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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All in vitro genotoxicity studies, the test item revealed clearly negative results. Therefore, it can be concluded that the test substance is not genotoxic in vitro.

Link to relevant study records

Referenceopen allclose all

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:
2006-09-09-2007-01-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study; GLP study without deviations
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Version / remarks:
(1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
other: Chinese hamster ovary (CHO) cells
Details on mammalian cell type (if applicable):
Species/cell type: CHO cells as originally isolated by Kao and Puck  (1968), obtained from Prof. Dr. A.T. Natarajan (University of Leiden, 
The  Netherlands)
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9-mix based on liver homogenate fraction  from male Wistar rats, induced with Aroclor 1254 (i.p.)
Test concentrations with justification for top dose:
up to 100 mg/l, see below
Vehicle / solvent:
Vehicle: dimethyl sulfoxide (DMSO)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
+S9-mix

Migrated to IUCLID6: 5 mg/l
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
vehicle (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
-S9-mix

Migrated to IUCLID6: Test 1: 0.1 mg/l; Test 2: 0.05 mg/l
Details on test system and experimental conditions:
- No. of metaphases analyzed: 100/culture = 200/concentration. At least  500 nuclei/slide examined for mitotic index

ADMINISTRATION: 
- Dosing:  
Stock solution (50 or 10 g/l) in vehicle dimethyl sulfoxide (DMSO).   
Solubility test: 1; 2; 3.9; 7.8; 15.6; 31.3; 62.5; 125; 250; 500 mg/l   
Test 1: 0.1; 0.2; 0.39; 0.78; 1.56; 3.13; 6.25; 12.5; 25; 50; 75; 100  mg/l   
Selected for sampling: 6.25; 12.5; 25; 50; 75; 100 mg/l   
Test 2: 30; 40; 50; 60; 80; 100 mg/l, all sampled. 2.5; 5; 10; 20 mg/l  only -S9, not sampled.   
Both for presence and absence of S9-mix, 3 relevant concentration levels per test were analyzed for chromosomal aberrations. The highest  level 
selected should have reduced the mitotic index by 50-70 %.
- Number of replicates: 2
- Application:    
Test 1: after 4 hours exposure visual inspection; replace medium with  test substance by medium without test substance;  
12 hours later (i.e. at 16 hours) visual inspection, addition of  colcemid (resulting concentration 0.1 mg/l);   
at 18 hours harvest   
Test 2: similar to Test 1, except continuos exposure for 18 hours (i.e.  no replacement of medium) of samples without S9-mix 
- Positive and negative control groups and treatment:    
negative: vehicle (DMSO)   
positive (+S9): cyclophosphamide (5 mg/l)   
positive (-S9): mitomycin C (Test 1: 0.1 mg/l, Test 2: 0.05 mg/l)
Evaluation criteria:
The study is considered valid if:
(1) the positive controls show a significant increase in the number of aberrant cells and   
(2) the negative controls are within historical ranges.

A response is considered to be positive if:
a statistically significant increase in the number of aberrant cells is observed which is   
(1) either concentration-related   
(2) or reproduced in the second experiment at a similar dose level.

A response is considered to be equivocal if:
0.05

Statistics:
Fisher's exact probability test (two-sided) for significant differences between treated and control cultures
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
approx. 100 mg/l (-S9); > 100 mg/l (+S9)
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first chromosomal aberration test, in both the absence and presence of a metabolic activation system, the test substance was weakly to
slightly toxic to the cells, at three concentrations analysed. In the second chromosomal aberration test, in the presence of S9-mix, the test
substance was not toxic to the cells at comparable dose levels. In the second chromosomal aberration test, in the absence of S9-mix, the test
substance was slightly toxic for the cells at two lowest concentrations analysed and clearly toxic at the highest concentration analysed.
Both chromosomal aberration tests did not induce a statistically significant increase in the number of aberrant cells, when compared to the
number of aberrant cells found in the negative (DMSO) control cultures.
Remarks on result:
other: other: Chinese hamster Ovary (CHO)
Remarks:
Migrated from field 'Test system'.

RESULT:


PRECIPITATION CONCENTRATION: 30 mg/l and higher
-------------------------------------------
MITOTIC INDEX AND CHROMOSOMAL ABERRATIONS (excl. gaps): 
-------------------------------------------
Concentration        % M.I.         % C.A.
-------------------------------------------
- Test 1, with S9
  neg. control        100            1.5
  100 mg/l             83            0.5
   75 mg/l             98            0.0
   50 mg/l             87            1.0
   25 mg/l            117       not inspected
  pos. control         38           29.5 ***
-------------------------------------------
- Test 1, without S9
  neg. control        100            0.0
  100 mg/l             91            0.0
   75 mg/l             97            0.0
   50 mg/l             78            0.0
   25 mg/l            111       not inspected
  pos. control        100           26.5 ***
-------------------------------------------
- Test 2, with S9
  neg. control        100            0.0
  100 mg/l            110            1.0
   80 mg/l            118            1.0
   60 mg/l            108       not inspected
   30 mg/l            127            1.0
  pos. control         51           39.5 ***
-------------------------------------------
- Test 2, without S9
  neg. control        100            0.0
  100 mg/l             53            0.5
   80 mg/l             73            0.0
   60 mg/l             92       not inspected
   30 mg/l             86            1.0
  pos. control         77           42.0 ***-
-------------------------------------------
*** p<=0.001 / ** p<=0.01 / * p<=0.05
-------------------------------------------
CYTOTOXIC CONCENTRATION: 
- With metabolic activation: > 100 mg/l
- Without metabolic activation: approx. 100 mg/l

Conclusions:
In both the first and second chromosomal aberration test, the test substance IPDI homopolymer did not induce a statistically significant increase in
the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in
the vehicle (DMSO) control cultures. Thus, under the conditions used in this study, IPDI homopolymer was not clastogenic for CHO cells.
Executive summary:

The test substance IPDI homopolymer was examined for its potential to induce structural chromosomal aberrations in Chinese Hamster Ovary (CHO) cells, in both the absence and presence of a metabolic activation system (S9 -mix). The study was carried out according to OECD method no. 473 in compliance with the current OECD Principles of Good Laboratory Practice. Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. Two separate chromosomal aberration tests were conducted. In all instances, duplicate cultures were used. The highest concentration tested was based on solubility of the test substance.

In both the first and second chromosomal aberration test, the test substance IPDI homopolymer did not induce a statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment periods analysed, when compared to the number of aberrant cells found in the vehicle (DMSO) control cultures.

The data obtained support the conclusion that, under the conditions used in this study, the test substance IPDI homopolymer was not clastogenic for Chinese Hamster Ovary (CHO) cells.

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:
2009-02-05 - 2009-05-13
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
1997
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
mutated gene loci resposible for histidine auxotropy
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
other: Tryptophan-independent
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine auxotroph
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/ß-Naphthoflavone induced rat liver S9; male Wistar rats
Test concentrations with justification for top dose:
Pre-Experiment/Experiment I and II:
3; 10; 33; 100; 333; 1000; 2500; and 5000 µg/plate
Vehicle / solvent:
DMSO (Dimethyl sulfoxid, CAS No. 67-68-5; purity > 99 %)
Untreated negative controls:
yes
Remarks:
untreated
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
for details see below
Positive control substance:
other: sodium azide; 4-nitro-o-phenylene-diamine; methyl methane sulfonate; 2-aminoanthracene
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Pre-Experiment / Experiment I: plate incorporation test (+/- metabolic activation)
Experiment II: pre-incubation assay (+/- metabolic activation)
- Metabolic activation assay: Phenobarbital/ß-Naphthoflavone induced Wistar rat liver S9 (protein concentration in S9: 30.7 mg/ml)
ADMINISTRATION
- Dosing: Pre-Experiment and Experiment I/II : 3 - 5000 µg/plate
- Number of replicates: 3
- Positive and negative control groups and treatment:
- without metabolic activation:
sodium azide for TA 1535 and TA 100
4-nitro-o-phenylene-diamine for TA 1537 and TA 98
methyl methane sulfonate for WP2 uvrA
- with metabolic activation:
2-aminoanthracene for all strains
- negative control: untreated; solvent control: DMSO
- Pre-incubation time: 60 min at 37 °C; incubation time: 48 h at 37 °C in the dark

Evaluation criteria:
CRITERIA FOR EVALUATING RESULTS:
mutagenic effects (i.e ratio of revertant rates treated/control >=2) at <= 5000µg/plate with generally positive dose-response relationship in any
strain
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory
Species / strain:
other: S. typhimurium TA 98; TA 100; TA1535 and TA 1537; E. coli WP2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000, 2500 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
GENTOXIC EFFECTS:
- With metabolic activation: None (even at cyctotoxic concentration)
- Without metabolic activation: None (even at cytotoxic concentration)
PRECIPITATION CONCENTRATION: > 1000 µg/plate
CYTOTOXIC CONCENTRATION (including effects on background lawn): see remarks on results

Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Cytotoxic effects occured in the test groups at the following concentrations (µg/plate)

 Strain

Experiment I   

Experiment II     


  without S9 mix  with S9 mix   without S9 mix  with S9 mix   
 TA 1535  5000  1000, 5000  / 5000   

 TA 1537

 2500 - 5000 2500 - 5000  2500 - 5000  5000    

 TA 98

 2500 - 5000 2500 - 5000   2500 - 5000   

 TA 100

 /  /  /   /  

 WP2 uvrA

 /

 /

 /

    /

/ = no toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5)

Conclusions:
Under the present conditions of this bacterial reverse mutation assay, IPDI homopolymer is considered to be non-mutagenic, both in presence and inabsence of metabolic activation (S9) at any dose level and for all test strains used in this study.
Executive summary:

The test substance IPDI homopolymer was examined for its potential to induce gene mutations in the plate incorporation test and the pre-incubation test using the Salmonella typhimurium strains TA 1535, TA1537, TA 98, and TA 100, and the Escherichia coli strain WP2 uvrA. The study was carried out according to OECD method 471 in compliance with the current OECD Principles of Good Laboratory Practice. Dose levels covering a total range of 3 to 5000 µg/plate, in triplicate both with and without the addition of a metabolising system (Phenobarbital/ß-Naphthoflavone induced rat liver S9 mix) were employed.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in strains TA 1535, TA 1537, and TA 98.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with IPDI homopolymer 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, IPDI homopolymer is considered to be non-mutagenic in this Salmonella typhimurium and E. coli reverse mutation assay.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2002-02-11 - 2002-07-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: only Ames-test screening
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
mutated gene loci responsible for histidine auxotropy
Species / strain / cell type:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and TA 102
Details on mammalian cell type (if applicable):
The original strains were obtained from Prof. Bruce Ames and arrived at Toxicology, Bayer AG, on August 15, 1997.
Additional strain / cell type characteristics:
other: histidine-auxotroph
Metabolic activation:
with and without
Metabolic activation system:
S9-mix based on liver homogenate fraction  from male Wistar rats, induced with Aroclor 1254 (i.p.)
Test concentrations with justification for top dose:
16, 50, 160, 500, 1600 and 5000 µg/plate
Vehicle / solvent:
DMSO (dried with a molecular sieve, 0.3 nm)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: sodium azide (TA 1535); Nitrofurantoin (TA 100); 4-Nitro-1,2-phenylene diamine (TA 1537 and TA 98); Mitomycin C (TA 102); Cumene hydroperoxide (TA 102); 2-Aminoanthracene (all strains);
Details on test system and experimental conditions:
Bacterial Reverse Mutation Test
SYSTEM OF TESTING
- Experiment I: plate incorporation test (+/- metabolic activation), incubation time: 48 h at 37 °C in the dark
Experiment II: independent repeat as pre-incubation assay (+/- metabolic activation); in water bath at 37°C for 20 minutes
- Metabolic activation assay: induced S9-mix based on liver homogenate fraction  from male Wistar rats, induced with Aroclor 1254 (i.p.)
ADMINISTRATION
- Dosing: Experiment I: 16 - 5000 µg/plate; Experiment II: the doses were determined on the basis of the results of the plate incorporation assay
(in µg/tube)
- Number of replicates: 3
- Positive and negative control groups and treatment:
- without metabolic activation:
sodium azide for TA 1535; µg/plate: 10
Nitrofurantoin for TA 100; µg/plate: 0.2
4-Nitro-1,2-phenylene-diamine for TA 1537 and TA 98; µg/plate: 10 and 0.2
Mitomycin C for TA 102 (in plate incorporation trials); µg/plate: 0.2
Cumene hydroperoxide for TA 102 (in preincubation trials); µg/plate: 50
- with metabolic activation:
2-aminoanthracene for all strains; µg/plate: 3
- negative control: untreated;
solvent control: none; sufficient evidence was available in the literature indicating that DMSO had no influence on the spontaneous mutant counts of the used strains

Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an
increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
Statistics:
According to the OECD Guideline 471, a statistical analysis of the data is not mandatory.
Species / strain:
other: S. typhimurium TA 1535, TA 100, TA 1537, TA 98 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
500, 1600 and 5000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
GENTOX EFFECTS:
- with metabolic activation: None (even at cytotoxic concentration)
- without metabolic activation: None (even at cyctotoxic concentration)
Precipitation Concentration: >= 1600 µg/plate
Cytotoxic Concentration: Doses up to and including 160 µg/plate did not cause any bacteriotox effects: Total bacteria counts remained unchanged
and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness
of this effect this range could nevertheless be used for assessment purpose.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

no remarks

Conclusions:
Under the present conditions of this bacterial reverse mutation assay, IPDI homopolymer (approx. 70% in solvent) is considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation screening at any dose level and for all test strains used in this study.
Executive summary:

IPDI homopolymer (approx. 70% in solvent) was initially screened with one plate per dose using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37 °C. Other conditions remained unchanged.

Doses up to and including 160 µg/plate did not cause any bacteriotox effects: Total bacteria counts remained unchanged and no inhibition of growth was observed. At higher doses, the substance had only a weak, strain-specific bacteriotoxic effect. Due to the weakness of this effect this range could nevertheless be used for assessment purpose. Substance precipitation occured at the dose

1600 µg per plate and above.

Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.

The positive controls sodium azide, nitrofurantoin, 4 -nitro-1,2 -phenylene diamine, mitomycin C, cumene hydroperoxide and

2 -aminoanthracene had a marked mutagenic effect, as seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.

Therefore, it can be concluded, that IPDI homopolymer (approx. 70% in solvent) was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation screening.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The test item did not induce gene mutations in bacteria (OECD TG 471; Harlan, Evonik Degussa GmbH, 2009) or in mammalian cells (OECD TG 476; Schulz and Hellwig, BASF, 2007) and demonstrate no potential to induce chromosome aberrations in Chinese Hamster Ovary cells in vitro (OECD TG 473; de Vogel, BASF, 2007) either with or without metabolic activation.

Results from genetic toxicity tests in vivo are not available.

Justification for classification or non-classification

Because all in vitro genotoxicity studies revealed clearly negative results, it can be concluded that the test item is not genotoxic in vitro and therefore must not be classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.