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Toxicological information

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:
2017 - 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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Details on test material:
Legal entity composition

Method

Species / strainopen allclose all
Species / strain / cell type:
E. coli WP2 uvr A
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
liver S9 fraction from rat
Test concentrations with justification for top dose:
Solubility of the test item was evaluated in a preliminary trial using DMSO (compatible with the survival of the bacteria and the S9 metabolic activity). An opaque preparation, suitable for treatment, was obtained at 100 µL/mL after sonication at 37°C for approximately 10 minutes and heating at 37°C for further 10 minutes . This result permitted a maximum concentration of 5.00 µL/plate to be used in the toxicity test.

Preliminary toxicity test:
50, 158, 500, 1580 and 5000 µL/plate
Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.

Main Assay I (on the basis of the results of the preliminary test):

Tester strain S9 Dose level (µL/plate)
TA1535, WP2 uvrA, TA98 ± 5.00, 2.50, 1.25, 0.625, 0.313
TA1537 ± 2.50, 1.25, 0.625, 0.313, 0.156
TA100 ± 2.50, 1.25, 0.625, 0.313, 0.156, 0.0781

No toxicity was observed at any dose level with any tester strain, in the absence or presenceof S9 metabolic activation
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments. Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.
The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.
Vehicle / solvent:
The test item was used as a solution in DMSO.
Controls
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Remarks:
water, DMSO
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2-aminoanthracene
Details on test system and experimental conditions:
Four strains of Salmonella typhimurium (TA1535, TA1537, TA98 and TA100) and a strain of Escherichia coli (WP2 uvrA) were used in this study. Permanent stocks of these strains are kept at -80°C in RTC. Overnight subcultures of these stocks were prepared for each day’s work PRELIMINARY TOXICITY TEST A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in the main assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. MAIN EXPERIMENTS Two experiments were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. In addition, plates were prepared to check the sterility of the test item solutions and the S9 mix and dilutions of the bacterial cultures were plated on nutrient agar plates to establish the number of bacteria in the cultures. The first experiment was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test item and S9 mix or phosphate buffer) were added to molten overlay agar and vortexed. The mixture was then poured onto the surface of a minimal medium agar plate and allowed to solidify prior to incubation. The second experiment was performed using a pre-incubation method. The components were added in turn to an empty test-tube. The incubate was vortexed and placed at 37°C for 30 minutes. Two mL of overlay agar was then added and the mixture vortexed again and poured onto the surface of a minimal medium agar plate and allowed to solidify. INCUBATION AND SCORING The prepared plates were inverted and incubated for approximately 72 hours at 37°C. After this period of incubation, plates were scored by counting the number of revertant colonies on each plate.
Evaluation criteria:
For the test item to be considered mutagenic, two-fold (or more) increases in mean revertant numbers must be observed at two consecutive dose levels or at the highest practicable dose level only. In addition, there must be evidence of a dose-response relationship showing increasing numbers of mutant colonies with increasing dose levels.
Statistics:
The regression analysis fits a regression line to the data by the least squares method, after square root transformation of the plate counts to satisfy normal distribution and homoscedasticity assumptions. The regression equation is expressed as:
y = a +bx
where:
y = transformed revertant numbers
a = intercept
b = slope value
x = dose level (in the units given).
The regression line does not include the untreated control data, but includes the solvent control data.
Regression lines are calculated using a minimum of the three lowest dose levels, and then including the further dose levels in turn. The correlation co-efficient (r), the value of students "t" statistic, and the p-value for the regression lines are also given.

Results and discussion

Test resultsopen allclose all
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
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
Additional information on results:
Preliminary toxicity test
A preliminary toxicity test was undertaken in order to select the concentrations of the test item to be used in theMain Assays. In this test a wide range of dose levels of the test item, set at half-log intervals, were used. Treatments were performed both in the absence and presence of S9 metabolism using the plate incorporation method; a single plate was used at each test point and positive controls were not included. Toxicity was assessed on the basis of a decline in the number of spontaneous revertants, a thinning of the background lawn or a microcolony formation. Neither toxicity, nor relevant increases in revertant numbers were observed with any tester strain, at any dose level, in the absence or presence of S9 metabolism.
Main Assays
Two Main Assays were performed including negative and positive controls in the absence and presence of an S9 metabolising system. Three replicate plates were used at each test point. On the basis of the results obtained in the preliminary toxicity test, in Main Assay I, using the plate incorporation method, the test item was assayed at 5.00, 2.50, 1.25, 0.625 and 0.313 µL/plate with all tester strains.
No toxicity was observed at any dose level with any tester strain, in the absence or presence of S9 metabolic activation.
As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.
Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism

Applicant's summary and conclusion

Conclusions:

The test item did not induce two-fold increases in the number of revertant colonies in the plate incorporation or pre-incubation assay, at any dose level, in any tester strain, in the absence or presence of S9 metabolism.
It is concluded that the test item Neopentyl Glycol Dipelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism,under the reported experimental conditions.
Executive summary:

The test item Neopentyl Glycol Dipelargonate was examined for the ability to induce gene mutations in tester strains of Salmonella typhimurium and Escherichia coli, as measured by reversion of auxotrophic strains to prototrophy. The five tester strains TA1535, TA1537, TA98, TA100 and WP2 uvrA were used. Experiments were performed both in the absence and presence of metabolic activation, using liver S9 fraction from rats pre-treated with phenobarbitone and betanaphthoflavone. The test item was used as a solution in DMSO.

As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed using the same concentrations and including a pre-incubation step for all treatments.

Neither toxicity, nor relevant increase in the number of revertant colonies was observed in the pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism.

No precipitation of the test item was observed at the end of the incubation period, at any concentration in any experiment.

Precipitation of the test item was observed at the end of the incubation period at the highest dose level both in the absence and presence of S9 metabolic activation in both experiments.

It is concluded that the test item Neopentyl Glycol Dipelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.