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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
Study period:
2008-12-02 - 2009-04-23
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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Health Effects Test Guidelines; United States Environmental Protection Agency; Prevention, Pesticides and Toxic Substances (7101); EPA712-C-98-247,
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(2,6-diisopropylphenyl)carbodiimide
EC Number:
218-487-5
EC Name:
Bis(2,6-diisopropylphenyl)carbodiimide
Cas Number:
2162-74-5
Molecular formula:
C25H34N2
IUPAC Name:
N,N'-bis(2,6-diisopropylphenyl)carbodiimide
Details on test material:
- Name of test material: Bis(2,6-diisopropylphenyl)carbodiimide
- Substance type: organic technical product
- Visual appearance: white crystalline clotted solid. Prior to the first use the test item was molten at 60°C and remained thereafter an aqueous clear colourless liquid does not reflect chemical composition)
- Batch number: 08100014
- Date of analysis: November 19, 2008 (identity in Leverkusen)
- Approved : until May 11, 2009
- Storage: at room temperature
- Indication: additive for plastics (antihydrolysis agent)
The batch used was approved for use for the test period prior to study initiation. A stability test in the solvent did not reveal significant degradation of the active ingredient (solvent is used as technical term, even if the formulation is a suspension or an emulsion).

The test item was dissolved in ethanol and formed clear colourless solutions. Mitomycin C was dissolved in deionized water. The other positive controls were dissolved in DMSO.
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethyl ether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature and from our own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.

According to these results the test item is stable in the solvent at room temperature at concentrations ranging from 0.01 mg/ml to 100 mg/ml for at least four hours, a time interval, which covers the time range from preparation of the formulation to last treatment.

Method

Target gene:
his-
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: histidine-auxotrophic strains
Species / strain / cell type:
S. typhimurium TA 102
Additional strain / cell type characteristics:
other: histidine-auxotrophic strains
Metabolic activation:
with and without
Metabolic activation system:
The rat S9 mix comprised 10% S9 fraction, 70% cofactor solution and 20% 0.15 M KCI.
Test concentrations with justification for top dose:
Doses up to and including 5000 µg per plate

The following doses of the test substance were evaluated (µg per plate):
Negative control: 0
test substance: 5000, 1581, 500, 158, 50, 10
Positive control:
Na-azide 10 (only TA 1535)
NF 0.2 (only TA 100)
4-NPDA 10 (only TA 1537)
4-NPDA 0.5 (only TA 98)
MMC 0.2 (only TA 102)
Cumene 50 (only TA 102)
2-AA: 3
Vehicle / solvent:
The amount of solvent for the test substance and for the controls in the plate incorporation part was 0.1 mL/plate. However, in the preincubation part the amount of solvent per plate used for the test substance and for the negative controls was 0.05 mL per plate due to the bacteriotoxic effect of the solvent in preincubation experiments. The lacking volume of 0.05 mL was replaced by 0.05 mL deionized water which was added directly to the tubes.

The test item was dissolved in ethanol and formed clear colourless solutions. Mitomycin C was dissolved in deionized water. The other positive controls were dissolved in DMSO.
The solvent used was chosen out of the following solvents, in the order given: water, DMSO, methanol, ethanol, acetone, ethylene glycol dimethyl ether (EGDE), and DMF according to information given by the sponsor. The order of these solvents is based on their bacteriotoxic effects in preincubation experiments.
No "untreated" negative control was set up for the used solvent, since sufficient evidence was available in the literature and from our own experience, indicating that this solvent had no influence on the spontaneous mutant counts of the used strains.
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
solvent (= ethanol) minus the test substance,
Positive controls:
yes
Remarks:
sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene
Positive control substance:
other: sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene
Details on test system and experimental conditions:
Mammalian metabolism is simulated by the 9000 g fraction of homogenized mammalian livers. Together with co-factors, this forms the "S9 mix" which represents the metabolic model in this test.

One 1 ml-portion was thawed for each test and strain, and quantities of 0.2 ml of the thawed culture were added to 10 ml nutrient broth. This culture was incubated overnight at 37°C and used only on the same day. A new, small stock culture, which had been checked for its properties directly before freezing, was thus available for each individual test. In general this obviated any need to re-check the genotype for each Salmonella/microsome test. This procedure is in accordance with the methods described by Ames et al. (1975) and Maron and Ames (1983).

For the mutant count, three plates were used, both with and without S9 mix, for each strain and dose. An equal number of plates, filled with the solvent minus the test substance, comprised the negative control. Each positive control also contained three plates per strain. In experiments without S9 mix buffer, was used as replacement.
The amount of solvent for the test substance and for the controls in the plate incorporation part was 0.1 ml/plate. However, in the preincubation part the amount of solvent per plate used for the test item and for the negative controls was 0.05 ml per plate due to the bacteriotoxic effect of the solvent in preincubation experiments. The lacking volume of 0.05 ml was replaced by 0.05 ml deionized water which was added directly to the tubes.
The doses for the first trial were routinely determined on the basis of a standard protocol: if not limited by solubility 5000 µg or 5 µl per plate were used as the highest dose. At least four additional doses were routinely used. If less than three doses were used for assessment, at least two repeats were performed. The results of the first experiment were then considered as a pre-test for toxicity. However, in case of a positive response or if at least three doses could be used for assessment, the first trial was included in the assessment. If the second test confirmed the results of the first, no additional repeat was performed. Doses of repeats were chosen on the basis of the results obtained in the first experiment.
Evaluation criteria:
Toxicity of the substance was assessed in 2 ways. First method was a gross appraisal of background growth on the plates for mutant determination. If a reduction in background growth was observed, it was indicated in the tables. Secondly, a toxic effect of the substance was assumed when there was a marked and dose-dependent reduction in the mutant count per plate, compared to the negative controls.

The following criteria determined the acceptance of an assay:
1. The negative controls had to be within the expected range, as defined by published data and/ or the laboratories' own historical data.
2. The positive controls had to show sufficient effects, as defined by the laboratories' experience.
3. Titre determinations had to demonstrate sufficient bacterial density in the suspension
Only trials which complied with all 3 criteria were accepted for assessment. Even if the criteria for points (b) and (c) were not met, a trial was accepted if it showed mutagenic activity of the test compound. Furthermore, an unacceptable trial would have been repeated.

A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA1535, TA100 & TA98 this increase should be about twice that of negative controls, whereas for TA1537, at least a threefold increase should be reached. For TA102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment. In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation was possible.
Statistics:
Data were processed Oracle based with the following released Perceptive Instruments Ltd. applications:
• Ames Study Manager 1.21
• Ames Report Generator 1.1
• Sorcerer 2.2
• Change Password Utility 1.0
• Password Management/User Administration 1.0

Results and discussion

Test resultsopen allclose all
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
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity, but tested up to precipitating concentrations
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 5000 µg per plate. Nevertheless, all doses could be used for assessment purposes.
Evidence of mutagenic activity of the test item was not seen. No biologically relevant increase in the mutant count, in comparison with the negative controls, was observed.
The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls.
Therefore, the test item was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

There was no indication of a bacteriotoxic effect of the test substance at doses of up to and including 5000 µg per plate. The total bacteria counts consistently produced results comparable to the negative controls, or differed only insignificantly. No inhibition of growth was noted as well. At 5000 µg per plate, the substance precipitated. Nevertheless, all doses could be used for assessment.

None of the five strains concerned showed in the plate incorporation test a dose-related and biologically relevant increase in mutant counts over those of the negative controls. This applied both to the tests with and without S9 mix and was confirmed by the results of the preincubation trials.

The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene increased mutant counts to well over those of the negative controls, and thus demonstrated the system's sensitivity and the activity of the S9 mix.

Table 1:

Summary of the Results in the Salmonella/Microsome Test
S9 mix TA 1535 TA 100 TA 1537 TA98 TA 102
without -ve -ve -ve -ve -ve
with -ve -ve -ve -ve -ve

-ve = negative

Table 2:

Tabulated Summary of Data 
Summary of Mean Values Without S9 Mix
Table and

    Strain        

Group TA1535 TA100 TA1537 TA98 TA102
         
1 - 5 µg/Plate          
0 9 114 6 23 217
50 8 78 4 17 204
158 6 86 6 25 228
500 8 94 7 25 200
1581 9 97 5 18 186
5000 7 102 8 22 205
Na-azide 859        
NF   445      
4-NPDA     39 101  
MMC         1078
         
6 - 10 µg/Tube          
0 6 122 6 15 227
50 8 133 9 17 263
158 6 130 8 13 240
500 10 85 7 13 165
1581 8 97 9 18 239
5000 10 91 7 19 195
Na-azide 749        
NF   475      
4-NPDA     56 85  
Cumene         593
Summary of Mean Values With S9 Mix
Table and

    Strain        

Group TA1535 TA100 TA1537 TA98 TA102
         
1 - 5 µg/Plate          
0 6 112 7 38 279
50 7 127 7 36 248
158 6 137 8 36 295
500 12 95 9 25 234
1581 9 77 8 23 169
5000 9 99 9 27 194
2-AA 89 2115 89 1862 728
         
6 -10 µg/Tube          
0 9 138 8 31 329
50 7 136 8 23 330
158 10 110 8 27 290
500 10 131 6 24 280
1581 7 110 7 15 310
5000 8 137 7 13 323
2-AA 80 2538 171 1108 883

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative with metabolic activation histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102
negative without metabolic activation histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102

The study was performed according to the OECD Guideline 471 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. The test material did not induce significant increases in the frequency of revertant colonies in any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of the test.
Executive summary:

The test substance was initially investigated using the Salmonella/microsome plate incorporation test for point mutagenic effects in doses of up to and including 5000 µg per plate on five Salmonella typhimurium LT2 mutants. These comprised the histidine-auxotrophic strains TA 1535, TA 100, TA 1537, TA 98 and TA 102. The independent repeat was performed as preincubation for 20 minutes at 37°C. Other conditions remained unchanged. Doses up to and including 5000 µg per plate did not cause any bacteriotoxic effects. Total bacteria counts remained unchanged and no inhibition of growth was observed. Substance precipitation occurred at the dose 5000 µg per plate. Nevertheless, all doses could be used for assessment purposes. Evidence of mutagenic activity of the compound was not seen. Evaluation of individual dose groups, with respect to relevant assessment parameters (dose effect, reproducibility) revealed no biologically relevant variations from the respective negative controls. The positive controls sodium azide, nitrofurantoin, 4-nitro-1,2-phenylene diamine, mitomycin C, cumene hydroperoxide and 2-aminoanthracene had a marked mutagenic effect, as was seen by a biologically relevant increase in mutant colonies compared to the corresponding negative controls. (demonstrating the system's high sensitivity). Despite this sensitivity, no indications of mutagenic effects of the test substance could be found at assessable doses of up to 5000 µg per plate in any of the Salmonella typhimurium strains used. Therefore, the test substance was considered to be non-mutagenic without and with S9 mix in the plate incorporation as well as in the preincubation modification of the Salmonella/microsome test.