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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance has proven not genotoxic in several in 2 Ames tests, 2 Chromosomal aberration tests in vitro and 1 Micronucleus test in vivo.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2006
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only the E. coli strain was tested, as the other TA strains were already tested in a pre-existing study.
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch number 9000631236
Purity 99.3%
Expiration date: August 28, 2006
Target gene:
Trp
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
3; 10; 33; 100; 333; 1000; 2500 and 5000 microg per plate
Vehicle / solvent:
Ethanol
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
with S9
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
The test item was found negative in this bacterial revers mutation assay in E. coli WP2.
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1989
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Strain E. Coli or TA 102 missing. The strain was tested in another study provided in this dossier.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Batch number 8906-87
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0.0005µL - 0.500 mL (background lawn reduction at highest doses)
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
water
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA 1535 and 100 (without S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
TA 1538 and 98 (without S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: quinacrine mustard
Remarks:
TA1537 (without S9)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-anthramine
Remarks:
all strains with S9
Species / strain:
other: S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
The test item was found negative in this bacterial revers mutation assay in S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2005
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch number 1100017369
Purity 98.8%
Expiration date January 26, 2005
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Scored up to:
50µg/mL (4hrs -S9)
1.88µg/mL (18hrs -S9)
7.50µg/mL (28hrs -S9)
150µg/mL (4hrs +S9)
100µg/mL (4hrs +S9)
High toxicity observed at higher doses.
Vehicle / solvent:
Ethanol was used as solvent
Untreated negative controls:
yes
Remarks:
Culture medium
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
yes
Remarks:
Culture medium
Negative solvent / vehicle controls:
yes
Remarks:
Ethanol
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
In conclusion it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells when tested up to cytotoxicity.
However, it is suspected to inhibit mitotic processes and to induce numerical chromosome aberrations (increased rate of polyploid cells), and to inhibit cell cycle progression (increased rate of cells with endoduplicate chromosome) in this chromosome aberration test in the absence of metabolic activation.
Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2014-2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Batch no. VE00340722
Expiry date 25 July 2015
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese hamster ovary cells were obtained from Dr. A.T. Natarajan (State
University of Leiden). This cell line derives from the CHO isolate originally
described by Kao and Puck (1968).
The karyotype, generation time and plating efficiency have been checked in
this laboratory. The cells are checked at regular intervals for the absence of
mycoplasmal contamination.
Permanent stocks of CHO cells are stored in liquid nitrogen, and subcultures
are prepared from the frozen stocks for experimental use.
Metabolic activation:
with and without
Metabolic activation system:
One batch of S9 tissue fraction, provided by Trinova Biochem GmbH, was used in this study and had the following characteristics:
Species: Rat
Strain: Sprague Dawley
Tissue: Liver
Inducing Agents: Phenobarbital – 5,6-Benzoflavone
Producer: MOLTOX, Molecular Toxicology, Inc.
Batch Number: 3350
Test concentrations with justification for top dose:
In the absence of S9 metabolic activation, using the short treatment time,
the experimental series considered valid was that performed as experiment
two using the following dose levels: 0.626, 0.501, 0.401, 0.321, 0.257, 0.205,
0.164, 0.131, 0.105, 0.0841 and 0.0673 mM.
In the presence of S9 metabolism, the experimental series considered valid
was that performed as experiment four using the following dose levels: 1.09,
0.952, 0.828, 0.720, 0.626, 0.544, 0.473, 0.412, 0.358 and 0.311 mM.
Cytotoxicity occured at higher doses.
Vehicle / solvent:
Solutions of the test item, as received, were prepared immediately before
use in DMSO.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Details on test system and experimental conditions:
Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively.
The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. Two previous experiments were performed in the absence and/or presence of S9 metabolism, where no adequate cell growth nor adequate toxicity was found and no dose level could be selected for the scoring of chromosomal aberrations. As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 20
hours was used. Solutions of the test item were prepared in dimethylsulfoxide (DMSO).
For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol and on the basis of the toxicity observed in the previous experiments. Dose levels of 0.626, 0.501, 0.401, 0.321, 0.257, 0.205, 0.164, 0.131, 0.105, 0.0841 and 0.0673 mM were used in the absence of S9 metabolism. Dose levels of 1.09, 0.952, 0.828, 0.720, 0.626, 0.544, 0.473, 0.412, 0.358 and 0.311 mM were used in the presence of S9 metabolism.
On the basis of results obtained in the first main experiment, dose levels of 0.320, 0.213, 0.142, 0.0948, 0.0632, 0.0421, 0.0281, 0.0187, 0.0125, 0.00832, 0.00554, 0.00370, 0.00247, 0.00164 and 0.00110 mM were used for the second main experiment.
Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.
Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of population doubling and mitototic index if considered necessary.
Rationale for test conditions:
According to guideline
Evaluation criteria:
In this assay, the test item is considered as clearly positive if the following criteria are met:
– Significant increases in the proportion of cells bearing aberrations (excluding gaps) over the concurrent controls occur at one or more concentrations.
– Any increases observed must be present in both replicate cultures.
– The proportion of aberrant cells exceeds the historical control range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as
positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies.
Statistics:
For the statistical analysis, Fisher’s Exact Test was used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. Bonferroni’s correction was applied for multiple comparisons. The analysis was performed using sets of data either including or excluding gaps. The results are presented in Table 4.
Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations (including or excluding gaps) over the control values, was observed at any dose level, in any experiment, in the absence or presence of S9 metabolism.
Incidences in endoreduplicated cells were significantly increased (at statistical analysis) both in the absence and presence of S9 metabolism. In addition, using the continuous treatment time, a statistically significant increase of polyploid cells was observed.
Key result
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
On the basis of the above mentioned results and in accordance with the criteria for outcome of the study, the test item was not considered to induce structural chromosomal aberrations in mammalian cells cultured in vitro.
The test item was considered to induce numerical changes of chromosomes.
Executive summary:

The test item FLORHYDRAL was assayed for the ability to induce chromosomal aberrations in Chinese hamster ovary cells, following in vitro treatment in the presence and absence of S9 metabolic activation. Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively. The harvest time of 20 hours, corresponding to approximately 1.5 cell cycle, was used. Two previous experiments were performed in the absence and/or presence of S9 metabolism, where no adequate cell growth nor adequate toxicity was found and no dose level could be selected for the scoring of chromosomal aberrations. As negative results were obtained in the first main experiment, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 20 hours was used. Solutions of the test item were prepared in dimethylsulfoxide (DMSO). For the first main experiment, the maximum dose level for treatment was selected in agreement with the Study Protocol and on the basis of the toxicity observed in the previous experiments. Dose levels of 0.626, 0.501, 0.401, 0.321, 0.257, 0.205, 0.164, 0.131, 0.105, 0.0841 and 0.0673 mM were used in the absence of S9 metabolism. Dose levels of 1.09, 0.952, 0.828, 0.720, 0.626, 0.544, 0.473, 0.412, 0.358 and 0.311 mM were used in the presence of S9 metabolism. On the basis of results obtained in the first main experiment, dose levels of 0.320, 0.213, 0.142, 0.0948, 0.0632, 0.0421, 0.0281, 0.0187, 0.0125, 0.00832, 0.00554, 0.00370, 0.00247, 0.00164 and 0.00110 mM were used for the second main experiment. Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point. Dose levels were selected for the scoring of chromosomal aberrations on the basis of the cytotoxicity of the test item treatments as determined by the reduction of population doubling and mitototic index if considered necessary.

On the basis of the obtained results, dose levels selected for scoring were as follows:

Experiment No.:       S9       Treatment time (hours)       Harvest time (hours)       Dose level (mM)

1                             −                     3                                         20                     0.401, 0.321 and 0.257

+                     3                                        20                     0.828, 0.720 and 0.626

2                            −                     20                                     20                     0.0632, 0.0421 and 0.0281

For the three selected doses, for the solvent and for the untreated controls, 100 metaphase spreads per cell culture were scored to assess the frequency of aberrant cells. Following treatment with the test item, no statistically significant increase in the incidence of cells bearing structural aberrations, including or excluding gaps, over the control value was observed at any dose level in any treatment series. Increases of cells bearing chromosomal numerical changes (mainly polyploid cells) over the control were observed. Statistically significant increases in the number of cells bearing aberrations (including and excluding gaps) were observed following treatments with the positive controls Cyclophosphamide and Mitomycin-C, indicating the correct functioning of the test system.

It is concluded that FLORHYDRAL does not induce structural chromosome aberrations in Chinese hamster ovary cells after in vitro treatment, under the reported experimental conditions.

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

Genetic toxicity in vivo

Description of key information

The substance was found not clastogenic and non aneugenic in a micronucleus test in vivo.

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian erythrocyte micronucleus test
Specific details on test material used for the study:
Batch number 185809
Purity 97.9%
Species:
mouse
Strain:
other: Fullinsdorf Moro Albino mice
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Standard Suspension Vehicle (SSV)
Details on exposure:
Once by oral gavage
Duration of treatment / exposure:
Once by oral gavage, exanimations at 24, 48 and 72 hours for the high dose and 24 hours only for the low dose
Frequency of treatment:
Single treatment
Post exposure period:
Up to 72 hours
Dose / conc.:
1 000 mg/kg bw/day
Dose / conc.:
2 000 mg/kg bw/day
No. of animals per sex per dose:
18 Males and 18 Females for the negative control
6M and 6F at 1000mg/kg -> 5M+5F were evaluated
18M and 18F at 2000mg/kg -> 15M and 15F were evaluated
6M and 6F for the positive control -> 5M and 5F were evaluated
Control animals:
yes, concurrent vehicle
Positive control(s):
Positive control: Procarbazine hydrochloride
Tissues and cell types examined:
Erythrocytes of the bone marrow of mice
Details of tissue and slide preparation:
Sampling times at 24, 48 and 72 hours for the high dose and 24 hours only for the low dose.
2 slides per animal treated were made.
1000 PCE were per animal.
Evaluation criteria:
The ratio of PCE/NCE was determined on 1000 counts of erythrocytes.
Statistics:
The observed yields of MN-PCE were evaluated by means of the Mann-Whitney-U-test.
Key result
Sex:
male/female
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Under the experimental conditions described in this report, the test item did not show any genotoxic activity in the mouse bone marrow cells.Thus the test item is is considered to show no genotoxic effects under the described experimental conditions..
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

According to the available test results, the substance does not need any classification for genotoxicity under CLP.