Registration Dossier

Toxicological information

Genetic toxicity: in vitro

Currently viewing:

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21st November 2017 - 26th March 2018
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 guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
EC Number:
916-328-0
Molecular formula:
C10H18O3
IUPAC Name:
Reaction mass of allyl (2-methylbutoxy)acetate and allyl (3-methylbutoxy)acetate
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: O’Laughlin (Nantong) Fine Chemicals Co., Ltd.; NTA375
- Expiration date of the lot/batch: Sep 25, 2020
- Purity: CAS No. 67634-00-8: 79.45 %; CAS No.: 67634-01-9 20.28 %

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Store in cool place. Keep container tightly closed in a dry and well-ventilated place.

Method

Species / strain
Species / strain / cell type:
other: S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E. coli WP2 uvrA
Metabolic activation:
with and without
Metabolic activation system:
Delor 106-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary test (plate-incorporation): 10 - 5000 µg/plate
Main test (plate-incorporation + and - S9): 10 - 1000 µg/plate
Main test (plate-incorporation + S9; E coli WP2 uvrA):100 - 1500 µg/plate

Preliminary test (pre-incubation): 250 - 1000 µg/plate
Main test (pre-incubation + and - S9): 10 - 200 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used:DMSO
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
2-acetylaminofluorene
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine: TA 98 without metabolic activation; 2-aminoanthracene : TA 1535, TA 1537, E.coli with metabolic activation; N-methyl-N´-nitro-N-nitrosoguanidine: E. coli without metabolic activation
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) for first main test; pre-incubation for confirmatory test; in agar (plate incorporation) for E. coli repeat test +S9.

DURATION
- Preincubation period: 30 minutes
- Exposure duration: 72 hours

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: changes in bacterial background and/or decreased number of revertants



Evaluation criteria:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods . After this rule the result is positive, if a reproducible dose-response effect occurs and/or a doubling of the ratio Rt/Rc is reached.

An increase is considered as ”biologically relevant“:
-if the number of reversions is at least twice as high as that in the solvent control for the strains having spontaneous reversion >10;
-if the number of reversions is at least three times as high as that in the solvent control for the strains having spontaneous reversion ≤10;

A test item producing neither a dose-related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the dose groups is considered to be non-mutagenic in this system

According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.
Statistics:
The main criterion for evaluation of results was modified two-fold increase rule, which is compatible with the application of statistical methods.
According to OECD TG 471, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Results and discussion

Test resultsopen allclose all
Species / strain:
S. typhimurium TA 98
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
1000 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
300 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
200 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Plate incorporation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
300 µg/plate
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Remarks:
Pre-incubation
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:
S. typhimurium TA 1537
Remarks:
Plate incorporation
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:
not valid
Species / strain:
S. typhimurium TA 1537
Remarks:
Pre-incubation
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
not valid
Untreated negative controls validity:
not valid
Positive controls validity:
not valid
Species / strain:
E. coli WP2 uvr A
Remarks:
Plate incorporation
Metabolic activation:
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
Remarks:
Plate incorporation
Metabolic activation:
with
Genotoxicity:
ambiguous
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
Remarks:
Plate incorporation (repeat)
Metabolic activation:
with
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
Remarks:
Pre-incubation
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:
RANGE-FINDING/SCREENING STUDIES:
The preliminary cytotoxicity test was performed in Salmonella typhimurium TA 98 without metabolic activation. Results of the toxicity testing are given in Table B. In the preliminary cytotoxicity test, signs of cytotoxicity occurred from 1000 µg per plate.

The second experiment was performed with pre-incubation, so a cytotoxicity test with pre-incubation was performed in Salmonella typhimurium TA 100 without metabolic activation. The test item was dosed in the volume of 50 µL. Pre-incubation was performed at 37±1°C and shaking for 30 minutes. Signs of cytotoxicity occurred from 250 µg per plate. Results are given in the Table C.

The additional experiment in E. coli with metabolic activation was performed with doses with expected effect - 100, 250, 500, 1000 and 1500 μg per plate. 1500 μg per plate was used as maximum because no toxicity was observed in this bacterial strain.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
Spontaneous reversions, solvent and positive controls were compared with historical controls in our laboratory. The concurrent positive controls verified the sensitivity of the assay and the metabolising activity of the liver preparations. Average revertant colony counts for the vehicle controls were within the current historical control range for the laboratory. The current ranges are given in the Table A.

Applicant's summary and conclusion

Conclusions:
In a reverse bacteria mutation study (Ames test), Allyl Amyl Glycolate, was non-mutagenic in S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA with and without metabolic activation.
Executive summary:

In a reverse gene mutation assay in bacteria (18 -122), strains of S. typhimurium TA 98, TA 100, TA 1535, TA 1537 and E. coli WP2 uvrA were exposed to Allyl Amyl Glycolate in DMSO at concentrations of 10 - 1000 µg/plate (plate incorporation), and 10 - 200 µg/plate (30 minute pre-incubation) in the presence and absence of mammalian metabolic activation (Delor 106-induced rat liver S9). The plate incorporation test was repeated in the E. coli WP2 uvrA strain using 100 - 1500 µg/plate with metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background in the S. typhimurium TA 98, TA 100, TA 1535 and TA 1537 strains using the plate incorporation or pre-incubation methods. An additional experiment with E.coli WP2 uvrA with metabolic activation performed to clarify the result of the first experiment, where Rt/Rc 1.5 and 2.1 were observed at 300 and 1000 μg per plate, respectively. No such increase was observed in the third experiment, and the experiment with pre-incubation was clearly negative, so mutagenicity of the test item was not confirmed in this strain.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.