4,4'-Methylenediphenyl diisocyanate, oligomers

Genetic toxicity: in vitro

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

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-Methylenediphenyl diisocyanate, oligomers

EC Number:

500-040-3

EC Name:

4,4'-Methylenediphenyl diisocyanate, oligomers

Cas Number:

25686-28-6

Molecular formula:

C14 H10 N O [C29 H20 N4 O2]n NCO, n= 0-2

IUPAC Name:

1,1'-methylenebis(4-isocyanatobenzene) homopolymer

Details on test material:

Batch number: 281118

Specific details on test material used for the study:

Batch RZW256

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:

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 150 or 500 μg per plate. Toxicity was observed beginning at 150, 500 or 1500 μg per plate with all test conditions except tester strain WP2 uvrA in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the confirmatory mutagenicity assay were 5000 μg per plate with tester strain WP2 uvrA in the presence of S9 activation, 2000 μg per plate with tester strains TA98, TA100 and TA1535 in the presence of S9 activation and 600 μg per plate with the remaining test conditions.

Vehicle / solvent:

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.

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, appropriate frozen permanent stock or with a lyophilized pellet 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 results

Species / strain:

other: TA98, TA100, TA1535, TA1537 and WP2 uvrA

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

In the confirmatory assay toxicity was observed beginning at 600 or 2000 µg per plate with all Salmonella tester strains in the absence of S9 activation.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Solubility Test
Anhydrous ethylene glycol dimethylether (EGDE) was selected as the solvent of choice based
on the request of the Sponsor (Seel et al., 1999 and Herbold et al., 1998), 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.

Sterility Results
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
Tester Strain
Experiment TA98 TA100 TA1535 TA1537 WP2 uvrA
Titer Value (x 109 cells per mL)
B1 1.4 2.3 1.1 0.5 3.4
B2 1.7 1.0 1.4 1.1 2.9

Initial Toxicity-Mutation Assay
The results of the initial toxicity-mutation assay are presented in Tables 1 through 10 and
summarized in Table 21. These data were generated in 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 150 or 500 μg per plate. Toxicity was observed beginning at 150, 500 or 1500 μg
per plate with all test conditions except tester strain WP2 uvrA in the presence of S9 activation.
Based on the findings of the initial toxicity-mutation assay, the maximum doses plated in the
confirmatory mutagenicity assay were 5000 μg per plate with tester strain WP2 uvrA in the
presence of S9 activation, 2000 μg per plate with tester strains TA98, TA100 and TA1535 in the
presence of S9 activation and 600 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.

Confirmatory Mutagenicity Assay
The results of the confirmatory mutagenicity assay are presented in Tables 11 through 20 and
summarized in Table 22. These data were generated in Experiment B2. The dose levels tested
were 60, 200, 600, 2000 and 5000 μg per plate with tester strain WP2 uvrA in the presence ofS9 activation, 6.0, 20, 60, 200, 600 and 2000 μg per plate with tester strains TA98, TA100 and
TA1535 in the presence of S9 activation and 2.0, 6.0, 20, 60, 200 and 600 μg per plate with the
remaining test conditions. Precipitate was observed beginning at 60, 200 or 600 μg per plate.
Toxicity was observed beginning at 600 or at 2000 μg per plate with all test conditions except
tester strain WP2 uvrA in the presence of S9 activation.
In Experiment B2 (Confirmatory Mutagenicity Assay), no positive mutagenic responses were
observed with any of the tester strains in either the presence or absence of S9 activation.

Any other information on results incl. tables

Bacterial Mutation Assay
Summary of Results - Initial Toxicity-Mutation Assay
Table 21

Study Number : AC26MM.503.BTL
Experiment No : B1
Average Revertants Per Plate ± Standard Deviation
Activation Condition : None
Dose (μg per plate) TA98 TA100 TA1535 TA1537 WP2 uvrA
Vehicle 14 ± 4 144 ± 8 16 ± 1 5 ± 0 33 ± 4
Untreated Control 18 ± 8 137 ± 0 12 ± 1 8 ± 1 33 ± 6
1.5 12 ± 1 150 ± 0 13 ± 1 7 ± 0 32 ± 6
5.0 16 ± 2 132 ± 1 17 ± 5 6 ± 1 37 ± 6
15 17 ± 4 143 ± 5 14 ± 1 3 ± 3 33 ± 6
50 16 ± 3 128 ± 21 13 ± 1 7 ± 1 33 ± 8
150 13 ± 4 136 ± 13 14 ± 3 3 ± 1 32 ± 6
500 0 ± 0 60 ± 25 5 ± 1 2 ± 1 23 ± 11
1500 0 ± 0 65 ± 4 2 ± 1 0 ± 0 21 ± 6
5000 0 ± 0 70 ± 1 2 ± 1 0 ± 0 14 ± 1
Positive 644 ± 250 663 ± 47 505 ± 134 1220 ± 22 603 ± 35

 

Activation Condition : Rat Liver S9
Dose (μg per plate) TA98 TA100 TA1535 TA1537 WP2 uvrA
Vehicle 22 ± 10 173 ± 3 11 ± 1 6 ± 1 33 ± 4
Untreated Control 19 ± 2 136 ± 3 12 ± 3 4 ± 1 39 ± 7
1.5 22 ± 11 164 ± 20 11 ± 1 5 ± 1 33 ± 6
5.0 16 ± 3 184 ± 1 15 ± 2 5 ± 3 36 ± 2
15 20 ± 2 163 ± 6 11 ± 2 5 ± 1 38 ± 6
50 24 ± 4 170 ± 3 13 ± 2 4 ± 3 33 ± 1
150 25 ± 1 129 ± 3 14 ± 10 2 ± 1 35 ± 4
500 16 ± 3 173 ± 48 10 ± 2 5 ± 1 30 ± 6
1500 12 ± 1 113 ± 14 10 ± 1 6 ± 3 28 ± 1
5000 9 ± 4 136 ± 15 7 ± 1 7 ± 1 18 ± 4
Positive 345 ± 57 1202 ± 33 188 ± 3 38 ± 6 365 ± 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 22

Study Number : AC26MM.503.BTL
Experiment No : B2
Average Revertants Per Plate ± Standard Deviation
Activation Condition : None
Dose (μg per plate) TA98 TA100 TA1535 TA1537 WP2 uvrA
Vehicle 19 ± 2 166 ± 19 17 ± 2 10 ± 2 54 ± 6
Untreated Control 19 ± 2 142 ± 4 14 ± 3 5 ± 1 57 ± 4
2.0 15 ± 2 141 ± 11 21 ± 6 8 ± 1 58 ± 8
6.0 16 ± 4 160 ± 28 17 ± 6 10 ± 3 58 ± 4
20 21 ± 1 143 ± 2 16 ± 5 12 ± 5 51 ± 2
60 13 ± 4 127 ± 11 20 ± 2 9 ± 2 52 ± 6
200 20 ± 5 101 ± 7 16 ± 4 8 ± 1 54 ± 4
600 10 ± 1 102 ± 9 13 ± 2 8 ± 3 41 ± 8
Positive 191 ± 51 678 ± 6 259 ± 35 1235 ± 187 642 ± 17

Activation Condition : Rat Liver S9
Dose (μg per plate) TA98 TA100 TA1535 TA1537 WP2 uvrA
Vehicle 21 ± 3 157 ± 8 15 ± 1 9 ± 2 53 ± 4
Untreated Control 28 ± 5 149 ± 8 21 ± 5 9 ± 4 52 ± 13
2.0 6 ± 1
6.0 23 ± 6 140 ± 12 18 ± 1 9 ± 4
20 26 ± 1 160 ± 9 15 ± 6 9 ± 1
60 26 ± 3 157 ± 5 14 ± 4 8 ± 3 51 ± 4
200 22 ± 6 149 ± 16 16 ± 3 8 ± 2 53 ± 5
600 21 ± 2 116 ± 6 14 ± 5 7 ± 2 54 ± 5
2000 18 ± 1 111 ± 14 18 ± 5 47 ± 3
5000 46 ± 5
Positive 624 ± 74 1657 ± 115 497 ± 29 139 ± 36 347 ± 31
Vehicle = Vehicle Control
Positive = Positive Control (50 μL plating aliquot)
Plating aliquot = 50 μL

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
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.

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

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 150 or 500 μg per plate. Toxicity was observed beginning at 150, 500 or 1500 μg per plate with all test conditions except tester strain WP2 uvrA in the presence of S9 activation. Based on the findings of the initial toxicity-mutation assay, the maximum doses
plated in the confirmatory mutagenicity assay were 5000 μg per plate with tester strain WP2 uvrA in the presence of S9 activation, 2000 μg per plate with tester strains TA98, TA100 and TA1535 in the presence of S9 activation and 600 μg per plate with the remaining test conditions.

In the confirmatory mutagenicity assay, no positive mutagenic response was observed. The dose levels tested were 60, 200, 600, 2000 and 5000 μg per plate with tester strain WP2 uvrA in the presence of S9 activation, 6.0, 20, 60, 200, 600 and 2000 μg per plate with tester strains TA98, TA100 and TA1535 in the presence of S9 activation and 2.0, 6.0, 20, 60, 200 and 600 μg per plate with the remaining test conditions. Precipitate was observed beginning at 60, 200 or 600 μg per plate. Toxicity was observed beginning at 600 or at 2000 μg per plate with all test conditions except tester strain WP2 uvrA in the presence of S9 activation.

Under the conditions of this study, test article Suprasec 2020 RZW 256 was concluded to be negative in the Bacterial Reverse Mutation Assay.