Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All three in vitro tests (Ames-Test, Chromosome aberration assay and HPRT gene mutation assay) were negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Experiment IIA: 0.03, 0.1, 0.3, 1, 3, 10, 33, 100 µg/plate
Pre-ExperimenUExperiment I and II: 3, 10, 33, 100, 333, 1000, 2500, 5000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: solubility properties and relatively non-toxic to the bacteria
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
for TA 1535 and TA 100 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 4-NOPD
Remarks:
for TA 1537 and TA 98 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
for TA 102 without metabolic activation
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
yes
Positive controls:
not specified
Positive control substance:
other: 2-aminoanthracene
Remarks:
for all strains with metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth
Evaluation criteria:
The Salmonella typhimurium reverse mutation assay is considered acceptable if it meets
the following criteria:
- regular background growth in the negative and solvent control
- the spontaneous reversion rates in the negative and solvent control are in the range of
our historical data
- the positive control substances should produce a significant increase in mutant colony
frequencies

A test item is considered as a mutagen if a biologically relevant increase in the number of
revertants exceeding the threshold of twice (strains TA 98, TA 100, and TA 102) or thrice
(strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is
observed.
A dose dependent increase is considered biologically relevant if the threshold is exceeded
at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically
relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is
regarded as an indication of a mutagenic potential if reproduced in an independent second
experiment. However, whenever the colony counts remain within the historical range of
negative and solvent controls such an increase is not considered biologically relevant.
Statistics:
According to the OECD guideline 471, a statistical analysis of the data is not mandatory.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid

The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test substance at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

Conclusions:
Based on the results of a study according to OECD Test Guideline 471 under GLP, the test substance is considered not to be mutagenic.
Executive summary:

The registered substance has been tested in a GLP-study according to OECD test guideline 471. The strain salmonella typhimurium TA 98, 100, 102, 1535 and 1537 were used without and with metabolic activation by S9. Appropriate, strain-specific controls were used giving valid results. The test substance was applied in suspension.

As the test substance did not give any positive result, it is considered not to be mutagenic.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosomal Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
n.a.
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:
Experiments without S9-mix:
IA: 0.1, 0.2, 0.7, 2.1, 6.2, 18.5, 55.6, 166.6, 500.0 ug/mL
IIA: 0.02, 0.04, 0.08, 0.16, 0.3, 0.6, 1.3, 2.5, 5.0 ug/mL
IIB: 0.3, 0.6, 1.3, 2.5, 5.0, 7.5, 10.0, 15.0 ug/mL

Experiments with S9-mix:
IA: 0.2, 0.5, 1.4, 4.1, 12.3, 37.0, 111.1, 333.3, 1000.0 ug/mL
IB: 0.8, 1.6, 3.1, 6.3, 9.4, 12.5, 18.8, 25.0, 50.0 ug/mL
IC: 1.6, 3.1, 6.3, 9.4, 12.5, 18.8, 25.0, 50.0 ug/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was a suitable vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
Reasons for the Choice of the Cell Line V79
The V79 cell line has been used successfully for many years in in vitro experiments. The high proliferation rate (doubling time of V79 cells in stock cultures: 14 hours, determined on April 18, 2008) and a reasonable plating efficiency of untreated cells (as a rule more than 70 %) both necessary for the appropriate performance of the study, support the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 ± 1.
Lacking metabolic activities of cells under in vitro conditions are a disadvantage of tests with cell cultures as many chemicals only develop a mutagenic potential when they are metabolized by the mammalian organism. However, metabolic activation of chemicals can be achieved at least partially by supplementing the cell cultures with liver microsome preparations (S9 mix).

Cell Cultures
Large stocks of the V79 cell line (obtained from Labor für Mutagenitätsprüfungen (LMP), Technical University Darmstadt, 64287 Darmstadt, Germany) were stored in liquid nitrogen in the cell bank of Harlan CCR, which allows the repeated use of the same cell culture batch in experiments. Before freezing each batch was screened for mycoplasma contamination and checked for karyotype stability. Consequently, the parameters of the experiments
remained similar because of the reproducible characteristics of the cells.
Thawed stock cultures were propagated at 37 °C in 80 cm2 plastic flasks. About 5 x 10e5 cells per flask were seeded in 15 mL of MEM (minimal essential medium) containing Hank’s salts
and 10 % (v/v) fetal bovine serum (FBS). Additionally, the medium was supplemented with neomycin (5 Mg/mL), Hepes (25 mM) and amphotericin B (2.5 Mg/mL). The cells were subcultured twice a week. The cell cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % carbon dioxide (98.5 % air).

Pre-experiment
A pre-test on cell growth inhibition was performed in order to determine the toxicity of the test item, the solubility during exposure and changes in osmolarity and pH value at experimental conditions. Due to known cytotoxicity properties and in agreement with the OECD guideline no. 473 the test item was applied up to a maximum concentration of 1000.0 Mg/mL. At the selected dose no influence on solubility, pH value, or osmolarity was
detected.
The experimental conditions in this pre-experiment were identical to those required and described below for the mutagenicity assay.

Dose Selection
The highest concentration used in the cytogenetic experiments was chosen considering the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced cell numbers or mitotic indices below 50 % of
control, whichever is the lowest concentration, and/or the occurrence of precipitation. In case of non-toxicity the maximum concentration should be 5 mg/mL, 5 ML/mL or 10 mM, whichever is the lowest, if formulation in an appropriate solvent is possible.
The highest applied concentrations of 500.0 Mg/mL (without S9 mix) and 1000.0 Mg/mL (with S9 mix) were applied as top concentrations for the cultures in the pre-test due to strong cytotoxic effects obtained in the performed parallel HPRT study. Test item concentrations between 0.1 and 0.7 Mg/mL (without S9 mix) or 0.2 and 12.3 Mg/mL (with S9 mix) were chosen for the evaluation of cytotoxicity. Higher concentrations could not be chosen for evaluation due to exceedingly strong cytotoxicity. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment IA.
To enable the evaluation of dose groups in the cytotoxic concentration range the experimental part with S9 mix was repeated in Experiment IB with a top concentration of 50.0 Mg/mL and closer spaced concentrations. To verify the positive results of Experiment IB a confirmatory Experiment IC with a top concentration of 50.0 Mg/mL was performed.
Dose selection of Experiment II was influenced by the results obtained in Experiments IA in the absence of S9 mix. Strong cytotoxic effects were observed at 2.1 Mg/mL and above.
Therefore, in Experiment IIA 5.00 Mg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. Due to missing cytotoxicity this experimental part was repeated with a top concentration of 15.0 Mg/mL.
The cytogenetic evaluation of higher concentrations in the respective intervals (with and without S9 mix) was impossible due to strong test item induced cytotoxic effects (reduced cell numbers and/or low metaphase numbers, partially paralleled by poor metaphase quality).

Culture Medium and Conditions
For seeding and treatment of the cell cultures the culture medium was MEM (minima essential medium) containing Hank’s salts, neomycin (5 Mg/mL), Hepes (25 mM) and amphotericin B (2.5 Mg/mL). All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Seeding of the Cultures
Seeding of the Cultures
Exponentially growing stock cultures more than 50 % confluent were rinsed with Ca-Mg-free salt solution containing 8000 mg/L NaCl, 200 mg/L KCl, 200 mg/L KH2PO4 and 150 mg/L Na2HPO4. Afterwards the cells were treated with trypsin-EDTA-solution at 37 °C for approx. 5 minutes. Then, by adding complete culture medium including 10 % (v/v) FBS the enzymatic treatment were stopped and a single cell suspension was prepared. The trypsin concentration for all sub-culturing steps was 0.5 % (w/v) in Ca-Mg-free salt solution. The cells were seeded into Quadriperm dishes, which contained microscopic slides. Into each chamber 1 x 10e4 – 6 x 10e4 cells were seeded with regard to the preparation time.

Treatment
Exposure period 4 hours
The culture medium of exponentially growing cell cultures was replaced with serum-free medium containing the test item. For the treatment with metabolic activation 50 ML S9 mix per mL culture medium were added.
Concurrent solvent and positive controls were performed. After 4 hours the cultures were washed twice with "Saline G" (pH 7.2) containing 8000 mg/L NaCl, 400 mg/L KCl, 1100 mg/L glucose • H2O, 192 mg/L Na2HPO4 • 2 H2O and 150 mg/L KH2PO4. The cells were then cultured in complete medium containing 10 % (v/v) FBS for the remaining culture time of 14 hours.
Exposure period 18 hours
The culture medium of exponentially growing cell cultures was replaced with complete medium containing 10 % (v/v) FBS including the test item without S9 mix. The medium was not changed until preparation of the cells. Concurrent solvent and positive controls were performed.
All cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 (98.5 % air).

Preparation of the Cultures
Colcemid was added to the culture medium (0.2 Mg/mL) 15.5 hours after the start of the treatment. The cells were treated, 2.5 hours later, on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with a mixture of methanol and glacial acetic acid (3:1 parts, respectively). After preparation the cells were stained with Giemsa and labelled with a computer-generated random code to prevent scorer bias.

Evaluation of Cell Numbers
The evaluation of cytotoxicity indicated by reduced cell numbers was made after the preparation of the cultures on spread slides. The cell numbers were determined microscopically by counting 10 defined fields per coded slide. The cell number of the treatment groups is given in percentage compared to the respective solvent control.

Analysis of Metaphase Cells
Evaluation of the cultures was performed according to the OECD guideline using NIKON microscopes with 100x objectives. Breaks, fragments, deletions, exchanges, and chromosome disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well but not included in the calculation of the aberration rates. At least 100 well spread metaphases per culture were evaluated for cytogenetic damage on coded
slides, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
Only metaphases with characteristic chromosome numbers of 22 ± 1 were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined.
In addition, the number of polyploid cells in 500 metaphases per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype). Additionally the number of endomitotic cells evaluated at the evaluation of polyploid cells was noticed and reported (% endomitotic metaphases).
Evaluation criteria:
Acceptability of the Test
The chromosome aberration test performed in our laboratory is considered acceptable, if it meets the following criteria:
a) The number of structural aberrations found in the solvent controls falls within the range of
the laboratory’s historical control data.
b) The positive control substances produce significant increases in the number of cells with structural chromosome aberrations, which are within the range of the laboratory’s historical control data.

Evaluation of Results
A test item is classified as non-clastogenic if:
the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of the laboratory’s historical control data.
and
no significant increase of the number of structural chromosome aberrations is observed.
A test item is classified as clastogenic if:
the number of induced structural chromosome aberrations is not in the range of the laboratory’s historical control data).
and
either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.
Statistical significance was confirmed by means of the Fisher’s exact test (p < 0.05).
However, both biological and statistical significance should be considered together. If the criteria mentioned above for the test item are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Although the inclusion of the structural chromosome aberrations is the purpose of this study, it is important to include the polyploids and endoreduplications. The following criterion is valid:
A test item can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of the laboratory’s historical control data.
Statistics:
Statistical analyses were conducted using Fisher’s exact test
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
Positive controls validity:
valid
Additional information on results:
The test item, dissolved in DMSO, was assessed for its potential to induce
structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in the
absence and presence of metabolic activation by S9 mix.
Five independent experiments were performed. In Experiment IA, the exposure period was
4 hours with and without metabolic activation. In Experiment IB and IC, the exposure period
was 4 hours with metabolic activation. In Experiment IIA and IIB the exposure period was
18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment
with the test item.
In each experimental group two parallel cultures were set up. At least 100 metaphases per
culture were evaluated for structural chromosome aberrations, except for the positive control
in Experiment I without metabolic activation, where only 50 metaphases were evaluated.
Precipitation of the test item in culture medium was observed after 4 hours treatment with
166.6 Mg/mL and above in the absence of S9 mix and 1000.0 Mg/mL and above in the
presence of S9 mix only in Experiment IA. No relevant influence of the test item on pH value
or osmolarity was observed (Exp. IA: solvent control 396 mOsm, pH 7.4 versus 393 mOsm
and pH 7.4 at 500.0 Mg/mL; Exp. IIA: solvent control 392 mOsm, pH 7.5 versus 399 mOsm
and pH 7.5 at 5.0 Mg/mL; Exp. IIB: solvent control 405 mOsm, pH 7.6 versus 383 mOsm and
pH 7.6 at 15.0 Mg/mL).
In Experiment IA moderate cytotoxic effects (mitotic index of 68.6 % of solvent control)
occurred in the experimental part without S9 mix at the highest evaluable concentration (0.7
Mg/mL). In the experimental part with metabolic activation no cytotoxicity was measured up
to a test item concentration of 12.3 Mg/mL. Higher concentrations could not be evaluated due
to exceedingly strong cytotoxicity. Therefore, Experiment IB was performed in the presence
of S9 mix with closer spaced does groups. However, no evaluation in the cytotoxic range
was possible. As the dose groups were very narrow spaced in the experimental part without
S9 mix (Experiment IA) no further experiment was performed without S9 mix and 4 hours
treatment. In the confirmatory experiment (Experiment IC) in the highest evaluable test item
concentration of 25.0 Mg/mL relevant cytotoxic effects (mitotic index of 52.8 % of solvent
control) were observed.
In Experiment IIA moderate cytotoxicity was observed in the highest applied concentration of
5.0 Mg/mL (mitotic index was 65.2 % of solvent control). Higher concentrations were not
evaluable due to exceedingly strong cytotoxic effects. In Experiment IIB no cytotoxicity was
observed in the highest evaluable concentration. However, the next higher and closely
spaced concentration of 10.0 Mg/mL was not evaluable due to exceedingly strong cytotoxic
effects.
In Experiment IA in the absence and presence of S9 mix and in Experiments IIA and IIB in
the absence of S9 mix, no biologically relevant increase in the number of cells carrying
structural chromosome aberrations was observed.
The aberration rates of the cells after treatment with the test item (1.0 - 2.5 % aberrant
cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.0 %
aberrant cells, excluding gaps) and within the range of the total range laboratory’s historical
control data (0.0 - 4.0 % aberrant cells, excluding gaps).
In Experiment IB, in the presence of S9 mix, statistically significant increases in the number
of aberrant cells, excluding gaps (9.5, 6.5, 9.5, 6.5 and 9.0 %, respectively) were observed
after treatment with 1.6, 6.3, 9.4, 12.5 and 18.8 Mg/mL. All values
exceeded the total range of the laboratory’s historical solvent control data (0.0 – 4.0 %
aberrant cells, excluding gaps). This increase of chromosomal aberrations was not dosedependant.
In the confirmatory Experiment IC the positive result obtained in Experiment IB was not
reproducible. No statistically significant or biologically relevant increase in the number of
aberrant cells, excluding gaps were observed. All values (1.0, 2.5,
2.0 and 1.5 % aberrant cells, excluding gaps, respectively) were in the total range of the
historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps). Therefore, the
observation obtained in Experiment IB is regarded as biologically irrelevant.
No biologically relevant increase in the rate of polyploid metaphases was found
after treatment with the test item (2.2 - 5.3 %) as compared to the rates of the solvent
controls (2.0 – 3.5 %). Several induced rates of polyploid metaphases were observed in
isolated dose groups were observed in the absence and presence of S9 mix. However,
these were not reproducible and are therefore regarded as biologically irrelevant.
In Experiments IA, IIA and IIB in the absence of S9 mix, no biologically relevant
increase in the rate of endomitotic metaphases was found after treatment with the test item.
In contrast, in Experiments IA, IB and IC in the presence of S9 mix the rate of endomitotic
metaphases was increased in all test groups (0.1 – 0.8 %). Generally, no endoreduplications
were observed in the control groups. As in Experiment IB 0.4 % of endoreduplications were
observed in the respective solvent group, the rate of endoreduplications in the test item dose
groups are regarded as biologically irrelevant.
In both experiments, either EMS (600 or 1000 Mg/mL) or CPA (1.4 Mg/mL) were used as
positive controls and showed distinct increases in the number of cells with structural
chromosome aberrations.
In conclusion, it can be stated that under the experimental conditions reported, the test item
did not induce structural chromosome aberrations in V79 cells (Chinese
hamster cell line) when tested up to the cytotoxic or highest evaluable concentrations.
Conclusions:
It can be stated that under the experimental conditions reported, the registered substance did not induce structural chromosome aberrations inV79cells (Chinese hamster cell line)in vitro.
Therefore, the registered substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest evaluable concentrations.
Executive summary:

The registered substance, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations inV79cells of the Chinese hamsterin vitroin the absence and presence of metabolic activation by S9 mix.

Five independent experiments were performed. In , the exposure period was 4 hours with and without metabolic activation. In Experiment IB and IC, the exposure period was 4 hours with metabolic activation. In Experiment IIA and IIB the exposure period was 18 hours without S9 mix. The chromosomes were prepared 18 hours after start of treatment with the test item.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment I without metabolic activation, where only 50 metaphases were evaluated.

Precipitation of the test item in culture medium was observed after 4 hours treatment with 166.6 µg/mL and above in the absence of S9 mix and 1000.0 µg/mL and above in the presence of S9 mix only in Experiment IA. No relevant influence of the test item on pH value or osmolarity was observed (Exp. IA: solvent control 396 mOsm, pH 7.4 versus 393 mOsm and pH 7.4 at 500.0 µg/mL; Exp. IIA: solvent control 392 mOsm, pH 7.5 versus 399 mOsm and pH 7.5 at 5.0 µg/mL; Exp. IIB: solvent control 405 mOsm, pH 7.6 versus 383 mOsm and pH 7.6 at 15.0 µg/mL).

Inmoderate cytotoxic effects (mitotic index of 68.6 % of solvent control) occurred in the experimental part without S9 mix at the highest evaluable concentration (0.7 µg/mL). In the experimental part with metabolic activation no cytotoxicity was measured up to a test item concentration of 12.3 µg/mL. Higher concentrations could not be evaluated due to exceedingly strong cytotoxicity. Therefore, Experiment IB was performed in the presence of S9 mix with closer spaced does groups. However, no evaluation in the cytotoxic range was possible. As the dose groups were very narrow spaced in the experimental part without S9 mix () no further experiment was performed without S9 mix and 4 hours treatment. In the confirmatory experiment (Experiment IC) in the highest evaluable test item concentration of 25.0 µg/mL relevant cytotoxic effects (mitotic index of 52.8 % of solvent control) were observed.

In Experiment II moderate cytotoxicity was observed in the highest applied concentration of 5.0 µg/mL (mitotic index was 65.2 % of solvent control). Higher concentrations were not evaluable due to exceedingly strong cytotoxic effects. In Experiment IIB no cytotoxicity was observed in the highest evaluable concentration. However, the next higher and closely spaced concentration of 10.0 µg/mL was not evaluable due to exceedingly strong cytotoxic effects (Table13and 16, page37and40).

Inin the absence and presence of S9 mix and in Experiments IIA and IIB in the absence of S9 mix, no biologically relevant increase in the number of cells carrying structural chromosome aberrations was observed. The aberration rates of the cells after treatment with the test item (1.0 - 2.5 % aberrant cells, excluding gaps) were close to the range of the solvent control values (1.0 - 2.0 % aberrant cells, excluding gaps) and within the range of the total range laboratory’s historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).

In Experiment IB, in the presence of S9 mix, statistically significant increases in the number of aberrant cells, excluding gaps (9.5, 6.5, 9.5, 6.5 and 9.0 %, respectively) were observed after treatment with 1.6, 6.3, 9.4, 12.5 and 18.8 µg/mL. All values exceeded the total range of the laboratory’s historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps). This increase of chromosomal aberrations was not dose-dependant.

In the confirmatory Experiment IC the positive result obtained in Experiment IB was not reproducible. No statistically significant or biologically relevant increase in the number of aberrant cells, excluding gaps were observed (see Table12, page 36). All values (1.0, 2.5, 2.0 and 1.5 % aberrant cells, excluding gaps, respectively) were in the total range of the historical solvent control data (0.0 – 4.0 % aberrant cells, excluding gaps). Therefore, the observation obtained in Experiment IB is regarded as biologically irrelevant.

No biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (2.2 - 5.3 %) as compared to the rates of the solvent controls (2.0 – 3.5 %). Several induced rates of polyploid metaphases were observed in isolated dose groups were observed in the absence and presence of S9 mix. However, these were not reproducible and are therefore regarded as biologically irrelevant.

In Experiments IA, IIA and IIB in the absence of S9 mix, no biologically relevant increase in the rate of endomitotic metaphases was found after treatment with the test item. In contrast, in Experiments IA, IB and IC in the presence of S9 mix the rate of endomitotic metaphases was increased in all test groups (0.1 – 0.8 %). Generally, no endoreduplications were observed in the control groups. As in Experiment IB 0.4 % of endoreduplications were observed in the respective solvent group, the rate of endoreduplications in the test item dose groups are regarded as biologically irrelevant.

In both experiments, either(600 or 1000 µg/mL) or CPA (1.4 µg/mL) were used as positive controls and showed distinct increases in the number of cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the registered substance did not induce structural chromosome aberrations inV79cells (Chinese hamster cell line) when tested up to the cytotoxic or highest evaluable concentrations.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12.2009 - 03.2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
none
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
HPRT
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:
Experiment I without S9
0.02 0.05 0.09 0.19 0.38 0.75 1.5 3 microg/mL
Experiment I with S9
1.6 3.1 6.3 12.5 25.0 37.5 50 100 microg/mL

Experiment II without S9
0.6 1.3 2.5 5 7.5 10 12.5 15 microg/mL
Experiment II with S9
5 10 20 25 30 35 microg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: DMSO was a suitable vehicle
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
7,12-dimethylbenzanthracene
ethylmethanesulphonate
Details on test system and experimental conditions:
After 24 h the medium was replaced with serum-free medium containing the test item, either without S9 mix or with 50 ul/mL S9 mix. Concurrent solvent and positive controls were treated in parallel. After 4 h this medium was replaced with complete medium following two washing steps with "saline G". In the second experiment the cells were exposed to the test item for 24 h in complete medium in the absence of metabolic activation.
The pH was adjusted to 7.2
The colonies used to determine the cloning efficiency (survival) were fixed and stained approx. 7 days after treatment as described below.
Three or four days after treatment 1.5×106 cells per experimental point were subcultivated in 175 cm² flasks containing 30 mL medium. Following the expression time of 7 days five 80 cm² cell culture flasks were seeded with about 3 - 5×105 cells each in medium containing 6-TG. Two additional 25 cm² flasks were seeded with approx. 500 cells each in non-selective medium to determine the viability.
The cultures were incubated at 37 °C in a humidified atmosphere with 1.5 % CO2 for about 8 days. The colonies were stained with 10 % methylene blue in 0.01 % KOH solution. The stained colonies with more than 50 cells were counted. In doubt the colony size was checked with a preparation microscope.
Evaluation criteria:
The gene mutation assay is considered acceptable if it meets the following criteria:
- the numbers of mutant colonies per 10e6 cells found in the solvent controls fall within the laboratory historical control data range.
- the positive control substances must produce a significant increase in mutant colony frequencies.
- the cloning efficiency II (absolute value) of the solvent controls must exceed 50 %.
A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration- related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequency at least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.
However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (0.6 – 31.7 mutants per 10e6 cells) a concentration-related increase of the mutations within this range has to be discussed. The variability of the mutation rates of solvent controls within all experiments of this study was also taken into consideration.
Statistics:
A linear regression (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA) statistics software. The number of mutant colonies obtained for the groups treated with the test item were compared to the solvent control groups. A trend is judged as significant whenever the p-value (probability value) is below 0.05. However, both, biological and statistical significance were considered together.
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
Positive controls validity:
valid
Additional information on results:
The test substance has been tested in accordance with OECD 476 and GLP requirements for its potential to induce gene mutations at the HPRT locus in V79 Chinese hamster lung fibroblasts.
The study was performed in two independent experiments using identical experimental procedures.
The cell cultures were evaluated at the following concentrations:
Experiment I without S9
0.02 0.05 0.09 0.19 0.38 0.75 1.5 3 microg/mL
Experiment I with S9
1.6 3.1 6.3 12.5 25.0 37.5 50 100 microg/mL
Experiment II without S9
0.6 1.3 2.5 5 7.5 10 12.5 15 microg/mL
Experiment II with S9
5 10 20 25 30 35 microg/mL
 
The maximum evaluated concentrations of the test item were limited by strong cytotoxicity and in addition by precipitation (experiment II only).
 
No relevant and reproducible increase in mutant frequencies compared to the historical range of solvent controls was observed in the main experiments up to the highest evaluable concentration. However, following 4 hours incubation two increased level mutant frequencies exceeded the historical data in the absence and presence of S9. As these values were not reproducible and were only slightly increased this effect is not considered to be biologically relevant. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment II with metabolic activation. This effect was judged to be based upon the rather low solvent control of 9 mutant colonies per 10E6 cells.
The positive controls and DMBA showed distinctive increases in mutant frequency.
 
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. The test substance is considered to be non-mutagenic.
Conclusions:
Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. The test substance is concidered to be non-mutagenic.
Executive summary:

The test substance C-SAT090071 has been tested in accordance with OECD 476 and GLP requirements for its potential to induce gene mutations at the HPRT locus in V79 Chinese hamster lung fibroblasts.

The study was performed in two independent experiments using identical experimental procedures.

The cell cultures were evaluated at the following concentrations:

Experiment I without S9

0.02 0.05 0.09 0.19 0.38 0.75 1.5 3 microg/mL

Experiment I with S9

1.6 3.1 6.3 12.5 25.0 37.5 50 100 microg/mL

Experiment II without S9

0.6 1.3 2.5 5 7.5 10 12.5 15 microg/mL

Experiment II with S9

5 10 20 25 30 35 microg/mL

 

The maximum evaluated concentrations of the test item were limited by strong cytotoxicity and in addition by precipitation (experiment II only).

 

No relevant and reproducible increase in mutant frequencies compared to the historical range of solvent controls was observed in the main experiments up to the highest evaluable concentration. However, following 4 hours incubation two increased level mutant frequencies exceeded the historical data in the absence and presence of S9. as these values were not reproducible and were only slightly increased this effect is not considered to be biologically relevant. The induction factor exceeded the threshold of three times the corresponding solvent control in the first culture of experiment II with metabolic activation. This effect was judged to be based upon the rather low solvent control of 9 mutant colonies per 10E6 cells.

The positive controlsand DMBA showed distinctive increases in mutant frequency.

 

Under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells. The test substance is considered to be non-mutagenic.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
at time of testing no guideline was available
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
uvrB
Species / strain / cell type:
S. typhimurium TA 1535
Species / strain / cell type:
S. typhimurium TA 1538
Species / strain / cell type:
S. typhimurium TA 98
Species / strain / cell type:
S. typhimurium TA 100
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
0.2, 2, 20, 500, 2000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) as described by Ames, B.N. et al. (1975), Mutation Research, 31, 347-364

NUMBER OF REPLICATIONS: 2
Evaluation criteria:
as described in the scientific publication by Dean et al. in Mutation Research, 153 (1985), pages 57-77
Statistics:
N/A
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Vehicle controls validity:
not specified
Untreated negative controls validity:
not applicable
Positive controls validity:
not specified
Conclusions:
Based on the results in a scientific publication, for which a test similar to OECD Test Guideline 471 was performed, the test substance is considered not to be mutagenic.
Executive summary:

As reported in the scientific publication by Dean et al. in Mutation Research, 153 (1985), pages 57-77, the mutagenic potential of the test substance, identified by the common name 2 -hydroxy-5 -tert-nonyl acetophenone oxime, was evaluated in a study similar to OECD Test Guideline 471 (Ames).

The substance as 50% active component (in hydrocarbon diluent) was dissolved in DMSO and applied to four Salmonella typhimurium strains (TA 1535, 1538, 98, 100) using the plate incorporation application method as described by Ames, B.N. et al. (1975), Mutation Research, 31, 347-364, with and without metabolic activation (S9). Vehicle and positive controls were included. The concentrations 0.2, 2, 20, 500, 2000 µg/plate were tested. The results were negative in the strains used. Therefore, the test substance is considered not to be mutagenic

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
see attached read-across justification in IUCLID dossier chapter 13
Reason / purpose:
read-across source
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Ames-Test

As reported in the scientific publication by Dean et al. in Mutation Research, 153 (1985), pages 57-77, the mutagenic potential of the test substance, identified by the common name 2 -hydroxy-5 -tert-nonyl acetophenone oxime, was evaluated in a study similar to OECD Test Guideline 471 (Ames).

The substance as 50% active component (in hydrocarbon diluent) was dissolved in DMSO and applied to four Salmonella typhimurium strains (TA 1535, 1538, 98, 100) using the plate incorporation application method as described by Ames, B.N. et al. (1975), Mutation Research, 31, 347-364, with and without metabolic activation (S9). Vehicle and positive controls were included. The concentrations 0.2, 2, 20, 500, 2000 µg/plate were tested. The results were negative in the strains used. Therefore, the test substance is considered not to be mutagenic.

The suitable read across substance Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched, which lacks a methyl group at the oxime carbon atom (CAS # 174333 -80-3) was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strains according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using the Salmonella typhimurium strains TA 1535, TA 1537, TA 98, TA 100, and TA 102.

The assay was performed with and without liver microsomal activation. Each concentration, including the controls, was tested in triplicate. Due to strong toxic effects in experiment II without S9 mix in strains TA 1535, TA 1537, TA 98 and TA 100 this part was repeated with reduced concentrations (reported as experiment Ila). The test item was tested at the following concentrations:

Pre-Experimen/Experiment I and II: 3; 10; 33; 100; 333; 1000; 2500; and 5000 μg/plate

Experiment II a: 0.03; 0. 1; 0.3; 1; 3; 10; 33; and 100 μg/plate

The plates incubated with the test item showed reduced background growth with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in the test groups with and without metabolic activation. No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with Benzaldehyde, 2-hydroxy-5-nonyl-, oxime, branched at any dose level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance.

Appropriate reference mutagens were used as positive controls and showed a distinct increase of induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Therefore, the test substance is considered to be non-mutagenic in this Salmonella typhimurium reverse mutation assay.

In vitro chromosome aberration test

The test substance, dissolved in DMSO, was assessed for its potential to induce structural chromosome aberrations in V79 cells of the Chinese hamster in vitro in five independent experiments.

In each experimental group two parallel cultures were set up. At least 100 metaphases per culture were evaluated for structural chromosome aberrations, except for the positive control in Experiment IA without metabolic activation, where only 50 metaphases were evaluated.

The highest applied concentrations of 500.0 ug/mL (without S9 mix) and 1000.0 ug/mL (with S9 mix) were chosen due to strong cytotoxic effects obtained in an HPRT study, which was performed in parallel with the test item and the same cellular system.

Dose selection for the cytogenetic experiments was performed considering the toxicity data.

No clear toxic effects indicated by reduced mitotic indices or reduced cell numbers of about or below 50 % of control were observed after treatment with the test item, except in Experiment IC in the presence of S9 mix. In this experimental part the mitotic index was reduced to 52.8 % of control at the highest evaluated concentration of 25.0 ug/mL. In Experiments IA and IIA the mitotic indices were reduced (68.6 and 65.2 % of control, respectively) at the highest evaluated concentrations in the absence of S9 mix.

No clastogenicity was observed without S9 mix. Statistically significant increases in the number aberrant cells obtained in Experiment IB in the presence of S9 mix. As these increases were not reproducible in the confirmatory Experiment IC this effect is regarded as biologically irrelevant.

No biologically relevant increase in polyploid or endomitotic metaphases was found after treatment with the test item as compared to the frequencies of the control cultures.

Appropriate mutagens were used as positive controls. They induced statistically significant increases (p < 0.05) in cells with structural chromosome aberrations.

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce structural chromosome aberrations in V79 cells (Chinese hamster cell line) in vitro.

Therefore, the test substance is considered to be non-clastogenic in this chromosome aberration test in the absence and presence of metabolic activation, when tested up to cytotoxic or the highest evaluable concentrations.

HPRT gene mutation assay

The study was performed to investigate the potential of the test substance to induce gene mutations at the HPRT locus in V79 cells of the Chinese hamster.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 hours. The second experiment was performed with a

treatment time of 4 hours with and 24 hours without metabolic activation.

The highest concentration (3340.0 ug/ml approx. 10 mM) used in the range finding pre-expenment was chosen with respect to the purity of the test item and the current OECD guideline 476. The dose range of the main experiments was limited by test item induced

cytotoxicity.

The dose ranges of the first main experiment (with and without S9 mix) and the second main experiment (without S9 mix) were limited by test item induced cytotoxicity. After metabolic activation the dose range of the second main experiment was limited by precipitation of the test item.

No substantial and reproducible dose dependent increase of the mutation frequency was observed in both main experiments.

Appropriate reference mutagens, used as positive controls, induced a distinct increase in mutant colonies and thus, showed the sensitivity of the test item and the activity of the metabolic activation system.

In conclusion it can be stated that under the experimental conditions reported the test item did not induce gene mutations at the HPRT locus in V79 cells.

Therefore, the test substance is considered to be non·mutagenic in this HPRT assay.


Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No indication of genotoxicity was observed in the Ames test (OECD 471, GLP), the HPRT Test (OECD 476, GLP) and the in vitro chromosome aberration assay (OECD 473, GLP). As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008,as amended for the fourteenth time in Regulation (EC) No. 2020/217.