Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

None of the available studies both on the target substance and on analogues show any effect in terms of genetic toxicity

Link to relevant study records

Referenceopen allclose all

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
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
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.
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.
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
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.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.

Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use cells were cleansed of pre-existing mutants.
Additional strain / cell type characteristics:
other: TK
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results obtained in the preliminary toxicity trial, 2 independent assays for mutation to trifluorothymidine resistance were performed with this concentration: Assay n°1: with and without S9: 2500, 791, 250, 79.1, 25.0 g/mL Assay n°2 : without S9: 2500, 1250, 625, 313, 156, 78.1 g/mL Assay n°3: with S9: 2500, 1250, 625, 313, 156, 78.1g/mL
Vehicle / solvent:
Solvent/vehicle:
- acetone batch no. : 07L110525 obtained from BDH and 12L110525 obtained from VWR
- DMSO batch no.: 1473729V obtained from Fluka
Test item was evaluated in a preliminary trial using DMSO and acetone. This solvent were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents. The test item is soluble in the acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Remarks:
Methylmethanesulphonate served as positive control in the absence of S9 and Benzo(a)pyrene (B(a)P) served as positive control in the presence of S9
Details on test system and experimental conditions:
1. Preparation of test cell cultures: A cell suspension (1X10^6 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

2. Cytotoxicity assay: a preliminary cytotoxicity test was performed in order to select appopriated dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatment were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9. Wells containing viable clones were identified by eye using background illumination and then counted.

3. Mutation assay
3.1. Treatment of cell cultures: Preparation of test cultures was performed as described above. Duplicate cultures were prepared at each test point, with the exception of the positive controls, which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment was performed, where a longer treatment time (24 hours) was used in the absence of S9 metabolic activation.
After washing in PBS, cells were re suspended in fresh complete medium and cell densities were determined. The cultures were incubated at 37°C in 5% CO2 atmosphere to allow for expression of the mutant phenotype.

3.2. Expression period: During the expression period, the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determine and adjusted.

3.3. Plating for 5-trifluorothymidine resistance: after dilution, the cell suspensions in complete medium were suplemented with trifluorothymidine and an estimated 2X 10^3 cells were plated in each well of four 96-well plates. Plate were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containg large colonies as well as the number of those containing small colonies were scored.

3.4. Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of 2 96-wells plates. These plates were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
1. Acceptance criteria: The assay was considered valid if the following criteria were met: - The cloning efficiencies at Day 2 in the solvent/vehicle control cultures in the absence of S9 metabolic activation fell within the range od 65-120% - The solvent/vehicle control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8-32 - The mutant frequencies in the solvent/vehicle control cultures fell within the range of 50-200X10^6 viable cells. - The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). 2. Criteria for OUTCOME of assay: For a test item to be considered mutagenic in this assay, it is required that: - The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126X10^-6) at one or more doses. - There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria are dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990)
Species / strain:
mouse lymphoma L5178Y cells
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'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item Trimethylolpropane Tripelargonate was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistance mutants in mouse lymphoma L5178 cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. This trial was performed in according to OECD 476 guidelines and GLP.

A preliminary solubility trial indicated that the maximum feasible concentration of the test item in the final treatment medium was 1250 µg/mL using acetone as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the presence and absence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, and 4.88 µg/mL.

No relevant toxicity was observed at any concentration tested, in any treatment series. Slight opacity was observed at the end of treatment period at the five highest concentrations. No precipitation was noted at any concentration tested.

Based on the results obtained in the preliminary toxicity trial, two independent assays for mutation to trifluorothymidine resistance were performed using the following dose levels:

Assay n°1 with and without S9, treatment of 3 hours doses: 2500, 791, 250, 79.1, 25 µg/mL

Assay n°2: without S9, treatment of 24 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

Assay n°3 with S9, treatment of 3 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

No relevant toxicity was observed at any concentration tested in any treatment series. in both experiments, at the end of the treatment time, test item particles in suspension were noted at the highest or two highest concentrations. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolim.

Negative and positive control treatments were included in each mutation experiment, both in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is conclude that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Remarks:
The two substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which are closely related to substances of known low toxicity.
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The source and the target substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which have no structural alerts for toxicity and which are closely related to substances of known low toxicity. More details are reported in the document attached in section 13.
Qualifier:
according to
Guideline:
other: OECD 487 (in vitro micronucleus test in human lymphocytes)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation
Species / strain / cell type:
other: peripheral blood for lymphocytes cultures
Details on mammalian cell type (if applicable):
For each experiment, whole blood was collected from healthy volunteer donors. The volunteers were non-smoker and were not receiving any medication or radiation exposure prior to the time of sampling.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First experiement: Dose levels 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 µg/mL
Second experiment: Dose levels 204, 117, 66.6, 38.1, 21.8, 12.4, 7.10 and 4.06 µg/mL
Vehicle / solvent:
DMSO and acetone. These solvents were selected since they are compatible with the survival of the cells and the S9 metabilic activity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Remarks:
Mitomycin C and colchicine in absence of S9 metabolic activation and Cyclophosphamide in presence of S9 metabolic activation
Details on test system and experimental conditions:

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolims, respectively.

The haverest time of 32 hours corresponding to approximately 2.0 cell cycles was used.
As a negative results were obtained, a second experiment was performed in the absence of S9 metabolism usung approximately the same haverst time. A continous treatment until harvest at 31 hours was used.

48 hours after initiation of lymphocytes cultures are initiated, they are centrifuged at 1000 rpm for 10 min and the culture medium is decanted and replaced with treatment medium.
After exposure period the lymphocytes cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.

A few drops of the cell suspension obtained in this way were dropped onto clean, wet grease-free glass slides.
Three slides were prepared for each test point and each was labelled with the identity of the culture.

The slides were allowed to air dry kept at room temperature prior to staining with a solution of Acridine Orange in PBS.
The slides will be randomly assigned code numbers by a person not subsequently involved in slide evaluation and any other identification marks will be concealed. For the three selected doses, for the solvent and for the negative and positive controls, at least 1000 binucleated cells per cell cultures will be scored to assess the frequency of micronucleated cells, but with the presence of cytokinesis block, a greater magnitude of response may be observed in mononucleated cells in compare with binucleated cells, it is why at least 1000 mononucleated cells per culture may be scored instead of binucleated cells.
Evaluation criteria:
The criteria for identifying micronuclei are as follows: (i) the micronucleus diameter must be less than 1/3 of the diameter of the nucleus, (ii) the micronucleus diameter must be greater than 1/16 of the diameter of the nucleus, (iii) no overlapping with the nucleus must be osberved, (iv) the aspect must be the same as the chromatin. In this assay, the test item is considered as clearly positive if the following criteria are met: - Significant increases in the proportion of micronucleus cells over the concurrent controls occur at one or more concentrations. - The proportion of micronucleus cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies. - There is a significant dose-relationship
Statistics:
For the statistical analysis, a modified chi-squared test was used tocompare the number of binucleated cells with micronuclei in control and treated cultures.
Species / strain:
lymphocytes: human lymphocytes
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: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

On the basis of this results, it is concluded that TMP perlargonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item TMP pelargonate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. This study was performed in accordance with OECD guideline 487 and GLP without significant deviation.

Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively.

The harvest time of 32 hours corresponding to approximately 2.0 cells cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 31 hours was used. Solutions of the test item were prepared in acetone.

For the main experiment, the maximum dose level for treatment was selected in agreement with the sutdy protocol and on the basis of the solubility of the test item. Dose levels of 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the first main experiment. Based on the results obtained, dose levels of 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the second main experiment.

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronuclei taking into account the cytotoxicity of the test item treatments, calculated by the cytokinesis-block proliferation index (CBPI), and the observed test item precipitation by the end of treatment.

1000 binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrend vehicule control value was observed at any dose level in any treatment series.

Statistically increase in the incidence of micronuclei cells were observed following treatments with the positive controls Cyclosphosphamide, Motomycin-C and Colchicine indicating the correct functioning of the test system.

It is concluded that TMP perlagonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Remarks:
The two substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which are closely related to substances of known low toxicity.
Adequacy of study:
key study
Study period:
2013-09-24 - 2013-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The source and the target substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which have no structural alerts for toxicity and which are closely related to substances of known low toxicity. More details are reported in the document attached in section 13.
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
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:
Toxicity test:

50, 158, 500, 1580 and 5000 µg/plate

Main Assay I:
5000, 2500, 1250, 625 and 313 µg/plate (-S9)
1600, 800, 400, 200, 100 µg/plate (+S9)

In Main Assay II, the test item was assayed at the same dose levels used in Main Assay I.
Vehicle / solvent:
The test item was used as a solution in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone, DMSO
Positive controls:
yes
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.
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:
TOXICITY TEST
The test item TMP pelargonate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, both in the absence or presence of S9 metabolism. ASSAY FOR REVERSE MUTATION
Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate (-S9) and 1600, 800, 400, 200, 100 μg/plate (+S9). No toxicity was observed with any tester strain at any dose level both in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations, both in the absence and presence of metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentrations in the presence of S9 metabolism only. No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Interpretation of results (migrated information):
negative

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 TMP pelargonate does not induce reverse
mutation in Salmonella typhimurium or Escherichia coli under the reported
experimental conditions.
Executive summary:

The test item TMP pelargonate 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 acetone.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

As no relevant increase in revertant numbers was observed at any concentration tested in Main Assay I, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I.

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 TMP pelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The source and the target substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which have no structural alerts for toxicity and which are closely related to substances of known low toxicity. More details are reported in the document attached in section 13.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
TK
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
L5178Y TK+/- (Clone 3.7.2C) mouse lymphoma cells were obtained from American Type Culture Collection, Rockville, Maryland (ATCC code: CRL 9518). The generation time and mutation rates (spontaneous and induced) have been checked in this laboratory. The cells are checked at regular intervals for the absence of mycoplasmal contamination.

Permanent stocks of the L5178Y TK+/- cells are stored in liquid nitrogen, and subcultures are prepared from the frozen stocks for experimental use. Prior to use cells were cleansed of pre-existing mutants.
Additional strain / cell type characteristics:
other: TK
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the results obtained in the preliminary toxicity trial, 2 independent assays for mutation to trifluorothymidine resistance were performed with this concentration: Assay n°1: with and without S9: 2500, 791, 250, 79.1, 25.0 g/mL Assay n°2 : without S9: 2500, 1250, 625, 313, 156, 78.1 g/mL Assay n°3: with S9: 2500, 1250, 625, 313, 156, 78.1g/mL
Vehicle / solvent:
Solvent/vehicle:
- acetone batch no. : 07L110525 obtained from BDH and 12L110525 obtained from VWR
- DMSO batch no.: 1473729V obtained from Fluka
Test item was evaluated in a preliminary trial using DMSO and acetone. This solvent were selected since they are compatible with the survival of the cells and the S9 metabolic activity. In addition, there are many historical control data demonstrating that no mutagenic effects are induced by these solvents. The test item is soluble in the acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
methylmethanesulfonate
Remarks:
Methylmethanesulphonate served as positive control in the absence of S9 and Benzo(a)pyrene (B(a)P) served as positive control in the presence of S9
Details on test system and experimental conditions:
1. Preparation of test cell cultures: A cell suspension (1X10^6 cells/mL) in complete medium was prepared. A common pool was used for each experiment to prepare the test cultures in appropriately labelled conical screw-cap tissue culture tubes.

2. Cytotoxicity assay: a preliminary cytotoxicity test was performed in order to select appopriated dose levels for the mutation assays. In this test a wide range of dose levels of the test item was used and the survival of the cells was subsequently determined. Treatment were performed in the absence and presence of S9 metabolic activation for 3 hours and for 24 hours only in the absence of S9. Wells containing viable clones were identified by eye using background illumination and then counted.

3. Mutation assay
3.1. Treatment of cell cultures: Preparation of test cultures was performed as described above. Duplicate cultures were prepared at each test point, with the exception of the positive controls, which were prepared in a single culture. In the first experiment, the cells were exposed to the test item for a short treatment time (3 hours). Since negative results were obtained, a second experiment was performed, where a longer treatment time (24 hours) was used in the absence of S9 metabolic activation.
After washing in PBS, cells were re suspended in fresh complete medium and cell densities were determined. The cultures were incubated at 37°C in 5% CO2 atmosphere to allow for expression of the mutant phenotype.

3.2. Expression period: During the expression period, the cell populations were subcultured in order to maintain them in exponential growth. At the end of this period, the cell densities of each culture were determine and adjusted.

3.3. Plating for 5-trifluorothymidine resistance: after dilution, the cell suspensions in complete medium were suplemented with trifluorothymidine and an estimated 2X 10^3 cells were plated in each well of four 96-well plates. Plate were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified by eye using background illumination and counted. In addition, the number of wells containg large colonies as well as the number of those containing small colonies were scored.

3.4. Plating for viability: After dilution, in complete medium A (20%), an estimated 1.6 cells/well were plated in each well of 2 96-wells plates. These plates were incubated at 37°C in a 5% CO2 atmosphere for 14 days and wells containing clones were identified as above and counted.
Evaluation criteria:
1. Acceptance criteria: The assay was considered valid if the following criteria were met: - The cloning efficiencies at Day 2 in the solvent/vehicle control cultures in the absence of S9 metabolic activation fell within the range od 65-120% - The solvent/vehicle control growth factor in the absence of S9 metabolic activation over 2 days fell within the range of 8-32 - The mutant frequencies in the solvent/vehicle control cultures fell within the range of 50-200X10^6 viable cells. - The positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value). 2. Criteria for OUTCOME of assay: For a test item to be considered mutagenic in this assay, it is required that: - The induced mutant frequency (IMF) is higher than the global evaluation factor (GEF) suggested for the microwell method (126X10^-6) at one or more doses. - There is a significant dose-relationship as indicated by the linear trend analysis. Results which only partially satisfy the above criteria are dealt with on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity require careful interpretation when assessing their biological significance. Any increase in mutant frequency should lie outside the historical control range to have biological relevance.
Statistics:
Statistical analysis was performed according to UKEMS guidelines (Robinson W.D., 1990)
Species / strain:
mouse lymphoma L5178Y cells
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'.
Conclusions:
Interpretation of results (migrated information):
negative

It is concluded that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.
Executive summary:

The test item Trimethylolpropane Tripelargonate was examined for mutagenic activity by assaying for the induction of 5 -trifluorothymidine resistance mutants in mouse lymphoma L5178 cells after in vitro treatment, in the absence and presence of S9 metabolic activation, using a fluctuation method. This trial was performed in according to OECD 476 guidelines and GLP.

A preliminary solubility trial indicated that the maximum feasible concentration of the test item in the final treatment medium was 1250 µg/mL using acetone as solvent. On the basis of this result, a cytotoxicity assay was performed. Both in the presence and absence of S9 metabolic activation, the test item was assayed at a maximum dose level of 1250 µg/mL and at a wide range of lower levels: 625, 313, 156, 78.1, 39.1, 19.5, 9.77, and 4.88 µg/mL.

No relevant toxicity was observed at any concentration tested, in any treatment series. Slight opacity was observed at the end of treatment period at the five highest concentrations. No precipitation was noted at any concentration tested.

Based on the results obtained in the preliminary toxicity trial, two independent assays for mutation to trifluorothymidine resistance were performed using the following dose levels:

Assay n°1 with and without S9, treatment of 3 hours doses: 2500, 791, 250, 79.1, 25 µg/mL

Assay n°2: without S9, treatment of 24 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

Assay n°3 with S9, treatment of 3 hours doses: 2500, 1250, 625, 313, 156, 78.1 µg/mL

No relevant toxicity was observed at any concentration tested in any treatment series. in both experiments, at the end of the treatment time, test item particles in suspension were noted at the highest or two highest concentrations. No increases in mutant frequencies were observed following treatment with the test item, in the absence or presence of S9 metabolim.

Negative and positive control treatments were included in each mutation experiment, both in the absence and presence of S9 metabolism. The mutant frequencies in the solvent control cultures fell within the normal range. Marked increases were obtained with the positive control treatments indicating the correct functioning of the assay system.

It is conclude that Trimethylolpropane Tripelargonate does not induce mutation at the TK locus of L5178Y mouse lymphoma cells in vitro in the absence or presence of S9 metabolic activation, under the reported experimental conditions.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The two substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which are closely related to substances of known low toxicity.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
other: OECD 487 (in vitro micronucleus test in human lymphocytes)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
The test item was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation
Species / strain / cell type:
other: peripheral blood for lymphocytes cultures
Details on mammalian cell type (if applicable):
For each experiment, whole blood was collected from healthy volunteer donors. The volunteers were non-smoker and were not receiving any medication or radiation exposure prior to the time of sampling.
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
First experiement: Dose levels 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 µg/mL
Second experiment: Dose levels 204, 117, 66.6, 38.1, 21.8, 12.4, 7.10 and 4.06 µg/mL
Vehicle / solvent:
DMSO and acetone. These solvents were selected since they are compatible with the survival of the cells and the S9 metabilic activity
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: colchicine
Remarks:
Mitomycin C and colchicine in absence of S9 metabolic activation and Cyclophosphamide in presence of S9 metabolic activation
Details on test system and experimental conditions:

In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolims, respectively.

The haverest time of 32 hours corresponding to approximately 2.0 cell cycles was used.
As a negative results were obtained, a second experiment was performed in the absence of S9 metabolism usung approximately the same haverst time. A continous treatment until harvest at 31 hours was used.

48 hours after initiation of lymphocytes cultures are initiated, they are centrifuged at 1000 rpm for 10 min and the culture medium is decanted and replaced with treatment medium.
After exposure period the lymphocytes cultures were centrifuged for 10 minutes at 1000 rpm and the supernatant was removed. The cells were resuspended in hypotonic solution. Fresh methanol/acetic acid fixative was then added. After centrifugation and removal of this solution, the fixative was changed several times by centrifugation and resuspension.

A few drops of the cell suspension obtained in this way were dropped onto clean, wet grease-free glass slides.
Three slides were prepared for each test point and each was labelled with the identity of the culture.

The slides were allowed to air dry kept at room temperature prior to staining with a solution of Acridine Orange in PBS.
The slides will be randomly assigned code numbers by a person not subsequently involved in slide evaluation and any other identification marks will be concealed. For the three selected doses, for the solvent and for the negative and positive controls, at least 1000 binucleated cells per cell cultures will be scored to assess the frequency of micronucleated cells, but with the presence of cytokinesis block, a greater magnitude of response may be observed in mononucleated cells in compare with binucleated cells, it is why at least 1000 mononucleated cells per culture may be scored instead of binucleated cells.
Evaluation criteria:
The criteria for identifying micronuclei are as follows: (i) the micronucleus diameter must be less than 1/3 of the diameter of the nucleus, (ii) the micronucleus diameter must be greater than 1/16 of the diameter of the nucleus, (iii) no overlapping with the nucleus must be osberved, (iv) the aspect must be the same as the chromatin. In this assay, the test item is considered as clearly positive if the following criteria are met: - Significant increases in the proportion of micronucleus cells over the concurrent controls occur at one or more concentrations. - The proportion of micronucleus cells at such data points exceeds the normal range. If the increases fall within the range of values normally observed in the negative control cultures, the test item can not be classified as positive. Any significant increases over the concurrent negative controls are therefore compared with historical control values derived from recent studies. - There is a significant dose-relationship
Statistics:
For the statistical analysis, a modified chi-squared test was used tocompare the number of binucleated cells with micronuclei in control and treated cultures.
Species / strain:
lymphocytes: human lymphocytes
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: strain/cell type: lymphocytes
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information):
negative

On the basis of this results, it is concluded that TMP perlargonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.
Executive summary:

The test item TMP pelargonate was assayed for the ability to induce micronuclei in human lymphocytes, following in vitro treatment in the presence and absence of S9 metabolic activation. This study was performed in accordance with OECD guideline 487 and GLP without significant deviation.

Two main experiments were performed. In the first experiment, the cells were treated for 3 hours in the presence and absence of S9 metabolism, respectively.

The harvest time of 32 hours corresponding to approximately 2.0 cells cycles was used. As negative results were obtained, a second experiment was performed in the absence of S9 metabolism using approximately the same harvest time. A continuous treatment until harvest at 31 hours was used. Solutions of the test item were prepared in acetone.

For the main experiment, the maximum dose level for treatment was selected in agreement with the sutdy protocol and on the basis of the solubility of the test item. Dose levels of 625, 357, 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the first main experiment. Based on the results obtained, dose levels of 204, 117, 66.6, 38.1, 21.8, 12.4, 7.11 and 4.06 g/mL were used for the second main experiment.

Each experiment included appropriate negative and positive controls. Two replicate cell cultures were prepared at each test point.

The actin polymerisation inhibitor cytochalasin B was added prior to the targeted mitosis to allow the selective analysis of micronucleus frequency in binucleated cells.

Dose levels were selected for the scoring of micronuclei taking into account the cytotoxicity of the test item treatments, calculated by the cytokinesis-block proliferation index (CBPI), and the observed test item precipitation by the end of treatment.

1000 binucleated cells per culture were scored to assess the frequency of micronucleated cells. Following treatment with the test item, no statistically significant increase in the incidence of micronucleated cells over the concurrend vehicule control value was observed at any dose level in any treatment series.

Statistically increase in the incidence of micronuclei cells were observed following treatments with the positive controls Cyclosphosphamide, Motomycin-C and Colchicine indicating the correct functioning of the test system.

It is concluded that TMP perlagonate does not induce micronuclei in human lymphocytes after in vitro treatment, under the reported experimental conditions.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2013-09-24 - 2013-09-25
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
The two substances share structural similarities with common functional groups, esters, and side chains varying in their length. Moreover, the side chains are chemically simple structures which are closely related to substances of known low toxicity.
Reason / purpose:
read-across source
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
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:
Toxicity test:

50, 158, 500, 1580 and 5000 µg/plate

Main Assay I:
5000, 2500, 1250, 625 and 313 µg/plate (-S9)
1600, 800, 400, 200, 100 µg/plate (+S9)

In Main Assay II, the test item was assayed at the same dose levels used in Main Assay I.
Vehicle / solvent:
The test item was used as a solution in acetone.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
acetone, DMSO
Positive controls:
yes
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.
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:
TOXICITY TEST
The test item TMP pelargonate was assayed in the toxicity test at a maximum dose level of 5000 μg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 μg/plate. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentration in the absence of metabolic activation and at the two highest concentrations in the presence of metabolic activation. No toxicity was observed with any tester strain at any dose level, both in the absence or presence of S9 metabolism. ASSAY FOR REVERSE MUTATION
Two experiments were performed. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate (-S9) and 1600, 800, 400, 200, 100 μg/plate (+S9). No toxicity was observed with any tester strain at any dose level both in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the two highest concentrations, both in the absence and presence of metabolic activation. As no relevant increase in revertant numbers was observed at any concentration tested, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I. No toxicity was observed with any tester strain at any dose level in the absence or presence of S9 metabolism. Precipitation of the test item, which did not interfere with the scoring, was observed at the end of the incubation period at the highest concentrations in the presence of S9 metabolism only. No relevant increase in the number of revertant colonies was observed in the plate incorporation or pre-incubation assay, at any dose level, with any tester strain, in the absence or presence of S9 metabolism. The sterility of the S9 mix and of the test item solutions was confirmed by the absence of colonies on additional agar plates spread separately with these solutions. Marked increases in revertant numbers were obtained in these tests following treatment with the positive control items, indicating that the assay system was functioning correctly.
Remarks on result:
no mutagenic potential (based on QSAR/QSPR prediction)
Conclusions:
Interpretation of results (migrated information):
negative

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 TMP pelargonate does not induce reverse
mutation in Salmonella typhimurium or Escherichia coli under the reported
experimental conditions.
Executive summary:

The test item TMP pelargonate 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 acetone.

No toxicity was observed with any tester strain at any dose level, in the absence or presence of S9 metabolism. On the basis of toxicity test results, in Main Assay I, using the plate incorporation method, the test item was assayed at the following dose levels: 5000, 2500, 1250, 625 and 313 μg/plate.

As no relevant increase in revertant numbers was observed at any concentration tested in Main Assay I, a pre-incubation step was included for all treatments of Main Assay II. The test item was assayed at the same dose levels used in Main Assay I.

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 TMP pelargonate does not induce reverse mutation in Salmonella typhimurium or Escherichia coli under the reported experimental conditions.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Justification for classification or non-classification

As no effects on Ames bacteria, human lymphocytes and observed micronuclei, the substance is not to be classified according to the criteria described in EU Regulation No. 12 72/2008 on the Classification, Labelling and Packaging of Substances and Mixtures (CLP).