Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a mammalian cell gene mutation assay (V79/HPRT-test) conducted according the OECD 476 guideline, in compliance with GLP, HDI Trimer MEKO blocked was found to be non-mutagenic.

In an in vitro chromosome aberration assay performed according to OECD 473 guideline in compliance with GLP, HDI Trimer MEKO blocked was cytotoxic but not clastogenic for Chinese Hamster Ovary (CHO).

In a reverse gene mutation assay in bacteria performed according to OECD guideline 471 and in compliance with GLP, DESMODUR BL3175 was not mutagenic in bacteria in the absence and the presence of activation sytem.

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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well conducted guideline, GLP with some deviations
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Only 4 strains tested instead of 5, no data available on analytical investigation of the compound, the signature who approved the protocol was undated.
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
Not applicable
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
not applicable
Metabolic activation:
with and without
Metabolic activation system:
liver homogenate derived from Aroclor1254 induced rats (S9 mix)
Test concentrations with justification for top dose:
Doses per plate used for the first assay: 0, 8, 40, 200, 1000 and 5000 µg/plate.
Doses per plate used for the repeat assay: 0, 8, 40, 200, 1000 and 3000 µg/plate.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:
The solvent employed for the DESMODUR BL 3175 is acetone.The solvent used for the positive control is DMSO.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide, Nitrofurantoin, 4-nitro-1,2-phenylene diamine, 2-aminoanthracene,
Remarks:
No remarks
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: 48 hours
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 4 tubes were plated by strain for each concentration and control. 3 tests have been performed. In the first assay, tubes
were plated immediately after addition of the last component whereas in the 2 repeat tests, all tubes were preincubated at 37°C for 30 min before
plating. Because of cytotoxicity in the first repeat dose, this assay has not been interpretable.

DETERMINATION OF CYTOTOXICITY

The total bacteria counts consistently produced results in the range of the negative controls, or differed only insignificantly.
No growth inhibition was observed.
Higher doses revealed a weak, strain-specific bacteriotoxic effect. Nevertheless, doses up to 5000 µg/plate could still be used for assessment.
In the first preincubation test, bacteriotoxic effects most likely due to the solvent acetone were observed for all plates including the solvent control
plates. The result of this first preincubation test was not interpretable. Therefore a second preincubation test was performed in which only 0.06 ml of acetone was used as a solvent. No solvent-induced bacteriotoxic effects were observed in this repeat experiment.

The substance precipitated at a dose of 5000 µg/plate. As a result the corresponding plates could not be evaluated.
Evaluation criteria:
A test is defined as being positive if a reproductible and dose-related increase of mutany colony numbers becomes apparent for at least one strain.
For TA 1535, TA 100 and TA 98 mutant colony numbers should increase by a factor of two or more over the negative control numbers, while at least a three-fold increase should be apparent for TA 1537. Otherwise, the result is judged as negative.
However, these guidelines may be overruled bu good scientific judgement.
Statistics:
No data
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:
no cytotoxicity, but tested up to precipitating concentrations
Vehicle controls validity:
not applicable
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
None of the four test strains showed a dose-related and biologically relevant increase of revertant colony numbers after exposure to the test
substance over negative control levels. This applied to the tests with and without S9 mix and was confirmed by the results of the repeat test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.6.1/1: Number of revertants per plate (mean of quadruplicates) in the absence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 100

TA 1537

TA 98

Mean

Mean

Mean

Mean

0

13

75

8

22

8

12

79

7

28

40

17

86

8

23

200

13

87

8

22

1000

14

72

7

21

5000

10

72

9

17

Na-azide

636

NF

271

4-NPDA

42

92

Table 7.6.1/2: Number of revertants per plate (mean of triplicates) in the absence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 100

TA 1537

TA 98

Mean

Mean

Mean

Mean

0

16

100

11

26

8

14

96

11

29

40

17

97

13

30

200

16

104

10

27

1000

15

105

8

26

5000

14

110

8

19

Na-azide

522

NF

 

499

 

 

4-NPDA

 

 

51

85

 

Table 7.6.1/3 Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (First test)

Test substance concentration

(µg/plate)

TA 1535

TA 100

TA 1537

TA 98

Mean

Mean

Mean

Mean

0

16

119

10

45

8

13

108

8

43

40

13

108

8

40

200

13

108

11

26

1000

13

114

5

31

5000

15

105

6

22

2-AA

152

917

205

693

 

Table 7.6.1/4 Number of revertants per plate (mean of triplicates) in the presence of metabolic activation (Second test)

Test substance concentration

(µg/plate)

TA 1535

TA 100

TA 1537

TA 98

Mean

Mean

Mean

Mean

0

18

147

11

32

8

15

138

14

42

40

18

152

14

45

200

18

160

16

40

1000

14

154

12

41

5000

15

125

10

35

2-AA

180

1317

302

1113

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the test conditions, DESMODUR BL 3175 was not mutagenic in bacteria.
Executive summary:

In a reverse gene mutation assay in bacteria performed according to OECD guideline 471 and in compliance with GLP, DESMODUR BL 3175 was tested in S. typhimurium TA 1535, TA 1537, TA 100, TA 98.

Two independent mutation tests were performed in the presence and the absence metabolic activation. The first test was a standard plate incorporation assay conducted at 0, 8, 40, 200, 1000 and 5000 µg/plate for DESMODUR BL 3175. The second assay involved a 30-minute pre-incubation at 37°C using 0, 8, 40, 200, 1000 and 3000 µg/plate for DESMODUR BL3175.

The substance precipitated at a dose of 5000 µg/plate.

The positive controls induced the appropriate responses in the corresponding strains.

Increase in the revertants was not observed in any tested strains following exposure to DESMODUR BL3175 at any concentrations in the absence and the presence of S9 -mix activation system.

Under the test conditions, DESMODUR BL3175 is not mutagenic in bacteria in the absence and the presence of activation sytem.

This study is considered as acceptable and satisfies the requirement for the bacterial reverse gene mutation endpoint.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
from 06 feb 2007 to 11 jul 2007
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted study, according to guideline, GLP, no deviation.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
The V79 cell line was originally derived from the lung of a male Chinese hamster (Chu and Mailing, 1968). The V79 cells used in this study (designa-
ted V79 in this study) were a kind gift from Prof. G. Speit, University of Ulm, Germany. These cells have since been recloned to maintain karyotypic
stability. They have a modal chromosome number of 22 and a rapid population doubling time (10 to 14 hours).
V79 cell stocks are stored in liquid nitrogen. Laboratory cultures were maintained in plastic tissue culture vessels at 37°C in a humidified atmosphere containing approximately 5% CO2. Exponential growth of cell cultures was maintained by subculturing at least twice a week. For cell detachment in
order to subculture, an adequate dilution (ranging between 1:2.5 and 1:5) in phosphate buffered saline (PBS) of a commercially available PBS solution consisting of 0.5% trypsin and 0.2% EDTA (ethylenediamine-N,N,N',N'-tetraacetic acid) has been employed.
The V79 cells were checked on January 31, 2007 for karyotype stability utilising a modified protocol of the method of Moorhead et al. (1960) and the karyotype (modal chromosomes number: 22) was confirmed. A routine check for mycoplasma was performed on August 10, 2006 (when the used
frozen permanent was prepared) using a DNA-Staining Kit (Biochrom) according to the method provided by the supplier. There was no evidence of
mycoplasma contamination.

To keep the number of spontaneous 6-TG resistant mutants at a low level, cell cultures were subcloned by plating about 1,000 cells per culture
vessel at least every two weeks. If necessary, the spontaneous frequency of HPRT-mutants was additionally reduced by supplementing the culture
medium with thymidine (9 pg/ml), hypoxanthine (10 pg/ml), glycine (22.5 pg/ml) and methotrexate (0.3 pg/ml). A 6-TG sensitive subclone was then used for the HPRT-test.
In all parts of this study incubation was performed at 37°C in a humidified atmosphere with about 5% CO2
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
from the livers of Aroclor 1254 induced male Sprague Dawley rats
Test concentrations with justification for top dose:
0, 12.5, 25, 50, 100, 200 and 400 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for the choice of solvent: HDI Trimer MEKO blocked was soluble up to 500 mg/ml in this solvent.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO (as negative control)
True negative controls:
no
Positive controls:
yes
Remarks:
EMS, DMBA
Positive control substance:
other: see test conditions
Remarks:
Ethyl methanesulfonate (EMS) (Sigma-Aldrich, order number M0880 or M-0880, batch 085K0733), is a directly alkylating agent which has been shown to be clearly mutagenic in V79 cells (Bradley et al., 1981). Because EMS is a liquid, no solvent was needed.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

Positive controls:
Ethyl methanesulfonate (EMS) (Sigma-Aldrich, order number M0880 or M-0880, batch 085K0733), is a directly alkylating agent which has been
shown to be clearly mutagenic in V79 cells (Bradley et al., 1981). Because EMS is a liquid, no solvent was needed. EMS was used at a final concentra-
tion of 900 µg/ml in non-activation trials.

Dimethylbenzanthracene (DMBA) (Sigma-Aldrich, order number D3254, batch 055K1360), requires as promutagen metabolic activation to induce
mutations in V79 cells (Bradley et al., 1981; Krahn and Heidelberger, 1977). DMBA was dissolved in DMSO and used at a final concentration of 20 µg/ml for trials with S9 mix.

Determination of Cytotoxicity:
Exponentially growing V79 cells were plated in 20 ml culture medium in a 75 cm² flask with a total of volume 275 ml, (4x10*E6cells per flask). For
each concentration one culture was available. After attachment (16-24 hours later), cells were exposed without S9 mix to vehicle alone and to a
range of concentrations of the test substance for 5 hours in 20 ml medium containing 2% FCS. In experiments with metabolic activation 1 ml of medium was replaced by 1 ml S9 mix. Thereafter, cell monolayers were washed with PBS, trypsinized and replated in 5 ml culture medium at a density of
200 cells into each of 3 Petri dishes (diameter: 60 mm). These dishes were incubated for 6 to 8 days to allow colony development.

Thereafter, colonies were fixed with 95% methanol, stained with Giemsa (Merck; stock solution diluted 1:5 with deionized water) and counted. If not
interfered e.g. by precipitation on the plates or coloration of the plates, colonies were counted automatically using an Artek counter, model 982B.
Data were transferred to a PC and processed with the released and DOS 6.0 based software HPRT-Test (V1.08) of Bayer HealthCare AG, which
includes also counting of mutant colonies.

Cytotoxicity was expressed by comparison of colonies in treated cultures versus negative control cultures (relative cloning efficiency).
Evaluation criteria:
- Mutant frequencies will only be used for assessment, if at least 5 dishes per culture were available and relative survival to treatment, relative population growth and absolute cloning efficiency were 10% or greater.
- A trial will be considered positive if a concentration-related and n parallel cultures reproductible in mutant frequencies is observed. To be relevant,
the increase in mutant frequencies should be at least two to three times that of the highest negative or negative control value observed in the
respective trial. If the result can be reproducted in a second trial, the substance is considered to be mutagenic.
- Despite these criteria, a positive result will only be considered relevant, if no significant change in the osmolality compared to the negative
control can be observed. Otherwise, unphysiological culture conditions may be the reason for the positive result (Scott et al, 1991).
- A test substance will be judged as equivocal if there is no strictly concentration related increase in mutation frequencies but if one or more
concentrations induce a reproductive and biologically relevant increase in mutant frequencies in all trials.
- An assay will be considered negative if no reproductible and relevant increases of mutant frequencies were observed.

However, these criteria may be overruled by good scientific judgement.
Statistics:
All acceptable groups are included in the weighted analysis of variance followed by pairwise comparisons to the negative control on a nominal signi-
ficance level of a = 0.05 using the Dunnett test (Dunnett, 1955). The regression analysis part is performed on the basis of the actual concentrations
thereby omitting the positive, negative and negative controls. If there is a significant concentration related increase of the mutant frequency
(a = 0.05) in the main analysis the highest concentration will be dropped and the analysis will be repeated. This procedure will be repeated until
p > 0.05. In that way eliminated concentrations are flagged correspondingly.
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the absence and in the presence of S9 mix Chinese hamster V79 cells were exposed to HDI Trimer MEKO-blocked at concentrations of up to and
including 400 µg/ml. Without and with S9 mix substance precipitation occurred in the medium at the concentration 200 µg/ml and above. Therefore, the test without S9 mix was no longer interpretable at 400 µg/ml.

The means of the absolute cloning efficiency for the negative controls in the mutation experiments were 56.8% and 61.6% in the experiments without activation. In experiments with metabolic activation 69.2% and 69.3% were observed. These results demonstrate good cloning conditions for the
experiments.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Table 7.1.6/3 Preliminary cytotoxicity Screen

 

Relative Cell Survival (%)

Metabolic activation

Test article

µg/mL

-

+

DMSO

100.0

100.0

Test substance

3.13

75.2

103.0

Test substance

6.25

80.7

109.3

Test substance

12.5

80.8

102.5

Test substance

25

80.7

102.3

Test substance

50

89.1

121.1

Test substance

100

99.1

123.0

Test substance

200P

79.7

109.1

Test substance

400P

69.5

109.7

Table 7.6.1/4a HPRT-Test 1 without metabolic activation

Concentration

in µg/ml

Survival to treatment in % of NC

Total number of Mutant colonies

(8 dishes)

Cloning Efficiency

Mutant Frequency *10-6

Untreated control

78.1

6

56.0 +/- 4.9

4.5

108.4

2

62.5 +/- 5.3

1.3

Negative control

100.0

2

54.7 +/- 3.3

1.5

DMSO anhydrous

100.0

4

58.8 +/- 8.8

2.8

Test substance

 

 

 

 

12.5

99.8

0

53.8 +/- 1.2

0.0

69.6

1

48.3 +/- 1.6

0.9

25

93.8

1

52.2 +/- 1.8

0.8

109.1

2

57.8 +/- 3.2

1.4

50

130.8

4

66.8 +/- 7.4

2.5

90.7

1

59.2 +/- 4.5

0.7

100

98.3

3

60.7 +/- 4.6

2.1

103.3

1

56.5 +/- 5.6

0.7

200

117.7

1

53.8 +/- 4.6

0.9

102.3

1

68.2 +/- 6.6

0.7

400

-

-

-

-

-

-

-

-

Positive control

 

 

 

 

EMS 900

36.9

697

46.0 +/- 7.4

631.3

33.2

562

33.3 +/- 3.0

702.5

Table 7.6.1/4b HPRT-Test 2 without metabolic activation

Concentration

in µg/ml

Survival to treatment in % of NC

Total number of Mutant colonies

(8 dishes)

Cloning Efficiency

Mutant Frequency *10-6

Untreated control

117.5

2

61.0 +/- 0.9

1.4

83.3

3

67.7 +/- 4.5

1.8

Negative control

100.0

4

77.0 +/- 5.5

2.2

DMSO anhydrous

100.0

1

61.5 +/- 3.1

0.7

Test substance

 

 

 

 

12.5

114.7

6

72.2 +/- 4.0

4.6

71.3

1

67.2 +/- 5.0

0.6

25

91.4

1

79.3 +/- 1.9

0.5

93.9

1

66.7 +/- 5.5

0.6

50

101.9

1

63.0 +/- 3.3

0.9

75.9

2

65.0 +/- 2.6

1.7

100

99.2

1

68.8 +/- 3.2

0.7

86.3

3

56.0 +/- 4.4

2.2

200

91.2

4

74.0 +/- 2.6

2.3

59.1

2

55.5 +/- 3.8

1.5

400

42.8

1

57.0 +/- 3.6

0.8

33.9

1

65.8 +/- 3.0

0.7

Positive control

 

 

 

 

EMS 900

56.4

89

658 +/- 3.4

56.3

66.8

49

53.2 +/- 3.4

43.9

 

Table 7.6.1/5a HPRT-Test 1 with metabolic activation

Concentration

in µg/ml

Survival to treatment in % of NC

Total number of Mutant colonies

(8 dishes)

Cloning Efficiency

Mutant Frequency *10-6

Untreated control

105.6

7

64.3 +/- 2.3

4.5

104.4

7

71.3 +/- 5.8

4.1

Negative control

100.0

5

71.0 +/- 4.4

2.9

DMSO anhydrous

100.0

2

67.3 +/- 2.70.88

1.2

Test substance

 

 

 

 

12.5

94.7

6

71.3 +/- 2.5

3.5

84.4

5

70.8 +/- 1.5

2.9

25

110.4

19

74.2 +/- 2.6

10.7

101.0

0

75.0 +/- 4.0

0.0

50

117.3

14

67.8 +/- 5.3

8.6

98.8

2

72.0 +/- 6.5

1.2

100

105.6

3

64.7 +/- 2.5

1.9

106.6

2

62.0 +/- 6.1

1.3

200

106.9

4

67.3 +/- 1.8

2.5

108.5

0

67.0 +/- 4.3

0.0

400

106.9

3

63.3 +/- 2.3

2.0

99.3

0

60.0 +/- 4.8

0.0

Positive control

 

 

 

 

EMS 900

94.7

135

58.7 +/- 5.0

95.9

88.6

66

-

-

Table 7.6.1/5b HPRT-Test 2 with metabolic activation

Concentration

in µg/ml

Survival to treatment in % of NC

Total number of Mutant colonies

(8 dishes)

Cloning Efficiency

Mutant Frequency *10-6

Untreated control

117.5

2

61.0 +/- 0.9

1.4

83.3

3

67.7 +/- 4.5

1.8

Negative control

100.0

4

77.0 +/- 5.5

2.2

DMSO anhydrous

100.0

1

61.5 +/- 3.1

0.7

Test substance

 

 

 

 

12.5

114.7

6

72.2 +/- 4.0

4.6

71.3

1

67.2 +/- 5.0

0.6

25

91.4

1

79.3 +/- 1.9

0.5

93.9

1

66.7 +/- 5.5

0.6

50

101.9

1

63.0 +/- 3.3

0.9

75.9

2

65.0 +/- 2.6

1.7

100

99.2

1

68.8 +/- 3.2

0.7

86.3

3

56.0 +/- 4.4

2.2

200

91.2

4

74.0 +/- 2.6

2.3

59.1

2

55.5 +/- 3.8

1.5

400

42.8

1

57.0 +/- 3.6

0.8

33.9

1

65.8 +/- 3.0

0.7

Positive control

 

 

 

 

EMS 900

56.4

89

65.8 +/- 3.4

56.3

66.8

49

53.2 +/- 3.4

43.9

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the test conditions, HDI Trimer MEKO-blocked in solvent naphta 100 (68.5% and 31.5% respectively) is evaluated as non-mutagenic in the V79/
HPRT Forward Mutation Assay, both in presence or absence of metabolic activation.
Executive summary:

In a mammalian cell gene mutation assay (V79/HPRT-test, Bayer, 2007) conducted according the OECD 476 guideline, in compliance with GLP, Chinese hamster lung fibroblasts cells cultured in vitro were exposed to HDI Trimer MEKO blocked in solvent naphta 100 (68.5% and 31.5% respectively) at concentrations ranging from 12.5 µg/ml to 400 µg/ml in the presence and absence of mammalian metabolic activation.

Under both activation conditions, cytotoxic effects of 80% to 90% were induced at the respectable highest assessable concentration. In addition, HDI Trimer MEKO-blocked in solvent naphta 100 was tested up to and over its limits of solubility in the medium. Due to the precipitation 400 µg/ml could not be used for assessment without S9 mix.

HDI Trimer MEKO-blocked in solvent naphta 100 induced no biologically relevant increases in mutant frequencies.

The positive controls EMS and DMBA had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant frequencies as compared to the corresponding untreated controls and thus demonstrated the sensitivity of the test system and the activity of the used S9 mix.

Despite this sensitivity, neither without nor with S9 mix, indications of mutagenic effects of HDI Trimer MEKO-blocked in solvent naphta 100 and therefore HDI Trimer MEKO blocked could be found in the V79/HPRT forward mutation assay. It could be concluded that as the test has been performed up to precipitation concentration and as the solvent is non mutagenic, HDI Trimer MEKO blocked in solvent naphta 100 is non-mutagenic in the V79/HPRT Forward Mutation Assay.

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well-conducted GLP study, according to guideline
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
dates of the study (no influence in the outcome of the study) and lack of information in the tables of results not influencable for interpretaion of the results
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. QA statement)
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):
The CHO cells (CHO K-1 line) were obtained from Prof. Dr. A.T. Natarajan, University of Leiden, The Netherlands. This cell fine derives from the CHO
cells originally isolated from an explant of the ovary of the Chinese hamster (Cricetulus griseus, 2n = 22) by Kao and Puck (1968). The modal
chromosome number of these cells is 20-22 (stable aneuploid karyotype). The cell-cycle time is 12-14 h. The cells are stored as frozen stock
cultures in liquid nitrogen. Subcultures were prepared from these stocks (passage 16) for experimental use. Each passage CHO cens in the liquid
nitrogen is checked for mycoplasma contamination and karyotype stability, which were absent and stable, respectively.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
The S9-mix consisted of a liver homogenate fraction (S9) and cofactors
Test concentrations with justification for top dose:
The first chromosome aberration test: in both presence and absence of metabolic activation, the treatment / harvesting time was 4/18 hours and the
concentrations selected were 62.5, 125, 250, µg/mL.

The second chromosomal aberration test: in both presence of metabolic activation, the treatment harvesting time was 4/18 hours and the
concentrations selected were 50, 200 and 300 µg/mL; in the absence of metabolic activation, the treatment harvesting time was 18/18 hours and the
concentrations selected were 50, 200 and 300 µg/mL.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Remarks:
Mitomycin C and Cyclophosphamide
Positive control substance:
other: see above
Remarks:
no remarks
Details on test system and experimental conditions:
Cell fixation, preparation of slides and scoring:
Two hours before the end of the total incubation period, the selected cell cultures for cell sampling, were arrested in the metaphase stage of mitosis by the addition of colcemid (final concentration: 0.1 µg/ml). At the end of the total incubation period the cells were harvested by trypsinization,
treated for 15 min at ca. 37°C with a hypotonic solution (1% sodium citrate), fixed with a 3:1 mixture of methanol: glacial acetic acid (two refresh-
ments of the fixative), and transferred to clean microscope slides. Two slides were prepared from each culture. The slides were stained in a 2%
solution of Giemsa, rinsed in water, air-dried and embedded. The slides were coded by a qualified person not involved in scoring the slides, to enable "blind" scoring. At least 1000 nuclei in each culture were examined (500 on each slide) to determine the mitotic index (percentage of cells in
mitosis). After the results of the mitotic index scoring, and the observations with respect to the quality of the metaphases, a selection of the
concentrations, to be analysed for chromosomal aberrations, was carried out.
In all treatment groups, in both the first and second (independent) chromosomal aberration test, three concentrations of the test substance together with the negative and positive controls were selected for the analysis of chromosomal aberrations. If possible, the highest concentration should
reduce the mitotic index at least 50% (but not more titan 70%), when compared to the negative control value or exhibit some other clear indication of
cytotoxicity. Subsequently, the cultures of the selected concentrations of the test substance, together with the negative and positive control cultures, were analysed for the induction of structural chromosomal aberration. For each treatment group, 200 well-spread metaphases per concentration
(100 metaphases per culture), each containing 20-22 centromeres, were analysed by microscopic examination for chromatid-type aberrations (gaps, breaks, fragments, interchanges) and chromosome-type aberrations (gaps, breaks, minutes, rings, dicentrics), according to the criteria
recommended by Savage (1975). If heavily damaged cells were observed, these cells were recorded but the cells were not counted and included in the 200 analysed cells. The Vernier readings of all aberrant metaphases were recorded.
Evaluation criteria:
The study is considered valid if the positive controls give statistically significant increase in the number of aberrant cells and if the negative controls are within the historical range.
A response is considered to be positive if a concentration-related increase or a reproducible increase in the number of cell with structural chromo-
somal aberrations is observed.
A test substance is considered to be clastogenic if a concentration-related increase in the percentage of cells with structural chromosomal aberra-
tions over the concurrent control frequencies is observed, or if a single positive test point is observed in both tests at approximately the same dose
level.
A response is considered to be negative in the chromosomal aberration test if it produces neither a dose-related increase in the number of structural chromosomal aberrations nor a reproductible positive response at any of the test points.
Statistics:
Data were analysed statistically by Fisher's exact probability test (two-sided) to determine 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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
In the first chromosomal aberration test:
with metabolic activation (S9-mix),
the mitotic index of the lowest concentration analysed (62.5 µg/ml) was reduced to 85%,
the mitotic index of the mid- concentration analysed (125 µg/ml) was reduced to 65% and
the mitotic index of the highest concentration analysed (250 µg/ml) was reduced to 41%, when compared to the mitotic index of the concurrent
(DMSO) control.
Without metabolic activation (S9-mix):
the mitotic index of the lowest concentration analysed (62.5 µg/ml) was reduced to 87%,
the mitotic index of the mid- concentration analysed (125 µg/ml) was reduced to 95% and
the mitotic index of the highest concentration analysed (250 µg/ml) was reduced to 65%, when compared to the mitotic index of the concurrent
(DMSO) control.
At higher concentrations (500 and 1000 µg/ml) for both test with and without metabolic activation (S9-mix), the test substance became too toxic to
the cells and were for this reason not selected for cell sampling.

In the second chromosomal aberration test:
With metabolic activation (S9-mix):
the mitotic index of the lowest concentration analysed (50 µg/ml) was reduced to 96%,
the mitotic index of the mid- concentration analysed (200 µg/ml) was reduced to 57% and
the mitotic index of the highest concentration analysed (300 µg/ml) was reduced to 31%, when compared to the mitotic index of the concurrent
(DMSO) control.
Without metabolic activation (S9-mix), in the continuous treatment group of 18 hours:
the mitotic index of the lowest concentration analysed (50 µg/ml) was reduced to 83%,
the mitotic index of the mid- concentration analysed (200 µg/ml) was reduced to 54% and
the mitotic index of the highest concentration analysed (300 µg/ml) was reduced to 33%, when compared to the mitotic index of the concurrent
(DMSO) control.
At the next higher concentration (400 µg/ml) for both test with and without metabolic activation (S9-mix) , the test substance became too toxic to the cells ans was for this reason not selected for chromosomal aberration analysis.

The test substance did not induce a statistically significant increase in the number of aberrant cells, at any of the dose level analysed, when
compared to the number of aberrant cells found in the concurrent (DMSO) control cultures.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Table 7.6.1/1a Chromosomal aberration analysis of Test 1 with S9

Treatment/ harvest time (h)

Dose level of test substance (µg/ml)

Number of cells showing structural chromosome aberrations

Number of cells with only gaps

Relative mitotic index (%)

Cells observed

Number of cells showing aberrations (%)

4/18

 

Neg. control (DMSO)

100

1

2

100

100

0

1

200

1(0.5)

3

62.5

100

0

3

85

100

0

2

200

0(0.0)

5

125

100

0

2

65

100

0

3

200

0(0.0)

5

250

100

1

1

41

100

0

1

200

1(0.5)

2

Pos. control: cyclophosphamide (5.0)

100

39

0

39

100

31

0

200

70(35)

0

 

Table 7.6.1/1b Chromosomal aberration analysis of Test 1 without S9

Treatment/ harvest time (h)

Dose level of test substance (µg/ml)

Number of cells showing structural chromosome aberrations

Number of cells with only gaps

Relative mitotic index (%)

Cells observed

Number of cells showing aberrations (%)

4/18

 

Neg. control (DMSO)

100

1

1

100

100

0

3

200

1(0.5)

4

62.5

100

0

2

87

100

0

2

200

0(0.0)

4

125

100

0

0

95

100

1

1

200

1(0.5)

1

250

100

0

3

65

100

0

1

200

0(0.0)

4

Pos. control: mitomycin C (0.1)

100

22

0

73

100

25

0

200

47(23.5)

0

 

Table 7.6.1/2a Chromosomal aberration analysis of Test 2 with S9

Treatment/ harvest time (h)

Dose level of test substance (µg/ml)

Number of cells showing structural chromosome aberrations

Number of cells with only gaps

Relative mitotic index (%)

Cells observed

Number of cells showing aberrations (%)

4/18

 

Neg. control (DMSO)

100

1

1

100

100

0

2

200

1(0.5)

3

62.5

100

0

2

96

100

0

2

200

0(0.0)

4

125

100

1

2

57

100

1

1

200

2(1.0)

3

250

100

0

1

31

100

1

0

200

1(0.5)

1

Pos. control: cyclophosphamide (5.0)

100

20

0

42

100

25

0

200

45(22.5)

0

 

Table 7.6.1/2b Chromosomal aberration analysis of Test 2 without S9

Treatment/ harvest time (h)

Dose level of test substance (µg/ml)

Number of cells showing structural chromosome aberrations

Number of cells with only gaps

Relative mitotic index (%)

Cells observed

Number of cells showing aberrations (%)

18/18

 

Neg. control (DMSO)

100

0

1

100

100

0

0

200

0(0.0)

1

62.5

100

1

2

83

100

0

2

200

1(0.5)

4

125

100

0

1

54

100

0

2

200

0(0.0)

3

250

100

1

0

33

100

0

3

200

1(0.5)

3

Pos. control: mitomycin C (0.05)

100

27

0

70

100

24

0

200

51(25.5)

0

 

Conclusions:
Interpretation of results (migrated information):
negative with and without metabolic activation

Under the conditions used in the study, HDI Trimer MEKO-blocked in Solventnaphta 100 (75/25% respectively) was cytotoxic but not clastogenic for
CHO cells.
Executive summary:

In an in vitro chromosome aberration assay performed according to OECD 473 guideline in compliance with GLP, Chineses Hamster Ovary (CHO) cells were exposed to HDI Trimer MEKO-blocked in Solventnaphta 100 (75/25%): in the first chromosome aberration test, in both presence and absence of metabolic activation (treatment harvesting time of 4/18 hours) concentrations selected were 62.5, 125, 250, µg/mL and in the second test: in the presence of metabolic activation (treatment harvesting time of 4/18 hours) concentrations selected were 50, 200 and 300 µg/mL and in the absence of metabolic activation (treatment harvesting time of 18/18 hours) at concentrations of 50, 200 and 300 µg/mL.

Dimethylsulfoxide (DMSO) was used as vehicle for the test substance. Two separate chromosomal aberration tests were conducted. In all instances, duplicate cultures were used. In the first chromosome aberration test, in both presence and absence of metabolic activation, the treatment harvesting time was 4/18 hours and the

concentrations selected were 62.5, 125, 250, µg/mL.

In both the first and second chromosomal aberration test, HDI Trimer MEKO  blocked in Solventnaphta 100 did not induce statistically significant increase in the number of aberrant cells, at any of the concentrations and treatment period analysed, when compared to the number of aberrant cells found in the vehicle (DMSO) control cultures.

In both chromosomal aberration tests, the numbers of aberrant cells, found in the vehicle (DMS0) control cultures, were within the historical range and the positive control substances Mitomycin C (without S9-mix) and cyclophosphamide (with S9-mix)induced the expected statistically

significant increases in the incidence of structural chromosomal aberrations. This demonstrates the validity of the assay.

Under the conditions, HDI Trimer MEKO blocked in Solventnaphta (75/25%) was cytotoxic but not clastogenic for Chinese Hamster Ovary (CHO).

The study is considered as acceptable and satisifies the requirement for the cytogenicity endpoint.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a mammalian cell gene assay performed according to the OECD guideline N° 476 in compliance with GLP (Herbold, 2007), Chinese Hamster lung fibroblasts were exposed to HDI Trimer MEKO blocked in Solventnaphta 100 at concentrations ranging from 12.5 to 400 µg/mL in the presence and absence of metabolic activation.

In an in vitro chromosome aberration test performed according to the OECD 473 in compliance with GLP (de Vogel, 2007), Chineses Hamster Ovary cells were exposed to HDI Trimer MEKO blocked in solventnaphta 100 within 2 tests. In the first chromosome aberration test, in both presence and absence of metabolic activation, the treatment harvesting time was 4/18 hours and the concentrations selected were 62.5, 125, 250, µg/mL and in the second chromosomal aberration test, in both presence of metabolic activation, the treatment harvesting time was 4/18 hours and the concentrations selected were 50, 200 and 300 µg/mL and in the absence of metabolic activation, the treatment harvesting time was 4/18 hours and the concentrations selected were 50, 200 and 300 µg/mL.

In a reverse gene mutation assay in bacteria performed according to the OECD 471 guideline, in compliance with GLP (Gahlmann, 1994),

S. Typhirium TA 1535, TA 1537, TA 98 and TA 100 were exposed to DESMODUR BL 3175 (HDI Trimer MEKO blocked in Solventnaphta 100) within 2 independant tests.The first test was conducted at 0, 8, 40, 200, 1000 and 5000 µg/plate for DESMODUR BL 3175. The second assay involved a 30-minute pre-incubation at 37°C using 0, 8, 40, 200, 1000 and 3000 µg/plate for DESMODUR BL3175.


Short description of key information:
In a mammalian cell gene assay performed according to the OECD guideline N° 476 in compliance with GLP, HDI Trimer MEKO blocked in
Solventnaphta 100 induced no biologically relevant increases in mutant frequencies at any concentrations in both presence and absence of metabolic activation.

In an in vitro chromosome aberration test performed according to the OECD 473 in compliance with GLP, HDI Trimer MEKO blocked in
solventnaphta 100 did not induce statistically significant increase in the number of aberrant cells at any concentrations in both absence and
presence of metabolic activation.

In a reverse gene mutation assay in bacteria performed according to the OECD 471 guideline, in compliance with GLP, DESMODUR BL 3175 (HDI
Trimer MEKO blocked in Solventnaphta 100) did not induce increase of revertants in any strains at any concentrations, in both absence and presence of metabolic activation.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In 3 in vitro genotoxicity studies, a mammalian cell gene assay in Chinese Hamster fibroblasts, an in vitro chromosome aberration test in Chinese Hamster Ovary cells and in a reverse gene mutation assay in bacteria, HDI Trimer MEKO blocked in Solvent naphta 100 showed neither significant nor relevant increase of mutant frequencies nor aberrant cells nor revertants.

Therefore, HDI Trimer MEKO blocked in Solventnaphta 100 is not classified for genetic toxicity according to the criteria of the Annex VI to the Directive 67/548/EC and CLP Regulation N°(1272/2008). As these tests have been performed either up to precipitation or cytoxicity with the preparation, HDI Trimer MEKO blocked is not considered to be mutagenic and claastogenic either.