Rape oil, reaction products with diethylenetriamine, di-Me sulfate-quaternized

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Based on the available study results from read across in vitro Ames assays, the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' is considered to be non-mutagenic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Study period:

From April 17, 2015 to May 01, 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with some test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
- In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with most test conditions.

Vehicle / solvent:

Ethanol (solution in EtOH at a concentration of approximately 500 mg/mL)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

- The assay was performed in two phases, using the plate incorporation method. The first phase (1.), the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase (2.), the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.
1. Vehicle control, positive controls and eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
2. Six dose levels of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.
- The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Rationale for test conditions:

Dose-range finding test
Cytotoxicity

Evaluation criteria:

- Scoring:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
- Evaluation:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Key result

Species / strain:

other: S. typhymurium TA98, TA100, TA1535 and TA1537 + E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. Initial Toxicity-Mutation Assay:
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with some test conditions.
2. Confirmatory Mutagenicity Assay:
No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with most test conditions.
- All criteria for a valid study were met as described in the protocol.

Conclusions:

Based on the results of the read across study, a similar absence of mutganic response in Ames test is expected for the test substance.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the read across substance, 'di-C16 and C18-unsatd. AAEMIM-MS', (active: 93.67%)according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 uvr A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The plates were inverted and incubated for 48 to 72 h at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL (in ethanol) and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.In the initial toxicity-mutation assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation(aroclor induced rat liver S9). Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with some test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay wasdetermined to be5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with most test conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.The negative and positive controls gave results within the expected range; hence the experiment was considered valid.Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation(Bioreliance, 2015). Based on the results of the read across study, a similar absence of mutganic response in Ames test is expected for the test substance,di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS.

Reference 2

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:

From June 16, 2009 to July 08, 2009

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

KL2 due to RA

Justification for type of information:

Refer to section 13 of IUCLID for details on the read-across justification. The study with the read across substance is considered sufficient to fulfil the information requirements as further explained in the provided endpoint summary.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9

Test concentrations with justification for top dose:

- In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 200 mg/mL and a 25 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No toxicity or positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate.
- In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 1500 μg per plate. No appreciable toxicity was observed.

Vehicle / solvent:

Tetrahydrofuran (THF) for the test substance and DMSO for the positives controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

- The assay was performed in two phases, using the plate incorporation method. The first phase (1.), the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase (2.), the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance.
1. Vehicle control, positive controls and eight dose levels of the test substance were plated, two plates per dose, with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9.
2. Six dose levels of test substance along with appropriate vehicle control and positive controls were plated with overnight cultures of TA98, TA100, TA1535, TA1537 and WP2 uvrA on selective minimal agar in the presence and absence of Aroclor-induced rat liver S9. All dose levels of test substance, vehicle control and positive controls were plated in triplicate.
- The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.

Rationale for test conditions:

Guidelines
Dose-range finding test
Cytotoxicity - Precipitation

Evaluation criteria:

- Scoring:
The condition of the bacterial background lawn was evaluated for evidence of test substance toxicity by using a dissecting microscope. Precipitate was evaluated after the incubation period by visual examination without magnification. Toxicity and degree of precipitation were scored relative to the vehicle control plate using the codes shown in the following table. As appropriate, colonies were enumerated either by hand or by machine.
- Evaluation:
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two increasing concentrations of test substance.

Statistics:

For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative 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:

cytotoxicity

Remarks:

at 1500 and 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

1. Initial Toxicity-Mutation Assay:
The test substance formed workable suspensions in THF from 60 to 200 mg/mL, a soluble but cloudy solution at 20 mg/mL and soluble and clear solutions from 0.060 to 6.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No background lawn toxicity was observed. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

2. Confirmatory Mutagenicity Assay:
The test substance formed workable suspensions in THF from 20 to 200 mg/mL, a soluble but cloudy solution at 6.0 mg/mL and a soluble and clear solution at 2.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.

- All criteria for a valid study were met as described in the protocol.

Conclusions:

Based on the results of the read across study, similar absence of mutagenic response in Ames test is expected for the test substance.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the read across substance, 'di-C18-22 AAEMIM-MS', in an Ames test, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 uvr A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No toxicity or positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. The test substance formed workable suspensions in the vehicle (tetrahydrofuran) from 20 to 200 mg/mL, a soluble but cloudy solution at 6.0 mg/mL and a soluble and clear solution at 2.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.The negative and positive controls gave results within the expected range; hence the experiment was considered valid. Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2009). Based on the results of the read across study, similar absence of mutagenic response in Ames test is expected for the test substance, di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Study 1:

An in vitro study was conducted to determine the mutagenic potential of the read across substance, 'di-C18-22 AAEMIM-MS', in an Ames test, according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 uvr A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The plates were inverted and incubated for 48 to 72 hours at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted.In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate. No toxicity or positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. Precipitate was observed beginning at 1500 μg per plate. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay was 5000 μg per plate. In the confirmatory mutagenicity assay, the dose levels tested were 50, 150, 500, 1500 and 5000 μg per plate. The test substance formed workable suspensions in the vehicle (tetrahydrofuran) from 20 to 200 mg/mL, a soluble but cloudy solution at 6.0 mg/mL and a soluble and clear solution at 2.0 mg/mL. Precipitate was observed beginning at 1500 μg per plate. No appreciable toxicity was observed. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.The negative and positive controls gave results within the expected range; hence the experiment was considered valid.Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2009).

Study 2:

An in vitro study was conducted to determine the mutagenic potential of the read across substance, 'di-C16 and C18-unsatd. AAEMIM-MS', (active: 93.67%)according to OECD Guideline 471, in compliance with GLP. Salmonella typhimurium strains TA 98, TA100, TA 1535 and TA 1537 as well as Escherichia coli strain WP2 uvr A were used in this experiment. The assay was performed in two phases, using the plate incorporation method. The first phase, the initial toxicity-mutation assay, was used to establish the dose-range for the confirmatory mutagenicity assay and to provide a preliminary mutagenicity evaluation. The second phase, the confirmatory mutagenicity assay, was used to evaluate and confirm the mutagenic potential of the test substance. The plates were inverted and incubated for 48 to 72 h at 37±2°C. Plates that were not counted immediately following the incubation period were stored at 2-8°C until colony counting could be conducted. In the initial toxicity-mutation assay, the maximum dose tested was 5000 μg per plate; this dose was achieved using a concentration of 100 mg/mL (in ethanol) and a 50 μL plating aliquot. The dose levels tested were 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg per plate.In the initial toxicity-mutation assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation(aroclor induced rat liver S9). Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with some test conditions. Based on the findings of the initial toxicity-mutation assay, the maximum dose plated in the confirmatory mutagenicity assay wasdetermined to be5000 μg per plate. In the confirmatory mutagenicity assay, no positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation. The dose levels tested were 15, 50, 150, 500, 1500 and 5000 μg per plate. Precipitate was observed at 5000 μg per plate. Toxicity was observed beginning at 1500 or at 5000 μg per plate with most test conditions. No positive mutagenic responses were observed with any of the tester strains in either the presence or absence of S9 activation.The negative and positive controls gave results within the expected range; hence the experiment was considered valid.Under the study conditions, the read across substance was determined to be non-mutagenic in Ames test, with or without metabolic activation(Bioreliance, 2015).

Based on the available study results from read across in vitro Ames assays, the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' is considered to be non-mutagenic.

Justification for classification or non-classification

Based on the available study results from read across in vitro Ames assays, the test substance, 'di-C16-18-satd. and C18-24-unsatd. AAEMIM-MS' does not warrant classification for genotoxicity, according to the EU CLP criteria (Regulation 1272/2008/EC).