Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Bacterial gene mutation assay (Ames test): not mutagenic (WoE, rel. 2)
Bacterial gene mutation assay (Ames test) QSAR prediction: not mutagenic (WoE, rel. 2)
Mammalian cell chromosome aberration study in CHO cells: not clastogenic (K, rel. 2)
Unscheduled DNA synthesis assay in mammalian liver cells: no induction of UDS (Read-across, K, rel. 2)
BlueScreen genotoxicity screening assay, in vitro mammalian gene mutation: Not genotoxic (Key, Rel. 2)

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:
From August 12 to September 01, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
GLP study conducted similarly to OECD test guideline No. 471 with deviation: S. typhimurium TA102 or E. coli WP2 uvrA strain not used. Insufficient number of strains but considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
(S. typhimurium TA102 or E. coli WP2 uvrA strain not used)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Histidine
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
Not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
3, 10 and 30 % S9-mix: S9 fraction from liver of rats treated with Arochlor 1254 (500 mg/kg bw, i.p) dissolved in corn oil.
Test concentrations with justification for top dose:
Preliminary toxicity assay: 5, 50, 500 and 5000 µg/plate in TA1535, TA1537, TA98 and TA100, without and with 10 % S9 mix. Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
First mutation assay: 5, 15, 50, 150 and 500 µg/plate in TA1535, TA1537, TA98 and TA100, without and with 3 and 10 % S9 mix.
Second mutation assay: 5, 15, 50, 150 and 500 µg/plate in TA1535, TA1537, TA98 and TA100, without and with 10 and 30 % S9 mix.
Five test item dose levels were selected in main tests in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to limited solubility of test material in water, DMSO was selected as the vehicle of choice.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
Remarks:
Without S9-mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9-mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: Preincubation

DURATION
- Preincubation period: 60 minutes at 37 °C
- Exposure duration: Approximately 2-3 days at 37 °C

NUMBER OF REPLICATIONS:
- Preliminary toxicity assay: One plate/dose
- Mutation assay: 3 plates/dose

DETERMINATION OF CYTOTOXICITY
- Method: Evaluation of the toxicity was performed on the basis of growth of the bacterial background lawn.
Rationale for test conditions:
Rationale for test conditions
Pre-test - Maximum concentration was 5000 μg/plate (the maximum recommended dose level).
Experiment 1 & 2 - Five test item dose levels were selected in Experiment 2 (main test) in order to achieve both four non-toxic dose levels and the toxic limit of the test item.
Evaluation criteria:
- The positive and negative control values should fall within specified ranges.
- The test substance will be considered to be mutagenic if a dose related increase in revertants, up to at least a doubling of the control values, is obtained in two independent tests.
Statistics:
None
Key result
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:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Not applicable
- Effects of osmolality: Not applicable
- Evaporation from medium: No data
- Water solubility: None
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES: Test material was toxic to all the tester strains at 5000 µg/plate, without and with S9-mix. Toxicity was also observed at 500 µg/plate in TA98, without S9-mix and in both TA98 and TA1537, with S9-mix.

OTHER:
- The sterility of the S-9 mix, solvent and test substance (500 µg/plate) was confirmed by the incubation of aliquots of the liquids on nutrient agar.

- See the attached file for table of results.

Conclusions:
Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.
Executive summary:

In a reverse gene mutation assay performed similarly to OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 were exposed to test material diluted in DMSO, both in the presence and absence of metabolic activation system (3, 10 and 30 % liver S9-mix). The dose range was determined in a preliminary toxicity assay and ranged between 5-500 µg/plate for all tester strains, without and with 3 and 10 % S-9 mix (first mutation assay). In the second mutation assay, dose ranged between 5-500 µg/plate for all tester strains, without and with 10 and 30 % S-9 mix. Vehicle (DMSO) and positive control groups were also included in mutagenicity tests.

The number of revertants for the vehicle, negative and positive controls was as specified in the evaluation criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Test material was toxic to all the tester strains at 5000 µg/plate, without and with S9-mix. Toxicity was also observed at 500 µg/plate in TA98, without S9-mix and in both TA98 and TA1537, with S9-mix. Test material did not induce significant number of revertant colonies with any of the four tester strains either in the presence or absence of S9-mix in both experiments.

Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100 according to the criteria of the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.

In this study, insufficient number of strains were used but the study was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
(Q)SAR
Adequacy of study:
weight of evidence
Study period:
27 March, 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
results derived from a valid (Q)SAR model and falling into its applicability domain, with adequate and reliable documentation / justification
Justification for type of information:
1. SOFTWARE
OASIS TIMES 2.27.16

2. MODEL (incl. version number)
Ames mutagenicity v.10.10

3. SMILES OR OTHER IDENTIFIERS USED AS INPUT FOR THE MODEL
CCCc1ccc(O)c(OC)c1

4. SCIENTIFIC VALIDITY OF THE (Q)SAR MODEL
See QMRF attached

- Defined endpoint:
Species: Salmonella typhimurium
Endpoint:In vitro: Ames mutagenicity with metabolic activation
According to JRC pre-classification list of endpoints:
No. 207 QMRF Human Health Effects, QMRF 4.10 Mutagenicity.
Endpoint units : Qualitative – positive/ negative
Dependent variable : Observed Mutagenicity with S9
Experimental protocol : Bacterial Reverse Mutation Assay (e.g. Ames test)
Endpoint data quality and variability : High quality, chemicals provided by National Toxicology Program (NTP), ECVAM database and Badische Anilin und Soba-Fabrik AG (BASF AG)

- Unambiguous algorithm:
Type of model : Structural alerts based model
Explicit algorithm : Prediction of Bacterial mutagenicity.
Mutagenicity of chemicals, we have combined the alerting group approach with a pattern recognition type of model to delineate reactivity of chemicals toward DNA within a given interaction mechanism. The explicit generation of metabolites allowed the DNA reactivity model to be applied not only to parent chemicals but also their stable metabolites.
Descriptors in the model : Name: Molecular weight (MW)

- Defined domain of applicability:
The domain consists of the following sub-domain layers:
1. General parametric requirements.
The variations of molecular parameters that may affect the quality of the measured endpoint significantly are included here (such as molecular weight, etc.). The domain of general parametric includes the range of variation of hydrophobicity (log KOW) and Molecular weight (MW) of chemicals in training set.
2. Structural domain.
The structural component of the model is based on the structural similarity between chemicals in the training set which were correctly predicted by the model. The structural neighborhood of atom-centered fragments (accounting for the first neighbours) extracted from correctly and incorrectly predicted parent structures from the training set is used to determine this similarity.
The target chemical could contain the following types of ACF:
o Fragments present in correctly predicted training chemicals only (i.e. correct fragments),
o Fragments found both in correctly and non-correctly predicted training chemicals (i.e. fuzzy fragments). These fragments are treated as correct fragments,
Fragments present in non-correctly predicted training chemicals only (i.e. incorrect fragments),
o Fragments not present in the training chemicals (i.e. unknown fragments).
A chemical belongs to the structural domain of the model if it could be partitioned only on correct fragments. The user is able to analyse how important are unknown and incorrect fragments (if present in the target) and to make a decision about their effect on the quality of prediction.The distribution of structural characteristics of the target chemical and accepted thresholds is used as a criterion to determine how well the target is represented in the structural space of correctly predicted chemicals. The accepted domain thresholds for Mutagenicity are as follows:
o Correct = 100%
o Incorrect = 0%
A chemical is considered In Domain if it is classified to belong to all sub-domain levels. The information implemented in the applicability domain is extracted from the correctly predicted training chemicals used to build the model and in this respect the applicability domain determines practically the interpolation space of the model.
- Method used to assess the applicability domain : The approach use to determine and assess the domain is described in:
Dimitrov S, Dimitrova G., Pavlov T., Dimitrova D., Patlewicz G., Niemela J., Mekenyan O., A stepwise approach for defining the applicability domain of SAR and QSAR models, J. Chem. Inf. Model., 45, 839-849 (2005).
- Software name and version for the applicability domain assessment
The LMC software OASIS Domain Manager v.1.10 (which is embedded in OASIS platform) is used to determine the applicability domain.
http://oasis-lmc.org/products/software/domain-manager.aspx
- Limits of applicability
General properties requirements
Property Domain Target chemical
log KOW [-13.155; 21.646] 2.06
MW, Da [18.014; 1560.198] 155

KOW is calculated by EPI Suite
Structural domain extracted from 3107 training chemicals contains:
o 8419 correct fragments,
o 1166 fuzzy fragments (treated as correct fragments),
o 1243 incorrect fragments.

- Appropriate measures of goodness-of-fit and robustness and predictivity:
Availability of the training set : The training set consisting of 3107 chemicals (of which 1528 are proprietary) is embedded in the software implementation of the model.
Available information for the training set : Chemical names, CAS numbers, SMILES, data source, conditions and strain information are available.
Data for the dependent variable for the training set : Data for the dependent variable of the training set are embedded in the software implementation of the model.
Statistics for goodness-of-fit : Statistics of the model:
·Sensitivity = (predicted positive/observed positive) = 77%
·Specificity = (predicted negative/observed negative) = 81%
·Concordance = (correct predicted positive and negative chemicals in respect to all training set chemicals) = 81%

- Mechanistic interpretation:
- Mechanistic basis of the model :
The reactivity model describing interactions of chemicals with DNA is based on an alerting group approach. Only those toxicophores extracted from the training set having clear interpretation for the molecular mechanism causing the ultimate effect are included in the model.
The derived model is combined with in vitro metabolic simulator used for predicting metabolic activation of chemicals with the S9 mix. The in vitro metabolic simulator is trained to reproduce documented maps for mammalian liver metabolism for 261 chemicals. Parent chemicals and each of the generated metabolites are submitted to a battery of models to screen for a general effect and mutagenicity mechanisms. Thus, chemicals is predicted to be mutagenic as parents only, parents and metabolites, and metabolites only. Mutagenicity could be due to the parent chemical only or as a result of its metabolic activation (i.e., the parent is inactive but it is transformed to a mutagenic metabolite), or both parent structure and metabolites could be mutagenic.
For more details see the reference in Section 2 (2.7).
- A priori or a posteriori mechanistic interpretation
The model building followed the traditional approach:
a. Building a hypothesis for the modelled event,
b. Defining the alerting groups based on parent structures,
c. Fitting of model variable to the observed data,
d. Verification of model quality,
e. Depending on the results found in step d model building could continue with step a, b or f,
f. Determination of the applicability domain and practical application of the model.

5. APPLICABILITY DOMAIN
See QPRF attached

- Descriptor domain: The chemical fulfils the general properties requirements
- Structural and mechanistic domains: The following ACF are identified:
Fragments in correctly predicted training chemicals – 100.00%
Fragments in non-correctly predicted training chemicals – 0.00%
Fragments not present in the training chemicals – 0.00%
The chemical is in the interpolation structural space
- Similarity with analogues in the training set: No additional comments on structural analogues are
provided by the author of prediction
- Other considerations (as appropriate): Comments on the mechanistic interpretation of the model
prediction are not provided by the author of prediction

6. ADEQUACY OF THE RESULT
OASIS TIMES evaluation showed no alerts for mutagenicity. The substance is predicted to be negative - not mutagenic to S. typhimurium in vitro. This result fits well with the results of an Ames tests performed with Strains S. typhimurium TA1535, TA1537, TA98 and TA100.
Reason / purpose for cross-reference:
reference to other study
Principles of method if other than guideline:
In vitro Ames Mutagenicity with S9 metabolic activation
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
other: S. typhimurium
Details on mammalian cell type (if applicable):
not applicable
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with
Test concentrations with justification for top dose:
not applicable
Vehicle / solvent:
not applicable
Details on test system and experimental conditions:
not applicable
Evaluation criteria:
not applicable
Statistics:
not applicable
Key result
Species / strain:
other: S. typhimurium
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: not applicable
Vehicle controls validity:
not applicable
Untreated negative controls validity:
not applicable
Positive controls validity:
not applicable
Additional information on results:
- Predicted value (model result): in vitro Ames negative
- Applicability domain (OECD Principle 3):
The chemical fulfils the general properties requirements
The following ACF are identified:
Fragments in correctly predicted training chemicals – 100.00%
Fragments in non-correctly predicted training chemicals – 0.00%
Fragments not present in the training chemicals – 0.00%

See attached documents:

- QMRF Ames mutagenicity with S9

- QPRF_TIMES_AMES_1analogues

- QPRF_TIMES_AMES_25analogues

Conclusions:
OASIS TIMES evaluation showed no alerts for mutagenicity. The substance is predicted to be negative - not mutagenic to S. typhimurium in vitro.
Executive summary:

OASIS TIMES 2.27.16.6 software was used to predict the mutagenicity of the substance (monoconstituent). No structural alert was identified for mutagenicity endpoint. The substance is predicted to be negative - not mutagenic to S. typhimurium in vitro.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From September 19 to October 20, 1988
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP study conducted according to OECD Guideline 473 with deviations: details of mycoplasma contamination, evaluation criteria and historical data not reported.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
details of mycoplasma contamination, evaluation criteria and historical data not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Source: BIBRA
- Type and identity of media: The cells were routinely grown and subcultured in Hams F12 medium supplemented with 5 % foetal calf serum at 37 °C in a humid atmosphere containing 5 % carbon dioxide in 175 cm2 plastic tissue culture flasks.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: No data
- Periodically checked for karyotype stability: No data
- Periodically "cleansed" against high spontaneous background: No data
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
10 % S9 mix: S9-mix from the livers of CD rats of Sprague-Dawley origin treated with Aroclor 1254 at 500 mg/kg bw.
Test concentrations with justification for top dose:
Preliminary toxicity test: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL, with and without S9-mix. All final concentration above 1000 µg/ml caused a precipitate to form in queous tissue culture medium. 1000 µg/ml is referred to as the maximum achievable concentration
Dose levels for metaphase analysis: 7.8, 15.6, 31.3 and 62.5 µg/mL, without S9-mix; 15.6, 31.3, 62.5 and 125 µg/mL, with S9-mix. The choice of concentrations was based on toxicity observed in the preliminary test.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test material preparation: Test material was diluted with DMSO immediately before use. Test material was miscible with the DMSO at a concentration of 166 mg/mL. All final concentration above 1000 µg/ml caused a precipitate to form in queous tissue culture medium. 1000 µg/ml is referred to as the maximum achievable concentration
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
Without S9-mix: 0.4 µg/mL of mitomycin C.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With S9-mix: 20 µg/mL of cyclophosphamide.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 h (without S9-mix); 6 h (with S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h

SPINDLE INHIBITOR (cytogenetic assays): Colchicine (0.25 µg/mL culture medium)
STAIN (for cytogenetic assays): 10 % Giemsa

NUMBER OF REPLICATIONS: Two cultures/dose for test material, 2 cultures for positive control, 4 cultures for negative control and vehicle control.

NUMBER OF CELLS EVALUATED: Approximately 100 metaphases per culture were evaluated.

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index

Other:
- Microscopical examination for mitotic index: Prepared slides were examined at magnification of x160 and the proportion of mitotic cells in each culture was recorded. The concentration of test compound causing a decline in mitotic index to between 20-50 % of the solvent control value (approximating to the EC50 value) was selected and this concentration was used as the highest dose level for metaphase analysis.
- Metaphase analysis: Metaphase spreads were identified using a magnification of x160 and examined at a magnification of x1000 using an oil immersion objective.
- Osmolality measurement: Following the exposure of the cells to the test material, a sample of the supernatants of the two highest doses and the control treatments was removed and a measurement of osmolality made by conventional techniques.
Rationale for test conditions:
The dose levels for metaphase analysis were based on toxicity observed in the preliminary test.
Evaluation criteria:
No data
Statistics:
Statistical significance was confirmed by means of the Fisher’s test (p< 0.001).
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No data
- Effects of osmolality: No significant difference from solvent control values.
- Evaporation from medium: No data
- Water solubility: Miscible with DMSO
- Precipitation: Yes; > 1000 µg/mL in culture medium.
- Other confounding effects: None

CYTOTOXICITY:
- Without S9-mix: Cultures treated with test material at 1000 µg/mL killed virtually all the cells; 500 µg/mL yielded no live cells; 250 µg/mL contained no mitotic cells; 125 µg/mL reduced the mitotic index value to approximately 9 % of that of the solvent control and 62.5 µg/mL caused a decline in mitotic index to approximately 53 % of the solvent control value. The five remaining concentrations of the test material did not reduce the mitotic index value to below 70 % of the solvent control. The concentrations selected for the metaphase analysis were 7.8, 15.6, 31.3 and 62.5 µg/mL.
- With S9-mix: Cultures treated with 1000 µg/mL of test material yielded no live cells, and those treated with 500 µg/mL contained no metaphase figures. 250 µg/mL of the test material caused a decline in mitotic index to approximately 6 % of the solvent control value, and 125 and 62.5 µg/mL reduced the mitotic index values to approximately 42 and 46 %, respectively, of that of the solvent control. The five remaining concentrations of the test material did not reduce the mitotic index value to less than 70 % of the solvent control. The concentrations selected for the metaphase analysis were 15.6, 31.3, 62.5 and 125 µg/mL.

None

Conclusions:
Under the test conditions, the test material is not considered as clastogenic according to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.
Executive summary:

In an in vitro chromosome aberration test performed according to OECD guideline No. 473 and in compliance with GLP, Chinese hamster ovary (CHO) cells of K1-BH4 strain were exposed to test material diluted in dimethylsulphoxide (DMSO) at the following concentrations: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL, without S9 mix (24 h exposure without recovery) and with S9-mix (6 h exposure and 18 h recovery). Two hours before the cells were harvested, mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations. Negative, solvent (DMSO) and positive control groups were also included in the experiment.

In the absence of metabolic activation, cultures treated with test material at 1000 µg/mL killed virtually all the cells; 500 µg/mL yielded no live cells; 250 µg/mL contained no mitotic cells; 125 µg/mL reduced the mitotic index value to approximately 9 % of that of the solvent control and 62.5 µg/mL caused a decline in mitotic index to approximately 53 % of the solvent control value. In the presence of metabolic activation, cultures treated with 1000 µg/mL of test material yielded no live cells, and those treated with 500 µg/mL contained no metaphase figures. 250 µg/mL of the test material caused a decline in mitotic index to approximately 6 % of the solvent control value, and 125 and 62.5 µg/mL reduced the mitotic index values to approximately 42 and 46 %, respectively, of that of the solvent control. The remaining concentrations of the test material did not reduce the mitotic index value to below 70-75 % of the solvent control. Dose levels for metaphase analysis: 7.8, 15.6, 31.3 and 62.5 µg/mL, without S9-mix; 15.6, 31.3, 62.5 and 125 µg/mL, with S9-mix. Test material caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in either the presence or absence of metabolic activation. Both positive control compounds, mitomycin C (0.4 µg/mL) and cyclophosphamide (20 µg/mL), caused statistically significant increases in the proportion of metaphase figures containing aberrations when compared with the relevant solvent controls. Thus, the sensitivity of the test system and the efficacy of the S-9 mix were validated.

Under the test condition, the test material is not considered as clastogenic according to the criteria of the Annex VI of the Regulation (EC) No.1272/2008 (CLP) and to the GHS.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study conducted similarly to OECD Guideline No. 482 with deviations: details of test material, individual and summary results not reported.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 482 (Genetic Toxicology: DNA Damage and Repair, Unscheduled DNA Synthesis in Mammalian Cells In Vitro)
Deviations:
yes
Remarks:
details of test material, individual and summary results not reported
Principles of method if other than guideline:
Not applicable
GLP compliance:
no
Type of assay:
DNA damage and repair assay, unscheduled DNA synthesis in mammalian cells in vitro
Target gene:
Not applicable
Species / strain / cell type:
hepatocytes: rat and mouse
Details on mammalian cell type (if applicable):
- Type and identity of media: William’s medium E supplemented with bovine calf serum (10 % for rats, 1 % for mice), and l-glutamine (200 mM) at 5 x 10^5 cells/mL.
- Properly maintained: Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
not applicable
Test concentrations with justification for top dose:
0.1, 1, 10, 100 and 1000 µM
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive control substance:
other: aminofluorene
Details on test system and experimental conditions:
PROCEDURE: UDS assay was conducted as described by McQueen (1989) and Williams (1977) with some modifications.

HEPATOCYTE ISOLATION: Rat and mouse were anesthetized with sodium pentobarbital prior to the isolation of hepatocytes. Isolations were performed using a two step perfusion technique as described by McQueen (1989). Briefly, the portal vein was cannulated and the liver perfused with HBSS buffered with EGTA and HEPES for 4 min, followed by a collagenase solution for 6.5 min for the rat, and for 5.5 min for the mouse. Flow rates for the rat perfusion were 40 and 20 mL/min for the HBSS solution and the collagenase solution, respectively. Flow rates for the mouse were 8 mL/min for each solution. The livers were removed from the animals and dissociated through sterile gauze. The hepatocytes were washed twice with media and counted. Only those cell preparations with 85 % viability or greater (as determined by trypan blue exclusion) were used for primary culture. Cells were suspended in William’s medium E supplemented with bovine calf serum (10 % for rats, 1 % for mice), and l-glutamine (200 mM) at 5 x 10^5 cells/mL.

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 18 h; incubations were conducted in 95 % air, 5 % CO2 at 37 °C.
- Selection time (if incubation with a selection agent): Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium).

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release. LDH activity was expressed as the percent of LDH released into the medium (LDH in the medium divided by total LDH in cells and medium, multiplied by 100). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes.

NUMBER OF REPLICATIONS: 3 independent experiments, triplicate wells for rat and duplicate wells for mouse.

PREPARATION AND SCORING OF SLIDES
- The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. Slides were examined under oil immersion. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus.

NUMBER OF CELLS EVALUATED: Twenty cells per slide, with three slides per dose level were counted when possible.
Rationale for test conditions:
Not reported
Evaluation criteria:
Positive grain counts indicate UDS. A compound is defined as positive if there is an increase in positive grain counts over two or more doses.
Statistics:
None
Key result
Species / strain:
hepatocytes: rat and mouse
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY MEASUREMENT: Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves.

UDS RESULTS: Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.
Executive summary:

In an UDS assay, rat and mouse hepatocytes were incubated with test material at the concentrations ranging from 0.1 to 1000 µM for 18 h. Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes. The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 500 to 5000 µM.

Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves. Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.

Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
Further information is included in Iuclid Section 13.

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
This read-across is based on the hypothesis that the source and target substances have similar physico-chemical, toxicological and environmental fate properties because of their structural similarity.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target and the source substances are both mono-constituent substances.

3. ANALOGUE APPROACH JUSTIFICATION
 In vitro gene mutation study in bacteria - Ames test
No (reverse) gene mutations were detected in the Ames test performed on both the source and the target substances.
 In vitro gene mutation study in mammalian cells
An unscheduled DNA synthesis (UDS) test was performed on the source substance (reaction-mass of cis- and trans-isomers instead of trans-isomer). The substances did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes, under the conditions of these tests.
The UDS test on the source substance provided in this dossier was performed similarly to OECD TG 482. The test material was not clearly identified (isomer ration and impurity profile not reported). It is however assumed to represent the source substance in terms of constituents and impurities. The result of the study is adequate for classification and labelling.
 In vitro cytogenicity study in mammalian cells
In the CAT performed on both the source and the source substances, no significant increases in the frequency of cells with aberrations was induced.
Therefore, based on the considerations above, it can be concluded that the result of the UDS conducted with the source substance is highly likely to predict the properties of the target substance and is considered as adequate to fulfil the information requirement of Annex VIII, 8.4.3.

4. DATA MATRIX
Cf. Iuclid Section 13.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across: supporting information
Key result
Species / strain:
hepatocytes: rat and mouse
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY MEASUREMENT: Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves.

UDS RESULTS: Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

None

Conclusions:
Based on data on the source substance, the target substance is not expected to induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.
Executive summary:

In an UDS assay, rat and mouse hepatocytes were incubated with test material at the concentrations ranging from 0.1 to 1000 µM for 18 h. Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes. The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 500 to 5000 µM.

Test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves. Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.

Based on data on the source substance, the target substance is not expected to induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Remarks:
No OECD test guideline and no internationally recognised validation. However, it has been well-validated and the validations have been published in high quality peer-reviewed journals.
Principles of method if other than guideline:
BlueScreen HC Assay was performed to assess the genotoxicity and cytotoxicity of 2-Methoxy-4-propylphenol with and without metabolic activation in an assay using 96-well microplates.
GLP compliance:
yes
Remarks:
The study according to the principles of GLP and the laboratory gained its GLP certificate in July 2014.
Type of assay:
other: BlueScreen HC Assay
Target gene:
GADD45a gene
Species / strain / cell type:
other: human lymphoblastoid TK6 cell strain (GLuc-T01)
Details on mammalian cell type (if applicable):
A genetically modified strain of cultured human lymphoblastoid TK6 cells is used (GLuc-T01).
Metabolic activation:
with and without
Metabolic activation system:
1% (v/v) S9: Aroclor-1254 induced rat liver S9 fraction mix
Test concentrations with justification for top dose:
9.77, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 μM. Concentrations were determined based on precipitate formation.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Test substance was combined with 100 % DMSO to give a 1250 mM stock solution. The compound precipitated when added to water therefore further dilution of the DMSO stock solution was required. The report does not state the final stock solution concentration but it was calculated to be 125 mM.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Remarks:
without metabolic activation; 0.125 and 0.5 μg/mL
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation; 5 and 25 μg/mL
Details on test system and experimental conditions:
APPARATUS
- 96-well, black microplate with an optically clear base

DURATION
- Exposure duration:
Without metabolic activation: Microplates were covered with a breathable membrane and incubated at 37 °C with 5% CO2 and 95% humidity for 48 hours.
With metabolic activation: Test substance was incubated with the test cells in the presence of 1% (v/v) S9 mix in exposure Medium at 37 °C (5% CO2, 95% humidity) for 3 hours. After this treatment time, cells were washed in phosphate buffered saline solution, harvested by centrifugation and allowed to recover in Recovery Medium for 45 hours at 37°C with 5% CO2 and 95% humidity.

NUMBER OF REPLICATIONS: Duplicate dilution series were performed within a single microplate assay.

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity is measured by lysis of the cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.

OTHER:
- Assay plates are then analysed using a microplate reader that provides measurements of fluorescence and flash luminescence for cells and solutions in the microplate wells.
- The luminescence and fluorescence data are automatically analysed using the BlueScreen HC software template to produce a result summary, with data presented both in tabulated and graphical formats, giving a semi-quantitative assessment of cytotoxicity and genotoxicity.
Rationale for test conditions:
Concentrations were determined based on precipitate formation.
Evaluation criteria:
Genotoxicity is evaluated by induction in GLuc expression. For this protocol the statistically defined threshold for a positive result is 1.8, i.e. 80% induction over and above the baseline for vehicle-treated control cells in the absence of metabolic activation and 1.5, i.e. 50% induction over and above the baseline for vehicle-treated control cells in the presence of metabolic activation.
Statistics:
Not applicable.
Key result
Species / strain:
other: human lymphoblastoid TK6 cell strain (GLuc-T01)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
CYTOTOXICITY:
Test substance was negative for cytotoxicity with and without S9 metabolic activation, i.e. did not reduce the relative cell density, compared to the vehicle-treated control either with or without metabolic activation.

None

Conclusions:
Under the test conditions, test substance is not genotoxic either with or without metabolic activation in human lymphoblastoid TK6 cell strain (GLuc-T01).
Executive summary:

In a BlueScreen HC Assay, human lymphoblastoid TK6 cell strain (GLuc-T01) was exposed to test substance at 9.77, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 μM with (3 hours exposure + 45 hours recovery) both with and without metabolic activation (48 hours). Microplates were incubated at 37 °C with 5% CO2 and 95% humidity. Cell density was determined by the subsequent lysis of cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence.

Test substance was negative for cytotoxicity in this assay with and without S9 metabolic activation, i.e. did not reduce the relative cell density, compared to the vehicle-treated control either with or without metabolic activation. Genotoxicity was evaluated by induction in GLuc expression. The statistically defined threshold for a positive result is 1.8, i.e. 80% induction over and above the baseline for vehicle-treated control cells (without metabolic activation); and a threshold of 1.5 i.e. a 50% induction over and above the baseline for vehicle-treated control cells (with metabolic activation). Test substance was classified negative for genotoxicity in the BlueScreen HC assay, both with and without metabolic activation.

The positive controls induced the genotoxicity and assay is considered valid.

 

Under the test conditions, test substance is not genotoxic either with or without metabolic activation in human lymphoblastoid TK6 cell strain (GLuc-T01).

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Gene Mutation Assay in Bacteria (Ames test)

In a reverse gene mutation assay performed similarly to OECD test guideline No. 471 and in compliance with GLP, S. typhimurium strains TA1535, TA1537, TA98 and TA100 were exposed to test material diluted in DMSO, both in the presence and absence of metabolic activation system (3, 10 and 30 % liver S9-mix). The dose range was determined in a preliminary toxicity assay and ranged between 5-500 µg/plate for all tester strains, without and with 3 and 10 % S-9 mix (first mutation assay). In the second mutation assay, dose ranged between 5-500 µg/plate for all tester strains, without and with 10 and 30 % S-9 mix.Vehicle (DMSO) and positive control groups were also included in mutagenicity tests. The number of revertants for the vehicle, negative and positive controls was as specified in the evaluation criteria. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated. Test material was toxic to all the tester strains at 5000 µg/plate, without and with S9-mix. Toxicity was also observed at 500 µg/plate in TA98, without S9-mix and in both TA98 and TA1537, with S9-mix. Test material did not induce significant number of revertant colonies with any of the four tester strains either in the presence or absence of S9-mix in both experiments. Under the test condition, test material is not mutagenic with and without metabolic activation in S. typhimurium strains TA1535, TA1537, TA98 and TA100.

In this study, insufficient number of strains were used but the study was considered sufficiently reliable in a weight of evidence for the purpose of hazard assessment.

 

QSAR Gene Mutatation in Bacteria (Ames test) Prediction

OASIS TIMES 2.27.17.6 software was used to predict the mutagenicity of the substance (monoconstituent). No structural alert was identified for mutagenicity endpoint. The substance is predicted to be negative - not mutagenic to S. typhimurium in vitro.

 

Mammalian Cell Chromosome Aberration test in vitro

In an in vitro chromosome aberration test performed according to OECD guideline No. 473 and in compliance with GLP, Chinese hamster ovary (CHO) cells of K1-BH4 strain were exposed to test material diluted in dimethylsulphoxide (DMSO) at the following concentrations: 2.0, 3.9, 7.8, 15.6, 31.3, 62.5, 125, 250, 500 and 1000 µg/mL, without S9 mix (24 h exposure without recovery) and with S9-mix (6 h exposure and 18 h recovery).Two hours before the cells were harvested, mitotic activity was arrested by addition of colchicine to each culture at a final concentration of 0.25 µg/mL. The cells were then treated with a hypotonic solution, fixed, stained and examined for mitotic indices and chromosomal aberrations.Negative, solvent (DMSO) and positive control groups were also included in the experiment. In the absence of metabolic activation, cultures treated with test material at 1000 µg/mL killed virtually all the cells; 500 µg/mL yielded no live cells; 250 µg/mL contained no mitotic cells; 125 µg/mL reduced the mitotic index value to approximately 9 % of that of the solvent control and 62.5 µg/mL caused a decline in mitotic index to approximately 53 % of the solvent control value. In the presence of metabolic activation, cultures treated with 1000 µg/mL of test material yielded no live cells, and those treated with 500 µg/mL contained no metaphase figures. 250 µg/mL of the test material caused a decline in mitotic index to approximately 6 % of the solvent control value, and 125 and 62.5 µg/mL reduced the mitotic index values to approximately 42 and 46 %, respectively, of that of the solvent control. The remaining concentrations of the test material did not reduce the mitotic index value to below 70-75 % of the solvent control. Dose levels for metaphase analysis: 7.8, 15.6, 31.3 and 62.5 µg/mL, without S9-mix; 15.6, 31.3, 62.5 and 125 µg/mL, with S9-mix. Test material caused no statistically significant increases in the proportion of metaphase figures containing chromosomal aberrations at any concentration in either the presence or absence of metabolic activation. Both positive control compounds, mitomycin C (0.4 µg/mL) and cyclophosphamide (20 µg/mL), caused statistically significant increases in the proportion of metaphase figures containing aberrations when compared with the relevant solvent controls. Thus, the sensitivity of the test system and the efficacy of the S-9 mix were validated.

Under the test condition, the test material is not considered as clastogenic.

 

Unscheduled DNA Synthesis (UDS) Assay in Rat and Mouse Hepatocytes.

In an UDS assay performed on the source substance Isoeugenol (reaction-mass of cis- and trans-isomers) (Burkey, 2000, Rel. 2), rat and mouse hepatocytes were incubated with the test material at the concentrations ranging from 0.1 to 1000 µM for 18 h. Hepatocytes were simultaneously exposed to [3H]thymidine (1 mL of 89.4 mCi/mL per 100 mL medium). Following incubation, cells were evaluated under an inverted microscope for gross evidence of cytotoxicity to the hepatocytes. The cells were treated with sodium citrate to swell the nuclei of the cells, fixed to the coverslips, and the coverslips exposed to photographic emulsion after being mounted to slides. The emulsion was developed after 6 days and the cells counterstained with a modified hematoxylin and eosin stain to facilitate counting. Results were quantified using an Artek colony counter. The number of silver grains per nucleus and three neighboring areas of cytoplasm in the same cell were counted. The difference between the number of silver grains in the nucleus and the highest cytoplasmic grain count was recorded. Results are expressed as mean grain counts per nucleus. Cytotoxicity was determined by measuring lactate dehydrogenase (LDH) release at the concentrations ranging from 500 to 5000 µM.

The test material had LC50 values of 300 µM for rat hepatocytes and 200 µM for mice, with both compounds showing relatively steep dose–response curves. Test material showed no increase in mean net grain counts at any of the concentrations tested in rats or mice.

Under the test conditions, test material did not induce unscheduled DNA synthesis (UDS) in rat or mouse hepatocytes.

The source substance is considered adequate for read-across purpose.

 

BlueScreen Genotoxicity Screening Assay

In a BlueScreen HC Assay (Gentronix, 2013, Rel.4), human lymphoblastoid TK6 cell strain (GLuc-T01) was exposed to test substance at 9.77, 19.5, 39.1, 78.1, 156.3, 312.5, 625 and 1250 μM with (3 hours exposure + 45 hours recovery) both with and without metabolic activation (48 hours). Microplates were incubated at 37 °C with 5% CO2 and 95% humidity. Cell density is determined by the subsequent lysis of cells and addition of a fluorescent DNA binding stain, followed by assessment of the resulting fluorescence. Test substance was negative for cytotoxicity in this assay with and without S9 metabolic activation, i.e. did not reduce the relative cell density, compared to the vehicle-treated control either with or without metabolic activation.Genotoxicity is evaluated by induction in GLuc expression. The statistically defined threshold for a positive result is 1.8, i.e. 80% induction over and above the baseline for vehicle-treated control cells (without metabolic activation); and a threshold of 1.5 i.e. a 50% induction over and above the baseline for vehicle-treated control cells (with metabolic activation). Test substance was classified negative for genotoxicity in the BlueScreen HC assay with and without metabolic activation. The positive controls induced the genotoxicity and assay is considered valid.  

The BlueScreen assay is considered to be an adequate test to meet the requirement for data on the gene mutation in mammalian cells.

Under the test conditions, test substance is not genotoxic either with or without metabolic activation in human lymphoblastoid TK6 cell strain (GLuc-T01).

Justification for classification or non-classification

Harmonized classification:

The test material has no harmonized classification for human health according to the Regulation (EC) No. 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding germ cell mutagenicity according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and to the GHS.