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Toxicological information

Genetic toxicity: in vitro

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Administrative data

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: Accetible standard publication

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1999

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
4-methyl-m-phenylene diisocyanate
EC Number:
209-544-5
EC Name:
4-methyl-m-phenylene diisocyanate
Cas Number:
584-84-9
Molecular formula:
C9H6N2O2
IUPAC Name:
2,4-diisocyanato-1-methylbenzene
Details on test material:
- Name of test material (as cited in study report): 2,4-diisocyanatotoluene (2,4-TDI); 2,6-diisocyanatotoluene (2,6-TDI); 80:20 mixture of 2,4- and 4,6-isomers of TDI (TDI 80)
This study was carried out using mixed isomers of TDI. As mixed isomers of TDI contains generally 655 or 80% of the 2,4-TDI isomer, (the remainder being 2,6-TDI), this study can also be considered to be a valid test of 2,4-TDI. No claim is made as to the contribution or otherwise of the 2,6-TDI isomer in this assay.

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Vehicle / solvent:
Ethyleneglycol dimethylether (EDGE), dimethylsulphoxide (DMSO)
Details on test system and experimental conditions:
Ames test

Results and discussion

Test results
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Additional information on results:
Positive (with S9 activation) - TA 100, TA 1537 and TA 98
Negative (with S9 activation) - TA 1535
Negative (without S9 activation) - all strains

2,4-TDI, 2,6-TDI, and TDI 80, all of which were dissolved in EGDE, showed a consistently negative response in the absence of S9 mix. Furthermore, no mutagenicity was observed in any of the TA 1535 strain experiments. Clearly positive results were obtained in the other strains after metabolic activation of 2,4-TDI,2,6-TDI, and TDI 80.

The HPLC analyses of the dissolved aromatic diisocyanates indicate that their degradation is considerably accelerated if DMSO is the solvent and may be completed before the Salmonella microsome test has even begun.

2,4-TDI, 2,6-TDI and TDI 80, all of which were dissolved in EGDE, showed a consistently negative response in the absence of S9 mix. Furthermore, no mutagenicity was observed in any of the TA 1535 strain experiments. With respect to these findings, the results of the TDIs were in complete agreement with those obtained for the MDIs. In contrast to the MDIs, however, clearly positive results were obtained in the other strains after metabolic activation of 2,4-TDI, 2,6-TDI and TDI 80. The results are summarised in Tables 1-3. Consistently positive results were obtained in strain TA 98 with all types of TDI tested. In TA 1537, clearly positive results were obtained for 2,4-TDI (Table 1) and weak effects were observed for TDI 80 whereas no effects were found for 2,6-TDI (Tables 2 and 3). Weak positive results were also obtained for TDI 80 with TA 100 (Table 3). S9 mixtures with varying amounts of the S9 fraction (10 and 30%) were used for 2,4- and 2,6-TDI. The results demonstrate (Table 1 and 2) that effects are slightly reduced in the presence of the S9 mix containing 30% of the S9 fraction. This reduction of effects may be examined with a selective or at least preferred reaction of the test samples with the proteins of the S9 fraction. [See remarks on results below for Tables 1-3)
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: Salmonella typhimurium strains TA1535, TA 100, TA 1537 and TA 98

Any other information on results incl. tables

See Additional information on results for Tables 1 -3.

Table 1. Results with 2,4-TDI (dissolved in EGDE) and metabolic activation

Strain S9,

ug/plate

TA 1537

TA 98

10% S9

30% S9

10% S9

30% S9

0

19

12

54

39

50

18

16

64

52

100

23

16

104*

76*

200

44*b

25

117*

76*

400

52*b

22b

153*b

87*b

600

51*b

45*b

172*b

93*b

800

49*bp

41*b

125*bp

128*bp

1000

41*bp

42*bp

117*bp

58*bp

AA3

233*

80*bp

1245*

509*

AA = 2-aminoanthracene * = mutagenic effect, b = background growth reduced, p = precipitation

Table 2. Results with 2,6-TDI (dissolved in EGDE) and metabolic activation

Strain S9,

ug/plate

TA 1537

TA 98

10% S9

30% S9

10% S9

30% S9

0

12

14

47

45

150

13

21

67

57

300

13

16

87*

61

600

15

15p

130*

73*

1200

7p

10p

166*p

97*p

2400

7p

9p

128*p

82*p

4800

p

p

p

p

AA 3

360*

66*

1538*

618*

AA = 2-aminoanthracene * = mutagenic effect, b = background growth reduced, p = precipitation

Table 3. Results with TDI 80 (dissolved in EGDE) and 10% S9 mix

Strain S9,

ug/plate

TA 100

TA 1537

TA 98

0

71

9

36

125

143

12

86*

250

188*

15

101*

500

211*p

21p

123*p

1000

57p

28*bp

88*p

2000

21bp

2bp

23bp

AA 3

869*

306*

888*

AA = 2-aminoanthracene * = mutagenic effect, b = background growth reduced, p = precipitation

Results

The stability of TDI solutions prior to salmonella /microsome tests was investigated (Table 4). The N=C=O content of 50-500 mg TDI, dissolved in 100 ml relatively 'dry' DMSO (0.02-0.03% water), dropped to 60% or less within the first 15 min of the test. Theoretically, the hydrolysis of 174 mg (1.0 mM) of TDI could consume 18 mg (1.0 mM) of water to form highly reactive intermediates, the aminoisocyanatotoluenes (TDAIs), and carbon dioxide. A homogeneous solution containing 0.02% (1.11 mM) water, as was the case for the 500 mg (2.86 mM) 2,4-TDI sample, would therefore convert about 40% of the TDI into reactive intermediates on a purely stoichiometric basis. These aminoisocyanates would then be available for further reactions with remaining 2,4-TDI or with themselves to produce a number of monomeric, oligomeric and polymeric ureas, which may be terminated by N=C=O and / or NH2 groups. At this point it is important to recall that a residual N=C=O content by no means indicates the presence of unmodified 2,4-TDI. The IR spectrum provides only insufficient information on the location of the N=C=O groups. The observed decline of isocyanate absorptions is, however, proof of the fact that chemical reactions have occurred.

In an additional experiment, 500 mg (2.86 mM) 2,4-TDI was dissolved in DMSO with an increased water content of 0.1% (5.56 mM), a level perfectly conceivable in practice. This amount of water led to an accelerated reduction of the N=C=O absorptions, so that after 15 min, only 43%, and after 4 h, 14% of the isocyanate groups could be detected.

The fate of aromatic diisocyanates in Salmonella/microsome tests were also investigated (Table 5). When a sample of 500 mg 2,6-TDI / 100 ml EGDE solution was mixed with the test ingredients or with water (0.1:2.6 ml), up to 6.6% of the TDI was converted into 2,6-TDA within 45s (Table 5). With respect to the amount of diamine produced, no difference was seen between the distilled water and the aqueous system of the Salmonella/microsome test. Concerning the decline of diisocyanate content, however, the transfer to a different environment became apparent. In water, the 2,6-TDI concentration dropped to around 60%, which is basically in agreement with the results of the 10-fold approaches shown in Table 4 for the different types of TDI. When the ingredients of the Salmonella/microsome test replaced water, more than 90% of the initial 2,6-TDI disappeared within 15-45s. In this case, not 500 ug, but less than 50 ug 2,6-TDI, enriched with around 25 ug 2,6-TDA and further unquantified products will be poured onto the plate.

Table 4a. Stability TDI (in EGDE) during the first minute of a simulated test(a); HPLC determination of residual TDI and its reaction products. [This table has been separated by TDI type : 2,4-TDI)

Diisocyanate

2,4-TDI

TDI in 100 ml EGDE

5000 mg

500 mg

Dose / plate

5000 ug

500 ug

Analysed products (b)

2,4-TDI [%]

2,4-TDA [%]

2,4-TDI [%]

2,4-TDA [%]

Start

100

nd

100

nd

After 15 s

95

0.35

57

3.9

After 30 s

83

0.65

48

5.7

After 45 s

93

0.60

41

6.6

After 60 s

93

0.65

35

7.4

nd: Not detectable, detection limit: 0.1%, e.g., 0.5 ug for the 500 mg/100 ml concentration.. na: Not available. (a) Simulating the mixing of dissolved TDI with the test ingredients (1 ml:26 ml mix with water). (b) Ureas and (insoluble) polyureas were not quantified.

<p>

Table 4b. Stability TDI (in EGDE) during the first minute of a simulated test(a); HPLC determination of residual TDI and its reaction products. [This table has been separated by TDI type : 2,6-TDI)

Diisocyanate

2,6-TDI

TDI in 100 ml EGDE

5000 mg

500 mg

Dose / plate

5000 ug

500 ug

Analysed products (b)

2,6-TDI [%]

2,6-TDA [%]

2,6-TDI [%]

2,6-TDA [%]

Start

100

nd

100

nd

After 15 s

91

0.27

67

3.5

After 30 s

92

0.56

50

6.6

After 45 s

92

0.81

37

9.5

After 60 s

90

0.98

40

10.7

nd: Not detectable, detection limit: 0.1%, e.g., 0.5 ug for the 500 mg/100 ml concentration.. na: Not available. (a) Simulating the mixing of dissolved TDI with the test ingredients (1 ml:26 ml mix with water). (b) Ureas and (insoluble) polyureas were not quantified.

<p>

Table 4c. Stability TDI (in EGDE) during the first minute of a simulated test(a); HPLC determination of residual TDI and its reaction products. [This table has been separated by TDI type : TDI 80)

Diisocyanate

TDI 80

TDI in 100 ml EGDE

5000 mg

500 mg

Dose / plate

5000 ug

500 ug

Analysed products (b)

2,4-TDI [%]

2,6-TDI [%]

2,4-TDA [%]

2,6-TDA [%]

Start

100

100

nd

nd

After 15 s

61

68

3.1

0.7

After 30 s

52

61

4.4

1.0

After 45 s

36

47

5.7

1.6

After 60 s

35

45

6.0

1.9

nd: Not detectable, detection limit: 0.1%, e.g., 0.5 ug for the 500 mg/100 ml concentration.. na: Not available. (a) Simulating the mixing of dissolved TDI with the test ingredients (1 ml:26 ml mix with water). (b) Ureas and (insoluble) polyureas were not quantified.

<p>

Table 5. Stability of 2,6-TDI during the first minute of the mutagenicity test(a): HPLC determination of residual 2,6-TDI and its reaction products

2,6-TDI in 100 ml solvent

500 mg EGDE

500 mg/EGDE

500 mg/DMSO

Reaction medium

Dist. water

Test ingredients(b)

Test ingredients(b)

2,6-TDI/plate

500 ug

500 ug

500 ug

Analysed products(c)

2,6-TDI [%]

2,6-TDA [%]

2,6-TDI [%]

2,6-TDA [%]

2,6-TDI [%]

2,6-TDA [%]

Start

100

nd

99.5

0.5

12.3

9.1

After 5 s

77.8

1.6

23.1

1.6

2.3

6.4

After 15 s

70.0

3.4

8.4

4.7

3.0

8.4

After 30 s

60.7

5.3

5.6

5.8

2.6

9.1

After 45 s

61.9

6.6

8.1

5.6

2.5

8.3

nd: Not detectable; detection limit: 0.1%, e.g. 0.5 ug. (a) Mixing 0.1 ml dissolved 2,6-TDI with 2.6ml water or 2.6ml test ingredients. (b) 2.0ml agar + 0.5 ml S9 mix + 0.1 ml nutrient broth. (c) Ureas and (insoluble) polyureas were not quantified.

Applicant's summary and conclusion

Conclusions:
The authors concluded that these results were in good agreement with their chemical stability and concluded that the mutagenicity was a consequence of breakdown of TDI and the formation of TDA in the aqueous environment of the test system.