Registration Dossier

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August - October 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

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
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
liquid
Details on test material:
Test item: Thuja oil
Identification/Synonym: BU-REACH-Thuja oil
Substance type: UVCB substance
CAS No.: 91770-83-1
Appearance: colourless to slightly yellow, mobile liquid
Batch No.: BU201806
Purity: 100% wt UVCB Substance
Recommended storage: Keep container tightly closed and in a well-ventilated place. Use local conditions and general ventilation. Store at temperatures not exceeding 25°C. Keep cool. Protect from sunlight
Stability under storage conditions: Stable
Manufactured date: July 2018
Expiry date: July 2020

Method

Target gene:
Histidine and tryptophan for S. typhimurium and E. coli, respectively.
Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and prepared according to the method of Ames et al from male Sprague Dawley rats induced with a single dose of Aroclor 1254.
Test concentrations with justification for top dose:
In order to determine the toxicity of BU-Reach-Thuja oil to the test bacteria, an initial plate incorporation experiment was carried out using TA98 and WP2 uvrA in the presence and absence of S 9 mix. The dose levels used were 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 or 5 µL/plate. Based on the range finder observations, dose levels for the main experiment were chosen to include the guideline regulatory maximum dose level of 5 µL/plate.
Vehicle / solvent:
Dimethylsulphoxide (DMSO), a commonly used vehicle for which extensive historical control data is available; at concentrations up to 50 mg/mL.
Controls
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
other: 2-Aminoanthracene (2AA)
Details on test system and experimental conditions:
Salmonella typhimurium and Escherichia coli strains were obtained from Molecular Toxicology Incorporated, Boone, NC, USA (MolTox™) and used in the Bacterial Reverse Mutation test.
The Salmonella typhimurium and Escherichia coli strains are defective in DNA repair (Δuvr B , A- respectively); this confers extra sensitivity to DNA damage. The Salmonella strains have a defective lipopolysaccharide barrier on the cell wall (rfa-) which affords greater permeability to large molecules. The strains TA98 and TA100 also contain a plasmid (pKM101) which enhances error prone repair and confers ampicillin resistance.
The strains are tested routinely for:
1. Auxotrophic control of growth, (histidine or tryptophan dependence).
2. Deep rough mutation (rfa) of the bacterial cell wall (cell membrane permeability, crystal violet sensitivity).
3. Plasmid presence (Ampicillin resistance).
4. Absence of uvrA or uvrB repair enzyme systems (sensitivity to UV irradiation).
TA1535, TA100 and WP2 uvrA are reverted to prototrophy by base substitution mutagens. TA1537 and TA98 are reverted by frame shift mutagens; TA100 can also be affected by these on occasion.
Before the experiment, 25 mL quantities of nutrient broth were inoculated with 100 µL from a freshly thawed vial of the appropriate strain, and incubated overnight at 37 °C to give a pure culture at approximately 10^9 cells per mL.
Rationale for test conditions:
Range finder: As all the Salmonella Escherichia strains show a similar toxic response to most chemicals, only one strain of each species, typically TA98 and WP2 uvrA, was tested by plate incorporation in this experiment.
A range of concentrations up to a maximum of 5000 μg/plate was used, with duplicate plates at each concentration, both with and without S-9.
The plates examined for integrity of the background lawns after approximately 48 hours ± 1 hour incubation.

Experiment I: Mutation experiment, plate incorporation : The mutation experiment was carried out using fresh bacterial cultures for each experiment.
The test item was tested at sufficient concentration levels (table1) to provide at least 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. The following was added to sterile disposable tubes containing 2 mL of L-histidine: D-biotin (Salmonella strains) or L-tryptophan (Escherichia strain) supplemented top agar. The contents of each tube was mixed and added to a Petri dish containing minimal agar. When the top agar has set, the Petri dishes will be inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour.

Experiment II: Mutation experiment, preincubation: The mutation experiment was carried out using fresh bacterial cultures for each experiment.
The test item was tested at sufficient concentration levels (table2) to provide 5 analysable concentrations, with triplicate Petri dishes prepared for each control and experimental point. Each tube was fitted with a sterile cap and incubated at approximately 37 °C with shaking, for 20 minutes.
At the end of this period, 2 mL of top agar (L-histidine: D-biotin for Salmonella strains or L-tryptophan for Escherichia strain) was added to each tube, the contents mixed and added to Petri dishes containing minimal agar. When the top agar has set, the Petri dishes are inverted and incubated at 37 °C ± 3 °C for approximately 66 hours ± 1 hour.
Evaluation criteria:
A test was considered to be positive if the test item induced dose related statistically significant increases in numbers of revertants over the range tested and/or a reproducible increase at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system, compared with negative Controls scored in 2 separate experiments. Biological relevance of the result should be considered. Statistical significance should not be the only determining factor for a positive response.

A test was considered to be negative if the test item produced no greater increases in revertants, than may be expected from normal variation in the negative Control number of revertants, for any strain, in either experiment.
Statistics:
Mean, standard deviation (s.d.) and Dunnett's t-statistic (t) were calculated for each group and bacterial strain

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
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
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Plate incorporation method:
All negative Controls gave mean counts of spontaneous revertant colonies within expected historical negative Control ranges obtained in this laboratory.
All positive Controls induced marked increases in the number of revertant colonies and gave mean counts of induced revertant colonies which were within historical positive Control ranges. The response to the positive Controls demonstrates that the bacteria were sensitive to the mutagens and that the S 9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
BU-Reach-Thuja oil was analysed up to the limit of toxicity of 0.05 µL/plate in TA1535, TA1537 and TA98 in the absence of S-9 mix, 0.15 µL/plate in TA100 in the absence of S-9 mix, 0.5 µL/plate in WP2 uvrA in the absence of S-9 mix and in all Salmonella strains in the presence of S-9 mix and the regulatory maximum dose level of 5 µL/plate in WP2 uvrA in the presence of S-9 mix under plate incorporation conditions.
Precipitation was noted at 1.5 and 5 µL/plate in WP2 uvrA in the presence of S-9 mix. Toxicity was demonstrated by a slightly reduced background bacterial lawn at 0.15 µL/plate in TA100 in the absence of S-9 mix, at 0.5 µL/plate in WP2 uvrA in the absence of S-9 mix and all Salmonella strains in the presence of S-9 mix, at 1.5 and 5 µL/plate in WP2 urvA in the presence of S-9 mix and by a reduced background bacterial lawn at 0.15 µL/plate in TA1535, TA1537, TA98 in the absence of S-9 mix and 1.5 µL/plate in all Salmonella strains in the presence of S 9 mix.
There was also a marked reduction (< 0.5-fold) in the mean colony count at 0.05 µL/plate in TA98 in the absence of S-9 mix, 0.5 µL/plate in TA1537 in the presence of S-9 mix and 1.5 and 5 µL/plate in WP2 uvrA in the presence of S-9 mix, indicating toxicity of the test item to the bacteria.

No increases in revertant numbers greater than twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537 were observed at any dose level of BU-Reach-Thuja oil, in the presence or absence of S 9 mix, under plate incorporation conditions.

Pre incubation method:
All negative Controls gave mean counts of spontaneous revertants within expected ranges obtained in this laboratory.
All positive Controls gave mean counts of induced revertants within historical positive control ranges obtained in this laboratory. The response to the positive Controls indicated that the bacteria were sensitive to the mutagens and that the S 9 mix was able to metabolise the pro-mutagen, 2AA, to a mutagen.
BU-Reach-Thuja oil was tested up to the limit of toxicity of 0.15 µL/plate in all Salmonella strains in the absence of S-9 mix, 0.5 µL/plate in WP2 uvrA in the absence of S 9 mix and all Salmonella strains in the presence of S-9 mix and 1.5 µL/plate in WP2 uvrA in the presence of S-9 mix, under pre-incubation conditions.
Toxicity was demonstrated by a slightly reduced background bacterial lawn at 0.05 and 0.15 µL/plate in all Salmonella strains in the absence of S-9 mix, 0.5 µL/plate in all Salmonella strains in the presence of S-9 mix and WP2 uvrA in the absence of S-9 mix and at 1.5 µL/plate in WP2 uvrA in the presence of S-9 mix.

There was also a marked reduction (< 0.5-fold) in the mean colony count in TA1535 at 0.05 µL/plate in the absence of S-9 mix, indicating toxicity of the test item to the bacteria.

No increases in revertant numbers greater than twice the negative Control value for TA98, TA100 or WP2 uvrA or three times the negative Control value for TA1535 or TA1537 were observed in any strain at any dose level of BU-Reach-Thuja oil, in the presence or absence of S 9 mix, under pre-incubation conditions

Any other information on results incl. tables

Table 3: TOXICITY RANGE-FINDER

After approximately 48 hours incubation in the presence of BU-Reach-Thuja oil, the following effects were observed:

Strain

Dose
µL/plate

0

0.0015

0.005

0.015

0.05

0.15

0.5

1.5

5

TA98

- S-9

+

+

+

+

+

srl

rl

rl

nl

+ S-9

+

+

+

+

+

+

+

rl

rl

WP2uvrA

- S-9

+

+

+

+

+

+

srl

rl

rl

+ S-9

+

+

+

+

+

+

+

+

srl

                  +:      normal growth of background bacterial lawn
                srl:      slightly reduced background bacterial lawn
                  rl:      reduced background bacterial lawn

Applicant's summary and conclusion

Conclusions:
the test item showed no mutagenic potential in the Bacterial Reverse Mutation Assay under the conditions of this test.
Executive summary:

In a reverse gene mutation assay in bacteria, performed according to the OECD Guideline 471 and in compliance with GLP, Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100, and Escherichia coli strain WP2uvrA were exposed to the test item at the following concentrations:

- Experiment 1 - Plate Incorporation Method: 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 µL/ plate for all salmonella strains and at 0.0015, 0.005, 0.015, 0.05, 0.15, 0.5 µL/plate for WP2 uvrA without S9-mix and at 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 µL/plate for all salmonella strains and at 0.015, 0.05, 0.15, 0.5, 1.5, 5  µL/plate for WP2 uvrA with S9-mix

- Experiment 2 - Pre-Incubation Method: 0.0005, 0.0015, 0.005, 0.015, 0.05, 0.15 µL/ plate for all salmonella strains and at 0.005, 0.015, 0.05, 0.15, 0.5µL /plate for WP2 uvrAwithout S9-mix and at 0.005, 0.015, 0.05, 0.15, 0.5 µL/plate for all salmonella strains and at 0.005, 0.015, 0.05, 0.15, 0.5, 1.5 µL/plate for WP2 uvrA with S9-mix

Rat liver homogenate (10% liver S9 in standard co-factors) was used as a metabolizing system. Vehicle control, negative (untreated) and positive control groups were also included in mutagenicity tests.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range.  All of the positive control chemicals used in the test induced marked increases in the frequency of revertant colonies, both with or without metabolic activation.  Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

There were no increases in revertant numbers greater than twice the negative Control value for TA98, TA100 or WP2uvrAor greater than three times the negative Control value for TA1535 or TA1537 observed at any dose level of, in the presence or absence of S‑9 mix, under either plate incorporation or pre-incubation conditions

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