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Diss Factsheets

Administrative data

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
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
Reference substance name:
Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine
EC Number:
291-909-3
EC Name:
Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine
Cas Number:
90506-47-1
Molecular formula:
complex substance
IUPAC Name:
Phosphoric acid, C8-16-alkyl esters, reaction products with (Z)-N-9-octadecenyl-1,3-propanediamine
Test material form:
liquid: viscous
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 80555E094
- Expiration date of the lot/batch: 05/12/2018
- Purity test date: 23/01/2018
- Date of receipt: 25/01/2018

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient

Method

Species / strain
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
The test item concentrations to be applied in the main experiments were chosen according to the results of the pre-experiment. 1000 μg/plate was selected as the maximum concentration.

The concentration range covered two logarithmic decades. Two independent experiments were performed with the following concentrations:
Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (TA 98, TA 100 with metabolic activation)
0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (TA 98, TA 100 without metabolic activation, TA1535, TA 1537 TA 102 with and without metabolic activation)
Experiment II:
0.50, 1.58, 5.00, 15.8, 50.0, 158, 500 and 1000 μg/plate
Vehicle / solvent:
The test item was dissolved in ethanol and diluted prior to treatment. The solvent was compatible with the survival of the bacteria and the S9 activity.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
sodium azide
methylmethanesulfonate
other: 4-NOPD; 4-nitro-o-phenylene-diamine & 2-AA; 2-aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
- Medium: in Vogel-Bonner Medium E agar plates with 2% glucose.
- Preparation of Bacteria: Samples of each tester strain were grown by culturing for 12 h at 37 °C in Nutrient Broth to the late exponential or early stationary phase of growth (approx. 10^9 cells/mL).
- S9 Homogenate: The S9 liver microsomal fraction was obtained from Trinova Biochem GmbH, Gießen, Germany. Male Sprague Dawley rats were induced with phenobarbital / β-naphthoflavone.
- Experimental Performance: For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 μL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 μL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 μL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 μL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.

NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.

DETERMINATION OF CYTOTOXICITY
Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or "B", respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.
Evaluation criteria:
Biologically relevant increases in revertant colony numbers.

Results and discussion

Test resultsopen allclose all
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 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 102
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:
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

Applicant's summary and conclusion

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, ARCOT 3135/384 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, ARCOT 3135/384 is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In order to investigate the potential of ARCOT 3135/384 for its ability to induce gene mutations the plate incorporation test (experiment I and II) was performed with the Salmonella typhimurium strains TA 98, TA 100, TA 1535, TA 1537 and TA 102.

In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:

Experiment I:

3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (TA 98, TA 100 with metabolic activation)

0.316, 1.00, 3.16, 10.0, 31.6, 100, 316 and 1000 μg/plate (TA 98, TA 100 without metabolic activation, TA1535, TA 1537 TA 102 with and without metabolic activation)

Experiment II:

0.50, 1.58, 5.00, 15.8, 50.0, 158, 500 and 1000 μg/plate

No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with ARCOT 3135/384 at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.