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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2009-04-30 to 2009-5-27
Reliability:
1 (reliable without restriction)

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

Constituent 1
Chemical structure
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 nmean = typical 0.04 – 0.23
IUPAC Name:
4,4'-Methylenediphenyl diisocyanate, oligomers

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver s9
Test concentrations with justification for top dose:
1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate
The initial toxicity-mutation assay was used to establish the dose-range for the confirmatory
mutagenicity assay and to provide a preliminary mutagenicity evaluation.
Vehicle / solvent:
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.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
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.
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.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium TA 98
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
Key result
Species / strain:
S. typhimurium 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:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
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
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
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

Applicant's summary and conclusion

Conclusions:
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, the 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 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, TA100and 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 the test material was concluded to benegative in the Bacterial Reverse Mutation Assay.