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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:
weight of evidence
Study period:
06.09.1991 to 04.02.1992
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1992
Report date:
1992

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OTS 798.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
EC Number:
204-794-1
EC Name:
2,2,2',2'-tetrakis(hydroxymethyl)-3,3'-oxydipropan-1-ol
Cas Number:
126-58-9
Molecular formula:
C10H22O7
IUPAC Name:
2,2'-[oxybis(methylene)]bis[2-(hydroxymethyl)propane-1,3-diol]
Specific details on test material used for the study:
- Name of test material (as cited in study report): dipentaerythritol
- Substance type: white powder
- Physical state: solid

Method

Target gene:
hisG46, hisC3076 and hisD3052.
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 dependent auxotrophic mutants
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
other: histidine dependent auxotrophic mutants
Metabolic activation:
with and without
Metabolic activation system:
-S9 mix obtained from the livers of Aroclor-1254 induced male Sprague-Dawley rats
- S9 mix contained: S-9 fraction (10% v/v), MgCl2 (8mgM), KCl (33mM). sodium phosphate buffer PH 7.4 (100mM), glucose-6-phosphate (5mM), NADP (4mM).
-the volume of S9 mix used in the culture is detailed in the "details on test system and experiment conditions" section below.

- quality controls of S9 : All the cofactors in S9 mix were filter sterilised before use. The S-9 fraction was tested with 7,12-dimethylbenxanthracene and 2-aminoanthracene before use.
Test concentrations with justification for top dose:
Range finding: solvent control, 5, 50, 500, 5000 µg/plate.
Mutation test: solvent control, 0, 50, 150, 500, 1500, 5000 µg/plate.

-Justification for the top dose: in the preliminary toxicity test, the test substance was not toxic towards the tester strains. therefore 5000 µg/plate was chose as the tope dose level in the mutation tests.
Vehicle / solvent:
dimethyl sulphoxide
- Vehicle(s)/solvent(s) used:DMSO
- Justification for choice of solvent/vehicle: Prior to commencing testing the solubility of the test material in solvents compatible with the test system was assessed. The chosen solvent was dimethyl sulphoxide.
Controlsopen allclose all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
dimethyl sulfoxide
True negative controls:
no
Positive controls:
yes
Positive control substance:
N-ethyl-N-nitro-N-nitrosoguanidine
Remarks:
Without S9: 5 µg/plate for TA1535, and 3 µg/plate for TA100
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
Remarks:
80 µg/plate for TA1537 without S9
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-nitrofluorene
Remarks:
Without S9; 2 µg/plate for TA1538, 1 µg/plate for TA98
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-aminoanthracene
Remarks:
With S9: 2 µg/plate for TA1535 and TA1537, 0.5 µg/plate for TA1538 and TA98, 1 µg/plate for TA100
Details on test system and experimental conditions:
The plate incorporation method was used. The Salmonella strains used in the experiment were: TA1535 hisG46 rfa uvrB, TA1537 hisC3076 rfa uvrB, TA1538 hisD3052 rfa uvrB, TA98 hisD3052 rfa uvrB pKM101, and TA100 hisG46 rfa uvrB pKM101.
Batches of the strains were stored at -80°C. Each frozen batch was tested for cell membrane permeability and, where applicable, for ampicillin resistance. The response of the strain to a series of diagnostic mutagens was also assessed.
Fuse in tests, an aliquot of frozen culture was added to 25 ml of nutrient broth (DAB 7, Merck) and incubated, with shaking, at 37°C for 10 hours. This culture provided approximately 2x10exp9 cells per ml which was checked photometrically.

Preliminary toxicity test
Four concentrations of test substance were assessed for toxicity using the five tester strains. The highest concentration was 5000 µg/plate. The chosen solvent was used as the negative control. 0.1 ml bacterial culture and 0.5 ml S9 mix or 0.5 ml 0.1M phosphate buffer (pH7.4) were placed in glass bottles. 0.1 ml of the test substance solution was added, following immediately by 2 ml of histidine deficient agar. The mixture was thoroughly shaken and overlaid onto previously prepared petri dishes containing 25 ml minimal agar. A single pertri dish was used for each dose level. Plates were also prepared without the addition of bacteria in order to test the sterility of the test substance, S9 mix and phosphate buffer. All plates were incubated at 37°C for 3 days. After this period the appearance of the background lawn was examined. Revertant colonies were counted using a Biotran Automatic Colony Counter.

Concentrations for the main study were chosen based on toxic effects as assessed by a substantial reduction in revertant colony counts or by the absence of a complete bacterial lawn.

Mutation test
The test substance was added to cultures of the five tester strains at five concentrations separated by c. half-log10 intervals. The highest concentration used was 5000 µg/plate. The negative control was the chosen solvent. The plate incubation method was used as in the preliminary toxicity test. Three petri dishes were used per dose level. The mutation test was independently repeated using the same concentration of the test substance.

Rationale for test conditions:
-Justification of the top does and vehicle choice were detailed in the relevant sections above.
-Justification of the number of plates per concentration: three petri dishes were used per does level which is aligned the OECD 471 suggestion.
Evaluation criteria:
The mean number of revertant colonies was compared between treated groups and the solvent control. The following criteria were used in assessing the mutagenic response:
- the substance was considered to be mutagenic if there was at least twice the number of revertant colonies compared to the control, with some evidence of a dose-response relationship, in two separate experiments, with any bacterial strain either in the presence or absence of S 9 mix. No statistical analysis is performed.


- the substance was considered to be non-mutagenic if there was no reproducible increase of at least 1.5 times to control values at any dose with any bacterial strain. No statistical analysis is performed.
Statistics:
When the results obtained failed to satisfy the evaluation criteria above for a clear positive or negative response. the test data maybe subjected to statistical analysis according to the study description.

As in the study no substantial increase of revertant colony numbers of any strains were observed, statistical analysis were not performed in the study.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
The mean number of the revertant colonies obtained in the first mutation test are shown in Table 2 in attachment. Positive control mutability checks are shown in Table 3.

The mean number of the revertant colonies obtained in the second mutation test are shown in Table 4 in attachment. Positive control mutability checks are shown in Table 5.

Any other information on results incl. tables

There were no substantial increases in revertant colony numbers of any of the tester strains following treatment with dipentaerythritol at any concentration, in either the presence or absence of S9 mix.

Applicant's summary and conclusion

Conclusions:
No evidence of mutagenic activity was seen at any dose level of dipentaerythritol both in the presence and absence of metabolic activation.
Executive summary:

In this in vitro assessment of the mutagenic potential of dipentaerythritol, histidine dependent auxotrophic mutants of Samonella typhimurium (strains TA1535, TA1537, TA1538, TA98 and TA100) were exposed to the test material, dissolved in dimethyl sulphoxide. In the preliminary dose range finding study with dose levels of up to 5000 µg/plate no toxicity was observed. A top dose level of 5000 µg/plate was chosen for the subsequent mutation study. Two independent mutation tests were performed, in the presence and absence of liver preparations from Aroclor 1254 -induced rats. No evidence of mutagenic activity was seen at any dose level of dipentaerythritol in either mutation test, when compared to the vehicle controls. The concurrent positive control compounds demonstrated the sensitivity of the assay and the metabolising activity of the liver preparations. It was concluded that dipentaerythritol was not mutagenic in this bacterial test system.

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