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Genetic toxicity in vitro

Description of key information

Two in vitro tests performed according to OECD 471 and 473 guidelines are available and did not show any genotoxic effect.

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
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
other: OCDE n° 471 Dir. 92/69/CEE : B13/B14
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
For Salmnonella typhimurium: histidine
For Escherichia coli: tryptophane
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: strain TA 98, TA 100 and E coli wp2 contains PKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9-Mix (10 % liver S9 in standard co-factors)
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 50 ... 5000 µg/plate
Concentration range in the main test (without metabolic activation): 50 ... 5000 µg/plate
Vehicle / solvent:
distilled water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: cis-diammineplatinium (II) dichloride
Remarks:
for E. coli strain
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 100 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
for TA 98 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
for TA 1537 strain, without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
for all S. typimurium strains, with S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: B-propiolactone
Remarks:
E. coli strain without S9
Details on test system and experimental conditions:
Assay without metabolic activation
For each of the strains, 100 μL of the bacterial suspension (1-2 x 109 bacteria/mL) and 100 μL of the test
substance are mixed with 2.0 mL of overlay agar and poured over the surface of a minimal agar plate (90
mm in diameter) (n = 3). The overlay agar is allowed to solidify before incubation.
Plates are incubated at 37°C over a 48 hour period. The number of revertant colonies per plate is counted.
Moreover the following controls are carried out :
- negative controls
. absolute negative control (spontaneous reversion rate),
. positive control solvent.
- test substance solvent if necessary.
- positive control

Assay with metabolic activation
Two tests can be performed using
· either a standard plate incorporation method where the protocol is similar to that described
above, except that just before pouring the mixture onto the plates, 500 μL of S-9 mix fraction is
quickly mixed,
· or the pre-incubation assay where the test substance is preincubated with the test strain, and
500 μL of S-9 mix fraction for 1 hour at 37°C prior to mixing with the overlay agar and pouring
onto the surface of the minimal agar plate. Tubes should be aerated during pre-incubation by
using a shaker. This method is known to increase the detection sensitivity of a number of
promutagens like alkaloïds, aliphatic N-Nitroso compounds (OECD n° 471* * * ).

Assay are conduced in triplicate
Evaluation criteria:
The result of the test is considered as negative if the revertant number is below three fold the
number of spontaneous reversions, for TA 1535 and TA 1537 strains, and below two fold the number of
spontaneous reversions for TA 98, TA 100 and Escherichia coli WP2 strains with and without metabolic
activation.

The result of the test is considered positive if a concentration –related in crease is obtained in one,
or several of the 5strains, with and/or without metabolic activation; a mutagenic effect is taken into
account for a given concentration of the test substance if the number of revertant colonies is at least two
fold that of spontaneous revertant colonies number for TA 98, TA 100 and Escherichia coli (WP2), and
three fold for TA 1535 and TA 1537.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/plate)
Vehicle 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: other: preliminary test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

There is no significant difference between the number of spontaneous reversions, the number of
reversions obtained in the positive controls (with and without metabolic activation), and the mean of
corresponding experimental historic values obtained in the laboratory.
There is no evidence of any increase in the number of revertant colonies in the presence of the test
substance (5 000 μg - 1 500 μg - 500 μg - 150 μg - 50 μg) with and without metabolic activation.
Results were confirmed in a second independent experiment.
Executive summary:

Analysis : Reverse mutation assay.

Test substance : LCA01006 LEMI code : DIB190601-2

Test procedure : Reverse mutation assay on “Salmonella typhimurium his-“ and “Escherichia coli” WP2 (pKM 101) strains according to the OECD guideline n° 471 (LEMI operating procedure MB08/45).

Test result : The product did not induce reverse mutation, Salmonella typhimurium and Escherichia coli WP2 (pKM 101) strains according to the OECD guideline n° 471 and according to the test method B13/B14 of the E.C. Directive n° 2000/32.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLP study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
2003-02-13
Type of assay:
other: in vitro mammalian cytogenicity (B10)
Species / strain / cell type:
other: human lyphocyte
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a
viral infection. The cell-cycle time for the lymphocytes from the donors used in this study were determined using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells and so calculate the average generation time (AGT). The
average AGT for the regular donors used in this laboratory has been determined to be approximately 17 hours under typical experimental exposure conditions.
Metabolic activation:
with and without
Metabolic activation system:
S9 miw was prepared frommicrosomal fraction of a rat liver de foie de rat exposed to a mix of phenobarbitone and beta-naphthoflavone.
Test concentrations with justification for top dose:
Concentration range in the main test (with metabolic activation): 156 ... 5000 µg/ml
Concentration range in the main test (with metabolic activation): 312 ... 5000 µg/ml
Concentration range in the main test (without metabolic activation): 156 ... 5000 µg/ml
Concentration range in the main test (without metabolic activation): 312 ... 5000 µg/ml
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Migrated to IUCLID6: without S9 activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with S9 activation
Details on test system and experimental conditions:
Exposure period (with metabolic activation): 4 hours
Exposure period (without metabolic activation): 24 hours

Expression time:
first assay: 4h- exposition with and without S9 mix.

second assay: 4h-exposition with S9 mix and 24 hours without S9 mix.

Selection time:
Premier essai : recueil 20 heures après la fin du traitement

avec et sans S9 mix.

Deuxième essai : recueil 20 heures après la fin du

traitement avec S9 mix et recueil à la fin du traitement

sans S9 mix.
Evaluation criteria:
Gaps (g)
Chromatid Breaks (ctb)
Chromosome Breaks (csb)
Exchanges (cte and cse)
Multiple Aberrations
Chromosome Number

A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship.
Statistics:
For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.

The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/ml)
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
not specified
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/ml)
Species / strain:
other: as specified above
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/ml)
Species / strain:
other: as specified above
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Remarks:
(up to 5000 µg/ml)
Remarks on result:
other: other: preliminary test
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

The test material was considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction.

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scott et al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Directive 2000/32/EC. The study design also meets the requirements of the UK Department of Health Committee on Mutagenicity Guidelines for the Mutagenicity Testing of Chemicals.

Methods.

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to three dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, ie. in Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hours exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results.

All vehicle (solvent) controls had frequencies of cells with aberrations within the range expected for normal human lymphocytes. All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system. The test material was non-toxic and did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments, using a dose range that included a dose level that was the maximum recommended dose level.

Conclusion.

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

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

Genetic toxicity in vivo

Description of key information
An in vivo micronucleus test (OECD 474) was performed and did not show any clastognenic effect, NOAEL = 2000 mg/kg
Link to relevant study records
Reference
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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: OECD guideline, GLp study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
Swiss
Sex:
male/female
Details on test animals and environmental conditions:
Preliminary toxicity test: 3 males and 3 females mice were used.
Main cytogenetic test: 56 mice, 28 males and 28 females were used.
Strain: Swiss Ico: OF1 (IOPS Caw).
Reason for this choice: rodent species generally accepted by regulatory authorities for this type ofstudy.
Breeder: Charles River Laboratories, l’Arbresle, France.
Age: on the day oftreatment, the animals were approximately 6 weeks old.
Weight: at the beginning oftreatment the mean body weight was 32.3 g for males (ranging from 29.7 to 33.6 g) and 26.3 g for females (ranging from 24.8 to 28.0 g).
Veterinary care at CIT: upon their arrival at CIT, the animals were given a complete examination to ensure that they were in good clinical conditions.
Acclimation: at least 5 days before the day of treatment except for females from the positive control (CPA) treated group and supplementary females from the high dose group which had only 4 days of acclirnation.
Constitution of groups: upon arrival, the animals were randomly allocated to the groups by sex. Subsequently, each group was assigned to a different treatment group.
Identification: individual tail marking upon treatment.
Route of administration:
oral: unspecified
Vehicle:
water
Details on exposure:
For the vehicle and the test item:
• Route: oral, since it is a possible route ofexposure in Man
• Frequency: two treatments separated by 24 hours
• Volume: 10 mL/kg

For the positive control (CPA):
• Route: oral
• Frequency: one treatment
• Volume: 10 mL/kg
Frequency of treatment:
2, spaced by 24 hours
Remarks:
Doses / Concentrations:
500 mg/kg bw
Basis:

Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:

Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:

No. of animals per sex per dose:
Male: mg/kg; No. of animals: ; Sacrifice time: hours
Male: 0 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 500 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 1000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Male: 2000 mg/kg; No. of animals: 5; Sacrifice time: 24 hours
Female: 0 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 500 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 1000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Female: 2000 mg/kg; No. of animals: 5; Sacrifice times: 24 hours
Control animals:
yes, historical
Positive control(s):
The positive control was cyclophosphamide (CPA, Endoxan, Baxter, Maurepas, France), batch No. 4A121 dissolved in distilled water at a concentration of 5 mg/mL.
The preparation was stored at -20°C and thawed irnrnediately before use.
Details of tissue and slide preparation:
Preparation of the bone marrow smears
At the time of sacrifice, all the animals were killed by C02 inhalation in excess. The femurs of the animals were removed and the bone marrow was flushed out using fetal calf serum. After centrifugation, the supernatant was removed and the cells in the sediment were resuspended by shaking. A drop of this cell suspension was placed and spread on a slide. The slides were air-dried and stained with Giemsa. The slides were coded 50 that the scorer is unaware of the treatment group of the slide under evaluation (“blind” scoring).

Microscopic examination of the slides
For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes; the polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).
The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close
Mumbles, Swansea SA3 4LY, UK), in compliance with GLP, and the Principal Investigator was
Natalie Danford.
Evaluation criteria:
All the individual data are presented in tabular form. The number of MPE/2000 PE and the
PEINE ratio are given for each animal.
The means and the standard deviations of the frequency of MPE/1000 PE and the PEINE ratio
are given for each experimental group.
Statistics:
Normality and homogeneity of variances will be tested using a Kolmogorov Smirnov test and a Bartlett test.
If normality and homogeneity of variances were demonstrated, the statistical comparisons was performed using a Student t-test (two groups) or a one-way analysis of variance ( three groups) followed by a Dunnett test (if necessary).
If normality or homogeneity of variances was not demonstrated, a Mann/Withney test (two groups) or a Kruskall Wallis test ( three groups) was performed followed by a Dunn test (ifnecessary).
All these analyses were performed using the software SAS Enterprise Guide V2 (2.0.0.4 17, SAS Institute mc), with a level of significance of 0.05 for all tests.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information): negative
Under our experimental conditions, the test item LCAO5 005 did not induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mglkglday.
Executive summary:

The objective of this study was to evaluate the potential of the test item LCAO5 005 to induce structural or numerical damage in bone marrow cells of mice. The study was performed according to the international guidelines (OECD 474 and Commission Directive No. B12) and in compliance with the Principles of Good Laboratory Practice Regulations.

Methods

A preliminary toxicity test was performed to define the dose-levels to be used for the cytogenetic study. In the main study, three groups of five male and five female Swiss Ico: OF1 (IOPS Caw) mice were given oral administrations of LCAO5005 at dose-levels of 500, 1000 or 2000 mg/kg/day, over a 2-day period. One group of five males and five females received the vehicle (water for injections) under the same experimental conditions, and acted as control group. One group of five males and five females received the positive control test item (cyclophosphamide) once by oral route at the dose-level of 50 mg/kg. The animals of the treated and vehicle control groups were killed 24 hours after the last treatment and the animals of the positive control group were killed 24 hours after the single treatment. Bone marrow smears were then prepared. For each animal, the number of the micronucleated polychromatic erythrocytes (MPE) was counted in 2000 polychromatic erythrocytes. The polychromatic (PE) and normochromatic (NE) erythrocyte ratio was established by scoring a total of 1000 erythrocytes (PE + NE).

Results

The highest dose-level for the cytogenetic test was selected according to the criteria specified in the international guidelines; since no toxic effects were observed, the highest dose-level was 2000 mg/kg/day. The two other selected dose-levels were 1000 and 500 mg/kg/day. For both males and females, the mean values of MPE as well as the PEINE ratio in the groups treated with the test item, were equivalent to those of the vehicle control group. The mean values of MPE as well as the PEINE ratio for the vehicle and positive controls were consistent with our historical data. Cyclophosphamide induced a significant increase in the frequency of MPE, indicating the sensitivity of the test system under our experimental conditions. The study was therefore considered valid.

Conclusion

Under our experimental conditions, the test item LCAO5 005 did flot induce any damage to the chromosomes or the mitotic apparatus of mice bone marrow cells after two oral administrations, at a 24-hour interval, at the dose-levels of 500, 1000 or 2000 mg/kg/day.

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

Additional information

Additional information from genetic toxicity in vivo:

Justification for selection of genetic toxicity endpoint
2 in vitro tests and 1 in vivo test are available. The in vivo test was selected as the key study to assess this endpoint.

Justification for classification or non-classification

The substance is not classified as genotoxic according to the criteria of the CLP regulation n°1272/2008/EC.