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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- positive in TA1535 and TA98, Bacterial mutation assay in S. typhimurium and E. coli (Ames test), Callander 1999

- equivocal in TA98, Bacterial mutation assay in S. typhimurium and E. coli, (Ames test), Callander 2004

- weakly positive, L5178Y TK +/- mouse lymphoma mutation assay, Clay 1999

- negative, In vitro cytogenetic assay in human lymphocytes, Fox 1999

- negative, 10 Batches of 4-Mesyl-2-nitrotoluene Reverse Mutation Assay “Ames Test” using Salmonella typhimurium, Thompson and Bowles 2012

- negative, Reverse Mutation Assay “Ames Test” using Salmonella typhimurium and Escherichia coli, Bowles and Thompson 2012

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:
from 2012-01-16 to 2012-02-02 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline-conform study under GLP without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine for Salmonella
Tryptophan for E. Coli
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli, other: WP2 uvrA pKM101, WP2 pKM101
Metabolic activation:
with and without
Metabolic activation system:
S9 Microsomal fraction
Test concentrations with justification for top dose:
Experiment I, without S9: 50, 150, 500, 1500, 5000 µg/plate
Experiment I, with S9: 50, 150, 500, 1500, 5000 µg/plate, 5 and 15 µg/plate in addition for strain TA1537 and WP2uvrA pKM101
Experiment II, without S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate, 1.5 µg/plate in addition for strain WP2uvrA pKM101 and WP2pKM1001
Experiment II, with S9: 5, 15, 50, 150, 500, 1500, 5000 µg/plate, 1.5 µg/plate in addition for strain WP2uvrA pKM101 and WP2pKM100
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide
- Justification for choice of solvent/vehicle: The test item was fully soluble in dimethyl sulphoxide at 50 mg/mL . The choice of solvent vehicle was based on its ability to solubilise the test item and lack of toxicity to the bacterial systems.
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
Experiment I with and without S9, Experiment II without S9: in agar (plate incorporation)
Experiment II with S9: preincubation

DURATION
- Preincubation period: 60 min (Experiment II with S9)
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: Toxic effects were evident as a reduction in the growth of the bacterial background lawns and/or number of revertants.
Evaluation criteria:
The test item will generally be considered mutagenic to bacteria if the following criteria are achieved. If the following criteria are not achieved then the test item will be considered non-mutagenic to bacteria.

i) In all strains a two-fold increase in the mean number of revertants per plate compared to the mean value of the concurrent vehicle control.
ii) Increases in revertant numbers for all strains must be related to increases in test item concentration.
iii) A positive response in one tester strain either with or without exogenous metabolic activation is sufficient to designate the test item as a bacterial mutagen.

Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit a definitive judgement about the test item activity. Results of this type will be reported as equivocal.
Statistics:
not performed
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Salmonella strain TA1537 in the presence of S9-mix (plate incorporation methodology).
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli, other: WP2 uvrA pKM101, WP2 pKM101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix.
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in either the presence or absence of S9-mix.

RANGE-FINDING/SCREENING STUDIES: Not performed

COMPARISON WITH HISTORICAL CONTROL DATA: THe number of revertants of the solvent control and the positive control lie within the hitsorical control range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The test item caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains in either the absence or presence of S9-mix. However, lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix (plate incorporation and pre-incubation methodology) and to Salmonella strain TA1537 in the presence of S9-mix ( plate incorporation methodology). The test item was tested up to the maximum recommended dose level.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

The test item was considered to be non-mutagenic under the conditions of this test.
Executive summary:

This GLP-study was performed to investigate the potential of the test item to induce gene mutation..The method followed was that outlined in OECD TG 471.

Salmonella typhimurium strains TA1535, TA1537, TA98, TA100 and Escherichia coli strains WP2uvrApKM101 and WP2pKM101 were treated with the test item using both the Ames plate incorporation and pre-incubation methods at seven dose levels, in triplicate, both with and without the addition of a rat liver homogenate metabolising system (10% liver S9 in standard co-factors). The dose range for the first experiment (performed using the plate incorporation method) ranged between 5 and 5000 µg/plate, depending on bacterial strain type and presence or absence of S9-mix. A second experiment (utilising the pre-incubation method dosed in the presence of S9 and plate incorporation dosed in the absence of S9) was performed at later dates using doses ranging from 1.5 to 5000 µg/plate, fresh cultures of the bacterial strains and fresh test item formulations. Additional dose levels and an expanded dose range were selected (where applicable) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. 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.

 The test item caused no visible reduction in the growth of the bacterial background lawns of any of the tester strains in either the absence or presence of S9-mix. However, lowered revertant counts (less than 0.5 fold compared to the concurrent vehicle controls) were noted at 5000 µg/plate to Escherichia coli strains WP2uvrApKM101 and WP2pKM101 in both the presence and absence of S9-mix (plate incorporation and pre-incubation methodology) and to Salmonella strain TA1537 in the presence of S9-mix ( plate incorporation methodology). The test item was tested up to the maximum recommended dose level. No test item precipitate was observed on the plates at any of the dose levels tested in either the presence or absence of S9-mix.

 No significant increases in the frequency of revertant colonies, in excess of twofold greater than the concurrent solvent controls, were recorded for any of the bacterial strains, for any dose level of the test item, either with or without metabolic activation.

The test item was considered to be non-mutagenic under the conditions of this test.

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:
18 June - 8 July 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Adopted at Step 4 of the ICH process 19 July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2A. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Adopted at Step 4 of the ICH process 16 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 [phenobarbital (80 mg/kg) and β-naphthoflavone (100 mg/kg) induced]
Test concentrations with justification for top dose:
100, 200, 500, 1000, 2500 and 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO or water (for mitomycin C and sodium azide); vehicles chosen due to solubilisation properties and lack of toxicity to the bacteria.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: With S9, all Salmonella strains and WP2P: 2-aminoanthracene, WP2P uvrA: benzo(a)pyrene. Without S9, TA1537: acridine mutagen ICR191, TA98: daunomycin HCI, WP2P uvrA: N-ethyl-N-nitro-N-nitrosoguanidine, WP2P: mitomycin C, TA1535 and TA100: sodium azide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
- A stock solution of 50 mg/L was prepared for each experiment in DMSO and serial dilutions made. All test and positive control substance dosing preparations were prepared as close to the time of culture treatment as possible and were dosed at a volume of 100 µL/plate.

DURATION
- Incubation period: 3 days (at 37°C)

NUMBER OF REPLICATIONS: 2
- NMST: 6 concentrations per strain, 3 plates per concentration
- Solvent control (DMSO, 100 µL): 5 plates
- Positive controls: 3 concentrations per strain, 2 plates per concentration
Evaluation criteria:
A positive response in a (valid) individual experiment is achieved when one or both of the following criteria are met:
• a significant, dose-related increase in the mean number of revertants is observed;
• a two-fold or greater increase in the mean number of revertant colonies (over that observed for the concurrent solvent control plates) is observed at one or more concentrations
A negative result in a (valid) individual experiment is achieved when:
• there is no significant dose-related increase in the mean number of revertant colonies per plate observed for the test substance; and
• in the absence of any such dose response, no increase in colony numbers is observed (at any test concentration) which exceeds 2x the concurrent solvent control.
For a positive response in an individual experiment to be considered indicative of an unequivocal positive, i.e. mutagenic, result for that strain/S9 combination, then the observed effect(s) must be consistently reproducible.
Statistics:
The assessment of statistical significance was carried out using a one-tailed Student's t-test (Ehrenberg L (1984). Aspects of statistical inference in testing for genetic toxicity. In: Handbook of Mutagenicity Test Procedures, second edition (eds Kilbey B J, Legator M, Nichols W and Ramel C). Elsevier, 1984, 775-822.). The corresponding probability for each dose level was derived by computer using the appropriate degrees of freedom.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: strain/cell type: TA98 and TA1537
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
positive

The test substance gave a positive, i.e. mutagenic response in S.typhimurium strains TA1537 and TA98 in both the presence and absence of S9-mix.
Executive summary:

The test substance was evaluated in a bacterial mutagenicity assay over a range of concentrations using four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100), and two strains of Escherichia coli (WP2P and WP2P uvrA) in the presence or absence of a rat liver derived metabolic activation system (S9-mix). The test substance concentrations used were, Phase 1 and Phase 2; 100, 200, 500, 1000, 2500 and 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9-mix). Maximum fold over control values observed in bacterial strains affording a positive response were; Phase 1 +S9-mix, TA1537 4.1 (5000 µg/plate); TA98 11.7 (5000 µg/plate); Phase 1 –S9-mix TA1537 8.0 (5000 µg/plate); TA98 35.3: Phase 2; +S9 -mix, TA1537 5.6 (5000 µg/plate); TA98 13.5 (5000 µg/plate); Phase 2 –S9-mix TA1537 10.4 (5000 µg/plate); TA98 31.4.

Thus, in two separate experiments, the test substance induced significant, reproducible increases in the observed numbers of revertant colonies in strains TA1537 and TA98, both in the presence or absence of S9-mix. The sensitivity of the test system and the metabolic activity of the S9-mix, were clearly demonstrated by the increases in the numbers of the revertant colonies induced by positive control substances. The test substance gave a positive, i.e. mutagenic response in S.typhimurium strains TA1537 and TA98 in both the presence and absence of S9-mix.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
26 March - 5 July 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Adopted at Step 4 of the ICH process 19 July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Adopted at Step 4 of the ICH process 16 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 [phenobarbital (80 mg/kg) and β-naphthoflavone (100 mg/kg) induced]
Test concentrations with justification for top dose:
250, 1000 and 2150 µg/mL (chromosomal aberration analysis)
10, 50, 100, 250, 500, 750, 1000, 1500, 1800 and 2150 µg/mL (cytogenetic tests)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO, the vehicle was chosen due to its solubilisation properties and lack of toxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9, 50 µg/mL in experiment 1; 75 µg/mL in experiment 2
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9, 0.75 µg/mL in experiment 1; 0.2 µg/mL in experiment 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substances at appropriate concentrations.
- Prior to treatment, the cultures to be treated for a 20 hour period were centrifuged and the culture medium was replaced with fresh supplemented RPMI-1640 culture medium. The cultures to be treated for a 3 hour period did not receive a medium change at this point.

DURATION
- Treatment of the cultures started approximately 48 hours after culture initiation.
- Aliquots of the test substance, solvent control or positive controls were administered to duplicate cultures. In addition, 200 µL of a 1: 1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. Cultures from both experiments in the presence of S9-mix and cultures from Experiment 1 in the absence of S9-mix were treated for a period of approximately 3 hours at 37°C, after which the culture medium was removed following centrifugation and replaced with fresh supplemented RPMI-1640 culture medium. The cultures were re-incubated at approximately 37°C for the remainder of the 68 hour growth period. Cultures from Experiment 2 in the absence of S9-mix were treated for a period of 20 hours until the end of the 68 hour growth period.

SPINDLE INHIBITOR (cytogenetic assays): Approximately 2 hours prior to harvesting, the cultures were treated with colcemid at a final concentration of 0.4 µg/mL.

CULTURE HARVESTING: Sixty-eight hours after culture establishment the cultures were centrifuged, the supernatant was removed and the cells were re-suspended in approximately 10 mL of 0.075M KCl at room temperature for approximately 10 minutes. The cultures were centrifuged, the supernatant was removed and the remaining cells were fixed in freshly prepared methanol/glacial acetic acid fixative (3:1 v/v) added dropwise and made up to a volume of approximately 10 mL. The fixative was removed following centrifugation and replaced with freshly prepared fixative. This process was repeated at least twice prior to slide preparation on clean, moist labelled microscope slides.

STAIN (for cytogenetic assays): The slides were air dried, stained in filtered Giemsa stain (10% Gurr's R66 in buffered [pH 6.8] double deionised water) for 7 minutes, rinsed in water, air-dried and mounted with coverslips.

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 100 (test groups)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index: Two independent cytogenetic experiments were carried out using several concentrations.
- The highest concentration selected for chromosomal aberration analysis was 2150 µg/mL which is equivalent to 10 mM, the limit concentration for the assay.
Evaluation criteria:
The percentages of aberrant metaphases and the number of aberrations per cell were calculated for each treatment scored, both including and excluding cells with only gap-type aberrations.
The Fisher Exact Probability Test (one-sided) was used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations). Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value. The data have been interpreted as follows:
a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values - NEGATIVE.
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range - NEGATIVE.
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values - POSITIVE.
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
precipitation was seen in cultures treated with NMST at concentrations of 100 µg/mL and above
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
The highest concentration selected for chromosomal aberration analysis was 2150 µg/mL which is equivalent to 10 mM, the limit concentration for this assay.

Dose related reductions in mitotic activity were noted in cultures from both cytogenetic experiments treated in the presence and absence of S9-mix. Significant reductions in mean mitotic activity, compared to the solvent control values, were observed in cultures from both Experiment 1 (41% +S9-mix; 34% -S9-mix) and Experiment 2 (52% +S9-mix; 44% -S9-mix) treated with the highest concentrations selected for chromosomal aberration analysis, thus demonstrating that the test substance is biologically active in this test system.

Precipitation of the test substance in the culture medium was noted in cultures treated at concentrations of 1000 µg/mL and above.

Treatment of the culture medium up to 10 mM (2150 µg/mL) had no significant effect on osmolality or pH.

In the chromosomal aberration analyses, a small but statistically significant increase in the percentage of aberrant cells was observed at the intermediate concentration in Experiment 1 in the absence of S9-mix; this value was within the concurrent historical control range. No statistically or biologically significant increases in the percentage of aberrant cells, above the solvent control values, were recorded in cultures treated in Experiment 1 in the presence of S9-mix or Experiment 2 in either the presence or absence of S9-mix.

The positive control materials, mitomycin C and cyclophosphamide induced statistically and biologically significant increases in the percentage of aberrant cells, compared to the solvent control cultures.

Table 1: Mean chromosomal aberrations and mitotic indices in the absence of metabolic activation (S9-mix)

Treatment

Mean % aberrant cells excluding gaps

Mean mitoyic index %

Experiment 1

 

 

Solvent control

10 µg/mL

0.00

13.3

Mitomycin C

0.75 µg/mL

56.00**

6.1∆

Test substance

2150 µg/mL

1000 µg/mL

250 µg/mL

1.00

2.50*

1.50

8.8

11.2

15.2

Experiment 2

 

 

Solvent control

10 µg/mL

1.50

8.0

Mitomycin C

0.2 µg/mL

32.00**

3.7∆

Test substance

2150 µg/mL

1000 µg/mL

250 µg/mL

2.50

1.00

1.00

4.5

3.3

7.4

* statistically significant increase in the percentage of aberrant cells at p<0.05 using Fisher’s Exact Test (one-sided)

** statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher’s Exact Test (one-sided)

∆ Positive control mitotic index and % aberrant cells are determined from a single culture

 

Table 2: Mean chromosomal aberrations and mitotic indices in the presence of metabolic activation (S9-mix)

Treatment

Mean % aberrant cells excluding gaps

Mean mitoyic index %

Experiment 1

 

 

Solvent control

10 µg/mL

1.00

9.8

Cyclophosphamide

0.50 µg/mL

7.00**

8.0∆

Test substance

2150 µg/mL

1000 µg/mL

250 µg/mL

2.00

1.50

1.50

5.8

6.8

10.4

Experiment 2

 

 

Solvent control

10 µg/mL

2.50

7.5

Cyclophosphamide

0.75 µg/mL

36.00**

4.1∆

Test substance

2150 µg/mL

1000 µg/mL

250 µg/mL

0.50

1.00

1.00

3.6

5.1

6.6

** statistically significant increase in the percentage of aberrant cells at p<0.01 using Fisher’s Exact Test (one-sided)

∆ Positive control mitotic index and % aberrant cells are determined from a single culture

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this assay, the test substance is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.
Executive summary:

The test substance was evaluated for its clastogenic potential in an in vitro cytogenetic assay using human lymphocytes in two separate experiments, up to a maximum concentration of 2150 µg/mL in both the presence and absence of S9-mix. Cultures were treated for 3 hours with or without S9 -mix in Experiment 1 and with S9 -mix in Experiment 2. In Experiment 2 the treatment time in the absence of S9 -mix was 20 hours.

No statistically or biologically significant increases in the percentage of aberrant cells, above the solvent control values, were recorded in cultures treated in Experiment 1 in the presence of S9-mix or Experiment 2 in either the presence or absence of S9-mix.

It is concluded that, under the conditions of this assay, the test substance is not clastogenic to cultured human lymphocytes treated in vitro in either the presence or absence of S9-mix.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10 August - 22 September 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Adopted at Step 4 of the ICH process 19 July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Adopted at Step 4 of the ICH process 16 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- L5178Y TK+/- 3.7.2.c cells (ex Dr J Cole, MRC, Sussex, UK)
- Stored in a liquid nitrogen freezer
- Checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9 [phenobarbital (80 mg/kg) and β-naphthoflavone (100 mg/kg) induced]
Test concentrations with justification for top dose:
125, 250, 500, 1000, 1500 and 2150 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethyl sulphoxide (BDH-Dried); the solvent was chosen for its solubilisation properties and lack of toxicity to the cells.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9, at a final concentration of 750 µg/mL (200 µg/mL for the 24 hr treatment)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
Remarks:
with S9, at a final concentration of 2 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Two series of exponentially growing suspension cultures of L5178Y cells were treated in duplicate with the solvent control, positive controls or a range of concentrations of the test substance in the presence and absence of S9-mix.

DURATION
- Exposure duration: 4 or 24 hrs; rotated on a roller apparatus in a 37°C hot room
- Expression time (cells in growth medium): 48 hrs in a 37°C hot room
- Selection time (if incubation with a selection agent): 10-13 days

SELECTION AGENT (mutation assays): trifluorothymidine

NUMBER OF REPLICATIONS: The substance was tested both in the presence and absence of S9-mix in two independent experiments. A 24 hour treatment period in the absence of S9-mix was used in the second experiment.

MUTATION ASSAY
- A dose-ranging study was performed to determine the concentrations of the test substance to be used in the main mutation assays.
- The cell density of each culture was determined and the cultures were then divided into two series of dilutions. The first was to form the cultures for assessment of mutants by TFT selection; the second was to assess the viability of the cultures (in the absence of TFT).
Evaluation criteria:
Cell growth in individual microwell plates was assessed after 10-13 days using a x10 dissecting microscope. The survival plates and viability plates were scored for the number of wells containing no cell growth (negative wells). The mutation plates were scored so that each well contained either a small colony (considered to be associated with clastogenic effects), a large colony (considered to be associated with gene mutation effects) or no colony.
For a positive response, a statistically significant dose-related increase in mutant frequency is required, but not only at concentrations eliciting excessive toxicity. The response must be reproducible.
A negative response is obtained when there is no reproducible statistically significant dose-related increase in mutant frequency.
Statistics:
Data were considered by logit regression using a complimentary log-log link function. Tests for trend and intergroup comparisons of log mutant frequency were performed. All tests were one-sided.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
acceptable levels of cytotoxicity, as evident by a reduction in survival levels, was observed at the maximum concentrations evaluated
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
acceptable levels of cytotoxicity, as evident by a reduction in survival levels, was observed at the maximum concentrations evaluated
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Precipitation of the test substance was observed at the end of the treatment period in all cultures treated with concentrations of the test substance of 1000µg/mL or higher, however it was not considered to interfere with the performance of the assay.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

2150 µg/mL of the test substance is approximately equivalent to 10 mM and as such is the limit concentration for this assay. Minimum survival levels, compared to the solvent control cultures, of 29% and 21% were observed in cultures treated with the maximum concentration of the test substance in the presence and absence of S9-mix respectively.

No significant increases in mutant frequency were observed in cultures treated with the test substance in the absence of S9-mix in either of the independent experiments. In the presence of S9-mix, treatment induced small, but reproducible increases in mutant frequency at the maximum concentration tested.

The positive controls induced increases in mutant frequency in all mutation experiments thus demonstrating the activity of the S9-mix and that the assay was capable of detecting known mutagens.

Table 1: Summary of data for experimental phase 1

Without S9 mix

With S9 mix

Concentration (µg/mL)

Mean % day 0 relative survival

Mean mutant frequency (x 10^4)

Concentration (µg/mL)

Mean % day 0 relative survival

Mean mutant frequency (x 10^4)

Test material

Test material

2150

21

7.3

2150

30

6.0*

1500

33

3.8

1500

32

2.8

1000

73

3.0

1000

76

3.6

500

77

4.5

500

88

3.3

250

88

6.3

250

93

4.0

125

91

4.1

125

101

4.8

Solvent control

Solvent control

DMSO (10 µl/mL)

100

4.2

DMSO (10 µl/mL)

100

2.6

Positive control

Positive control

EMS 750

41

14.1**

BP 2

7

18.8**

*Statistically significant p<0.05

**Statistically significant p<0.01

 

Table 2: Summary of data for experimental phase 2

Without S9 mix (24 hour)

With S9 mix

Concentration (µg/mL)

Mean % day 0 relative survival

Mean mutant frequency (x 10^4)

Concentration (µg/mL)

Mean % day 0 relative survival

Mean mutant frequency (x 10^4)

Test material

Test material

2150

0

b

2150

29

6.2*

1500

0

b

1500

41

3.4

1000

5

b

1000

66

2.5

500

33

3.0

500

83

2.6

250

67

3.2

250

91

2.6

125

76

5.2

125

84

2.4

Solvent control

Solvent control

DMSO (10 µl/mL)

101

3.7

DMSO (10 µl/mL)

100

2.5

Positive control

Positive control

EMS 200

67

13.2**

BP 2

9

18.2**

*Statistically significant p<0.05

**Statistically significant p<0.01

b – not counted due to excessive toxicity

Conclusions:
Interpretation of results (migrated information):
positive with metabolic activation
negative without metabolic activation

It is concluded that, under the conditions of this assay, the test substance is weakly mutagenic in L5178Y TK+/- cells treated in vitro in the presence of S9-mix.
Executive summary:

The test substance was tested up to a maximum concentration of 2150 µg/mL in both the presence and absence of S9-mix. Minimum survival levels, compared to the solvent control cultures, of 29% and 21% were observed in cultures treated with the maximum concentration in the presence and absence of S9-mix respectively.

No significant increases in mutant frequency were observed in cultures treated with the test substance in the absence of S9-mix in either of the independent experiments. In the presence of S9-mix, treatment induced small, but reproducible increases in mutant frequency at the maximum concentration tested. It is concluded that, under the conditions of this assay, the test substance is weakly mutagenic in L5178Y TK+/- cells treated in vitro in the presence of S9-mix.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Non-GLP, non-guideline study, only one strain, minor restrictions in design and notable limitations in reporting.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
investigative study using S typhimurium TA98 without metabolic activation
Principles of method if other than guideline:
Bacterial gene mutation assay using Salmonella typhimurium strain TA98. Three different samples (sources) of the test substance tested together with a "class" control, under the same conditions. Standard plate-incorporation assay protocol in the absence of any rat liver-derived metabolic activation system. Plates were assessed after 3 days incubation and results confirmed in an independent experiment.
GLP compliance:
no
Remarks:
protocol, experimental phases and report not subject to Quality Assurance audit.
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
without
Test concentrations with justification for top dose:
100-5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dried dimethylsulphoxide
- Justification for choice of solvent/vehicle: the vehicle was chosen due to its solubilisation properties and lack of toxicity to the bacteria
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
True negative controls:
no
Positive controls:
yes
Remarks:
known positive batch
Positive control substance:
other: 4-mesyl-2-nitrotoluene
Details on test system and experimental conditions:
no further details reported
Evaluation criteria:
not reported
Statistics:
not reported
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
ambiguous
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Remarks on result:
other: other: Batch 1
Remarks:
Migrated from field 'Test system'.

Batch

TA98 without metabolic activation

result

maximum increases over background mutation rate

experiment 1

experiment 2

Batch 1

equivocal

1.9

2.0

Batch 2

negative

1.6

1.5

Batch 3

positive

3.5

3.0

Class control

positive

58.6

31.2

Conclusions:
Interpretation of results (migrated information):
other: Result is potentially dependant on the substance impurity profile

Batch 2 tested negative, Batch 1 tested equivocal, and Batch 3 tested positive. The reported findings indicate that the test substance may not be intrinsically genotoxic in vitro in the bacterial mutation assay.
Executive summary:

In this study, three samples (sources) of the test substance were tested in Salmonella typhimurium strain T98 without metabolic activation to see whether gene mutation was induced. Under these limited assay conditions, Batch 2 gave a negative response, Batch 1 gave an equivocal response and Batch 3 gave a positive response.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
from 2011-11-15 to 2011-11-19 (experimental phase)
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study is well documented and follows methodology that is standard for Ames studies. Fewer strains than requested by the OECD guideline were tested. However, the strains have been chosen on a scientific basis and are the ones known to be the most responsive to this type of material. The test was performed without metabolic activation only.
Qualifier:
no guideline followed
Principles of method if other than guideline:
The purpose of the study was to evaluate 10 batches of the test item for the ability to induce reverse mutations, directly, at the histidine locus in the genome of two strains of bacteria. The study was based on the in vitro technique described by Ames et al (1975), Maron and Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimurium to various concentrations of the test item batches.
Salmonella typhimurium strains TA1537 and TA98 were treated against 10 different batches of the test item using the Ames plate incorporation method at six dose levels, in triplicate without the addition of a rat liver homogenate metabolising system.
Fewer strains than requested by the OECD guideline were tested. However, the strains have been chosen on a scientific basis and are the ones known to be the most responsive to this type of material. The test was performed without metabolic activation only, since this is not generally required for this sort of agent where a positive effect is seen.


- Ames, B N, McCann J, and Yamasaki E (1975), Methods for detecting carcinogens and mutagens with the Salmonella/mammalian microsome mutagenicity test, Mutation Research, 31, 347-364.
- Maron D M and Ames B N (1983), Revised Methods for the Salmonella mutagenicity test, Mutation Research, 113, 173 - 215.
- Mortelmans K, Zeiger E (2000), The Ames Salmonella/microsome mutagenicity assay, Mutation Research, 455, 29-60.
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1537
Species / strain / cell type:
S. typhimurium TA 98
Metabolic activation:
without
Test concentrations with justification for top dose:
Experiment I: 100, 200, 500, 1000, 2500 and 5000 μg/plate
Experiment II: 100, 200, 500, 1000, 2500 and 5000 μg/plate
Experiment III: 100, 200, 500, 1000, 2500 and 5000 μg/plate (plates were not scored and were discarded once the incubation period was
complete because there was concordance between the results of Experiments I and II, thereby confirming the definitive result of the test.)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Each batch of test item was soluble in DMSO at 50 mg/mL.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: approximately 48 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: visible reduction in the growth of the bacterial background lawns
Evaluation criteria:
The criteria for a positive result will be a two-fold increase (accurate to 1 decimal place), at any dose level, in the number of revertant colonies when compared to the solvent control (mean value of 15 plates). If the first two experiments give non-concordant results then the third experiment will determine the overall result. The overall conclusion for each batch of test item is "pass" if both strains pass and "fail" if either/both strains fail.
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No test item precipitate was observed on the plates at any of the doses tested in the absence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The frequency of revertant colonies for the negative, solvent and positive controls lie within the historical range.

ADDITIONAL INFORMATION ON CYTOTOXICITY: The 10 batches of test item caused no visible reduction in the growth of the bacterial background lawns of either tester strain.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Experiments 1& 2, Batch 20111008036

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

35

21

22

1.1

20

17

17

1.5

26

27

29

1.0

12

11

9

0.9

-

200

26

20

28

1.1

17

20

13

1.4

33

27

27

 

1.1

9

10

10

0.8

-

500

22

24

30

1.1

11

19

17

1.3

35

33

29

1.2

12

11

7

0.8

-

1000

21

33

20

1.1

16

11

22

1.3

26

31

33

1.2

9

11

12

0.9

-

2500

24

11

21

0.8

10

15

16

1.2

32

29

26

1.1

8

11

9

0.8

-

5000

17

28

16

0.9

5

16

13

0.9

20

23

33

1.0

8

10

12

0.8

0.0: Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20110906046

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

21

34

29

1.2

20

12

8

1.1

27

30

29

1.1

13

14

15

1.2

-

200

29

28

36

1.3

18

21

14

1.5

35

30

33

1.3

15

15

13

1.2

-

500

28

27

24

1.1

15

16

12

1.2

33

34

34

1.3

15

14

13

1.2

-

1000

21

32

22

1.1

11

11

15

1.0

35

30

29

1.2

14

15

13

1.2

-

2500

21

24

20

1.0

9

16

15

1.1

32

32

29

1.2

15

14

14

1.2

-

5000

12

12

16

0.6

10

13

16

1.1

32

27

32

1.2

14

16

11

1.2

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20110926106

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

20

29

38

1.3

18

11

18 (

1.3

31

35

33

1.3

13

12

15

1.1

-

200

18

29

28

1.1

16

13

13

1.2

27

33

29

1.2

14

14

15

1.2

-

500

28

33

28

1.3

7

22

9

1.1

32

22

27

1.0

14

15

15

1.3

-

1000

27

21

27

1.1

18

8

14

1.1

34

27

23

1.1

14

16

15

1.3

-

2500

18

17

28

0.9

12

10

6

0.8

31

22

22

1.0

15

12

12

1.1

-

5000

15

13

11

0.6

14

11

12

1.0

22

21

21

0.8

12

14

12

1.1

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20111008033

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

20

38

40

1.4

15

15

10

1.1

30

26

31

1.1

10

12

12

0.9

-

200

24

14

19

0.8

13

20

14

1.3

34

27

31

1.2

13

14

14

1.2

-

500

27

25

25

1.1

12

10

10

0.9

24

25

27

1.0

14

13

12

1.1

-

1000

15

16

27

0.8

15

14

7

1.0

24

30

27

1.0

13

12

13

1.1

-

2500

21

19

18

0.8

17

9

7

0.9

24

27

29

1.0

12

12

13

1.0

-

5000

27

7

14

0.7

13

13

7

0.9

21

25

21

0.8

10

11

11

0.9

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batches 20110917091

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

31

36

37

1.5

18

11

11

1.1

35

29

24

1.1

13

16

11

1.1

-

200

31

37

20

1.3

12

12

9

0.9

29

30

24

1.1

12

13

11

1.0

-

500

24

33

22

1.1

20

16

11

1.3

26

30

25

1.0

9

13

9

0.8

-

1000

18

35

18

1.0

21

17

19

1.6

29

34

29

1.2

15

9

13

1.0

-

2500

26

22

15

0.9

14

16

19

1.3

31

33

37

1.3

11

14

11

1.0

-

5000

19

11

19

0.7

10

7

9

0.8

26

20

20

0.8

12

14

14

1.1

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batches 20110905039

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

36

27

24

1.3

16

10

16

1.2

30

25

29

1.1

16

9

11

1.0

-

200

34

32

29

1.4

20

15

18

1.5

25

30

21

1.0

12

12

12

1.0

-

500

29

32

31

1.3

10

10

16

1.0

29

30

30

1.2

16

10

16

1.2

-

1000

22

18

14

0.8

13

10

19

1.2

30

29

30

1.2

14

14

11

1.1

-

2500

21

14

18

0.8

9

18

12

1.1

23

29

25

1.0

12

14

13

1.1

-

5000

13

20

18

0.7

12

14

15

1.2

29

23

27

1.0

13

15

12

1.1

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20110906044

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

26

44

30

1.4

14

16

20

1.4

34

32

31

1.2

11

12

12

1.0

-

200

27

19

27

1.0

10

13

21

1.3

24

30

22

1.0

13

14

14

1.2

-

500

17

21

28

1.0

16

18

12

1.3

24

30

32

1.1

12

12

7

0.8

-

1000

26

28

22

1.1

12

13

15

1.1

24

21

24

0.9

12

12

14

1.1

-

2500

16

21

19

0.8

11

6

18

1.0

29

23

29

1.0

13

14

13

1.1

-

5000

9

12

14

0.5

7

10

12

0.8

18

19

22

0.8

13

14

12

1.1

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batches 20110925104

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

25

29

21

1.1

17

16

11

1.3

34

24

24

1.0

15

13

8

1.0

-

200

22

19

16

0.8

16

25

16

1.6

29

30

26

1.1

12

13

8

0.9

-

500

26

18

20

0.9

8

12

10

0.8

25

26

29

1.0

8

11

14

0.9

-

1000

22

18

24

0.9

11

20

8

1.1

23

26

31

1.0

11

14

13

1.1

-

2500

20

21

14

0.8

17

18

14

1.3

25

29

30

1.1

14

15

13

1.2

-

5000

7

14

15

0.5

14

16

15

1.3

20

18

22

0.8

15

14

15

1.3

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20110906042

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

22

36

30

1.3

18

16

11

1.3

29

35

29

1.2

14

19

12

1.3

-

200

24

31

24

1.1

13

14

11

1.1

33

26

29

1.1

13

13

13

1.1

-

500

27

16

35

1.1

17

18

14

1.3

29

30

31

1.2

14

15

15

1.3

-

1000

20

27

24

1.0

16

12

18

1.3

29

34

29

1.2

15

15

11

1.2

-

2500

21

24

25

1.0

11

12

14

1.0

25

25

29

1.0

14

13

14

1.2

-

5000

10

16

17

0.6

4

13

12

0.8

33

29

29

1.2

14

13

13

1.1

0.0:Fold increase over concurrent solvent control

Experiments 1& 2, Batch 20111009038

With or without S9-Mix

Test substance concentration (µg/plate)

Number of revertants (mean number of colonies per plate) Frameshift Type

Experiment 1 TA98 TA1537

Experiment 2 TA98 TA1537

-

100

29

30

32

1.3

16

16

15

1.3

27

21

32

1.0

12

12

12

1.0

-

200

25

33

31

1.3

15

14

15

1.3

22

30

34

1.1

13

12

11

1.0

-

500

26

24

30

1.2

17

11

16

1.3

24

27

29

1.0

13

12

11

1.0

-

1000

26

33

32

1.3

15

16

19

1.4

24

30

26

1.0

13

11

11

1.0

-

2500

15

11

27

0.8

13

13

9

1.0

25

27

29

1.0

11

14

11

1.0

-

5000

15

16

16

0.7

14

8

7

0.8

26

24

26

1.0

10

13

12

1.0

0.0:Fold increase over concurrent solvent control

 

Conclusions:
Interpretation of results (migrated information):
positive without metabolic activation

The 10 batches of the test item were considered to be non-mutagenic under the conditions of this test.
Executive summary:

Salmonella typhimurium strains TA1537 and TA98 were treated against 10 different batches of the test item using the Ames plate incorporation method at six dose levels, in triplicate without the addition of a rat liver homogenate metabolising system. The dose range for the first experiment was 100, 200, 500, 1000, 2500 and 5000 μg/plate. A second experiment was performed using the same dose levels, fresh cultures of the bacterial strains and fresh test item formulations. Results from a third experiment were not required because the results of Experiment 1 and 2 were in concordance.

The vehicle (dimethyl sulphoxide) control plates gave counts of revertant colonies within the normal range. Both positive control chemicals used in the test induced marked increases in the frequency of revertant colonies without metabolic activation. Thus, the sensitivity of the assay was confirmed.

The 10 batches of test item caused no visible reduction in the growth of the bacterial background lawns of either tester strain. The test item was tested up to the maximum recommended dose level (5000 μg/plate). No test item precipitate was observed on the plates at any of the doses tested in absence of S9-mix.

No toxicologically significant increases in the frequency of revertant colonies, in excess of twofold greater than the concurrent solvent controls, were recorded for either of the bacterial strains, for any dose level of each batch of test item.

The 10 batches of the test item were considered to be non-mutagenic under the conditions of this test.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

- negative, Rat bone marrow micronucleus test in vivo, Fox 2005

- negative, In vivo Unscheduled DNA Synthesis in rat hepatocytes, Bohnenberger 2013

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Remarks:
Type of genotoxicity: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012-05-09 to 2013-01-19 (experimental phase)
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline conform study without deviations conducted under GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 486 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells in vivo)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.39 (Unscheduled DNA Synthesis (UDS) Test with Mammalian Liver Cells In Vivo)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
unscheduled DNA synthesis
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 8 to 11 weeks
- Weight at study initiation: males, first experiment: 253.3 g ± 10.9 g (range 228 to 267.6 g); females, first experiment: 159.9 g ± 5.4 g (range 150.5 to 169.9 g); males, second experiment: 241.5 g ± 7.4 g (range 231 to 254.1 g)
- Assigned to test groups randomly: yes
- Fasting period before study: not reported
- Housing: in groups in Makrolon Type III/IV cages with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 to > 90 during acclimatization; Relative humidity 45 - 65% during experimental performance
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hours dark, 12 hours light

IN-LIFE DATES: From: To:
Route of administration:
oral: gavage
Vehicle:
1% carboxymethylcellulose (CMC)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: on the day of the experiment the test item was suspended in 1% CMC. Grinding in a mortar was necessary to formulate the test item. The test substance was administered as suspension in 1% CMC. the administration volume was 15 mL/kg bw.
Duration of treatment / exposure:
Animals received a single dose of substance in vehicle and were observed for up to 16 hours
Frequency of treatment:
Single application
Post exposure period:
Up to 16 hours
Remarks:
Doses / Concentrations:
Males: 400 mg/kg and 800 mg/kg (additional group of 200 mg/kg was included due to high mortality in the 16-hours post-treatment group receiving 800 mg/kg); Females: 250 mg/kg and 500 mg/kg
Basis:
actual ingested
No. of animals per sex per dose:
Four
Control animals:
yes, concurrent vehicle
Positive control(s):
4 hours preparation interval: N,N'-dimethylhydrazinedihydrochloride (DMH) at 80 mg/kg bw
16 hours preparation interval: 2-acetylaminofluorene (2-AAF)
Tissues and cell types examined:
Isolated hepatocytes from the livers of test animals
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: a maximum tolerated dose was determined in two pre-experiments, in which the substance was administered by oral gavage to two male and two female animals under the same conditions that were applied in the main test

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
The washed hepatocytes were centrifuged and transferred into Williams medium E supplemented with:
Hepes 2.38 mg/mL
L-Glutamine 0.29 mg/mL
Penicillin 100 units/mL
Insulin 0.50 µg/mL
Streptomycin 0.10 mg/mL
Fetal calf serum (FCS) 100 µL/mL
This complete medium was adjusted to pH 7.6. Three cultures were established from each animal. Aliquots of 2.5 mL with freshly isolated hepatocytes in complete culture medium (200000 viable cells/mL) were added to 35 mm six-well dishes containing one 25 mm round plastic coverslip per well coated with gelatine.

After an attachment period of approximately 1.5 h in a 95 % air/ 5 % CO2 humidified incubator at 37° C the culture medium was discarded. The cell layer was then rinsed once with physiologically buffered saline (PBS) to remove non-adherent cells. Subsequently, 3HTdR (5 µCi/mL, specific activity 70 - 90 Ci/mmol) in 2.0 mL culture medium (Williams medium E (WME), 1 % (v/v) fetal calf serum (FCS)) was added to the cultures. After a labeling time of 4 h the cells were washed twice with WME supplemented with 1 % (v/v) FCS and 0.25 mM unlabeled thymidine. Cultures were incubated overnight using the same medium. To prepare for autoradiography the medium was replaced by a hypotonic solution of 1 % (w/v) sodium citrate for 10 minutes to swell the nuclei for better grain detection. The cells on the coverslips were then fixed by three changes of methanol:acetic acid (3:1 v/v) for 20 minutes each, rinsed with 96 % (v/v) ethanol, and air-dried.


DETAILS OF SLIDE PREPARATION:
The cover slips were mounted on glass slides, cell side upwards and coated with KODAK NTB photographic emulsion in the dark. The coated slides were stored in light-proof boxes in the presence of a drying agent for 14 days at 4°C . The photographic emulsion was then developed at room temperature, fixed in Fixer and stained with hematoxylin/eosin.

METHOD OF ANALYSIS:
Evaluation was performed microscopically on coded slides using microscopes with oil immersion objectives. Slides were examined to ensure sufficient cells of normal morphology were present before analysis. The cells for scoring were randomly selected according to a fixed scheme. The number of silver grains in the nuclear area was counted automatically using the Sorcerer UDS device. In addition, the number of grains of the most heavily labeled nuclear-sized cytoplasm area adjacent to the nucleus was counted. Two slides per animal and 50 cells per slide were evaluated (except for the positive control group of the females with a total of 151 evaluated cells per animal on three slides). Heavily radio-labeled cells undergoing replicative DNA synthesis were excluded from counting.

All animals per group were evaluated as described above, except for the mid dose group of the 4 h preparation interval in male rats and the high dose group of the 16 h preparation interval in female rats where three male animals instead of four animals were evaluated, due to technical reasons during the liver perfusion.

DATA RECORDING
The data generated were recorded in the raw data. The results were presented in tabular form, including experimental groups with the test item, vehicle and positive controls.

The nuclear and cytoplasmic grain counts, the net grain counts (nuclear minus cytoplasmic grains) as well as the mean and percentage of cells in repair (cells with a net grain count larger than 5) is reported separately. Individual slide and animal data are provided. The mean counts with standard deviation are used to describe the distribution of 3HTdR incorporation in the nucleus, the cytoplasm and for the net grains, respectively.
Evaluation criteria:
Nuclear and net grain counts are estimated together. Increased net grains should be based on enhanced nuclear grain counts rather than on decreased cytoplasmic grain counts.

A test item is classified as positive if the mean number of net grains is higher than five per nucleus at one of the test points.

A group average between 0 and 5 net grains is considered as a marginal positive response. A dose-related increase in nuclear and net grains and/or a substantial shift of the percentage distribution of the nuclear grain counts to higher values provide additional information to confirm a positive response with less than 5 net grains.

Statistical significance may give further evidence for a positive evaluation. Statistical significance can be evaluated by means of the non-parametric Mann-Whitney test.

A test item producing net grains not greater than 0 or not significantly greater than the concurrent control, at any of the test points is considered negative in this system.
Statistics:
A statistical evaluation of the results was not necessary to perform as the number of net grain counts of the groups treated with the test item were in the range of the corresponding vehicle controls.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: first pre-test with 1250 mg/kg in males and 800 mg/kg in females; a second pre-test was conducted due to severe clinical signs occurring in the first pre-test and doses of 800 mg/kg and 500 mg/kg were administered to two male and two female rats, respectively
- Solubility: the substance was suspended in the vehicle
- Clinical signs of toxicity in test animals: first pre-test caused severe clinical signs in animals and two males were sacrificed after 2 to 4 hours and the females were sacrificed moribund after 6 hours
- Rationale for exposure: the oral route was used as this is of relevance to human risk assessment

RESULTS OF DEFINITIVE STUDY
- Appropriateness of dose levels and route: Severe toxicity was noted in the male 800 mg/kg b.w. 16 h treatment group. On this basis additional male treatment groups were dosed at 200 mg/kg b.w., for both time points. Associated positive and negative control groups were also used. Hence, male rats receiving a single oral dose of the substance at dose levels of 200, 400 and 800 mg/kg showed a number of clinical symptoms including reduction of spontaneous activity, abdominal position, ruffled fur, tumbling, apathy, sunken flanks and death (800 mg/kg 16 h only). Female rats receiving a single oral dose of the substance at dose levels of 250 and 500 mg/kg showed a number of clinical symptoms including reduction of spontaneous activity, ruffled fur, sunken flanks, abdominal posture, eyelid closure, tumbling, lacrimation and gasping. the toxic effects seen indicated systemic exposure to the test substance. The animals in the negative and positive control groups did not show any signs of toxicity.
- Statistical evaluation: no statistical evaluation of the results was necessary.

Group means of nucleus, cytoplasmic area and net grains of males

 

Nuclear Grain Count

Cytoplasmic Grain Count

Net Grain Counts

Nuclear Grain Counts of Cells in Repair

Cells in Repair

Test Group

Mean

SD

Mean

SD

Mean

SD

Mean

SD

%

 

4 h Preparation Interval

Vehicle control (CMC 1 %)
1st experiment

22.58

11.28

38.85

14.61

-16.27

11.97

7.67

1.62

5

Vehicle control (CMC 1 %) 2nd experiment

15.31

7.08

30.00

10.63

-14.69

9.78

2.88

0.88

1

200 mg/kg b.w. test substance 2nd experiment

20.08

7.94

32.89

12.13

-12.81

9.93

5.40

1.23

3

400 mg/kg b.w. test substance 1st experiment*

19.29

6.99

37.54

12.67

-18.25

11.95

8.83

1.18

2

800 mg/kg b.w. test substance 1st experiment

17.07

6.60

32.62

12.14

-15.55

11.18

6.06

0.58

3

positive control (DMH)
1st experiment

55.90

19.65

28.01

11.64

27.88

16.88

30.05

15.42

92

positive control (DMH)
2nd experiment

72.82

21.60

32.17

11.84

40.65

18.40

42.19

16.97

96

 

16 h Preparation Interval

Vehicle control (CMC 1 %)
1st experiment

29.33

11.54

44.45

15.66

-15.13

14.34

14.46

9.76

7

Vehicle control (CMC 1 %) 2nd experiment

24.32

10.45

41.27

14.73

-16.95

13.76

11.06

7.06

8

200 mg/kg b.w. test substance 2nd experiment

24.14

9.12

33.31

11.09

-9.17

10.65

10.08

2.95

8

400 mg/kg b.w. test substance 1st experiment

29.04

11.70

51.79

18.50

-22.76

16.90

18.40

3.22

4

800 mg/kg b.w. test substance 1st experiment

invalid dose group due to high mortality of the treated animals

positive control (2-AAF)
1st experiment

74.68

22.82

22.50

7.90

52.18

20.94

52.18

20.94

100

positive control (2-AAF)
2nd experiment

33.38

11.26

21.11

9.38

12.27

10.19

16.05

8.43

76

SD=Standard deviation. The standard deviation shown for each animal is the deviation between the 100 analysed cells. The deviation shown for the mean of each group is the mean of the standard deviation obtained for each animal for a group consisting of four animals (* three animals) (test item groups) or two animals (control groups).

Group means of nucleus, cytoplasmic area and net grains of females

 

Nuclear Grain Count

Cytoplasmic Grain Count

Net Grain Counts

Nuclear Grain Counts of Cells in Repair

Cells in Repair

Test Group

Mean

SD

Mean

SD

Mean

SD

Mean

SD

%

 

4 h Preparation Interval

Vehicle control (CMC 1 %)

10.24

4.54

23.17

6.64

-12.93

7.27

2.56

1.25

2

250 mg/kg b.w. test substance

13.69

5.84

22.95

7.83

-9.26

8.06

8.22

1.81

4

500 mg/kg b.w. test substance

16.36

6.67

28.68

9.41

-12.32

9.60

5.52

1.77

4

positive control (DMH)

65.65

16.83

19.12

6.40

46.54

16.61

46.72

16.44

100

 

16 h Preparation Interval

Vehicle control (CMC 1 %)

17.83

9.07

36.21

12.59

-18.38

10.96

2.75

0.50

2

250 mg/kg b.w. test substance

18.11

8.04

31.56

10.63

-13.45

10.91

8.44

1.56

4

500 mg/kg b.w. test substance*

16.80

7.14

30.36

11.17

-13.56

10.09

4.92

1.06

3

positive control (2-AAF)

31.83

12.31

27.88

10.27

3.95

10.78

13.48

7.37

44

SD=Standard deviation. The standard deviation shown for each animal is the deviation between the 100 analysed cells. the deviation shown for the mean of each group is the mean of the standard deviations obtained for each animal for a group consisting of four animals (* three animals) (test item groups) or two animals (control groups).

Viability and number of hepatocytes for the males, 4 h timepoint

Treatment

Period

Animal no.

Viability*[%]

Number of isolated cells [x106]

1 % CMC (1stmain experiment)

4 h

1

62

158

2

78

351

1 % CMC (2ndmain experiment)

4 h

49

69

267

50

78

246

200 mg/kg b.w. Test substance

4 h

51

89

318

52

77

244

53

76

211

54

78

160

400 mg/kg b.w. Test substance

4 h

3

81

88.5

4

68

233

5

73

210

6

Perfusion failed

800 mg/kg b.w. Test substance

4 h

7

62

212

8

65

374

9

70

186

10

68

281

80 mg/kg b.w. DMH (1stmain experiment)

4 h

11

75

255

12

85

302

80 mg/kg b.w. DMH (2ndmain experiment)

4 h

55

78

265

56

75

233

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the males, 16 h timepoint

Treatment

Period

Animal no.

Viability*[%]

Number of isolated cells [x106]

1 % CMC (1stmain experiment)

 

16 h

13

72

306

 

14

90

266

 

1 % CMC (2ndmain experiment)

 

16 h

57

83

251

 

58

80

228

 

200 mg/kg b.w. Test substance

 

 

 

16 h

59

82

223

 

60

81

134

 

61

78

425

 

62

73

226

 

400 mg/kg b.w. Test substance

 

 

 

16 h

15

90

232

 

16

82

215

 

17

83

255

 

18

88

282

 

800 mg/kg b.w. Test substance

 

 

 

16 h

19

Invalid dose group due to high mortality of the treated animals

 

20

 

21

 

22

 

100 mg/kg b.w. 2-AAF (1stmain experiment)

 

16 h

23

86

275

 

24

83

278

 

100 mg/kg b.w. 2-AAF (2ndmain experiment)

 

16 h

63

75

306

 

64

65

258

 

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the females, 4 h timepoint

Treatment

Period

Animal no.

Viability*[%]

Number of isolated cells [x 106]

1 % CMC

4 h

25

81

75

26

71

147

250 mg/kg b.w. Test substance

 

 

 

4 h

27

57

85.5

28

78

318

29

59

103

30

79

154

500 mg/kg b.w. Test substance

 

 

 

4 h

31

72

205

32

82

269

33

74

196

34

65

83

80 mg/kg b.w. DMH

4 h

35

90

196

36

80

270

* Viability determined by means of trypan blue dye exclusion assay

Viability and number of hepatocytes for the females, 16 h timepoint

Treatment

Period

Animal no.

Viability*[%]

Number of isolated cells [´ 106]

1 % CMC

16 h

37

85

230

 

38

75

174

 

250 mg/kg b.w. Test substance

 

 

 

16 h

39

85

159

 

40

79

142

 

41

80

130

 

42

80

128

 

500 mg/kg b.w. Test substance

 

 

 

16 h

43

81

152

 

44

Perfusion failed

 

45

62

174

 

46

80

162

 

100 mg/kg b.w. 2-AAF

16 h

47

75

203

 

48

66

127

 

* Viability determined by means of trypan blue dye exclusion assay

Conclusions:
Interpretation of results (migrated information): negative
Under the experimental conditions, i.e. oral administration up to 800 mg/kg for males and up to 500 mg/kg for females, the test item did not induce DNA-damage leading to increased repair synthesis in the hepatocytes of the treated rats. Therefore, the substance is considered to be non-genotoxic in this in vivo UDS test system.
Executive summary:

The substance was assessed under GLP in this valid in vivo UDS assay for its potential to induce unscheduled DNA repair (UDS) in the hepatocytes of rats according to OECD TG 486 with doses of 200, 400 and 800 mg/kg bw for males and 250 and 500 mg/kg bw for females after post treatment intervals of 4 and 16 hours. The highest dose, i.e. the maximum tolerated dose leading to acceptable clinical symptoms, was established in two pre-experiments. The test item was suspended in 1% carboxymethylcellulose, which was also used as the vehicle control. The volume administered orally was 15 mL/kg body weight. After single oral treatment and a post-treatment period of 4 or 16 hours the animals were sacrificed by terminal anaesthesia. The livers were then perfused. Primary hepatocytes wee established and exposed for 4 hours to 3HTdR, which is incorporated if UDS occurs.

The viability of the hepatocytes was not substantially affected by the in vivo treatment with the test substance at any of the treatment periods or dose groups. The interindividual variations obtained for the numbers and the viability of the isolated hepatocytes were in the range of the laboratory historical control data. All viabilities were greater than the OECD TG recommended viability of > 50%.

No UDS induction in the hepatocytes of the male and female rats treated with a single oral dose of the test substance as compared to the animals of the concurrent vehicle controls was observed at 200, 400 and 800 mg/kg bw for male rats and at 250 and 500 mg/kg bw for female rats. The nuclear grain counts and the resulting net grain counts were not distinctly enhanced due to the in vivo treatment of the animals with the test item after 4 hours and 16 hours post treatment. Therefore, the mean net grain counts obtained after treatment with the the substance were consistently negative. As no UDS response was observed statistical analysis of the data was not performed. No substantial shift to higher values was obtained in the percentage of cells in repair, which confirms the lack of UDS.

Appropriate reference mutagens (N,N'-dimethylhydrazinedihydrochloride at 80 mg/kg bw and 2-acetylaminofluorene at 100 mg/kg bw) produced distinct increases in the number of nuclear and net grain counts, indicating UDS. Additionally, the percentage of cells in repair was significantly increased, which is consistent with a UDS response. The positive control at the 16 hour preparation interval of the female animals showed a mean net grain count of 3.95 being below the optimal value of 5 net grain counts indicating nominally used in this laboratory to indicate a positive result but within the laboratory historical control data range for similar studies and distinctly different to the strongly negative values observed for the negative control groups. Additionally, as 44% of the cells were in repair (compared to 1.5% of the cells in the vehicle control) the positive control response is considered as valid.

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21 February - 24 March 2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, no restrictions, fully adequate for assessment
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2A. Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals. Adopted at Step 4 of the ICH process 19 July 1995
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guideline S2B. Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceuticals. Adopted at Step 4 of the ICH process 16 July 1997
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: 5-7 weeks 
- Weight at study initiation: 200-272 g 
- Housing: in cages suitable for rats of this strain and weight range expected during the course of the study (individually or by group not reported)
- Diet: Rat and Mouse No.1 maintenance diet ad libitum
- Water: mains water ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25°C 
- Humidity: 30-70%
- Air changes: at least 15 per hr
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: From: 23 February 2005 To: 24 March 2005
Route of administration:
oral: gavage
Vehicle:
- Vehicle(s)/solvent(s) used: 1% w/v aqueous CMC (carboxymethyl cellulose), the vehicle was chosen due to its lack of toxicity
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
An individual stock suspension of the test substance was prepared in CMC for each group of animals. The positive control substance was prepared as a solution in sterile double deionised water.
All test and positive control substance dosing preparations were prepared as close to the time of dosing as possible. The test substance, vehicle and positive control substance were dosed at a volume of 10 mL/kg bodyweight.



Duration of treatment / exposure:
Single dose.
Frequency of treatment:
Once.
Post exposure period:
Bone marrow samples were taken 24 or 48 hours after dosing.
Remarks:
Doses / Concentrations:
500, 1000 or 2000 mg/kg
Basis:
other: single gavage dose
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Positive control(s):
cyclophosphamide
- Route of administration: oral (gavage)
- Doses / concentrations: 20 mg/kg
Tissues and cell types examined:
Two thousand immature erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity.
Details of tissue and slide preparation:
CRITERIA FOR DOSE SELECTION: Phase I involved the determination of a maximum tolerated dose (MTD), based on patterns of lethalities or severe toxicity observed over a four-day observation period following a single oral dose.
After acclimatisation, the rats for Phase II were randomly distributed on to racks and the animals were identified by cage cards and by ear punching (Phase I) or tail marking (Phase II).
In Phase II, animals were weighed and given a single oral dose of CMC, cyclophosphamide or the test substance.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields): The animals were killed by over exposure to halothane Ph. Eur. followed by cervical dislocation 24 and 48 hours after receiving a single oral dose of the test substance.

DETAILS OF SLIDE PREPARATION: Femurs were removed and stripped clean of muscle. The iliac end of the femur was removed and a fine paint brush was rinsed in saline, wiped to remove the excess and wetted with a solution of albumin (6% w/v in physiological saline). This was then dipped into the marrow canal and two smears were painted on an appropriately labelled clean, dry microscope slide. This procedure was repeated to give four smears of marrow per slide. The slides were air dried and fixed in solvent methanol for at least 10 minutes. The slides were dipped in phosphate buffer then stained with a solution of acridine orange (0.125 mg/mL) for 1 minute, then placed in fresh phosphate buffer for 10 minutes and then in a fresh batch of buffer for a further 15 minutes. After staining the slides were air dried and wet mounted in buffer prior to analysis.

METHOD OF ANALYSIS: Two thousand immature erythrocytes were examined for the presence of micronuclei for each animal. The slides were also examined for evidence of cytotoxicity, which may be manifest by alterations in the ratio of different cell types in the bone marrow. This was assessed by counting the ratio of immature to mature erythrocytes in a sample of 1000 erythrocytes.

OTHER: All animals were examined internally for abnormalities to organs/tissues. Slides were coded and scored blind.
The criteria for identification of cell types and micronuclei were based on those of Tinwell H and Ashby J (Comparison of acridine orange and giemsa stains in several mouse bone marrow micronucleus assays – including a triple dose study. Mutagenesis 1989, 4:476)
a) Immature erythrocytes - bright orange fluorescence
b) Mature erythrocytes - dull khaki/green colour
c) Micronuclei - bright yellow/green fluorescence.
Evaluation criteria:
The incidence of micronucleated immature erythrocytes and percentage immature erythrocytes in the erythrocyte sample, were considered by analysis of variance at 24 and 48 hours. The data for the incidence of micronucleated immature erythrocytes were transformed using a square root transformation, prior to analysis. The data for the percentages of immature erythrocytes were transformed using the double arcsine transformation of Freeman and Tukey (1950, Transformations related to the angular and the square root. Annals. of Maths stats. 21, 607), prior to analysis. Analyses were carried out using the MIXED procedure in SAS (2004). Each treatment group mean was compared with the control group mean at the corresponding sampling time using a one-sided Student's t-test, based on the error mean square in the analysis. The data have been interpreted as follows:-
a) No statistically significant increase in the incidence of micronucleated immature erythrocytes above concurrent vehicle control incidences - NEGATIVE.
b) A statistically significant increase in the incidence of micronucleated immature erythrocytes above the concurrent vehicle control incidences but which falls within the laboratory historical vehicle control range - NEGATIVE.
c) A statistically and biological significant increase in the incidence of micronucleated immature erythrocytes which is in excess of a three-fold increase when compared with both historical and concurrent vehicle control incidences - POSITIVE.
d) An incidence of micronucleated immature erythrocytes which is statistically significantly different from the concurrent vehicle control incidences, but less than 3-fold in excess of both historical and concurrent vehicle control incidences may require further evaluation.
e) For a test to be considered valid, the positive control group should show an appropriate increase, compared to vehicle control values, in the incidence of micronucleated immature erythrocytes.
Sex:
male
Genotoxicity:
negative
Toxicity:
yes
Remarks:
clinical observations and findings post mortem
Vehicle controls validity:
valid
Negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: no deaths, but clinical signs including reduced splay reflex, pinched in sides, lachrymation, decreased activity and salivation were observed at the limit dose level of 2000 mg/kg therefore this dose level together with two lower dose levels of 1000 and 500 mg/kg, were administered in Phase II.

RESULTS OF DEFINITIVE STUDY
- Clinical signs of toxicity in test animals: Clinical signs observed for males dosed at 2000 mg/kg included decreased activity, chromodacryorrhea of one or both eyes, salivation, pinched in sides and laboured breathing. Salivation and pinched in sides were also observed in animals dosed at 1000 mg/kg. The animals dosed at 500 mg/kg showed decreased activity.
- Examination of the internal organs showed three animals treated at 2000 mg/kg, five animals at 1000 mg/kg and two animals at 500 mg/kg to have stomachs which were enlarged to approximately three times the normal size and three animals at 500 mg/kg to have slightly enlarged stomachs. The contents of the stomachs were noted to be compacted in three animals at 2000 mg/kg and three at 1000 mg/kg. The intestine of one animal at 2000 mg/kg was filled with gas and the caeca of three animals at 1000 mg/kg were enlarged to approximately twice the normal size.

No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes, over the vehicle control values, were observed at either sampling time. No statistically significant differences in the percentage of immature erythrocytes, between the vehicle control and test substance treated animals, were observed at either sampling time. The test system positive control, cyclophosphamide, induced statistically and biologically significant increases in the frequency of micronucleated immature erythrocytes at the 24 hour sampling time.

Table 1: Mean incidence of micronucleated immature erythrocytes – group mean animal data

Group

Treatment

Dose

Mean incidence of MIE/1000 IE ± SD

 

 

 

24 hours

48 hours

11

Vehicle control

10 mL/kg

1.5 ± 0.6

0.9 ± 0.7

12

Cyclophosphamide

20 mg/kg

36.1 ± 3.7**

 

13

Test substance

2000 mg/kg

0.5 ± 0.5

1.6 ± 0.8

14

Test substance

1000 mg/kg

0.9 ± 0.5

 

15

Test substance

500 mg/kg

0.8 ± 0.7

 

IE = immature erythrocytes

MIE = micronucleated immature erythrocytes

SD = standard deviation

**statistically significant increase in micronucleated immature erythrocytes at p<0.01 in the Student’s t-test (one-sided) on transformed data

2000 immature erythrocytes were counted per animal, all means are based on the counts from 5 animals

 

Table 2: Mean percentage of micronucleated immature erythrocytes – group mean animal data

Group

Treatment

Dose

Mean % of immature erythrocytes ± SD

 

 

 

24 hours

48 hours

11

Vehicle control

10 mL/kg

36.7 ± 2.4

51.6 ± 15.3

12

Cyclophosphamide

20 mg/kg

41.7 ± 5.6

 

13

Test substance

2000 mg/kg

43.8 ± 11.1

48.0 ± 11.1

14

Test substance

1000 mg/kg

43.7 ± 15.8

 

15

Test substance

500 mg/kg

42.6 ± 12.9

 

SD = standard deviation

1000 erythrocytes were counted per animal, all means are based on the counts from 5 animals

Conclusions:
Interpretation of results (migrated information): negative
Under the conditions of test, the test substance is not clastogenic in the rat bone marrow micronucleus test.
Executive summary:

The test substance was evaluated for its ability to induce micronucleated immature erythrocytes in the bone marrow of Alpk:APfSD rats. A single oral dose was given to groups of male rats at dose levels of 2000, 1000 and 500 mg/kg. The highest dose level represents the maximum tolerated dose (MTD) based on patterns of clinical signs over a four day observation period and is the limit dose level of the assay. Bone marrow samples were taken 24 and 48 hours after dosing. No statistically or biologically significant increases in the incidence of micronucleated immature erythrocytes, over the vehicle control values, were seen at either of the sampling times investigated. Comparison of the percentage of immature erythrocytes showed no statistically or biologically important differences at either of the sampling times between the vehicle control animals and those treated with the test substance. The test system positive control, cyclophosphamide, induced statistically significant and biologically meaningful increases in micronucleated immature erythrocytes, compared to the vehicle control values, thus demonstrating the sensitivity of the test system to a known clastogen. Under the conditions of test, the test substance is not clastogenic in the rat bone marrow micronucleus test.

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

Additional information

Additional information from genetic toxicity in vitro:

A battery of standard genotoxicity assays has been conducted to assess in vitro and in vivo genotoxic responses. All the assays are assessed and considered to be key studies.

Bacterial mutation assay, positive (Callander 1999): The test substance was assessed using a standard Ames protocol in bacterial strains TA1535, TA1537, TA98, TA100, WP2P and WP2P uvrA to a maximum concentration of 5000 µg/plate. The test substance afforded a positive response in strains TA1537 and TA98, both in the presence and absence of metabolic activation and was considered to be mutagenic in this assay.

Bacterial mutation assay, equivocal (Callander 2004): This study used only a single bacterial tester strain, TA98 in the absence of metabolic activation to investigate the response of a number of different batches of the test substance in one of the bacterial strains testing positive in the original Ames study. The different batches afforded mixed outcomes, from negative to weakly positive. These data contradict, in part, the findings of the original Ames study (Callander 1999).

Mouse lymphoma mutation assay, weakly positive (Clay 1999): The test substance induced small, but reproducible increases in mutant frequency at the maximum concentration tested, 2150 µg/mL, approximately 10 mM, the limit concentration for the assay. It is concluded the test substance is weakly mutagenic to L5178Y TK +/- cells in vitro in the presence of metabolic activation.

In vitro cytogenetic assay, negative (Fox 1999): The test substance was negative in the in vitro chromosome aberration assay up to the maximum concentration tested, 2150 µg/mL, approximately 10 mM, the limit concentration for the assay. It is concluded the test substance is not clastogenic in vitro.

Reverse Mutation Assay, negative (Thompson 2012): This study used two bacterial tester strains, TA98 and TA1537 in the absence of metabolic activation to investigate the response of a number of different batches of the test substance, when tested to a maximum concentration of 5000 µg/plate, in the bacterial strains testing positive in the original Ames study. All the batches afforded a negative, non-mutagenic, result.

Reverse Mutation Assay, negative (Bowles 2012): The test substance was assessed using a standard Ames protocol in bacterial strains TA1535, TA1537, TA98, TA100, WP2pKM101 and WP2 uvrApKM101 to a maximum concentration of 5000 µg/plate. The test substance afforded a negative response in all strains both in the presence and absence of metabolic activation and is considered to be non-mutagenic in this assay.

Additional information from genetic toxicity in vivo:

Rat bone marrow micronucleus test, in vivo, negative (Fox 2005): In the in vivo rat bone marrow micronucleus assay the test substance gave a negative result when tested up to the limit dose for the assay; 2000, 1000 and 500 mg/kg. From gross investigations, post mortem, and the presence of clinical signs in the animals dosed with the test substance, the test substance is bioavailable. It is concluded the test substance is not clastogenic in vivo.

In vivo unscheduled DNA synthesis in rat hepatocytes, negative (Bohnenberger 2013): The test substance was assessed in the in vivo unscheduled DNA synthesis (UDS) assay for its potential to induce DNA repair in the hepatocytes of rats when tested up to the maximum tolerated dose level in both male (800, 400 and 200 mg/kg) and female (500 and 250 mg/kg) animals. From the presence of clinical signs in the animals dosed with the test substance, the test substance is considered to be bioavailable. None of the dose levels tested revealed UDS induction in the hepatocytes of the treated animals. Therefore, the test substance is considered to be non-genotoxic in this in vivo UDS test system.


Justification for selection of genetic toxicity endpoint
The test substance afforded positive results in initial in vitro bacterial gene mutation assays, and a weak positive result was obtained in an in vitro mammalian cell gene mutation assay. A subsequent in vitro bacterial gene mutation assay gave negative results. A chromosome aberration assay in vitro was negative, an in vivo mammalian erythrocyte micronucleus assay was negative, and an in vivo unscheduled DNA synthesis assay was negative. Based on the whole dataset, the test substance is not considered to be mutagenic.

Justification for classification or non-classification

The test substance afforded positive results in an in vitro bacterial gene mutation assay, and a weak positive result was obtained in an in vitro mammalian-cell gene mutation assay. A subsequent in vitro bacterial gene mutation assay gave negative results. A chromosome aberration assay in vitro was negative and an in vivo mammalian erythrocyte micronucleus assay was negative. An in vivo unscheduled DNA synthesis assay was negative. In both in vivo assays the test substance was shown to be bioavailable. In accordance with Regulation (EC) No. 1272/2008, Annex I, 3.5.2, based on the available data the test substance is not considered to be classified for germ cell mutagenicity. The endpoint is considered to be conclusive.