1-Propanaminium, 3-amino-N-ethyl-N,N-dimethyl-, N-rape-oil acyl derivs., Et sulfates

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the results of the Ames test with the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES', is considered to be non-mutagenic, with and without metabolic activation.

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From July 10, 2017 to July 11, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

S9

Test concentrations with justification for top dose:

Main experiment I:
Dose level (µg/plate)
TA1535 (S±) 400, 200, 100, 50.0, 25.0, 12.5
TA1537 (S-): 100, 50.0, 25.0, 12.5, 6.25
TA1537, TA100 (S+): 200, 100, 50.0, 25.0, 12.5
WP2 uvrA (S±): 800, 400, 200, 100, 50.0, 25.0
TA98
(S−): 1600, 800, 400, 200, 100, 50.0
(S+): 3200, 1600, 800, 400, 200
TA100 (S-): 100, 50.0, 25.0, 12.5, 6.25, 3.13

Main experiment II:
TA1535, WP2 uvrA (S−): 200, 100, 50.0, 25.0, 12.5, 6.25
TA1535 (S+): 400, 200, 100, 50.0, 25.0, 12.5
TA1537, TA100 (S−): 50.0, 25.0, 12.5, 6.25, 3.13, 1.56
TA1537 (S+): 200, 100, 50.0, 25.0, 12.5
WP2 uvrA (S+): 800, 400, 200, 100, 50.0, 25.0
TA98 (S ±): 1600, 800, 400, 200, 100, 50.0
TA100 (S+): 100, 50.0, 25.0, 12.5, 6.25, 3.13

Main experiment III:
TA1535 (S−): 20.0, 10.0, 5.00, 2.50, 1.25
TA1537 (S−): 2.50, 1.25, 0.625, 0.313, 0.156
WP2 uvrA (S−): 10.0, 5.00, 2.50, 1.25, 0.625
TA98 (S−): 40.0, 20.0, 10.0, 5.00, 2.50, 1.25

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

Details on test system and experimental conditions:

Solutions of the test substance, as received, were prepared immediately before use in DMSO. Solutions were prepared on a weight/volume basis without correction for the displacement due to the volume of the test substance. Concentrations were expressed in terms of material as received.

Preliminary toxicity test

A preliminary toxicity test was undertaken in order to select the concentrations of the test substance to be used in the Main Assays. In this test a wide range of dose levels of the test substance, set at half-log intervals, was 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 Assays

Three 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. In addition, plates were prepared to check the sterility of the test substance 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.

Main Assay I was performed using a plate-incorporation method. The components of the assay (the tester strain bacteria, the test substance 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 overlay mixture was composed as follows:
Overlay agar (held at 45°C) 2.0mL
Test or control item solution 0.1mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL
Bacterial suspension 0.1mL

Main Assay II andMain assay III were performed using a pre-incubation method. The components
were added in turn to an empty test-tube:

Bacterial suspension 0.1mL
Test item solution or control item solution 0.05mL
S9 mix or phosphate buffer (pH 7.4, 0.1 M) 0.5mL

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 from the preliminary toxicity test and Main assay I were held at 4°C for approximately 24 hours before scoring, while plates from Main Assay II and Main Assay III were immediately scored by counting the number of revertant colonies on each plate.

Rationale for test conditions:

Solubility of the test substance was evaluated in a preliminary trial using DMSO. This solvent was selected since it is compatible with the survival of the bacteria and the S9 metabolic activity. The test substance was found to be soluble at 100mg/mL. This result permitted a maximum concentration of 5000 µg/plate to be used in the toxicity test by adding 0.1 mL of the test substance solution at 50 mg/mL.

Evaluation criteria:

Results show that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each tester strain. The study was accepted as 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

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

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Toxicity test

The test substance 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.
No precipitation of the test substance was observed at the end of the incubation period at any concentration. Dose related toxic effects, as indicated by complete lack of microbial growth, microcolony formation, thinning of the background lawn and/or reduction in revertant numbers, were observed with all tester strains both in the absence and presence of S9 metabolism.
No relevant increase in revertant numbers was observed with any tester strain, at any dose level, in the absence or presence of S9 metabolic activation.

The maximum concentration of the test substance to be used in the main experiments was selected as the concentration which elicits a moderate toxicity. The number of lower dose levels included in each treatment series was selected in order to have a sufficient number of analysable concentrations.

Main Assay I

Dose related toxic effects, as indicated by clearance of the background lawn and/or reduction in revertant colonies, were observed at higher dose levels. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. The dose range used was modified to take into account the toxicity observed in the first assay.

Main assay II:

No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of S9 metabolism. Precipitation of the test substance, which did not interfere with the scoring, was noted in the presence of S9 metabolism at 1600, 800 and 400 µg/plate. Dose related toxicity was observed with all tester strain/activation condition combinations. However, in the absence of S9 metabolism, treatments following pre-incubation proved to be more toxic than plate incorporation treatments with all tester strains with the exception of TA100 and toxic effects were observed at almost all concentrations tested. Since mutagenic effects have to be evaluated at not toxic dose levels, an additional experiment was performed with these tester strains using a lower concentration range.

Main assay III:

A sufficient number of analysable dose levels was obtained and no increase in revertant numbers was noted with any tester strain, at any concentration tested. The sterility of the S9 mix and of the test substance 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 substances, indicating that the assay system was functioning correctly.

Conclusions:

Under the study conditions, it is concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of S9 metabolism.

Executive summary:

A study was conducted to determine the genotoxic potential of the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%) according to OECD Guideline 471 and EU Method B.13/14, using Ames test, in compliance with GLP. The test was performed using Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA strains, both in the absence and presence of metabolic activation. The test substance (a solution in dimethysulfoxide) was assayed in the preliminary toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Based on results of the preliminary test, Main Assay I was performed, using the plate incorporation method and selected test substance concentrations ranging from 3.13 to 3200 µg/plate with and without metabolic activation. Dose related toxic effects, as indicated by clearance of the background lawn and/or reduction in revertant colonies, were observed at higher dose levels. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. The dose range used (3.13 to 1600 µg/plate) was modified to take into account the toxicity observed in the first assay. No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of metabolic activation. Precipitation of the test substance, which did not interfere with the scoring, was noted in the presence of metabolic activation at 1600, 800 and 400 µg/plate. Dose related toxicity was observed with all tester strain/activation condition combinations. However, in the absence of metabolic activation, treatments following pre-incubation proved to be more toxic than plate incorporation treatments with all tester strains with the exception of TA100 and toxic effects were observed at almost all concentrations tested. Since mutagenic effects have to be evaluated at not toxic dose levels, an additional experiment was performed with these tester strains using a lower concentration range. Main assay III was performed using the pre-incubation method and selected test substance concentration ranged from 0.156 to 40 µg/plate, with and without metabolic activation. A sufficient number of analysable dose levels were obtained and no increase in revertant numbers was noted with any tester strain, at any concentration tested. Results showed that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each tester strain. No plates were lost through contamination or cracking. Therefore study had met all the validity criteria. The test substance 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 metabolic activation. Under the study conditions, it is concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of metabolic activation (RTC, 2017).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

A study was conducted to determine the genotoxic potential of the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES' (active: 104%) according to OECD Guideline 471 and EU Method B.13/14, using Ames test, in compliance with GLP. The test was performed using Salmonella typhimurium TA1535, TA1537, TA98, TA100 and Escherichia coli WP2 uvrA strains, both in the absence and presence of metabolic activation. The test substance (a solution in dimethysulfoxide) was assayed in the preliminary toxicity test at a maximum concentration of 5000 µg/plate and at four lower concentrations spaced at approximately half-log intervals: 1580, 500, 158 and 50.0 µg/plate. Based on results of the preliminary test, Main Assay I was performed, using the plate incorporation method and selected test substance concentrations ranging from 3.13 to 3200 µg/plate with and without metabolic activation. Dose related toxic effects, as indicated by clearance of the background lawn and/or reduction in revertant colonies, were observed at higher dose levels. As no relevant increase in revertant numbers was observed at any concentration tested, a Main Assay II was performed including a pre-incubation step for all treatments. The dose range used (3.13 to 1600 µg/plate) was modified to take into account the toxicity observed in the first assay. No increase in the revertant colonies was noted with any tester strain, at any concentration tested, in the absence or presence of metabolic activation. Precipitation of the test substance, which did not interfere with the scoring, was noted in the presence of metabolic activation at 1600, 800 and 400 µg/plate. Dose related toxicity was observed with all tester strain/activation condition combinations. However, in the absence of metabolic activation, treatments following pre-incubation proved to be more toxic than plate incorporation treatments with all tester strains with the exception of TA100 and toxic effects were observed at almost all concentrations tested. Since mutagenic effects have to be evaluated at not toxic dose levels, an additional experiment was performed with these tester strains using a lower concentration range. Main assay III was performed using the pre-incubation method and selected test substance concentration ranged from 0.156 to 40 µg/plate, with and without metabolic activation. A sufficient number of analysable dose levels were obtained and no increase in revertant numbers was noted with any tester strain, at any concentration tested. Results showed that mean plate counts for untreated and positive control plates fell within the normal range based on historical control data. The estimated numbers of viable bacteria/plate (titre) fell in the range of 100 - 500 million for each tester strain. No plates were lost through contamination or cracking. Therefore study had met all the validity criteria. The test substance 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 metabolic activation. Under the study conditions, it is concluded that the test substance does not induce reverse mutation in Salmonella typhimurium or Escherichia coli in the absence or presence of metabolic activation (RTC, 2017).

Justification for classification or non-classification

Based on the results of the Ames test with the test substance, 'C18-unsatd and C22-unsatd. AAP EDM-ES', is concluded not to warrant classification for genotoxicity according to EU CLP criteria (Regulation 1272/2008/EC).