Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames according to OECD 471: positive results in both assays


 


HPRT according to OECD 476: negative


 


Chromosome abberation according to OECD 473: negative

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
10-May-2012 to 31-May 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Vehicle / solvent:
- Vehicle used: DMSO
- Justification for choice of vehicle:
A homogeneous suspension could be obtained in DMSO. Test compound was stable in DMSO and DMSO has been accepted and approved by authorities and international guidelines.

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9
(650 µg/plate in DMSO for TA100)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
without S
(10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537)
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 S9
(10 µg/plate in DMSO for WP2uvrA)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
without S9
(5 µg/plate in saline for TA1535)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene in DMSO for all tester strains
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.
Evaluation criteria:
A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
valid
Positive controls validity:
not examined
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: Precipitation was only observed at the dose level of 5000 µg/plate in tester strain TA1535.

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity was observed up to and including the top dose of 5000 µg/plate
Conclusions:
Based on the results of this study it is concluded that C.I. Leuco Sulphur Brown 37 at concentrations up to 5000 µg/plate is mutagenic in the tester strains TA1537, TA98 and TA100 of the Salmonella typhimurium reverse mutation assay. C.I. Leuco Sulphur Brown 37 is not mutagenic in the other Salmonella typhimurium tester strain (TA1535) or Escherichia coli strain reverse mutation assay using strain WP2uvrA. The mutagenicity was confined only to incubations with metabolic activation.
Executive summary:

 


In the presence of S9-mix, C.I. Leuco Sulphur Brown 37 induced dose related increases in three out of the five tester strains. The increases observed in the tester strains TA1537, TA98 and TA100 were above our laboratory historical control data range and 4.8 to 27-fold the concurrent control.


 


The tester strains TA1535 and WP2uvrA in the absence and presence of S9-mix and the tester strains TA1537, TA98 and TA100 in the absence of S9-mix showed negative responses over the entire dose range, i.e. no dose-related, increase in the number of revertants.


 


Since 4.8 to 27-fold, dose related increases were observed in three tester strains in the presence of S9-mix, these increases are considered biologically relevant, C.I. Leuco Sulphur Brown 37 is mutagenic in the presence of S9-mix.


 


In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.


 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11-Jun-2012 to 12-Feb-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Hypoxanthine-Guanine Phosphoribosyl Transferase (HPRT) in V79 cells
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Minimum Eagles Medium with Earle’s salts (MEME, Invitrogen Corporation), supplemented with 5 ml of MEM non-essential amino acids solution (Invitrogen Corporation), 0.11 g of sodium pyruvate (Invitrogen Corporation), 0.292 g of L-glutamine (Invitrogen Corporation) and 2.2 g of sodium bicarbonate (Merck) per litre. Final concentrations of streptomycin and penicillin G (Invitrogen Corporation) were 50 µg/mL and 50 U/ml, respectively.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3 hours treatment: 1.5, 5, 15, 51, 154 and 512 µg/mL
Without S9-mix, 24 hours treatment: 1.5, 5, 15, 51, 154 and 512 µg/ml
Experiment 1:
Without and with S9-mix, 3 hours treatment: 0.05, 0.15, 0.5, 1.5, 5, 15, 51 and 154 µg/mL

Experiment 2
Without S9-mix, 24 hours treatment: 0.05, 0.15, 0.5, 1.5, 5, 15, 51 and 154 µg/mL

Vehicle / solvent:
- Vehicle/solvent used: In the dose range finding study, dimethyl sulfoxide was used as solvent and in the main study the solvent was dimethylformamide (DMF).
- Justification for choice of solvent/vehicle:
A homogeneous suspension could be in obtained in both DMSO as DMF and these solvents are accepted and approved by authorities and international guidelines
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
3-methylcholanthrene
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time: 6 days
- Selection time: 7 days

SELECTION AGENT: 5 µg/mL thioguanine (6-TG)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 1.5 x 10E6 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)
Evaluation criteria:
ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute colony forming efficiency of the solvent controls should be between 60 and 120%
b) At least seven doses of the test substance should have an acceptable number of surviving cells (106) which could be analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the solvent-treated control will be < 6 per 10E5 clonable cells.
d) The positive control substances induced significant (at least three-fold) increases in the mutant frequency.
e) The selected dose range has to include a clearly toxic concentration (10 to 20%) of the average of solvent controls or should exhibit limited solubility or should extend to 5 mg/plate.


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces at least a three-fold increase in the mutation frequency compared to the solvent control in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations show a mutation frequency of at least three-fold compared to the solvent control.
b) The results are confirmed in an independently repeated test.
Key result
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Remarks:
Toxicity was only observed after the prolonged treatment period
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at dose levels of 154 µg/mL and above. Except in the experiment with the polonged treatment period in which the test substance already precipitated at 51 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was only observed at the highest dose level after the prolonged treatment period.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Toxicity was only observed at the highest dose level after the prolonged treatment period.
Conclusions:
It was concluded that C.I. Leuco Sulphur Brown 37 is not mutagenic in the gene mutation test with V79 Chinese hamster cells.
Executive summary:

 


The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.


 


Mutation frequencies in cultures treated with positive control chemicals were increased 22- and 28-fold for Ethylmethanesulphonate (EMS) in the absence of S9-mix, and by 9.1-fold for 3-Methylcholanthrene(3-MCA) in the presence of S9-mix, in the first and second experiment, respectively. It was therefore concluded that the test conditions, both in the absence and presence of S9-mix, were appropriate and that the metabolic activation system (S9-mix) functioned properly.


 


In the absence of S9-mix, C.I. Leuco Sulphur Brown 37 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent repeat experiment with modifications in the duration of treatment time.


 


In the presence of S9-mix, C.I. Leuco Sulphur Brown 37 did not induce a significant increase in the mutation frequency.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30-May-2012 to 04-Oct-2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: Cultured peripheral human lymphocytes
Details on mammalian cell type (if applicable):
- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.
Metabolic activation:
with and without
Metabolic activation system:
Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.
Test concentrations with justification for top dose:
Dose range finding test:
Without and with S9-mix, 3hr exposure; 24 hr fixation: 2, 5, 16, 53 and 160 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 2, 5, 16, 53, 160 and 533 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time:8, 27 and 80 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 8, 27 and 80 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 16, 48 and 320 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 16, 48 and 160 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 16, 48 and 160 µg/mL
Vehicle / solvent:
- Vehicle/solvent used:
Dose range finding test: DMSO
Main experiments: dimethylformamide (DMF)
- Justification for choice of solvent/vehicle:
A homogeneous suspension or solution could be obtained in DMSO or DMF. Both solvents are accepted and approved by authorities and international guidelines

Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
(in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
(in Hank's Balanced Salt Solution: 10 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR: colchicine
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
Evaluation criteria:
A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
Statistics:
The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.
Key result
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: tested up to cytotoxicity or up to a precipitating dose level
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: In the dose range finding, precipitation in the exposure medium was observed at dose levels of 160 µg/ml and above. Further investigation, in the main studies, showed that a concentration of 80 µg/ml already precipitated in the culture medium.


RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 16 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 533 and 53 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr, respectively and at dose levels of 53 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity or a dose level with precipitate was reached and selected for scoring.
Conclusions:
It is concluded that C.I. Leuco Sulphur Brown 37 C.I. Leuco Sulphur Brown 37 is not clastogenic in human lymphocytes.
Executive summary:

 


The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.


 


C.I. Leuco Sulphur Brown 37 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.


 


No effects of C.I. Leuco Sulphur Brown 37 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that C.I. Leuco Sulphur Brown 37 does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations under the experimental conditions described in this report.


 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Genetic toxicity in vitro


Two gene mutation assays in bacteria gave positive results in 3 of 5 strains (TA 1537, TA98 & TA 100) only in the presence of an external activation system. Two strains showed no mutagenicity (TA 1535 & E. coli WP 2uvrA) at all. However, the positive result could not be confirmed in a mammalian cell line (V79) in the HPRT assay. Additionally, a chromosomal aberration test in human lymphocytes also did not show any mutagenic potential of the test material. This is further supported by the supposed polymeric structure of the test material and its poor solubility in aqueous media.


Therefore, it is assumed that both conducted AMES assays lead to false positive results, supported by the available experimental data on test systems with higher human relevance (i.e. mammalian cells).


However, further investigations will be performed in vivo (refer to section 7.6.2). Based on the experimental outcome of the proposed test, a re-evaluation of the mutagenicity potential of the test material will be completed.




Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008


Available experimental data on test systems with higher human relevance (i.e. mammalian cells) did not show a mutagenic potential with regard to gene mutation as well as chromosomal damage.


However, the available experimental test data are considered not be sufficient for classification purposes under Regulation (EC) No 1272/2008. Therefore classification will be re-evaluated after finalization of the proposed study.