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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report date:
2008

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 OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Remarks:
The Department of Health of the Government of the United Kingdom, GLP Monitoring Authority, UK
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Propylidynetrimethanol, propoxylated
EC Number:
500-041-9
EC Name:
Propylidynetrimethanol, propoxylated
Cas Number:
25723-16-4
Molecular formula:
C3H5(CH2OR)3 R= (C2H3(CH3)O)nH sum of n: >1 - <6.5 mol PO
IUPAC Name:
Propylidynetrimethanol, propoxylated
Details on test material:
Identity: Polyol R3530
Appearance: Colourless liquid
Storage conditions: Room temperature in the dark, dry
Batch number: 3568023
Expiry date: Indefinite
Purity/Assay: Hydroxyl number, 542 mg KOH/g

Method

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:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: tryptophan-dependent mutant
Metabolic activation:
with and without
Metabolic activation system:
liver preparations (S9 mix) from rats treated with phenobarbital and 5,6-benzoflavone
Test concentrations with justification for top dose:
5, 15, 50, 150, 500, 1500, 5000 µg/plate; the top dose is the standard limit concentration as recommended in the regulatory testing guidelines.
Vehicle / solvent:
The test substance was partly miscible with water. It was found to be miscible at 50 mg/mL with water. Water (purified in-house by reverse osmosis) was, therefore, used as the vehicle for this study. The highest concentration of the test substance in this study was 50 mg/mL in the chosen vehicle, which provided a final concentration of 5000 µg/plate. This is the standard limit concentration as recommended in the regulatory testing guidelines. The highest concentration in each test was diluted with water to produce a series of lower concentrations, separated by approximately half-log10 intervals.
Controls
Untreated negative controls:
yes
Remarks:
water
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene, 4-Nitroquinoline-1-oxide, 2-Aminoanthracene, Benzo[a]pyrene
Details on test system and experimental conditions:
Preparation of S9 fraction
S9 fraction, prepared from male Sprague-Dawley derived rats, dosed with phenobarbital and 5,6-benzoflavone to stimulate mixed-function oxidases in the liver, was purchased from a commercial source and stored at approximately -80°C.

Preparation of S9 mix
The S9 mix contained: S9 fraction (10% v/v), MgCl2 (8 mM), KCl (33 mM), sodium phosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADPH (4 mM) and NADH (4 mM) in water. All the cofactors were filter-sterilised before use.

Mutation test procedure: first test (plate incorporation test)
Aliquots of 0.1 mL of the test substance solutions (seven concentrations up to 5000 μg/plate), positive control or negative control were placed in glass vessels. S9 mix (0.5 mL) or 0.1 M pH 7.4 phosphate buffer (0.5 mL) was added, followed by 0.1 mL of a 10-hour bacterial culture and 2 mL of agar containing histidine (0.05 mM), biotin (0.05 mM) and tryptophan (0.05 mM). The mixture was thoroughly shaken and overlaid onto previously prepared Petri dishes containing 25 mL minimal agar. Each Petri dish was individually labelled with a unique code, identifying the contents of the dish. Three Petri dishes were used for each treatment. Plates were also prepared without the addition of bacteria in order to assess the sterility of the test substance, S9 mix and sodium phosphate buffer. All plates were incubated at approximately 37°C for ca 72 h. After this period, the appearance of the background bacterial lawn was examined and revertant colonies counted using an automated colony counter (Perceptive Instruments Sorcerer). Any toxic effects of the test substance would be detected by a substantial reduction in mean revertant colony counts or by a sparse or absent background bacterial lawn. In the absence of any toxic effects, the maximum concentration selected for use in the second test would be the same as that used in the first. If toxic effects were observed at more than one concentration, a lower concentration might be chosen, ensuring that signs of bacterial inhibition were present at this maximum concentration. Ideally, a minimum of four non-toxic concentrations should be obtained. If precipitate were observed on the plates at the end of the incubation period, at least four non-precipitating concentrations should be obtained.

Mutation test procedure: second test (pre-incubation test)
As a clear negative response was obtained in the first test, a variation to the test procedure was used for the second test. The variation used was the pre-incubation assay in which the tubes, which contained mixtures of bacteria, buffer or S9 mix and test dilution, were incubated at 37°C for 30 min with shaking before the addition of the agar overlay. The maximum concentration chosen was again 5000 μg/plate, but only five concentrations were used.
Evaluation criteria:
For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the 99% confidence limits of the current historical control range of the laboratory. The historical range is maintained as a rolling record over a maximum of five years. Also, the positive control compounds must induce an increase in mean revertant colony numbers of at least twice (three times in the case of strains TA1535 and TA1537) the concurrent vehicle controls. Mean viable cell counts in the 10-hour bacterial cultures must be at least 10E9/mL.
Statistics:
The mean number and standard deviation of revertant colonies were calculated for all groups. The “fold-increases” relative to the vehicle controls were calculated in order to compare the means for all treatment groups with those obtained for the vehicle control groups.

Results and discussion

Test resultsopen allclose all
Key result
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 nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
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:
valid
True negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Results
The absence of colonies on sterility check plates confirmed the absence of microbial contamination of the S9 mix, buffer and test substance formulation. The total colony counts on nutrient agar plates confirmed the viability and high cell density of the cultures of the individual organisms. The mean revertant colony counts for the vehicle controls were within or close to the 99% confidence limits of the current historical control range of the laboratory. Appropriate positive control chemicals (with S9 mix where required) induced substantial increases in revertant colony numbers with all strains in all reported tests, confirming sensitivity of the cultures and activity of the S9 mix.

First test (plate incorporation test)
No evidence of toxicity was observed following exposure to the test substance. A maximum exposure concentration of 5000 μg/plate was, therefore, selected for use in the second test. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains following exposure to the test substance at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Second test (pre-incubation test)
Based on the results of the first assay, the highest test concentration was maintained at 5000 µg/plate also for the second assay. No evidence of toxicity was noted following exposure to the test substance. No substantial increases in revertant colony numbers over control counts were obtained with any of the tester strains at any concentration up to 5000 μg/plate in either the presence or absence of S9 mix.

Any other information on results incl. tables

Additional data supporting the information is attached below under 'Attached background material'.

Applicant's summary and conclusion

Conclusions:
It is concluded that propylidentrimethanol, propoxylated showed no evidence of mutagenic activity in bacteria under the test conditions used.