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)

HPRT test: non mutagenic (OECD 476, GLP, K, rel.1)

Micronucleus test: ambiguous results (OECD 487, GLP, K, rel. 1): increases in micronuclei considered of questionable biological relevance in cultured human peripheral blood lymphocytes following 3-hour treatment in the absence metabolic activation system (S‑9) and small increases in micronuclei considered of questionable biological importance were observed following both 3-hour treatment in the presence of S-9 and 24 hour treatment in the absence of S-9

Link to relevant study records

Referenceopen allclose all

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.
Qualifier:
according to
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
Target gene:
hypoxanthine-guanine phosphoribosyl transferase (hprt) locus
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.
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
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.
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.

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

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.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 20 March 2017 to 5 June 2017.
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.
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
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:
Not applicable.
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Blood from two healthy, non-smoking female volunteers from a panel of donors at Covance.
- Sex, age and number of blood donors : female, 27 and 33 years for the range finder and 28 and 33 years for the main experiment.
- For each experiment, an appropriate volume of whole blood was drawn from the peripheral circulation into heparinised tubes within two days of culture initiation. Blood was stored refrigerated and pooled using equal volumes from each donor prior to use.

MEDIA USED
- Type and identity of media: HEPES-buffered RPMI medium containing 10% (v/v) heat inactivated foetal calf serum and 0.52% penicillin / streptomycin, so that the final volume following addition of S-9 mix/KCl and the test article in its chosen vehicle was 10 mL. The mitogen Phytohaemagglutinin (PHA, reagent grade) was included in the culture medium at a concentration of approximately 2% of culture to stimulate the lymphocytes to divide.
Additional strain / cell type characteristics:
not applicable
Cytokinesis block (if used):
Cytochalasin B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.
Metabolic activation:
with and without
Metabolic activation system:
The mammalian liver post-mitochondrial fraction (S-9) was prepared from male Sprague Dawley rats induced with Aroclor 1254. The final S-9 volume in the test system was 1% (v/v).
Test concentrations with justification for top dose:
Preliminary Toxicity Test:
- 0, 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432, 720, 1200 and 2000 µg/mL (3-hour exposure to the test item without S9-mix or with S9-mix (1%))
- 0, 7.256, 12.09, 20.16, 33.59, 55.99, 93.31, 155.5, 259.2, 432, 720, 1200 and 2000 µg/mL (24-hour continuous exposure to the test item without S9-mix)

Main Experiment:
3-hour exposure to the test item without S9-mix: 0, 25, 75, 100, 150, 200, 250, 275, 300, 325, 350, 375 and 400 µg/mL.
3-hour exposure to the test item with S9-mix (2%): 0, 25, 75, 100, 150, 200, 250, 275, 300, 325, 350, 375 and 400 µg/mL.
24-hour continuous exposure to the test item without S9-mix: 0, 20, 40, 60, 80, 100, 120, 140, 160, 180, 200, 250 and 300 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethylformamide (DMF)
- Justification for choice of solvent/vehicle: Preliminary solubility trials indicated that DMF was the most suitable vehicle. Labdanum gum formed a fine, homogeneous suspension at concentrations up to approximately 200 mg/mL that were considered suitable for use. The solubility limit in culture medium was in the range of 125 to 500 µg/mL, as indicated by visible precipitation at the higher concentration which persisted following a 26 hour incubation period. A maximum concentration of 2000 µg/mL was selected for the cytotoxicity Range-Finder Experiment, in order that treatments were performed up to a regulatory suitable maximum.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9 mix - CPA: 2 - 3 µg/mL for 3-hour exposure.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMF
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
mitomycin C
other:
Remarks:
Without S9 mix - Mitomycin C: 0.2 - 0.3 µg/mL for 3-hour exposure; Vinblastine: 0.04 - 0.06 µg/mL for 24-hour continuous exposure
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h (± S9) and 24 h continuous exposure (-S9) in preliminary toxicity test; 3 h (± S9) and 24 h continuous exposure (-S9) in main experiment
- At the end of the exposure period, the cell cultures were pelleted and washed twice with sterile saline and resuspented in fresh pre-warmed medium and then incubated for a further 21 h in the presence of Cytochalasin B for 3 h (± S9) and 24 h continuous exposure (-S9) respectively.

SPINDLE INHIBITOR (cytogenetic assays): Prior to the mitosis (after exposure of the test substance), Cytochalasin-B (formulated in DMSO) was added to post wash-off culture medium to give a final concentration of 6 µg/mL per culture.

STAIN (for cytogenetic assays): Slides were stained with Acridine orange solution (12.5 µg/mL using purified water) for 10 minutes

NUMBER OF REPLICATIONS:
- Preliminary toxicity test: Single culture for test item and untreated control. Duplicate for vehicle control.
- Main test: Duplicate cultures per dose for test item, untreated control and positive controls. Quadruplicate for vehicle control (DMF).

NUMBER OF CELLS EVALUATED:
- Cytotoxicity: A minimum of approximately 500 cells per culture were scored for the incidence of mononucleate, binucleate and multinucleate cells and the Replication index value expressed as a percentage of the vehicle controls.
- Scoring of Micronuclei: A minimum of one thousand binucleate cells from each culture (2000 per concentration) were analysed for micronuclei. The number of cells containing micronuclei and the number of micronuclei per cell on each slide was recorded.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of test item in the lymphocyte cultures was determined using the the replication index (RI):
RI, which indicates the relative number of nuclei compared to vehicle controls was determined using the formula as follows:
RI = number binucleate cells + 2 (number multinucleate cells)/total number of cells in treated cultures
Relative RI (expressed in terms of percentage) for each treated culture was calculated as follows:
Relative RI (%) = (RI of treated cultures/RI of vehicle controls )x100
Cytotoxicity (%) is expressed as (100 – Relative RI).


Evaluation criteria:
For valid data, the test article was considered to induce clastogenic and/or aneugenic events if:
1. A statistically significant increase in the frequency of MNBN cells at one or more concentrations was observed
2. An incidence of MNBN cells at such a concentration that exceeded the normal range in both replicates was observed
3. A concentration-related increase in the proportion of MNBN cells was observed (positive trend test).
* The test article was considered positive in this assay if all of the above criteria were met.
* The test article was considered negative in this assay if none of the above criteria were met.
* Results which only partially satisfied the above criteria were dealt with on a case-by-case basis. Evidence of a concentration-related effect was considered useful but not essential in the evaluation of a positive result (Scott et al., 1990). Biological relevance was taken into account, for example consistency of response within and between concentrations, or effects occurring only at very toxic concentrations (Thybaud et al., 2007).
Statistics:
The proportions of MNBN cells in each replicate were used to establish acceptable heterogeneity between replicates by means of a binomial dispersion test (Richardson et al., 1989).
The proportion of MNBN cells for each treatment condition were compared with the proportion in vehicle controls by using Fisher's exact test (Richardson et al., 1989). A Cochran-Armitage trend test was applied to each treatment condition. Probability values of p≤0.05 were accepted as significant
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
ambiguous
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: no marked changes in pH (shifts of greater than 1 pH unit) was observed in the citotoxicity Range finder Experiment
- Effects of osmolality: no marked changes in osmolality (shifts of greater than 50 mOsm/kg) was observed in the citotoxicity Range finder Experiment


RANGE-FINDING/SCREENING STUDIES (See Tables 1, 2 and 3):
- The dose range for the Preliminary Toxicity Test was 7.256 to 2000 µg/mL.
- A precipitate of the test item was observed from 93.31 µg/mL (with and without S9/3 hours exposure) and 259.2 µg/mL (without S9/24 hours exposure).
- The selection of the maximum dose level for the Main Experiment was based on toxicity in the exposure groups in the absence of S9-mix and the lowest precipitating dose level for the exposure group in the presence of S9.

COMPARISON WITH HISTORICAL CONTROL DATA:
- The concurrent vehicle and positive controls were within the laboratory historical control data range.

ADDITIONAL INFORMATION ON GENOTOXICITY/CYTOTOXICITY:
Main experiments:
- The 3-hour treatment of cells with Labdanum gum in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the highest two concentrations analysed (350 and 375 µg/mL) with a clear concentration related response apparent (statistically significant linear trend). The MNBN cell frequencies of both Labdanum gum treated cultures at both of these concentrations and single cultures at lower concentrations of 250 and 300 µg/mL exceeded the historical vehicle control (95th percentile of the observed) range. These data indicated a positive induction of MNBN cells.
- The 3-hour +S-9 treatment of cells with Labdanum gum resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle control cultures for all five concentrations analysed. Frequencies of MNBN cells exceeded the normal range for both treated cultures at concentrations of 275 and 375 µg/mL, and in single cultures for concentrations of 325 and 350 µg/mL though the magnitude of these increases was not large and similar in magnitude to slight increases observed in vehicle control. No notable concentration related effect was apparent (linear trend test was negative) such that these slight increases in MNBN cell frequency were considered of questionable biological relevance.
- Extended 24-hour treatment in the absence of S-9 resulted in small but significantly (p≤0.05) elevated frequencies of MNBN cells for two intermediate concentrations analysed (120 and 160 µg/mL). However, these increases were small with the MNBN cell values of single cultures marginally exceeding normal ranges. No such increase was observed in the replicate cultures at either of these concentrations, or for any other Labdanum gum treated culture at lower or higher concentrations analysed (80 and 180 µg/mL inducing 12% and 46% cytotoxicity respectively). The small increases observed (poorly reproduced between replicate cultures) were therefore considered of questionable biological importance.

Table1: Data for 3-Hour Treatments -S-9, Range-Finder:

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity Based on RI (%)

% Excluded Cells

Vehicle (Total)

A/B

121

273

7

401

0.72

-

-

UTC

A

63

135

2

200

0.70

-

10%

7.256

A

NSc

-

-

-

-

-

-

12.09

A

NSc

-

-

-

-

-

-

20.16

A

43

155

2

200

0.80

0

10%

33.59

A

66

131

3

200

0.69

4

10%

55.99

A

55

143

2

200

0.74

0

10%

93.31

A

73

126

1

200

0.64

11E

10%

155.5

A

76

124

0

200

0.62

13E

20%

259.2

A

119

81

0

200

0.41

43PE

30%

432

A

195

5

0

200

0.03

97PE

80%

720

A

T

-

-

-

-

-PEH

-

1200

A

T

-

-

-

-

-PEH

-

2000

A

T

-

-

-

-

-PEH

-

Table2: Data for 3-Hour Treatments +S-9:

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity Based on RI (%)

% Excluded Cells

Vehicle (Total)

A/B

127

256

17

400

0.73

-

NR

UTC

A

70

125

5

200

0.78

-

NR

7.256

A

NSc

-

-

-

-

-

-

12.09

A

NSc

-

-

-

-

-

-

20.16

A

51

137

12

200

0.81

0

NR

33.59

A

42

153

5

200

0.82

0

10%

55.99

A

61

136

3

200

0.71

2

10%

93.31

A

60

140

0

200

0.70

3E

10%

155.5

A

81

117

2

200

0.61

17E

10%

259.2

A

107

90

3

200

0.48

34PE

30%

432

A

194

6

0

200

0.03

96PE

70%

720

A

T

-

-

-

-

-PEH

-

1200

A

T

-

-

-

-

-PEH

-

2000

A

T

-

-

-

-

-PEH

-

Table3: Data for 24-Hour Treatments -S-9

Treatment

(µg/mL)

Replicate

Mono

Bi

Multi

Total

RI

Cytotoxicity Based on RI (%)

% Excluded Cells

Vehicle (Total)

A/B

132

261

7

400

0.69

-

 

UTC

A

24

150

26

200

1.09

-

NR

7.256

A

56

140

4

200

0.74

0

5%

12.09

A

57

141

2

200

0.73

0

5%

20.16

A

67

129

4

200

0.69

0

5%

33.59

A

71

126

3

200

0.66

4

5%

55.99

A

82

116

2

200

0.60

13

5%

93.31

A

122

77

1

200

0.40

43

10%

155.5

A

135

64

1

200

0.33

52

20%

259.2

A

189

11

0

200

0.06

92P

80%

432

A

T

-

-

-

-

-P

-

720

A

T

-

-

-

-

-PH

-

1200

A

T

-

-

-

-

-PEH

-

2000

A

T

-

-

-

-

-PEH

-

UTC = Untreated control

NSc = Not scored 

P = Precipitation observed at treatment

E = Precipitation observed at the end of treatment incubation

H = Precipitation observed at harvest

T = Toxic

Mono = Mononucleate

Bi = Binucleate

Multi = Multinucleate

RI = Replication index

 Table 4: Micronucleus data:

Treatment

Concentration (µg/ml)

Cytotoxicity (%)***

Mean MNBN Cell Frequency (%)

Historical Control Range (%)

Statistical Significance

3-hour -S-9

Vehicle*

-

0.73

0.20-1.00

-

 

UTC

-

0.80

 

NS

 

150

15

0.65

 

NS

 

250

28

0.95

 

NS

 

300

41

1.15

 

NS

 

350

32

1.40

 

p<0.01

 

375****

38

1.65

 

p<0.001

 

**MMC, 0.20

42

4.75

 

p<0.001

3-hour +S-9

Vehicle*

-

1.08

0.20-1.07

-

 

UTC

-

0.60

 

NS

 

150

11

0.75

 

NS

 

275

19

1.15

 

NS

 

325

20

0.95

 

NS

 

350

24

1.35

 

NS

 

375****

27

1.30

 

NS

 

**CPA, 2*

31

1.85

 

p<0.01

24-hour-S-9

Vehicle

-

0.38

0.10 – 0.90

-

 

UTC

-

0.25

 

NS

 

80

12

0.65

 

NS

 

120

26

0.75

 

p<0.05

 

160

46

0.95

 

p<0.01

 

180****

47

0.45

 

NS

 

**VIN, 0.04

39

6.20

 

p<0.001

*Vehicle control was DMF

**Positive control

***Based on replication index (RI)

NS: Not significan

**** Maximum concentrations analysed were limited by cytotoxicity to the maximum practicable concentrations that could be scored. Test article treatment at high concentrations impacted on  the cellular cytoplasm. Cells without cytoplasm are excluded from RI analysis such that under these circumstances RI may underestimate the true levels of cytotoxicity.

Conclusions:
Under the test conditions of this study, the test item induced increases in micronuclei in cultured human peripheral blood lymphocytes following 3-hour treatment in the absence of metabolic activation system (S-9), considered of questionable biological relevance. In the same test system, small increases in micronuclei considered of questionable biological importance were observed following both 3-hour treatment in the presence of S-9 and 24-hour treatment in the absence of S-9. The maximum concentrations analysed under each treatment condition were limited by cytotoxicity. However, this cytotoxicity may have been underestimated, as evidenced with the increase in the percentage of excluded cells (effects on cytoplasm indicating probable excessive cytotoxicity, membrane effects) at higher concentrations (from 200-250 µg/mL) in the preliminary and the main expriments as well as with the flat Replication Index (RI) dose-response in the main study (RI not appropriate for this substance). In addition, discordant precipitate observations were recorded in 3+21h +/- S9 conditions between the preliminary and main tests, with precipitate observed from 93 µg/mL vs. no precipitate at the end of the treatment up to 375 µg/mL, respectively. Now, in the HPRT study with the same test item, 200 µg/mL was the limit dose in the presence of S9 because of precipitate at the end of the exposure period. The possible underestimation of cytotoxicity and the discrepancy in precipitate observations weaken the relevance of the effects observed from 200 µg/mL in all treatment conditions.
Executive summary:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 3hour exposure in the presence and absence of a standard metabolizing system (S-9 at a 1% final concentration) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 21 or 24  hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the toxicity and the precipitation of the test item both in the absence and presence of S9-mix. The dose levels selected for the Main Test were as follows:

3-hour without S9: 0, 150, 250, 300, 350 and 375 μg/mL

3-hour with S9 (1%): 0, 150, 275, 325, 350 and 375 μg/mL

24-hour without S9: 0, 80, 120, 160 and 180 μg/mL

All vehicle (DMF) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

- The 3- hour treatment of cells with the test item in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the highest two concentrations analysed (350 and 375 µg/mL) with a clear concentration related response apparent (statistically significant linear trend). The MNBN cell frequencies of both test item treated cultures at both of these concentrations and single cultures at lower concentrations of 250 and 300 µg/mL exceeded the historical vehicle control (95th percentile of the observed) range. These data indicated a positive induction of MNBN cells.

- The 3-hour + S-9 treatment of cells with test item resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle control cultures for all five concentrations analysed. Frequencies of MNBN cells exceeded the normal range for both treated cultures at concentrations of 275 and 375 µg/mL, and in single cultures for concentrations of 325 and 350 µg/mL though the magnitude of these increases was not large and similar in magnitude to slight increases observed in vehicle control. No notable concentration related effect was apparent (linear trend test was negative) such that these slight increases in MNBN cell frequency were considered of questionable biological relevance.

- Extended 24-hour treatment in the absence of S-9 resulted in small but significantly (p≤0.05) elevated frequencies of MNBN cells for two intermediate concentrations analysed (120 and 160 µg/mL). However, these increases were small with the MNBN cell values of single cultures marginally exceeding normal ranges. No such increase was observed in the replicate cultures at either of these concentrations, or for any othertest item culture at lower or higher concentrations analysed (80 and 180 µg/mL inducing 12% and 46% cytotoxicity respectively). The small increases observed (poorly reproduced between replicate cultures) were therefore considered of questionable biological importance.

Under the test conditions of this study, the test item induced increases in micronuclei in cultured human peripheral blood lymphocytes following 3-hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S‑9). In the same test system, small increases in micronuclei considered of questionable biological importance were observed following both 3-hour treatment in the presence of S-9 and 24 hour treatment in the absence of S-9. The maximum concentrations analysed under each treatment condition were limited by cytotoxicity. However, this cytotoxicity may have been underestimated, as evidenced with the increase in the percentage of excluded cells (effects on cytoplasm indicating probable excessive cytotoxicity, membrane effects) at higher concentrations (from 200-250 µg/mL) in the preliminary and the main expriments as well as with the flat Replication Index (RI) dose-response in the main study (RI not appropriate for this substance). In addition, discordant precipitate observations were recorded in 3+21h +/- S9 conditions between the preliminary and main tests, with precipitate observed from 93 µg/mL vs. no precipitate at the end of the treatment up to 375 µg/mL, respectively. Now, in the HPRT study with the same test item, 200 µg/mL was the limit dose in the presence of S9 because of precipitate at the end of the exposure period. The possible underestimation of cytotoxicity and the discrepancy in precipitate observations weaken the relevance of the effects observed from 200 µg/mL in all treatment conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2014 - March 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline 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
GLP compliance:
yes (incl. certificate)
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
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:50, 150, 500, 1500 and 5000 μg/plate in all strains with and without S9-mix;
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- 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 dimethyl sulphoxide, therefore, this solvent was selected as the vehicle.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulphoxide
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
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:
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and
Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase 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.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:

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 lawn at any dose level, either in the presence or absence of metabolic activation (S9-mix), in the first mutation test (plate incorporation method) and consequently the same maximum dose level was used in the second mutation test. Similarly, there was no visible reduction in the growth of the bacterial background lawn at any dose level, either in the presence or absence of metabolic activation, in the second mutation test (pre-incubation method). An opaque test item film was noted at and above 1500 μg/plate in Experiment 1 (plate incorporation method) and at 5000 μg/plate in Experiment 2 (pre-incubation method).

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.
Conclusions:
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: 50, 150, 500, 1500 and 5000 μg/plate in all strains without S9-mix

Negative, vehicle ( dimethyl sulphoxide) 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 were no 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 S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method).

 

Under the test condition, test material is not mutagenic with and without metabolic activation inS. typhimurium (strains TA1535, TA1537, TA98 and TA100) andE.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.

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 ascertain 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:50, 150, 500, 1500 and 5000μg/plate inall strains without S9-mix

Negative, vehicle (dimethyl sulphoxide) 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 were no 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 S9-mix in Experiment 1 (plate incorporation method). Similarly, no significant increases in the frequency of revertant colonies were recorded for any of the bacterial strains, with any dose of the test item, either with or without S9-mix in Experiment 2 (pre-incubation method).

 

Under the test condition, test material is not mutagenic with and without metabolic activation inS. typhimurium (strains TA1535, TA1537, TA98 and TA100) and E.coli WP2 uvrA.

Micronucleus test:

In an in vitro micronucleus test performed according to OECD Guideline 487 and in compliance with GLP, cultured peripheral human lymphocytes were exposed to test item in the presence and absence of a metabolic activation system. Three exposure conditions in a single experiment were used for the study using a 3hour exposure in the presence and absence of a standard metabolizing system (S-9 at a 1% final concentration) and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 21 or 24  hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by the toxicity and the precipitation of the test item both in the absence and presence of S9-mix. The dose levels selected for the Main Test were as follows:

3-hour without S9: 0, 150, 250, 300, 350 and 375μg/mL

3-hour with S9 (1%): 0, 150, 275, 325, 350 and 375μg/mL

24-hour without S9: 0, 80, 120, 160 and 180μg/mL

All vehicle (DMF) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

- The 3-hour treatment of cells with the test item in the absence of S-9 resulted in frequencies of MNBN cells which were significantly (p≤0.05) higher than those observed in concurrent vehicle controls for the highest two concentrations analysed (350 and 375 µg/mL) with a clear concentration related response apparent (statistically significant linear trend). The MNBN cell frequencies of both test item treated cultures at both of these concentrations and single cultures at lower concentrations of 250 and 300 µg/mL exceeded the historical vehicle control (95th percentile of the observed) range. These data indicated a positive induction of MNBN cells.

- The 3-hour + S-9 treatment of cells with test item resulted in frequencies of MNBN cells that were similar to and not significantly (p≤0.05) higher than those observed in concurrent vehicle control cultures for all five concentrations analysed. Frequencies of MNBN cells exceeded the normal range for both treated cultures at concentrations of 275 and 375 µg/mL, and in single cultures for concentrations of 325 and 350 µg/mL though the magnitude of these increases was not large and similar in magnitude to slight increases observed in vehicle control. No notable concentration related effect was apparent (linear trend test was negative) such that these slight increases in MNBN cell frequency were considered of questionable biological relevance.

- Extended 24-hour treatment in the absence of S-9 resulted in small but significantly (p≤0.05) elevated frequencies of MNBN cells for two intermediate concentrations analysed (120 and 160 µg/mL). However, these increases were small with the MNBN cell values of single cultures marginally exceeding normal ranges. No such increase was observed in the replicate cultures at either of these concentrations, or for any othertest item culture at lower or higher concentrations analysed (80 and 180 µg/mL inducing 12% and 46% cytotoxicity respectively). The small increases observed (poorly reproduced between replicate cultures) were therefore considered of questionable biological importance.

Under the test conditions of this study, the test item induced increases in micronuclei in cultured human peripheral blood lymphocytes following 3-hour treatment in the absence of an Aroclor-induced rat liver metabolic activation system (S‑9). In the same test system, small increases in micronuclei considered of questionable biological importance were observed following both 3-hour treatment in the presence of S-9 and 24 hour treatment in the absence of S-9. The maximum concentrations analysed under each treatment condition were limited by cytotoxicity.However, this cytotoxicity may have been underestimated, as evidenced with the increase in the percentage of excluded cells (effects on cytoplasm indicating probable excessive cytotoxicity, membrane effects) at higher concentrations (from 200-250 µg/mL) in the preliminary and the main expriments as well as with the flat Replication Index (RI) dose-response in the main study (RI not appropriate for this substance). In addition, discordant precipitate observations were recorded in 3+21h +/- S9 conditions between the preliminary and main tests, with precipitate observed from 93 µg/mL vs. no precipitate at the end of the treatment up to 375 µg/mL, respectively. Now, in the HPRT study with the same test item, 200 µg/mL was the limit dose in the presence of S9 because of precipitate at the end of the exposure period. The possible underestimation of cytotoxicity and the discrepancy in precipitate observations weaken the relevance of the effects observed from 200 µg/mL in all treatment conditions.

HPRT test:

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:

 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.

Justification for classification or non-classification

The registered substance was negative in an Ames test (Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100 and Escherichia coli strain WP2 uvrA-; with and without S9) and in an in vitro HPRT test in mouse lymphoma cells. Increases of questionable biological relevance were observed in an in vitro micronucleus test. An in vivo micronucleus test performed in animals used for the preliminary study of the OECD TG 422 study is planned in order to ascertain the biological relevance of the ambiguous effects observed in vitro. Therefore the registered substance is considered, at this stage, as not requiring classification for mutagenicity.