Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: non mutagenic (OECD 471, GLP, K, rel. 1)

Link to relevant study records

Referenceopen allclose all

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:
November 07, 2014 to January 12, 2015
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.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
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
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Program (inspected on March 12 to 14, 2014 / Signed on May 12, 2014)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine and tryptophan.
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:
10% S9: S9-mix from the livers of male rats treated with phenobarbitone/β-naphthoflavone (80/100 mg/kg bw/day by oral route).
Test concentrations with justification for top dose:
Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix
Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Tetrahydrofuran
- Justification for choice of solvent/vehicle: The test item was insoluble in sterile distilled water, dimethyl sulphoxide, dimethyl formamide and acetonitrile at 50 mg/mL, acetone at 100 mg/mL and tetrahydrofuran at 200 mg/mL in solubility checks performed in–house. The test item formed the best doseable suspension in tetrahydrofuran, therefore, this solvent was selected as the vehicle.
- Preparation of test materials: The test item was accurately weighed and approximate half-log dilutions prepared in tetrahydrofuran by mixing on a vortex mixer and sonication for 30 minutes at 40 °C on the day of each experiment. Tetrahydrofuran is toxic to the bacterial cells at and above 50 μL (0.05 mL), therefore all of the formulations were prepared at concentrations four times greater than required on Vogel-Bonner agar plates. To compensate, each formulation was dosed using 25 μL (0.025 mL) aliquots. Tetrahydrofuran is considered an acceptable vehicle for use in this test system (Maron et al., 1981). All formulations were used within four hours of preparation and were assumed to be stable for this period.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9-mix
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
Tetrahydrofuran
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:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Exposure duration: Plates were incubated at 37 °C ± 3 °C for approximately 48 hours

NUMBER OF REPLICATIONS: Triplicate plates per dose level.

DETERMINATION OF CYTOTOXICITY
- Method: The plates were viewed microscopically for evidence of thinning (toxicity).

OTHERS:
After incubation, the plates were assessed for numbers of revertant colonies using an automated colony counting system. Manual counts were performed at and above 500 μg/plate because of test item precipitation.
Evaluation criteria:
There are several criteria for determining a positive result. Any, one, or all of the following can be used to determine the overall result of the study:

- A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
- A reproducible increase at one or more concentrations.
- Biological relevance against in-house historical control ranges.
- Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
- Fold increases greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out of historical range response (Cariello and Piegorsch, 1996)).

A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgment about test item activity. Results of this type will be reported as equivocal.
Statistics:
Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
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:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.
- Other confounding effects: None

COMPARISON WITH HISTORICAL CONTROL DATA: All tester strain cultures exhibit a characteristic number of spontaneous revertants per plate in the vehicle and positive controls. The comparison was made with the historical control ranges for 2013 and 2014 of the corresponding Testing Laboratory.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- There was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the initial second experiment, the toxicity of the test item yielded results that differed from Experiment 1 (due to a change in test methodology from plate incorporation to pre-incubation) and consequently, there were an insufficient number of non-toxic dose levels for the majority strains in both the absence and presence of S9-mix (the data is not given in this report). Therefore, a repeat second experiment was performed employing additional dose levels and an expanded dose range. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology.

OTHERS:
- Prior to use, the master strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The test material formulation, amino acid supplemented top agar and S9-mix used in this experiment were shown to be sterile.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

See the attached document for information on tables of results

Conclusions:
negative with metabolic activation
negative without metabolic activation

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).
Executive summary:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP, Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle 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 was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant 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 in Experiment 1 (plate incorporation method). Similarly, no toxicologically 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 in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA- according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for reverse gene mutation endpoint.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2017-January 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Well conducted and well described study in accordance with GLP and OECD Guideline 487 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
29 July 2016
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
None
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: from the European Collection of Authenticated Cell Cultures (ECACC) and maintained at Sequani Limited
- Methods for maintenance in cell culture if applicable: The cell stocks were stored ≤ -140 °C and a fresh culture was established for a minimum of one week before starting an experiment.
- Normal (negative control) cell cycle time: calculated as 13.5 hours

MEDIA USED
- Type and identity of media : RPMI 1640 containing 10 % (v/v) foetal bovine serum (FBS), 100 units/mL penicillin and 100 µg/mL streptomycin (R10)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
(10%) S-9 mix, a liver post-mitochondrial fraction derived from the livers of Aroclor 1254 treated rats
Test concentrations with justification for top dose:
Range-finder experiment: 0, 0.65, 2, 6.5, 20, 65, 200, 650 or 2000 µg/mL (treatment times were 3 hours in the presence of S9 mix and 3 hours and 27 hours in the absence of S9, serum content during treatment: 10% (v/v) FBS)

First experiment (main experiment): Treatment time: 3 hours, Recovery period: 41 hours and, Harvest time (hours after treatment began): 44 hours
in presence of S9-mix (10%): 0, 20, 65, 100, 150, 175, 200, 225 and 250 µg/mL.
in absence of S9-mix: 0, 5, 20, 65, 80, 95, 110, 125, 140 and 155 µg/mL.

Second experiment (main experiment): Treatment time: 27 hours, Recovery period: none and, Harvest time (hours after treatment began): 27 hours, serum content during treatment: 10% (v/v) FBS
in absence of S9-mix: 0, 2, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- The test item was formulated on the day of use as a solution in dimethylsulphoxide (DMSO) at concentrations up to 200 mg/mL.
- Preparations of CISTUS CONCRETE were made at 100 times the desired final concentration and added to the culture medium at 1 % (v/v). Preparations of CISTUS CONCRETE were made immediately before use.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 1% (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9-mix (10%v/v), 2.0 and 2.5 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 1% (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9-mix, 0.1 µg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO 1% (v/v)
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: vinblastine
Remarks:
without S9-mix, 0.0005 and 0.00075 µg/mL
Details on test system and experimental conditions:
RANGE-FINDER EXPERIMENT
Following the approximately 24-hour incubation period after culture establishment, single cultures were set up for each experimental point containing approximately 30 x 10^4 cells/mL. A metabolic activation system (S-9) was added to the appropriate cultures. Cultures were then dosed at 1 % final volume with dimethylsulphoxide (negative Control), or solutions of the test item and returned to the incubator at 37 °C ± 3 °C and approximately 5 % CO2. At the completion of treatment, the treatment media from the 3-hour treatments were removed and the cells washed with phosphate buffered saline (PBS). Fresh R10 was added and the cultures were returned to the incubator until harvesting. Cultures treated in the absence of S 9 mix for 27 hours were harvested at the end of the treatment period.
Treatment times were 3 hours in the presence of S-9 mix and 3 hours and 27 hours in the absence of S 9.
Osmolality and pH measurements were taken for the negative Control and preparations of CISTUS CONCRETE in treatment medium, without serum and without metabolic activation.
The Relative Increase in Cell Count (RICC) was calculated for each culture using a Countess II automated viable cell counter.

FIRST EXPERIMENT
Bulk cell cultures from a working cell stock were established and incubated at 37 °C ± 3 °C and approximately 5 % CO2 for approximately 24 hours before treatment. Individual aliquots of bulk culture were added to tubes to give a concentration of 30 x10^4 cells/mL.
The treatment solutions contained dimethylsulphoxide (negative Controls), solutions of the test item at the appropriate concentrations, or positive Control chemicals as appropriate. Treatment in the presence and absence of S 9 mix was for 3 hours and was carried out in serum-free medium. S 9 mix (10 % v/v) was added to the cultures requiring metabolic activation at 10 % of final volume. All cultures were then returned to the incubator at 37 °C ± 3 °C and approximately 5 % CO2.
Sufficient cultures were set-up to provide duplicate cultures (designated Cultures A and B) for each treatment.
At the completion of treatment, the media were removed and the cells washed with PBS. The supernatant was removed and fresh R10 medium was then added and the cultures were returned to the incubator until harvesting at approximately 44 hours after start of treatment. At harvest, cells in each culture were counted using a Countess II automated viable cell counter and toxicity was measured by calculation of the RICC.

SECOND EXPERIEMENT
This experiment was conducted similarly to the first experiment, but used a set of cultures treated for 27 hours (equivalent to 1.5 – 2 cell cycles) in the absence of S 9 mix and harvested at the end of treatment. After dosing, all cultures were returned to the incubator at 37 °C ± 3 °C.
Cultures were harvested at the end of the treatment time and toxicity was measured by calculation of the RICC.
Evaluation criteria:
The effect of the test item was evaluated using two statistical approaches:
1. A linear trend test, built as a linear combination of the logs of the proportions of micronuclei, for dose-response relationship investigation.
2. Pairwise comparisons to the negative Control, using the one-sided Likelihood Ratio test, to detect the doses significantly different from the Control.
Statistics:
Data were analysed using a Poisson model with a log link function using GENMOD procedure in SAS.

All the tests were one-sided, a decreasing trend being not of interest. In case of a decrease and calculated p value < 0.5, the final p value was estimated as 1-[p value].

Over-dispersion of the data was estimated by the deviance of the model divided by the associated degrees of freedom. Where over-dispersion was detected (dispersion parameter >1), a correction was applied on the model.

Probability values of less than 5% were regarded as providing sufficient evidence to reject the null hypothesis. Statistical significance therefore was identified at the p<0.05 level and presented as follows:

NA (Not applicable) for the negative Control group,
NS = non significant at 5 % level,
* = significant at 5 % level,
** = significant at 1 % level.
*** = significant at 0.1 % level

The positive Control group was not included in the model with the treated groups, but was calculated separately in a model that contained only the two Control groups.
Key result
Species / strain:
human lymphoblastoid cells (TK6)
Remarks:
3-hour exposure
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:
human lymphoblastoid cells (TK6)
Remarks:
27-hour exposure
Metabolic activation:
without
Genotoxicity:
positive
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
There were no changes in the osmolality or pH of the treatment media, at the test concentration assessed for micronuclei, which were considered capable of producing artifactual aberrations due to the physical environment of the test cells.

RANGE-FINDER TEST:
Based on the RICC observed in the range-finder, the test concentrations selected for the first experiment in the presence and absence of S 9 mix were as follows:
- 20, 65, 100, 150, 175, 200, 225 or 250 µg/mL for 3-hour treatment in the presence of S-9 mix and 5, 20, 65, 80, 95, 110, 125, 140 or 155 µg/mL for 3-hour treatment in the absence of S-9 mix.
- For 27-hour treatment in the absence of S-9 mix the test concentrations selected were 2, 20, 25, 30, 35, 40, 45, 50, 55 or 60 µg/mL.

FIRST EXPERIMENT
All negative Controls gave mean counts within expected ranges.
The positive Control chemicals caused statistically significant increases in the number of MN scored. This showed the cells to be sensitive to the effects of known clastogens and that the S9 metabolic activation system was able to activate a pro-mutagen to a mutagen.
In the absence of S-9 mix, the highest treatment level analysed was 140 µg/mL, which had a RICC of 44 %.
The highest treatment level analysed in the presence of S 9 mix was 225 µg/mL, which had a RICC of 40 %.
There were no statistically significant increases in % MN at any dose level of CISTUS CONCRETE in the presence or absence of S-9 mix, with a 3-hour treatment.

SECOND EXPERIMENT
All negative Controls gave mean counts within expected ranges.
The positive Control chemical caused statistically significant increases in the number of MN scored. This showed the cells to be sensitive to the effects of a known aneugen.
The highest treatment level analysed was 45 µg/mL, which had a RICC of 42 %.
There was a statistically significant increase in % MN at 2 µg/mL, 20 µg/mL, 35 µg/mL and 45 µg/mL in the absence of S-9 mix after a 27-hour treatment. The increase at 2 µg/mL was within historical control data; however, the increases in mean % MN at 20 µg/mL and both mean and individual counts at 35 µg/mL and 45 µg/mL were outside the historical control range. As the % MN values obtained for the 20 µg/mL culture were inconsistent, it was excluded from the data for the trend analysis. The trend analysis was statistically significant, indicating a dose response.

Details of relative RICC and % MN are reported in section "Attached background material".

Table 7.6.1/1: pH and osmolality

Dose1 of Cistus concrete (µg/mL) pH  Osmolarity (mOsm/kg) 
 0  7.26  445
 0.65  7.28  450
 2  7.24  451
 6.5  7.26  451
 20  7.27  449
 65  7.26  444
 200  7.29  446
 650  7.29  432
 2000  7.28  379

1 = 3 -hour treatment medium without metabolic activation or serum

  

Conclusions:
With or without S9-mix with a 3-hour treatment under the test conditions: negative
Without S9-mix with a 27-hour treatment under the test conditions: positive

CISTUS CONCRETE was considered to be either clastogenic or aneugenic under the conditions of this test, requiring no metabolic activation to induce such effects in TK6 cells.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, human lymphoblastoid cells (TK6) were exposed to test item, Cistus concrete, in the presence and absence of a metabolic activation system. Three exposure conditions in two experiments were used for the study using a 3‑hour exposure in the presence and absence of a standard metabolizing system (S9 at a 10% final concentration) and a 27 -hour exposure in the absence of metabolic activation. 

The dose levels used in the Main Experiment were selected using data from the range-finder experiment based on the RICC observed in the range-finder. The dose levels selected for the Main Test were as follows:

First experiment (main experiment): Treatment time: 3 hours, Recovery period: 41 hours and, Harvest time (hours after treatment began): 44 hours

- in presence of S9-mix (10%): 0, 20, 65, 100, 150, 175, 200, 225 and 250 µg/mL.

- in absence of S9-mix: 0, 5, 20, 65, 80, 95, 110, 125, 140 and 155 µg/mL.

Second experiment (main experiment): Treatment time: 27 hours, Recovery period: none and, Harvest time (hours after treatment began): 27 hours, serum content during treatment: 10% (v/v) FBS

- in absence of S9-mix: 0, 2, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL.

 

All negative Controls gave mean counts within expected ranges. The positive Control chemicals consistently caused statistically significant increases in the number of micronuclei (MN) scored. This showed the cells to be sensitive to the effects of known clastogens and an aneugen and that the S‑9 metabolic activation system was able to activate a pro-mutagen to a mutagen.


There was a statistically significant increase in % micronucleated (MN) cells at 2 µg/mL, 20 µg/mL, 35 µg/mL and 45 µg/mL in the absence of S-9 mix after a 27-hour treatment. The increase at 2 µg/mL was within the historical control data range; however, the increases in mean % MN at 20 µg/mL and both mean and individual counts at 35 µg/mL and 45 µg/mL were outside the historical control range. As the % MN values obtained for the 20 µg/mL culture were inconsistent, it was excluded from the data for the trend analysis. The trend analysis was statistically significant, indicating a dose response.

 

There were no statistically significant increases in % MN in either the presence or absence of S-9 mix after a 3-hour treatment period.

Under the test conditions, Cistus concrete was considered to be either clastogenic or aneugenic under the conditions of this test, requiring no metabolic activation to induce such effects in TK6 cells.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
March - June 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
hprt locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
originated from Dr Donald Clive, Burroughs Wellcome Co.
MEDIA USED
- Type and identity of media including CO2 concentration:
RPMI A: Horse serum (heat inactivated, 0 % v/v). Penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 10: Horse serum (heat inactivated, 10 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL), Sodium pyruvate acid (0.2 mg/mL) and pluronic (0.5 mg/mL)
RPMI 20: Horse serum (heat inactivated, 20 % v/v), penicillin (100 units/mL), streptomycin (100 μg/mL), amphotericin B (2.5 μg/mL) and Sodium pyruvate acid (0.2 mg/mL)
RPMI 5 consisted of RPMI 10 diluted with RPMI A [prepared as RPMI 10 but with no serum added] to give a final concentration of 5% serum

CELL CULTURES
- For each experiment, at least one vial was thawed rapidly, the cells diluted in RPMI 10 and incubated at 37 ± 1 °C. When the cells were growing well, subcultures were established in an appropriate number of flasks.
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: Yes
- Periodically 'cleansed' against high spontaneous background: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
2 % S9 (final concentration); S9 fraction was prepared from liver homogenates of male Sprague Dawley rats treated with Aroclor 1254.
Test concentrations with justification for top dose:
Range-Finder Experiment: 15.63, 31.25; 62.5; 125; 250; 500; 1000; and 2000 μg/mL, with and without S9 mix
Justification: A maximum concentration of 2000 μg/mL was therefore selected for the cytotoxicity Range-Finder Experiment in order that treatments were performed up to a precipitating treatment concentration (OECD, 2016).

Mutation Experiment 1 :
Without S9: 25;;50; 75; 100; 125; 150; 175; 200; 225 and 250 μg/mL
With S9: 25;;50; 75; 100; 150; 200; 250; 275; 300; 350; 400; 450 and 500 μg/mL
Justification: Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Mutation Experiment 2 :
Without S9: 25;;50; 75; 100; 125; 150; 160; 170; 180; 190; 200 and 250 μg/mL
With S9: 25;;50; 75; 100; 150; 200; 250; 260; 270; 280; 290 and 300 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: Preliminary solubility data indicated ethanol was the most suitable vehicle. Cistus concrete formed a suitable, homogeneous suspension at concentrations up to approximately 200 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
ethanol diluted 100-fold in the treatment medium.
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
benzo(a)pyrene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; RPMI 1640 media supplied containing L-glutamine and HEPES

- Cell density at seeding:
Cytotoxicity Range-Finder Experiment: Cell concentrations were adjusted to 8 cells/mL and, for each concentration, 0.2 mL was plated into each well of a 96-well microtitre plate for determination of relative survival.
Mutation Experiment: At least 10^7 cells in a volume of 18.8 mL of RPMI 5 were placed in a series of sterile disposable 50 mL centrifuge tubes.

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 14 days
- Plating for Survival: 7 days
- Plating for viability: 8
- Plating for 6TG resistance: 12 days
- All incubations were performed at 37 ± 1 °C in a humidified incubator gassed with 5 ± 1 % v/v CO2 in air.

SELECTION AGENT (mutation assays): 6-thioguanine (6TG) at a final concentration of 15 μg/mL

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single cultures/dose for test item and vehicle control
- Main test: Two cultures for vehicle control and test item; single culture for positive control

NUMBER OF CELLS EVALUATED: 1.6, 1.6 and 20000 cells per well plated for survival, viability and 6TG resistance respectively.

DETERMINATION OF CYTOTOXICITY
- Method: Percentage Relative Survival
Cloning efficiency (CE) = P / No of cells plated per well; and as an average of 1.6 cells/well were plated on all survival and viability plates, CE = P/1.6.
Percentage relative survival (% RS) = [CE (test)/CE (control)] x 100.
Adjusted % RS = % RS x (Post-treatment cell concentration for test article treatment / Post-treatment cell concentration for vehicle control)

- OTHER:
Mutant Frequency (MF) per 10^6 viable cells for each set of plates was calculated as: MF = [CE (mutant)/CE (viable)] x 10^6.
Evaluation criteria:
For valid data, the test article was considered to be mutagenic in this assay if:
1. The MF at one or more concentrations was significantly greater than that of the negative control (p≤0.05)
2. There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
3. If both of the above criteria were fulfilled, the results should exceed the upper limit of the last 20 studies in the historical negative control database (mean MF +/ 2 standard deviations.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.
Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines (Robinson et al., 1990). The control log mutant frequency (LMF) was compared with the LMF from each treatment concentration and the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
not biologically relevant
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration tested, compared to the concurrent vehicle controls.
- Precipitation: Yes
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 2000 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations in the absence and presence of S-9 (250 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (500 to 2000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentrations to give 10% RS were 125 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S 9, which gave 70% and 31% RS, respectively

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g
. 95%)
The historical control ranges for the last 20 experiments performed in this laboratory are as follows:
- Negative (solvent/vehicle) historical control data:
Vehicle Controls
In the absence of S-9
Mean: 4.52 mutants per 10^6 viable cells, Range* = 0.80 to 8.24 mutants per 10^6 viable cells.
In the presence of S-9
Mean: 5.26 mutants per 10^6 viable cells, Range* = 1.30 to 9.22 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.
- Positive historical control data:
NQO 0.15 μg/mL in the absence of S-9:
Mean: 43.04 mutants per 10^6 viable cells, Range* = 1.08 to 84.99 mutants per 10^6 viable cells.
NQO 0.2 μg/mL in the absence of S-9:
Mean: 56.23 mutants per 10^6 viable cells, Range* = 7.85 to 104.62 mutants per 10^6 viable cells.
B[a]P 2 μg/mL in the presence of S-9:
Mean: 28.32 mutants per 10^6 viable cells, Range* = 0 to 58.85 mutants per 10^6 viable cells.
B[a]P 3 μg/mL in the presence of S-9:
Mean: 42.66 mutants per 10^6 viable cells, Range* = 8.06 to 77.25 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

MUTATION EXPERIMENT
-In Mutation Experiment 1, twelve concentrations ranging from 25 to 350 µg/mL in the absence of S-9 and 25 to 500 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 350 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 500 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence of S-9 (250 to 350 µg/mL) and at the high concentration only (500 µg/mL) in the presence of S-9. Therefore, in the absence of S-9 the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentrations discarded. Seven days after treatment, the highest three concentrations (200 to 250 µg/mL) in the absence of S-9 and the highest four concentrations (350 to 500 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 175 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 16% and 10% RS, respectively
- In Mutation Experiment 2, twelve concentrations ranging from 25 to 250 µg/mL in the absence of S-9 and 25 to 350 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 250 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 350 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the high concentration only (250 µg/mL) in the absence of S-9 and at the highest two concentrations (300 and 350 µg/mL) in the presence of S-9. Therefore, in the presence of S-9, the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentration discarded. Seven days after treatment, concentrations of 25 and 75 µg/mL in the absence of S-9 and 25 µg/mL in the presence of S-9 were not selected to determine viability and 6TG resistance as sufficient non-toxic concentrations were available. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 250 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 12% and 35% RS, respectively

Table 7.6.1/1: Range-finder experiment - 3 h treatment in the absence and presence of S-9

 

Concentration (μg/mL)

%RS (Percent Relative Survival)

3 hour treatment –S-9

3 hour treatment +S-9

0

100

100

15.63

94

84

31.25

112

99

62.5

127

97

125

70

66

250 P

1

31

500 P, PP

0

0

P Precipitation noted at time of treatment

PP Precipitation noted at end of treatment incubation period

 

Table 7.6.1/2: Mutation experiment 1- 3 h treatment in the absence and presence of S-9

 

3 hour treatment –S-9

3 hour treatment +S-9

Concentration (μg/mL)

%RS (Percent Relative Survival)

MF §

Concentration (μg/mL)

%RS (Percent Relative Survival)

MF §

0

100

5.27

0

100

4.76

25

92

7.88 NS

25

86

6.82 NS

50

75

8.12 NS

50

87

9.47*

75

74

9.47*

100

59

6.28 NS

100

73

7.70NS

150 P

67

9.96*

125

61

6.14 NS

200 P

63

8.23 NS

 150

 43

5.98 NS 

250 P

47

6.67 NS

175

16

9.16 NS

275 P

28

3.55 NS

300 P

10

15.28*

 

NQO 0.15

71 63.70  B[a]P2  61  52.14
 NQO 0.2  49  84.23  B[a]3  59  75.05

Linear trend: Not Significant (negative trend) – 3 hour absence of S-9

Linear trend: Not Significant – 3 hour presence of S-9

§                          6‑TG resistant mutants/106viable cells 7 days after treatment

%RS                    Percent relative survival adjusted by post treatment cell counts

P                          Precipitation noted at time of treatment

NS                       Not significant

*                           Comparison of each treatment with control: Dunnett's test (one-sided), significant at  5% level

Table 7.6.1/3: Mutation experiment 2- 3 h treatment in the absence and presence of S-9

3 Hour Treatment –S-9

3 Hour Treatment +S-9

Concentration

%RS

MF §

Concentration

%RS

MF §

µg/mL

 

 

µg/mL

 

 

0

100

3.45

0

100

4.06

UTC

82

2.06

UTC

90

2.21

50

94

2.93 NS

50

97

4.51 NS

100

69

3.92 NS

100

87

7.68*

125 P

72

4.06 NS

150 P 

70

4.35 NS

150 P

63

3.92 NS

200 P

60

3.97 NS

160 P

56

6.69*

250 P

58

3.15 NS

170 P

48

7.25*

260 P

51

4.88 NS

180 P

53

4.48 NS

270 P

47

3.86 NS

190 P

54

2.38 NS

280 P

45

3.29 NS

200 P

41

2.47 NS

290 P

40

3.24 NS

250 P PP

12

9.71*

300 P PP

35

3.40 NS

NQO 0.15

51

66.14

B[a]P 2

81

40.63

NQO 0.20

42

83.36

B[a]P 3

52

55.07

Linear trend tests on mutant frequency - S-9: Not significant

Linear trend tests on mutant frequency - S-9: Not significant, negative trend

 

UTC                    Untreated control

§                          6‑TG resistant mutants/106 viable cells 7 days after treatment

%RS                    Percent relative survival adjusted by post treatment cell counts

P                          Precipitation noted at time of treatment

NS                       Not significant

*                           Comparison of each treatment with control: Dunnett's test (one-sided), significant at 5% level

Conclusions:
Under the test conditions, test item did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of toxicity, in the absence and presence of a rat liver metabolic activation system.
Executive summary:

In a nin vitro mammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item for 3 h at the following concentrations:

Range-Finder Experiment: 15.63, 31.25; 62.5; 125; 250; 500; 1000;  and 2000 μg/mL, with and without S9 mix

Mutation Experiment 1 :

Without S9: 25; 50; 75; 100; 125; 150; 175; 200; 225 and 250 μg/mL

With S9: 25; 50; 75; 100; 150; 200; 250; 275; 300; 350; 400; 450 and 500 μg/mL

Mutation Experiment 2 :

Without S9: 25; 50; 75; 100; 125; 150; 160; 170; 180; 190; 200 and 250 μg/mL

With S9: 25; 50; 75; 100; 150; 200; 250; 260; 270; 280; 290 and 300 μg/mL

Vehicle, negative and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 2000 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations in the absence and presence of S-9 (250 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (500 to 2000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentrations to give 10% RS were 125 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S 9, which gave 70% and 31% RS, respectively

 

-In Mutation Experiment 1, twelve concentrations ranging from 25 to 350 µg/mL in the absence of S-9 and 25 to 500 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 350 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 500 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence of S-9 (250 to 350 µg/mL) and at the high concentration only (500 µg/mL) in the presence of S-9. Therefore, in the absence of S-9 the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentrations discarded. Seven days after treatment, the highest three concentrations (200 to 250 µg/mL) in the absence of S-9 and the highest four concentrations (350 to 500 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 175 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 16% and 10% RS, respectively

- In Mutation Experiment 2, twelve concentrations ranging from 25 to 250 µg/mL in the absence of S-9 and 25 to 350 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 250 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 350 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the high concentration only (250 µg/mL) in the absence of S-9 and at the highest two concentrations (300 and 350 µg/mL) in the presence of S-9. Therefore, in the presence of S-9, the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentration discarded. Seven days after treatment, concentrations of 25 and 75 µg/mL in the absence of S-9 and 25 µg/mL in the presence of S-9 were not selected to determine viability and 6TG resistance as sufficient non-toxic concentrations were available. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 250 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 12% and 35% RS, respectively

Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

 

In the absence of S-9 inExperiment 1, a statistically significant (P0.05) increase in mutation frequency (MF) over the concurrent control was observed at 75 µg/mL. Although there was no accompanying statistically significant linear trend, the mutation frequency at 75 µg/mL (9.47per 106viable cells) did exceed the mean historical vehicle control MF for the last 20 studies +2SD (8.68 per 106viable cells). It was observed however that the untreated control also exhibited a MF of 9.41 per

106viable cells and therefore, the statistical increase in MF at 75 µg/mL was considered to be of no biological relevance.

In the presence of S-9inExperiment 1, statistically significant (P0.05) increases in MF over the concurrent control were observed at 50, 150 and 300 µg/mL. However, there was no accompanying statistically significant linear trend. The MF at 50, 150 and 300 µg/mL were 9.47, 9.96 and 15.28per 106viable cells respectively and all exceeded the mean historical vehicle control MF for the last 20 studies +2SD (9.08 per 106viable cells).

A second experiment in the absence and presence of S-9 was performed in order to assess the reproducibility of the results observed in Experiment 1.

In the absence of S-9 inExperiment 2, statistically significant (P0.05) increases in MF over the concurrent control were observed at 160, 170 and 250 µg/mL. Although there was no accompanying statistically significant linear trend, the MF at 250 µg/mL (9.71per 106viable cells) did also exceed the mean historical vehicle control MF for the last 20 experiments +2SD (8.74 per 106viable cells).

In the presence of S-9 inExperiment 2, there was a statistically significant (P0.05) increase in MF over the concurrent control observed at 100 µg/mL. However, there was no accompanying statistically significant linear trend and none of the MF values exceeded the mean historical vehicle control MF for the last 20 experiments +2SD (9.29 per 106viable cells).

The data of both experiments showed sporadic increases in MF after exposure to Cistus concrete, both in the absence and presence of S-9. However, the effects were poorly reproducible between dose concentrations and experiments and there was no clear evidence of a concentration related-response, except at dose concentrations at the upper limit of acceptable toxicity. Therefore, the data do not fulfil the criteria for a clear positive result as defined in OECD Guideline 476 and the increases in MF were considered to be spurious.

It is concluded that the observed increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells after exposure to Cistus concrete, tested to a toxic and/or precipitating concentration for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), were not reproducible and not dose related under the experimental conditions employed, and consequently were considered to be not biologically relevant. 

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

An in vivo micronucleus test is on-going, combined with the preliminary study of the OECD TG 422 study, in order to confirm the relevance of the results obtained in the in vitro micronucleus test.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose:
reference to same study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Biolandes / L1117/1
- Appearance: Brown-red, solid
- Expiration date of the lot/batch: november 2019

STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material:
Species:
rat
Strain:
other: Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Age at study initiation: Males: Approximately 71 days old; Females: Approximately 85 days old.
- Weight at study initiation: Males: 339-379 g; Females: 241-302 g
- Housing: Solid (polycarbonate) bottom cages were used during the acclimatization, pre-pairing, gestation, littering and lactation periods. Grid bottomed polypropylene cages were used during pairing; Cages comprised of a polycarbonate body with a stainless steel mesh lid.
- Number of animals per cage: Pre-pairing: up to five animals of one sex; Pairing one: male and one female; Males after mating: up to five animals; Gestation: one female; Lactation: one female + litter
- Diet: SDS VRF1 Certified powdered diet, ad libitum (removed overnight before blood sampling for hematology and blood chemistry investigations and during urine collection)
- Water: Potable water from the public supply via polycarbonate bottles with sipper tubes, ad libitum (removed overnight during urine collection)
- Acclimation period: Females: six days prior to the commencement of estrous cycle evaluation; Males: six days prior to the commencement of treatment.

ENVIRONMENTAL CONDITIONS
- Temperature: 20-24 °C
- Humidity: 40-70 %
- Air changes: Filtered fresh air which was passed to atmosphere and not recirculated.
- Photoperiod: Artificial lighting, 12 h light : 12 h dark
- Environmental Enrichment
Aspen chew block: A soft white untreated wood block; provided to each cage throughout the study (except during pairing and lactation) and replaced when necessary.
Plastic shelter: Provided to each cage throughout the study (except during pairing and lactation) and replaced at the same time as the cages.

IN-LIFE DATES:
Route of administration:
oral: feed
Vehicle:
Corn oil
Details on exposure:
DIET PREPARATION
- Diet: SDS VRF1 Certified powdered diet
- Correction factor: A correction factor was not required.
- Stabilizer: Corn oil (test material to corn oil ratio 5:1).
- Method of preparation: The test substance was incorporated into the diet to provide the required concentrations by initial preparation of a premix. On each occasion of the preparation of the premix, the required amount of test substance and corn oil were weighed into a suitable container. An amount of sieved diet that approximately equalled the weight of test substance was added and the mixture stirred together. A further amount of sieved diet (approximately equal to the weight of this mixture) was added and it was stirred well. This doubling up process was repeated until half of the final weight of the premix was achieved. This mixture was then ground using a mechanical grinder after which it was made up to the final weight of the premix with plain diet. This premix was mixed in a Turbula mixer for 100 cycles to ensure the test substance was dispersed in the diet. Aliquots of the premix were then diluted with further quantities of plain diet to produce the required dietary concentrations. Each batch of treated diet was mixed for a further 100 cycles in a Turbula mixer.
For the control diet, an amount of diet was added directly to the corn oil and then prepared as indicated for the premix.
- Frequency of preparation: Weekly.
- Storage of formulation: Deep-frozen (nominally -30 to -10 °C) until the day before use. Formulations were used within 28 h of removal from the freezer.
Duration of treatment / exposure:
21 days
Frequency of treatment:
Continuous
Post exposure period:
None
Dose / conc.:
15 000 ppm
Dose / conc.:
0 ppm
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
Five bone marrow smears previously prepared from rats administered cyclophosphamide (Envigo Study Number ) will be stained and coded along with the bone marrow smears prepared in this study.
Tissues and cell types examined:
Bone marrow smears
Details of tissue and slide preparation:
- Preparation of bone marrow smears
The following will be performed by Necropsy personnel: On completion of the treatment period, the first five surviving Toxicity phase males and females from each group will be euthanised by carbon dioxide inhalation, followed by exsanguination. One femur will be dissected from each animal and the proximal head removed.
The following will be performed by Cell and Molecular Sciences personnel: The contents of the femur from each animal will be flushed out and pooled in a total volume of 3 mL pre-filtered foetal calf serum. The cell suspension will be sedimented by centrifugation, the supernatant will be discarded and the cells will be re-suspended in a small volume of fresh foetal calf serum. A small drop of the suspension will be transferred to a glass microscope slide and a smear prepared. At least four smears will be prepared from each animal. At least one smear from each animal will be stained and examined, the remaining smears being held temporarily in reserve in case of technical problems with the first smear.
- Fixation and staining of slides
Fixed for a minimum of 10 minutes in methanol and allowed to air-dry. Rinsed in purified water. Stained in acridine orange solution (0.0125 mg/mL using purified water) for 4 minutes. Washed in purified water for 5 minutes. Rinsed in cold tap water for 2 minutes. Stored at room temperature until required. Immediately prior to scoring, slides are wet mounted with glass coverslips using purified water.
- Microscopic examination
At least 5 slides (prepared in a separate study (Envigo Study Number )) from animals treated with Cyclophosphamide, a well characterised clastogen, will be stained and coded along with the bone marrow smears prepared from this study.
Coded slides will be examined by fluorescence microscopy and 4000 polychromatic erythrocytes per animal will be examined for the presence of micronuclei. Usually only one smear is examined per animal, any remaining smears being held temporarily in reserve in case of technical problems with the first smear.
The proportion of polychromatic erythrocytes will be assessed by examination of a total of at least 1000 erythrocytes per animal and the number of micronucleated normochromatic erythrocytes will be recorded.
Evaluation criteria:
Providing that all acceptability criteria are fulfilled, the test article is considered clearly negative if, in all experimental conditions examined:
a) None of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) There is no dose-related increase when evaluated by an appropriate trend test,
c) All results are inside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits),
and d) Bone marrow exposure to the test substance(s) occurred.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly positive if:
a) At least one of the treatment groups exhibits a statistically significant increase in the frequency of micronucleated polychromatic erythrocytes compared with the concurrent negative control,
b) This increase is dose-related when evaluated with an appropriate trend test,
and c) Any of these results are outside the distribution of the historical negative control data (e.g. Poisson-based 95% control limits).
When conducting a dose-response analysis, at least three treated dose groups should be analysed. Statistical tests should use the animal as the experimental unit. Positive results in the micronucleus test indicate that a test chemical induces micronuclei, which are the result of chromosomal damage or damage to the mitotic apparatus in the erythroblasts of the test species.
Statistics:
Analysis of data: The results obtained for each treatment group will be compared with the results obtained for the vehicle control group using non-parametric statistical methods. Analysis will be performed for the study using data from all animals that survive to scheduled termination to maximise the power of statistical analysis. If less than five animals per group survive to scheduled termination the study may be repeated.
For the proportion of polychromatic erythrocytes at 24 hours, an asymptotic one-tailed Jonckheere’s test for trend (Jonckheere 1954) with “step-down” will be used on Groups 1 to 4 for a decrease from control. Exact one-tailed Wilcoxon pairwise tests (Wilcoxon 1945), for a decrease from control, will also be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides).
For incidences of micronucleated polychromatic erythrocytes at 24 hours, an exact one-tailed Linear-by-Linear association test (Cytel 1995) with “step-down” will be used on Groups 1 to 4 for an increase from control. Also, exact one-tailed pairwise Permutation tests (Cytel 1995), for an increase from control, will be carried out on Group 1 (control) versus Groups 2, 3, 4 and 5 (positive control slides). Statistical significance will be declared at the 5% level for all tests.
Quasar (version 1.4) and/or SAS (version 9.1.3) and/or StatXact 3 in-house statistical analysis packages may be used for statistical analysis.
Sex:
male/female
Genotoxicity:
not determined
Remarks:
on-going study
Toxicity:
not specified
Remarks:
on-going study
Vehicle controls validity:
not specified
Remarks:
on-going study
Negative controls validity:
not examined
Positive controls validity:
not specified
Remarks:
on-going study

Additional information

Ames test:

In a reverse gene mutation assay performed according to the OECD test guideline No. 471 and in compliance with GLP,Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA- were exposed to test material both in the presence and absence of metabolic activation system (10% liver S9 in standard co-factors).

Test for Mutagenicity (Experiment 1) – Plate Incorporation Method: 1.5, 5, 15, 50, 150, 500, 1500 and 5000 µg/plate in all strains with and without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.015, 0.05, 0.15, 0.5, 1.5, 5, 15 and 50 μg/plate in TA 100, TA 1535 and TA 1537 strains without S9-mix; 0.15, 0.5, 1.5, 5, 15, 50 and 150 μg/plate in TA 98 and WP2uvrA strains without S9-mix

Test for Mutagenicity (Experiment 2) – Pre-Incubation Method: 0.15, 0.5, 1.5, 5, 15, 50, 150 and 500 μg/plate in TA 100, TA 1535 and TA 1537 strains with S9-mix; 15, 50, 150, 500, 1500 and 5000 μg/plate in TA 98 and WP2uvrA strains with S9-mix

Negative, vehicle (tetrahydrofuran) and positive control groups were also included in mutagenicity tests.

The vehicle 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 was no visible reduction in the growth of the bacterial background lawns at any dose level, either in the presence or absence of S9-mix, in the first mutation test (plate incorporation method) and consequently the same maximum dose level was originally selected for the second mutation test. In the second mutation test (pre-incubation method) the test item induced a stronger toxic response with weakened bacterial background lawns initially noted in the absence of S9-mix from 5 μg/plate (TA1535), 15 μg/plate (TA1537), 50 μg/plate (TA100) and 150 μg/plate (TA98 and WP2uvrA). In the presence of S9-mix weakened lawns were initially noted at 50 μg/plate (TA100 and TA1537) and 500 μg/plate (TA1535). No toxicity was noted to TA98 or WP2uvrA at any test item dose level in the presence of S9-mix. The sensitivity of the bacterial tester strains to the toxicity of the test item varied slightly between strain type, exposures with or without S9-mix and experimental methodology. A test item precipitate (greasy and particulate in appearance) was noted at and above 500 μg/plate; this observation did not prevent the scoring of revertant colonies.

 

There were no toxicologically significant 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 in Experiment 1 (plate incorporation method). Similarly, no toxicologically 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 in the second mutation test (pre-incubation method). Small, statistically significant increases in revertant colony frequency were observed in the first Experiment at 15 μg/plate (TA98 dosed in the absence of S9-mix) and in the second experiment at 15 μg/plate (TA100 dosed in the presence of S9-mix). These increases were considered to be of no biological relevance because there was no evidence of a dose-response relationship or reproducibility. Furthermore, the individual revertant counts at the statistically significant dose levels were within the in-house historical untreated/vehicle control range for each tester strain and the maximum fold increase was only 1.5 times the concurrent vehicle controls.

 

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coliWP2 uvrA.

HPRT test:

In a nin vitromammalian cell gene mutation test performed according to OECD Guideline 476 and in compliance with GLP, L5178Y tk+/-(3.7.2C) mouse lymphoma cells were exposed to test item for 3 h at the following concentrations:

Range-Finder Experiment:15.63, 31.25; 62.5; 125; 250; 500; 1000;  and 2000 μg/mL,with and without S9 mix

Mutation Experiment 1 :

Without S9: 25;;50; 75; 100; 125; 150; 175; 200; 225 and 250 μg/mL

With S9: 25;;50; 75; 100;  150; 200; 250; 275; 300; 350; 400; 450 and 500 μg/mL

Mutation Experiment 2 :

Without S9: 25;;50; 75; 100; 125; 150; 160; 170; 180; 190;  200 and 250 μg/mL

With S9: 25;;50; 75; 100;  150; 200; 250; 260; 270; 280; 290 and 300 μg/mL

Vehicle, negative and positive control groups were also included in each mutagenicity test. Metabolic activation system used in this test was 2 % S9 mix (final concentration). S9 fraction was prepared from liver homogenates of rats treated with Aroclor 1254.

 

In the cytotoxicity Range-Finder Experiment, eight concentrations were tested in the absence and presence of S-9 ranging from 15.63 to 2000 μg/mL. Upon addition of the test article to the cultures, precipitate was observed at the highest four concentrations in the absence and presence of S-9 (250 to 2000 µg/mL). Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence and presence of S-9 (500 to 2000 µg/mL). The lowest concentration at which precipitate was observed at the end of the treatment incubation period in the absence and presence of S-9 was retained and higher concentrations were discarded. The highest concentrations to give 10% RS were 125 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S 9, which gave 70% and 31% RS, respectively

 

-In Mutation Experiment 1, twelve concentrations ranging from 25 to 350 µg/mL in the absence of S-9 and 25 to 500 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 350 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 500 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the highest three concentrations in the absence of S-9 (250 to 350 µg/mL) and at the high concentration only (500 µg/mL) in the presence of S-9. Therefore, in the absence of S-9 the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentrations discarded. Seven days after treatment, the highest three concentrations (200 to 250 µg/mL) in the absence of S-9 and the highest four concentrations (350 to 500 µg/mL) in the presence of S-9 were considered too toxic for selection to determine viability and 6TG resistance. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 175 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 16% and 10% RS, respectively

- In Mutation Experiment 2, twelve concentrations ranging from 25 to 250 µg/mL in the absence of S-9 and 25 to 350 µg/mL in the presence of S-9, were tested. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (125 to 250 µg/mL) in the absence of S-9 and at the highest nine concentrations (150 to 350 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at the high concentration only (250 µg/mL) in the absence of S-9 and at the highest two concentrations (300 and 350 µg/mL) in the presence of S-9. Therefore, in the presence of S-9, the lowest concentration at which precipitate was observed at the end of the treatment incubation period was retained and the higher concentration discarded. Seven days after treatment, concentrations of 25 and 75 µg/mL in the absence of S-9 and 25 µg/mL in the presence of S-9 were not selected to determine viability and 6TG resistance as sufficient non-toxic concentrations were available. All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 250 µg/mL in the absence of S-9 and 300 µg/mL in the presence of S-9, which gave 12% and 35% RS, respectively

Vehicle and positive control treatments were included in the Mutation Experiments in the absence and presence of S-9. Mutant frequencies (MF) in vehicle control cultures fell within acceptable ranges and clear increases in mutation were induced by the positive control chemicals 4-nitroquinoline 1-oxide (NQO) (without S-9) and benzo(a)pyrene (B[a]P) (with S-9). Therefore the study was accepted as valid.

 

In the absence of S-9 inExperiment 1, a statistically significant (P0.05) increase in mutation frequency (MF) over the concurrent control was observed at 75 µg/mL. Although there was no accompanying statistically significant linear trend, the mutation frequency at 75 µg/mL (9.47per 106viable cells) did exceed the mean historical vehicle control MF for the last 20 studies +2SD (8.68 per 106viable cells). It was observed however that the untreated control also exhibited a MF of 9.41 per

106viable cells and therefore, the statistical increase in MF at 75 µg/mL was considered to be of no biological relevance.

In the presence of S-9inExperiment 1, statistically significant (P0.05) increases in MF over the concurrent control were observed at 50, 150 and 300 µg/mL. However, there was no accompanying statistically significant linear trend. The MF at 50, 150 and 300 µg/mL were 9.47, 9.96 and 15.28per 106viable cells respectively and all exceeded the mean historical vehicle control MF for the last 20 studies +2SD (9.08 per 106viable cells).

A second experiment in the absence and presence of S-9 was performed in order to assess the reproducibility of the results observed in Experiment 1.

In the absence of S-9 inExperiment 2, statistically significant (P0.05) increases in MF over the concurrent control were observed at 160, 170 and 250 µg/mL. Although there was no accompanying statistically significant linear trend, the MF at 250 µg/mL (9.71per 106viable cells) did also exceed the mean historical vehicle control MF for the last 20 experiments +2SD (8.74 per 106viable cells).

In the presence of S-9 inExperiment 2, there was a statistically significant (P0.05) increase in MF over the concurrent control observed at 100 µg/mL. However, there was no accompanying statistically significant linear trend and none of the MF values exceeded the mean historical vehicle control MF for the last 20 experiments +2SD (9.29 per 106viable cells).

The data of both experiments showed sporadic increases in MF after exposure to Cistus concrete, both in the absence and presence of S-9. However, the effects were poorly reproducible between dose concentrations and experiments and there was no clear evidence of a concentration related-response, except at dose concentrations at the upper limit of acceptable toxicity. Therefore, the data do not fulfil the criteria for a clear positive result as defined in OECD Guideline 476 and the increases in MF were considered to be spurious.

It is concluded that the observed increases in mutation frequency at the hprt locus of L5178Y mouse lymphoma cells after exposure to Cistus concrete, tested to a toxic and/or precipitating concentration for 3 hours in the absence and presence of a rat liver metabolic activation system (S-9), were not reproducible and not dose related under the experimental conditions employed, and consequently were considered to be not biologically relevant.

 

Micronucleus test:

In anin vitromicronucleus test performed according to OECD Guideline 487 and in compliance with GLP, human lymphoblastoid cells (TK6) were exposed to test item, Cistus concrete, in the presence and absence of a metabolic activation system. Three exposure conditions in two experiments were used for the study using a 3‑hour exposure in the presence and absence of a standard metabolizing system (S9 at a 10% final concentration) and a 27 -hour exposure in the absence of metabolic activation. 

The dose levels used in the Main Experiment were selected using data from the range-finder experimentbased on the RICC observed in the range-finder. The dose levels selected for the Main Test were as follows:

First experiment (main experiment): Treatment time: 3 hours, Recovery period: 41 hours and, Harvest time (hours after treatment began): 44 hours

- in presence of S9-mix (10%): 0, 20, 65, 100, 150, 175, 200, 225 and 250 µg/mL.

- in absence of S9-mix: 0, 5, 20, 65, 80, 95, 110, 125, 140 and 155 µg/mL.

Second experiment (main experiment): Treatment time: 27 hours, Recovery period: none and, Harvest time (hours after treatment began): 27 hours, serum content during treatment: 10% (v/v) FBS

- in absence of S9-mix: 0, 2, 20, 25, 30, 35, 40, 45, 50, 55 and 60 µg/mL.

 

All negative Controls gave mean counts within expected ranges. The positive Control chemicals consistently caused statistically significant increases in the number of micronuclei (MN) scored. This showed the cells to be sensitive to the effects of known clastogens and an aneugen and that the S‑9 metabolic activation system was able to activate a pro-mutagen to a mutagen.


There was a statistically significant increase in % micronucleated (MN) cells at 2 µg/mL, 20 µg/mL, 35 µg/mL and 45 µg/mL in the absence of S-9 mix after a 27-hour treatment. The increase at 2 µg/mL was within the historical control data range; however, the increases in mean % MN at 20 µg/mL and both mean and individual counts at 35 µg/mL and 45 µg/mL were outside the historical control range. As the % MN values obtained for the 20 µg/mL culture were inconsistent, it was excluded from the data for the trend analysis. The trend analysis was statistically significant, indicating a dose response.

 

There were no statistically significant increases in % MN in either the presence or absence of S-9 mix after a 3-hour treatment period.

Under the test conditions,Cistus concrete was considered to be either clastogenic or aneugenic under the conditions of this test, requiring no metabolic activation to induce such effects in TK6 cells.

Justification for classification or non-classification