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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:
From September 29th, 2008 to October 6th, 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study performed according to OECD 471 and according to GLP Due to organization error, the test was performed with 4 strains of S. typhimurium and one of E. coli, instead of 5 S. typhimurium claimed in the study plan. However, strains TA 102 and WP2 uvrA (pKM101) have the same type of mutation

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:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
GLP according to Directive 88/320 EEC, Directive 99/11/EC
Type of assay:
bacterial reverse mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Propanediol Dicaprylate
- Substance type: Organic
- Physical state: Liquid
- Analytical purity: > 95%
- Lot/batch No.: 08058601
- Expiration date of the lot/batch: June 3, 2009
- Storage condition of test material: In original container, at room temperature in the dark

Method

Target gene:
Histidine (his- to his+) and tryptophan (tryp- to tryp+) genes in Salmonella typhimurium and Escherichia coli respectively
Species / strainopen allclose all
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Metabolic activation system:
S9 microsome fraction of Aroclor 1254 induced rat liver
Test concentrations with justification for top dose:
Test substance concentrations (microL/plate): 5, 1.67, 0.56, 0.19 and 0.06
Number of replica: 3
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: the test item is stable for 96h in corn oil
Controls
Untreated negative controls:
yes
Remarks:
solvent control
Negative solvent / vehicle controls:
yes
Remarks:
Corn oil
True negative controls:
yes
Positive controls:
yes
Positive control substance:
other: Without metabolic activation: 2-nitrofluorene (TA98), sodium azide (TA1535 and TA100), 4-nitroquinoline-N-oxide (WP2 uvrA), and 9-aminoacridine (TA 1537) / With metabolic activation: 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: direct plate incorporation and pre incubation methods

Length of the experimental part: 8 days
Metabolic activation system: S9 fraction from Aroclor induced rats (Moltox, USA)
Incubation time: 72 hours
Cytoxicity test: Observation of dose-dependent effects
Repetition test: triplicates for each strain and for negative and positive controls. Performed independently with the preincubation method
Counting: Automatic colony counter

Concentrations tested: 0.06, 0.19, 0.56, 1.67 and 5 microliters/plate

Cultures of bacteria were incubated overnight with nutrient broth up to an absorbance (660 nm) of approximately 1.2-1.4 OD. This OD indicates that bacteria are growing in the late exponential or early stationary phase of growth (approximately 10^9 bacteria/mL).
Plates were prepared with minimal agar medium. Medium was mixed and preheated to about 45ºC, and then poured into the plate and cooled at roomtemperature. Each bacterial strain was tested by triplicate in the presence and absence of the metabolic system (S9). The bacterial suspension, the test item and PBS (-S9) or metabolic activation system mix (+S9) were mixed and tempered at about 37ºC.
The suspension was mixed with top agar and poured over minimal agar medium plate. The top agar was allowed to solidify at room temperature before final incubation.
Plates were incubated at about 37ºC for about 72 hours. Two controls were included in the experiment:
Negative control: Control cultures were treated with solvent.
Positive control: Control mutagens were used for each strain and experimental condition.

An independent confirmation test was performed with the test item according to the preincubation procedure. After the bacterial suspension, the test item and PBS (-S9) or metabolic activation system mix (+S9) were mixed and the mixture was incubated at about 37ºC for about 20 minutes. Thereafter, the study was performed in the same way as the first test.


Due to organization error, the test was performed with 4 strains of S. typhimurium and one of E. coli, instead of 5 S. typhimurium claimed in the study plan. However, strains TA 102 and WP2 uvrA (pKM101) have the same type of mutation
Evaluation criteria:
A test substance is considered positive in the test if: a dose-response in the range tested and/or a reproducible increase in the ratio (number of revertant colonies in the presence of test item/number of revertant colonies in the absence of test item) below 2.5 at one or more concentrations in the number of revertant colonies per plate in at least one strain with or without metabolic activation system is observed.

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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
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
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

The controls of the test were in concordance with the expected results: Sterility test showed no contamination during the study.

No cytotoxic effect was observed. All positive controls performed showed valid ratios (R) above 2.5. Positive and negative controls showed mean numbers of revertant colonies comparable to historical data. These results validate the assay.

No concentration of the test item showed a biological significant increase (R = 2.5) of the number of revertant either with or without S9 metabolic activation.

No dose response was observed in none of the tested bacterial strains.

No experiment with the test item showed ratios (R) above 2.5 as compared to the negative control, either with or without S9 metabolic activation.

No dose response was observed in none of the tested bacterial strains with the test item.

Based on the results obtained in this study, the test item PROPANEDIOL DICAPRYLATE was found to be NON MUTAGENIC and NON PRO-MUTAGENIC under the test conditions.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative not mutagenic with and without metabolic activation

Propanediol Dicaprylate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.
Executive summary:

The present Bacterial Reverse Mutation Test (Ames test) was performed in order to evaluate the mutagenic potential of the test item.

The test was performed in accordance with OECD Guideline 471 for the Testing of Chemicals (Bacterial Reverse Mutation Test. Adopted 21st July 1997) and the test Method B13/B14 of Commission Directive 2000/32/EC.

Doses ranging from 5µL to 0.06µL per plate were tested. No cytotoxicity was observed at any dose. Suspensions of 4 amino-acid requiring strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537) and one Escherichia coli WP2 uvrA (pKM101) strain auxotroph for an amino acid were exposed by the direct plate incorporation method to five doses of the test item in the presence and in the absence of an exogenous metabolic activation system. Both tests were repeated with the pre-incubation method.

The bacteria were allowed to grow for 72h. Only revertant bacteria due to point or frameshiftmutations at specific locus are able to grow, forming colonies. These colonies were counted and compared to the number of spontaneous revertant colonies on solvent control plate (negative control). Similarly, specific standard mutagens were tested and used as positive controls.

Based on the results obtained in this study, the test item PROPANEDIOL DICAPRYLATE was found to be NON MUTAGENIC and NON-PROMUTAGENIC under the test conditions