Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report Date:
2009

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Test Article I.D.: Suprasec 2029 RZW 91
Chemical Name: Reaction mass of 1,2-Propanediol, polymer with 1,1'-methylenebis[isocyanatobenzene] and 1,3-Butanediol, polymer with 1-isocyanato-2- [(4-isocyanatophenyl) methyl] benzene and 1,1'-methylenebis[4-isocyanato benzene] and 4,4'-Methylenediphenyl diisocyanate, oligomeric reaction products with 2,4'- diisocyanatodiphenylmethane and 4,4'- Methylenediphenyldiisocyanate, oligomeric reaction products with 2,4'- diisocyanatodiphenylmethane and [(methylethylene)bis(oxy)]dipropanol and 4,4'- methylenediphenyl diisocyanate and o-(pisocyanatobenzyl) phenyl isocyanate
Test Article Batch No.: RZW 91
Test Article CAS No.: 4,4’-MDI(homopolymer)/2,4’-MDI/1,3-
BG/TPG/PG (CAS No. 500-312-1)
Test Article Description: clear colorless liquid
Storage Conditions: room temperature, stored under nitrogen in the
dark without desiccant

Method

Target gene:
Each S. typhimurium tester strain contains, in addition to a mutation in the histidine operon, additional mutations that enhance sensitivity to some mutagens. The rfa mutation results in a cell wall deficiency that increases the permeability of the cell to certain classes of chemicals such as those containing large ring systems that would otherwise be excluded. The deletion in the uvrB gene results in a deficient DNA excision repair system. Tester strains TA98 and TA100 also contain the pKM101 plasmid (carrying the R factor). It has been suggested that the plasmid increases sensitivity to mutagens by modifying an existing bacterial DNA repair polymerase complex involved with the mismatch repair process.

TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. TA100 is reverted by both frameshift and base substitution mutagens and TA1535 is reverted only by mutagens that cause base substitutions.
The E. coli tester strain has an AT base pair at the critical mutation site within the trpE gene (Wilcox et al., 1990). Tester strain WP2 uvrA has a deletion in the uvrA gene resulting in a deficient DNA excision repair system. Tryptophan revertants can arise due to a base change at the originally mutated site or by a base change elsewhere in the chromosome causing the original mutation to be suppressed. Thus, the specificity of the reversion mechanism is sensitive to base pair substitution mutations (Green and Muriel, 1976).
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.
Vehicle / solvent:
The vehicle used to deliver the test article to the test system was anhydrous ethylene glycol dimethylether (EGDE, CAS No. 110-71-4, Lot No. 79097MJ, Exp. Date: March 2012), obtained from Sigma-Aldrich. Test article dilutions were prepared immediately before use and delivered to the test system at room temperature under yellow light.

There is adequate evidence to demonstrate that use of EDGE as solvent is appropriate whereas DMSO can give rise to false positives due to solvent-catalysed conversion to MDA. The latter solvent should not be used for MDI in these assays. (Herbold, B. et al. 1998. Studies on the effect of the solvents dimethylsulfoxide and ethyleneglycoldimethylether on the mutagenicity of four types of diisocyanates in the Salmonella/microsome test. Mutation Research. 412:167-175.)
Details on test system and experimental conditions:
The tester strains used were the Salmonella typhimurium histidine auxotrophs TA98, TA100, TA1535 and TA1537 as described by Ames et al. (1975) and Escherichia coli WP2 uvrA as described by Green and Muriel (1976). Salmonella tester strains were received from Dr. Bruce Ames’ designated distributor, Discovery Partners International, San Diego, California. E. coli tester strains were received from the National Collection of Industrial and Marine Bacteria, Aberdeen, Scotland.

Tester strains TA98 and TA1537 are reverted from histidine dependence (auxotrophy) to histidine independence (prototrophy) by frameshift mutagens. Tester strain TA1535 is reverted by mutagens that cause basepair substitutions. Tester strain TA100 is reverted by mutagens that cause both frameshift and basepair substitution mutations. Specificity of the reversion mechanism in E. coli is sensitive to basepair substitution mutations, rather than frameshift mutations (Green and Muriel, 1976).

Overnight cultures were prepared by inoculating from the appropriate master plate or from the appropriate frozen permanent stock into a vessel containing ~50 mL of culture medium. To assure that cultures were harvested in late log phase, the length of incubation was controlled and monitored. Following inoculation, each flask was placed in a resting shaker/incubator at room temperature. The shaker/incubator was programmed to begin shaking at approximately 125 rpm at 37±2°C approximately 12 hours before the anticipated time of harvest. Each culture was monitored spectrophotometrically for turbidity and was harvested at a percent transmittance yielding a titer of greater than or equal to 0.3x109 cells per milliliter. The actual titers were determined by viable count assays on nutrient agar plates.

Metabolic Activation System
Aroclor 1254-induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from male Sprague-Dawley rats induced with a single intraperitoneal injection of Aroclor 1254, 500 mg/kg, five days prior to sacrifice. The lots of S9 were prepared by and purchased from MolTox (Boone, NC). Upon arrival at BioReliance, the S9 was stored at -60°C or colder until used. Each bulk preparation of S9 was assayed for its ability to metabolize at least two promutagens to forms mutagenic to Salmonella typhimurium TA100.

The S9 mix was prepared immediately before its use and contained 10% S9, 5 mM glucose-6-phosphate, 4 mM ß-nicotinamide-adenine dinucleotide phosphate, 8 mM MgCl2 and 33 mM KCl in a 100 mM phosphate buffer at pH 7.4. The Sham S9 mixture (Sham mix), containing 100 mM phosphate buffer at pH 7.4, was prepared immediately before its use. To confirm the sterility of the S9 and Sham mixes, a 0.5 mL aliquot of each was plated on selective agar.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test article to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test article.

Data sets for tester strains TA1535 and TA1537 were judged positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 3.0-times the mean vehicle control value. Data sets for tester strains TA98, TA100 and WP2 uvrA were judged
positive if the increase in mean revertants at the peak of the dose response was equal to or greater than 2.0-times the mean vehicle control value.
An equivocal response is a biologically relevant increase in a revertant count that partially meets the criteria for evaluation as positive. This could be a dose-responsive increase that does not achieve the respective threshold cited above or a non-dose responsive increase that is equal to or
greater than the respective threshold cited. A response will be evaluated as negative, if it is neither positive nor equivocal.
Statistics:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
The primary computer or electronic systems used for the collection of data or analysis included but were not limited to the following:
Minicount Colony Counter (Imaging Products International), LIMS Labware Version 5 (BioReliance), Excel 2003 (Microsoft Corporation) and Kaye Lab Watch Monitoring System (Kaye GE).

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
not determined
Positive controls validity:
valid
Additional information on results:
Anhydrous ethylene glycol dimethylether (EGDE) was selected as the solvent of choice based on the request of the Sponsor, solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in EGDE at 500 mg/mL, the maximum concentration tested.
No contaminant colonies were observed on the sterility plates for the vehicle control, the test article dilutions and the S9 and Sham mixes.

Tester Strain Titer Results
Experiment Tester Strain
TA98 TA100 TA1535 TA1537 WP2 uvrA
Titer Value (x 10e9 cells per mL)
B1 2.8 1.4 2.6 0.7 3.6
B2 2.0 1.2 1.2 1.2 4.2
B3 -- -- -- 1.1 --

Initial Toxicity-Mutation Assay
Experiment B1: In the initial toxicity mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. Precipitate was observed beginning at concentrations ranging from 50 to 1500 µg per plate. Toxicity was observed beginning at concentrations ranging from 50 to 5000 µg per plate with all Salmonella tester strains in the presence and absence of S9 activation. Based on the findings of the initial toxicity mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 1500 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation and 5000 µg per plate with the remaining test conditions.
In Experiment B1 (Initial Toxicity-Mutation Assay), no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. (Table 1)
Confirmatory Mutagenicity Assay
Experiments B2 and B3:
In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA100, TA1535 and WP2 uvrA in the absence of S9 activation.(Table 2) The dose levels tested were 5.0, 15, 50, 150, 500 and 1500 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation and 15, 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 500 or 1500 µg per plate. Toxicity was observed beginning at 500 or 1500 µg per plate. Due to excessive toxicity (beginning at 50 µg per plate), tester strain TA1537 in the absence of S9 activation was not evaluated but was retested in Experiment B3 with an adjustment in dose levels.
In Experiment B3 (Retest of the Confirmatory Mutagenicity Assay), no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation.(Table 2). The dose levels tested were 0.50, 1.5, 5.0, 15, 50, 150, 500 and 1500 µg per plate. Precipitate was observed beginning at 150 µg per plate. Toxicity was observed beginning at 500 µg per plate

Historical Negative and Positive Control Values
2005 – 2007

revertants per plate
Strain Control Activation
None Rat Liver
Mean SD Min Max Mean SD Min Max
TA98 Neg 17 6 5 56 25 8 6 60
Pos 189 112 30 1812 526 343 73 2669
TA100 Neg 133 30 52 250 143 32 67 263
Pos 548 148 112 4349 698 343 232 2652
TA1535 Neg 20 7 4 53 16 5 4 45
Pos 420 146 54 1019 104 59 18 985
TA1537 Neg 7 3 1 20 8 3 1 29
Pos 740 356 14 2514 88 87 13 1297
WP2 uvrA Neg 21 9 5 69 23 10 4 65
Pos 214 137 28 1086 268 174 29 1178
SD=standard deviation; Min=minimum value; Max=maximum value; Neg=negative control (including but not limited to deionized water, dimethyl sulfoxide, ethanol and acetone); Pos=positive control

Any other information on results incl. tables

Bacterial Mutation Assay

Summary of Results - Initial Toxicity-Mutation Assay

Table 1

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test Article Id

:

Study Number

:

AC26MP.503.BTL

Experiment No

:

B1

 Average Revertants Per Plate ± Standard Deviation

Activation Condition

:

None

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Vehicle

20

±

1

85

±

3

16

±

4

8

±

3

45

±

1

Untreated

15

±

1

82

±

7

16

±

2

8

±

1

35

±

4

1.5

14

±

3

73

±

7

13

±

0

11

±

2

45

±

8

5.0

15

±

5

91

±

6

21

±

10

4

±

1

40

±

5

15

15

±

4

89

±

13

13

±

7

12

±

2

33

±

4

50

9

±

3

97

±

9

15

±

1

6

±

1

34

±

8

150

14

±

4

78

±

4

12

±

1

4

±

1

38

±

4

500

14

±

1

98

±

25

10

±

1

2

±

1

36

±

5

1500

11

±

4

78

±

13

8

±

1

0

±

0

22

±

1

5000

2

±

2

68

±

19

7

±

1

2

±

2

29

±

6

Positive

158

±

86

545

±

9

112

±

3

1411

±

42

384

±

10

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Activation Condition

:

Rat Liver S9

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Vehicle

30

±

1

100

±

8

13

±

1

12

±

1

34

±

4

Untreated

22

±

1

83

±

3

16

±

3

7

±

1

50

±

5

1.5

23

±

1

126

±

4

16

±

5

10

±

3

43

±

13

5.0

25

±

3

97

±

7

18

±

5

8

±

3

45

±

10

15

20

±

1

95

±

8

18

±

2

7

±

5

48

±

5

50

20

±

4

116

±

16

15

±

4

10

±

2

43

±

3

150

25

±

6

89

±

13

16

±

0

12

±

4

38

±

9

500

22

±

3

100

±

1

13

±

1

7

±

3

34

±

3

1500

28

±

1

76

±

3

10

±

0

7

±

2

31

±

6

5000

10

±

1

60

±

13

10

±

0

4

±

0

29

±

3

Positive

437

±

6

792

±

263

87

±

8

52

±

16

175

±

8

Vehicle = Vehicle Control

Positive = Positive Control (50 μL plating aliquot)

Plating aliquot = 50 µL

Bacterial Mutation Assay

Summary of Results - Confirmatory Mutagenicity Assay

Table 2

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Test Article Id

:

Suprasec 2029 RZW 91

Study Number

:

AC26MP.503.BTL

Experiment Nos

:

B2 and B3

 

 

 

 

 

 

 

 

 Average Revertants Per Plate ± Standard Deviation

Activation Condition

:

None

Dose (µg per plate)

TA98

TA100

TA1535

TA1537a

WP2uvrA

Vehicle

22

±

2

157

±

14

21

±

3

11

±

2

51

±

4

Untreated

18

±

4

136

±

10

20

±

2

7

±

3

39

±

10

0.50

 

 

 

 

 

 

 

 

 

7

±

1

 

 

 

1.5

 

 

 

 

 

 

 

 

 

7

±

4

 

 

 

5.0

20

±

7

157

±

17

21

±

2

7

±

1

 

 

 

15

20

±

2

156

±

11

20

±

6

10

±

1

43

±

7

50

19

±

7

139

±

2

21

±

3

7

±

2

40

±

3

150

16

±

2

128

±

13

16

±

3

5

±

2

48

±

2

500

7

±

1

102

±

7

13

±

2

4

±

1

51

±

6

1500

6

±

1

115

±

19

7

±

3

5

±

1

26

±

3

5000

 

 

 

 

 

 

 

 

 

 

 

 

28

±

6

Positive

252

±

35

727

±

37

510

±

45

1024

±

228

574

±

54

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Activation Condition

:

Rat Liver S9

Dose (µg per plate)

TA98

TA100

TA1535

TA1537

WP2uvrA

Vehicle

24

±

6

156

±

17

14

±

1

5

±

2

50

±

1

Untreated

16

±

3

164

±

17

17

±

3

6

±

2

53

±

8

5.0

 

 

 

 

 

 

15

±

4

5

±

2

 

 

 

15

22

±

1

147

±

15

15

±

6

5

±

2

50

±

4

50

23

±

4

158

±

8

18

±

3

8

±

3

42

±

5

150

30

±

4

157

±

9

17

±

3

7

±

1

49

±

6

500

26

±

3

148

±

9

14

±

9

5

±

1

52

±

6

1500

24

±

3

138

±

21

10

±

2

5

±

2

48

±

4

5000

15

±

1

147

±

9

 

 

 

4

±

1

39

±

8

Positive

470

±

117

1050

±

71

205

±

20

60

±

7

235

±

20

Vehicle = Vehicle Control

Positive = Positive Control (50 μL plating aliquot)

Plating aliquot = 50 µL

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative Under the conditions of this study, test article did not cause a positive mutagenic response in either the presence or absence of Aroclor-induced rat liver S9.

All criteria for a valid study were met as described in the protocol. The results of the Bacterial Reverse Mutation Assay indicate that, under the conditions of this study, test article did not cause a positive mutagenic response in either the presence or absence of Aroclor induced rat liver S9.
Executive summary:

The test article was tested in the Bacterial Reverse Mutation Assay using Salmonella typhimurium tester strains TA98, TA100, TA1535 and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of Aroclor‑induced rat liver S9. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose‑range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test article.

Anhydrous ethylene glycol dimethylether (EGDE) was selected as the solvent of choice based on the request of the Sponsor, solubility of the test article and compatibility with the target cells. The test article formed a soluble and clear solution in EGDE at 500 mg/mL, the maximum concentration tested.

In the initial toxicity-mutation assay, the maximum dose tested was 5000 µg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 µL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 µg per plate. In the initial toxicity‑mutation assay, no positive mutagenic response was observed. Precipitate was observed beginning at concentrations ranging from 50 to 1500 µg per plate. Toxicity was observed beginning at concentrations ranging from 50 to 5000 µg per plate with all Salmonella tester strains in the presence and absence of S9 activation. Based on the findings of the initial toxicity‑mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 1500 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation and 5000 µg per plate with the remaining test conditions.

In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in the presence of S9 activation or with tester strains TA98, TA100, TA1535 and WP2uvrA in the absence of S9 activation. The dose levels tested were 5.0, 15, 50, 150, 500 and 1500 µg per plate with all Salmonella tester strains in the absence of S9 activation and tester strain TA1535 in the presence of S9 activation and 15, 50, 150, 500, 1500 and 5000 µg per plate with the remaining test conditions. Precipitate was observed beginning at 500 or 1500 µg per plate. Toxicity was observed beginning at 500 or 1500 µg per plate. Due to excessive toxicity (beginning at 50 µg per plate), tester strain TA1537 in the absence of S9 activation was not evaluated but was retested with an adjustment in dose levels.

In the retest of the confirmatory mutagenicity assay, no positive mutagenic response was observed with tester strain TA1537 in the absence of S9 activation. The dose levels tested were 0.50, 1.5, 5.0, 15, 50, 150, 500 and 1500 µg per plate. Precipitate was observed beginning at 150 µg per plate. Toxicity was observed beginning at 500 µg per plate. 

Under the conditions of this study, the test article was concluded to be negative in the Bacterial Reverse Mutation Assay.