Gum of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by extraction with alkaline solution

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

TBC

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP study conducted according to OECD Guideline 476 without any deviation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2016

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Type of assay:

other: mammalian cell gene mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Gum of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by extraction with alkaline solution

EC Number:

946-963-9

Molecular formula:

not applicable for UVCB

IUPAC Name:

Gum of Cistus ladaniferus (Cistaceae) obtained from stems and leaves by extraction with alkaline solution

Test material form:

solid

Details on test material:

Test item: Labdanum gum (Cistus ladaniferus)
Other: Labdanum gum spain

Content: 100 %
Appearance: Dark brown solid
Storage conditions: stored at 15 25°C, protected from light

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (hprt) locus

Species / strain

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Dr Donald Clive, Burroughs Wellcome Co.
- Suitability of cells:
Mouse lymphoma L5178Y systems are statistically more sensitive than CHO and V79 systems.

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
- 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 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
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 2000 µg/mL (the maximum recommended concentration according to current regulatory guidelines).

Mutation Experiment:
Without S9: 25, 50, 100, 150, 175, 200, 210, 220, 230, 240, 250 and 300 µg/mL
With S9: 25, 50, 100, 150, 175, 200, 250, 260, 270, 280, 290 and 300 µg/mL
Justification: Concentrations selected for the Mutation Experiment were based on the results of this cytotoxicity Range-Finder Experiment.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Preliminary solubility trials indicated that dimethylformamide (DMF) was the most suitable vehicle. Labdanum gum formed a suitable, homogeneous suspension at concentrations up to approximately 200 mg/mL. The solubility limit in culture medium was in the range of 125 to 250 µg/mL, as indicated by precipitation at the higher concentration which persisted for at least 3 hours after test article addition.
- Test article stock solutions were prepared by suspending Labdanum gum under subdued lighting in DMF, with the aid of vortex mixing, warming at 37°C and ultrasonication (where required), to give the maximum required concentration. Subsequent dilutions were made using DMF. The test article suspensions were protected from light and used within approximately 2 hours of initial formulation.

Controls

Controls 1

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF diluted 100 fold in treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

Without S9

Controls 2

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMF diluted 100-fold in the treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

With S9

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 (cells in RPMI 10 diluted with RPMI A [no serum] to give a final concentration of 5% serum) 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): 12 to 14 days
- Plating for Survival: 7 days
- Plating for viability: 8 to 9 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
- Main test: Two cultures for vehicle, test article, culture medium for the UTC or positive control solution; single cultures for positive control solution

NUMBER OF CELLS EVALUATED: 192 wells averaging 1.6 cells/well, 192 wells averaging 1.6 cells/well and 384 wells at 2 x 104 cells/well 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 induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
- The MF at one or more concentrations was significantly greater than that of the vehicle control (p≤0.05)
- There was a significant concentration-relationship as indicated by the linear trend analysis (p≤0.05)
- 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.

Results and discussion

Test results

Key result

Species / strain:

mouse lymphoma L5178Y cells

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 and osmolality: No marked changes in osmolality or pH were observed in the Range-Finder at the highest concentration analysed (250 µg/mL), compared to the concurrent vehicle controls.
- Precipitation: Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest four concentrations (250-2000 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 the higher concentrations discarded.
- 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 (an acceptable maximum concentration for in vitro genetic toxicology studies according to current regulatory guidelines). Upon addition of the test article to the cultures and following the 3 hour treatment incubation period, precipitate was observed at the highest four concentrations (250-2000 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 the higher concentrations discarded. The highest concentration to give >10% RS was 125 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S-9, which gave 69% and 16% 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 the laboratory are as follows:
- Negative (solvent/vehicle) historical control data:
Vehicle Controls
In the absence of S-9
Mean: 4.91 mutants per 10^6 viable cells, Range* = 1.15 to 8.68 mutants per 10^6 viable cells.
In the presence of S-9
Mean: 5.12 mutants per 10^6 viable cells, Range* = 1.23 to 9.01 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: 43.86 mutants per 10^6 viable cells, Range* = 1.51 to 86.22 mutants per 10^6 viable cells.
NQO 0.20 µg/mL in the absence of S-9
Mean: 56.98 mutants per 10^6 viable cells, Range* = 9.44 to 104.52 mutants per 10^6 viable cells.
B[a]P 2 µg/mL in the presence of S-9
Mean: 26.89 mutants per 10^6 viable cells, Range* = 0 to 55.24 mutants per 10^6 viable cells.
B[a]P 3 µg/mL in the presence of S-9
Mean: 41.64 mutants per 10^6 viable cells, Range* = 7.71 to 75.57 mutants per 10^6 viable cells.
*Range = Mean ± 2 x SD.

MUTATION EXPERIMENT
- In the Mutation Experiment, twelve concentrations, ranging from 25 to 300 µg/mL, were tested in the absence and presence of S-9. Upon addition of the test article to the cultures, precipitate was observed at the highest eight concentrations (175 to 300 µg/mL) in the absence of S-9 and at the highest seven concentrations (200 to 300 µg/mL) in the presence of S-9. Following the 3 hour treatment incubation period, precipitate was observed at 300 µg/mL in the absence of S-9 and in the highest seven concentrations (200 to 300 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 concentrations discarded. Seven days after treatment, in the absence of S-9, the highest five concentrations (220 to 300 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance (RS <10%). All other concentrations were selected in the absence and presence of S-9. The highest concentrations analysed were 210 µg/mL in the absence of S-9 (limited by cytotoxicity) and 200 µg/mL in the presence of S-9 (limited by the appearance of post-treatment precipitate) which gave 14% and 66% RS.
- No statistically significant increases in MF were observed following treatment with Labdanum gum at any concentration tested in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Any other information on results incl. tables

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

 

Concentration	3 h treatment –S-9	3 h treatment +S-9
µg/mL	% RS	% RS
0	100	100
UTC	107	99
15.63	73	87
31.25	60	101
62.5	75	75
125	69	75
250 P PP	0	16

UTC: Untreated control

% RS: Percent relative survival adjusted by post treatment cell counts

P: Precipitation observed at the time of treatment

PP: Precipitation noted at end of treatment incubation period

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

 

3h treatment -S9			3h treatment +S9
Concentration (µg/mL)	% RS	MF §	Concentration (µg/mL)	% RS	MF §
0	100	4.50	0	100	7.06
0 UTC	112	7.74	0 UTC	108	7.17
25	94	7.40 NS	25	90	6.82 NS
50	103	6.11 NS	50	89	5.33 NS
100	105	5.90 NS	100$	101	6.50 NS
150	74	3.16 NS	150	81	4.52 NS
175 P	38	7.17 NS	175	75	5.44 NS
200 P	19	7.95 NS	200 P PP	66	5.48 NS
210 P	14	7.79 NS	B[a]P 2	81	53.15
NQO 0.150	70	46.73	B[a]P 3	55	57.92
NQO 0.200	50	43.97	-	-	-

Linear trend tests on mutant frequency +/-S-9: Not significant (negative trends) 

UTC: Untreated control

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

$: Heterogeneity observed between repllicate cultures

% RS: Percent relative survival adjusted by post treatment cell counts

P: Precipitation noted at time of treatment

PP: Precipitation noted at end of treatment incubation period

NS: Not significant

Applicant's summary and conclusion

Conclusions:

Under the test conditions, test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the presence of a rat liver metabolic activation system (S-9) and up to toxic concentrations for 3 hours in the absence of S-9.

Executive summary:

In an in 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 substance 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

Mutation Experiment:

Without S9: 25, 50, 100, 150, 175, 200, 210, 220, 230, 240, 250 and 300 µg/mL

With S9: 25, 50, 100, 150, 175, 200, 250, 260, 270, 280, 290 and 300 µg/mL

 

Negative (untreated culture media), vehicle 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 2000µg/mL (an acceptable maximum concentration forin vitrogenetic toxicology studies according to current regulatory guidelines).Post-treatment precipitate was observed in the four highest concentrations (250 to 2000 µg/mL). The highest concentration to give>10% relative survival (RS) was 125 µg/mL in the absence of S-9 and 250 µg/mL in the presence of S-9, which gave 69% and 16% RS, respectively.

In the Mutation Experiment, twelve concentrations, ranging from 25 to 300 µg/mL, were tested in the absence and presence of S-9. Post-treatment precipitation was observed at 300 µg/mL in the absence of S-9 and in the highest seven concentrations (200 to 300 µg/mL) in the absence of S-9. Seven days after treatment, the highest concentrations analysed to determine viability and 6TG resistance were 210 µg/mL in the absence of S-9 (limited by cytotoxicity) and 200 µg/mL in the presence of S-9 (limited by the appearance of post-treatment precipitate) which gave 14% and 66% RS, respectively.

Vehicle and positive control treatments were included in the Mutation Experiment 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.

No statistically significant increases in MF were observed following treatment withLabdanum gumat any concentration analysed in the absence and presence of S-9 and there were no statistically significant linear trends, indicating a clear negative result.

Under the test conditions, test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested up to the limit of solubility, for 3 hours in the presence of a rat liver metabolic activation system (S-9) and up to toxic concentrations for 3 hours in the absence of S-9.