of this assay, the test item gave a negative, i.e. non-mutagenic response in S.typhimurium strains TA98, TA100, TA1535, TA1537 and E.coli strain WP2uvrA- in the presence and absence of S-9 mix.