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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:
23 August 2017 - 06 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

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
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Reference substance name:
Not available
Cas Number:
2135769-54-7
Molecular formula:
NA
IUPAC Name:
Not available
Test material form:
liquid: viscous
Details on test material:
Vapour pressure: 3.9 x 10-3 Pa
Water solubility: TBC
Density: 1.0
Appearance: black viscous liquid
Melting point: -52 to -21 °C
Boiling point: no boiling and no decomposition up to 300°C
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
Identification: Tall Oil Pitch, reaction product with triethylene glycol
CAS: 2135769-54-7
CAS Name: Tall-oil pitch, ester with triethylene glycol
Batch: LABO 16-02
Physical state: Brown viscous liquid
Expiry date: 14/11/2018
Storage Conditions: room temperature in the dark under nitrogen
Purity: 100%

Method

Vehicle / solvent:
Identity: Tetrahydrofuran
Supplier: Sigma Aldrich
Batch number (purity): STBG9857 (>99.9%), Expiry: 07/2022
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: 2-Aminoanthracene
Details on test system and experimental conditions:
Microsomal Enzyme Fraction:
The S9 Microsomal fractions were pre-prepared using standardized in-house procedures.

S9-Mix and Agar
The S9-mix was prepared before use using sterilized co-factors and maintained on ice for the
duration of the test. A 0.5 mL aliquot of S9-mix and 2 mL of molten, trace histidine or tryptophan supplemented, top agar were overlaid onto a sterile Vogel-Bonner Minimal agar plate in order to assess the sterility of the S9-mix. This procedure was repeated, in triplicate, on the day of each experiment.

Media
Top agar was prepared using 0.6% Bacto agar (lot number 6221620 05/21) and 0.5% sodium
chloride with 5 mL of 1.0 mM histidine and 1.0 mM biotin or 1.0 mM tryptophan solution
added to each 100 mL of top agar.

Bacteria
Salmonella typhimurium
TA1537 his C 3076; rfa-; uvrB-:
TA98 his D 3052; rfa-; uvrB-;R-factor
TA1535 his G 46; rfa-; uvrB-:
TA100 his G 46; rfa-; uvrB-;R-factor

Escherichia coli
WP2uvrA trp-; uvrA-:


Evaluation criteria:
A test item will be considered non-mutagenic (negative) in the test system if the following criteria are not met.
1. A dose-related increase in mutant frequency over the dose range tested (De Serres and Shelby, 1979).
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al., 1989).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response (Cariello and Piegorsch, 1996)).
Statistics:
Statistical significance was confirmed by using Dunnetts Regression Analysis (* = p < 0.05) for those values that indicate statistically significant increases in the frequency of revertant colonies compared to the concurrent solvent control.

Results and discussion

Test resultsopen allclose all
Key result
Species / strain:
S. typhimurium 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
Key result
Species / strain:
S. typhimurium TA 1535
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
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
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:
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
Additional information on results:
A test item precipitate (greasy in appearance) was noted at 5000 µg/plate in both experiments with and without S9, this observation did not prevent the scoring of revertant colonies.
Remarks on result:
other: NEGATIVE
Remarks:
NEGATIVE

Applicant's summary and conclusion

Conclusions:
The test material Tall Oil Pitch, reaction product with triethylene glycol was considered to be non-mutagenic under the conditions of this test.
Executive summary:

The Bacterial Reverse Mutation Test was performed to assess the potential mutagenicity of the test material Tall Oil Pitch, reaction product with triethylene glycol.

 

The test was performed according to the OECD 471 Guideline and under GLP conditions. The study was based on the in vitro technique described by Ames et al., (1975), Maron and

Ames (1983) and Mortelmans and Zeiger (2000), in which mutagenic effects are determined by exposing mutant strains of Salmonella typhimuriumTA1537, TA98, TA1535, TA100to various concentrations of the test Item. Additionally, a mutant strain of Escherichia coli (WP2uvrA) which requires tryptophan and can be reverse mutated by base-pair substitution to tryptophan independence (Green and Muriel, 1976 and Mortelmans and Riccio, 2000) is used to complement the Salmonella strains.

 

The test item was tested using the following method. The maximum concentration was 5000µg/plate (the maximum recommended dose level). Eight concentrations of the test item (1.5, 5, 15, 50, 150, 500, 1500 and 5000µg/plate) were assayed in triplicate against each tester strain, using the direct plate incorporation method.The dose range used for Experiment 2 was determined by the results of Experiment 1 and was 15, 50, 150, 500, 1500, 5000 µg/plate.

 

No increases in the frequency of revertant colonies was recorded for any of the bacterial strains, with any dose of the test item, either with or without metabolic activation

(S9-mix) in Experiment 1 and 2.

 

In conclusion, the test material Tall Oil Pitch, reaction product with triethylene glycol was considered to be non-mutagenic under the conditions of this test.