Fatty acids, rape-oil, hydrogenated, 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 in vitro assays with the test as well as read across substance, the test substance, 'di-C18-22 AAEMIM-MS' is considered to be non-genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 16, 2009 to July 08, 2009

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

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:

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:
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:

Under the study conditions, the test substance did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor-induced rat liver S9.

Executive summary:

An in vitro study was conducted to determine the mutagenic potential of the test 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 test substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2009).

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From December 22, 2003 to January 12, 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

February 1988

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254-induced rat liver S9

Test concentrations with justification for top dose:

Preliminary toxicity assay:
Without metabolic activation (4 h treatment): 2.5, 5, 10, 20, 30 40 and 50 µg/mL
With metabolic activation (20 h treatment): 0.625, 1.25, 2.5, 5, 10 and 20 µg/mL
With metabolic activation (4 h treatment): 2.5, 5, 10, 20, 30, 40 and 50 µg/mL

Vehicle / solvent:

Ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

- Exposure of 4 or 20 hours
- Concentrations from 0 to 100 μg/mL
- With or without metabolic activation (S9 mix)
- Negative (vehicle) and positive controls included

Test system: Chinese hamster ovary (CHO-K1) cells (repository number CCL 61) were obtained from American Type Culture Collection, Manassas, VA, on 29 May 1997. In order to assure the karyotypic stability of the cell line, working cell stocks were not used beyond passage 20. The frozen lot of cells was tested using the Hoechst staining procedure and found to be free of mycoplasma contamination. This cell line has an average cell cycle time of 10-14 h with a modal chromosome number of 20. The use of CHO cells has been demonstrated to be an effective method of detection of chemical clastogens.

Rationale for test conditions:

- Guideline
- Cytotoxicity

Evaluation criteria:

- Chromosome damages
- Number of chromosomal aberrations

The toxic effects of treatment were based upon cell growth inhibition relative to the solvent-treated control and are presented for the toxicity and aberration studies. The number and types of aberrations found, the percentage of structurally and numerically damaged cells (percent aberrant cells) in the total population of cells examined, and the mean aberrations per cell were calculated and reported for each group. Chromatid and isochromatid gaps are presented in the data but are not included in the total percentage of cells with one or more aberrations or in the frequency of structural aberrations per cell. Statistical analysis of the percent aberrant cells was performed using the Fisher's exact test. Fisher's exact test was used to compare pairwise the percent aberrant cells of each treatment group with that of the solvent control. In the event of a positive Fisher's exact test at any test substance dose level, the Cochran-Armitage test was used to measure dose-responsiveness.

All conclusions were based on sound scientific basis; however, as a guide to interpretation of the data, the test substance was considered to induce a positive response when the percentage of cells with aberrations is increased in a dose-responsive manner with one or more concentrations being statistically significant. However, values that are statistically significant but do not exceed the range of historic solvent controls may be judged as not biologically significant. Test substances not demonstrating a statistically significant increase in aberrations will be concluded to be negative. Negative results with metabolic activation may need to be confirmed on a case-by-case basis. In those cases where confirmation of negative results is not necessary, justification will be provided.

Statistics:

According to guideline

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not valid

Positive controls validity:

valid

 Summary:

Treatment time	Recovery time	Harvest time	S9	Toxicity* at highest dose scored (µg/mL)	Mitotic index reduction **	LED^1for structural aberrations (µg/mL)	LED^1for numerical aberrations
4 h	16 h	20 h	`-	58% at 30	29%	None	None
20 h	0 h	20 h	`-	52% at 20	10%	None	None
4 h	16 h	20 h	`+	53% at 100	35%	None	None

 

* Cell growth inhibition

** Relative to solvent control at high dose evaluated for chromosome aberrations

1 LED = Lowest effective dose

Summary of results:

Treatment	S9 activation	Treatment time (h)	Mean mitotic index	Cells scored	Abberrations per cell (Mean +/- SD)	Cells with abberration
						Numerical (%)	Structural (%)
Ethanol	`-	4	7.9	200	0.005 ±0.071	1.5	0.5
Test substance
5	`-	4	6.1	200	0.005 ±0.071	3	0
10	`-	4	6.5	200	0.005 ±0.071	2.5	1.5
30	`-	4	5.6	200	0.005 ±0.071	2	2.5
MMC 0.2	`-	4	7	100+	0.3 ±0.0628	1.5	23**
Ethanol	`-	4	8.5	200	0.000 ±0.000	2.5	0
Test substance
5	`-	4	7.3	200	0.010 ±0.10	2.5	1
10	`-	4	6.8	200	0.015 ±0.1222	2.5	1.5
30	`-	4	5.5	200	0.005 ±0.071	3	0.5
CP 10	`-	4	7.3	100+	0.3 ±0.0628	2	24**
Ethanol	`-	20	7.2	200	0.005 ±0.071	2	1.5
Test substance
5	`-	20	7.2	200	0.015 ±0.1222	2.5	1.5
10	`-	20	6.4	200	0.030 ±0.198	3	2.5
30	`-	20	6.5	200	0.040 ±0.373	2	2
MMC 0.1	`-	20	6.3	200	0.085 ±0.280	2.5	8.5**

** Numerical aberrations are out of 200 cells scored.

Treatment:Cells from all treatment conditions were harvested 20 h after the initiation of the treatments.

Aberrations per cell:Severely damaged cells were counted as aberrations

Percent aberrant cells:*p≤0.05; **, p≤0.01; using Fishers exact test

Conclusions:

Under the study conditions, the substance was not clastogenic in Chinese Hamster Ovary (CHO-K1) cells.

Executive summary:

An in vitro study was conducted to determine clastogenic potential of a formulation containing 39% of the test substance, ‘C18-22 AAEMIM-MS’, in a chromosomal aberration assay, according to OECD Guideline 473, in compliance with GLP. A preliminary toxicity test was performed to establish the dose range for the assay. In the preliminary toxicity assay, the maximum dose tested was 5000 µg/mL. Visible precipitate was observed in the treatment medium at dose level ≥ 15 µg/mL. Dose levels ≤ 5 µg/mL were soluble in treatment medium. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity was observed at dose levels ≥ 50 µg/mL with and without S9, in the 4 h exposure group, and at dose level ≥ 15 µg/mL without S9, in the 20 h continuous exposure group. Based on these findings, the doses chosen for the main assay ranged from 2.5 to 50 µg/mL for both with and without S9, 4 h exposure groups, and ranged from 0.625 to 20 µg/mL for without S9, 20 h continuous exposure group. In the initial chromosome aberration assay, the cells were treated for 4 h and 20 h without S9 and for 4 h with S9. All cells were harvested 20 h after treatment initiation. Visible precipitate observed at dose ≥ 40µg/mL. Selection of doses for microscopic analysis was based on toxicity without S9, 4 and 20 h exposure groups. Due to lack of 50% toxicity, as measured by cell growth or mitotic inhibition, at any dose level in the with S9, 4 h exposure group, this portion of the chromosome aberration assay was repeated at dose levels 20, 30, 40, 50, 75, 100, 125 and 150 µg/mL. In the repeat chromosome aberration assay, the cells were treated for 4 h with S9. All cells were harvested 20 h after treatment initiation. Visible precipitate was observed in treatment medium at dose levels ≥ 40µg/mL. Dose levels ≤30 µg/mL was soluble in treatment medium. Selection of doses for microscopic analysis was based on toxicity in with S9 activated, 4 h exposure group. The percentage of cells with structural or numerical aberrations in test substance treated group was not significantly increased above that of the solvent control at any dose level. Under the study conditions, the substance was considered to be not clastogenic in Chinese Hamster Ovary (CHO-K1) cells, with and without metabolic activation (Bioreliance, 2004).

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

From October 11, 2007 to November 22, 2007

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 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Directive 2000/32/EC, L 1362000, Annex 4A: ”Mutagenicity – In vitro Mammalian Chromosome Aberration Test“, May 19, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Data submitter is data owner or has letter of access

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Chromosome aberration assays detect the induction of chromosome breakage (clastogenesis). Although mutagenic substances produce structural
chromosome aberrations by a variety of mechanisms, the endpoint is a discontinuity in the chromosomal DNA which is left unrejoined, or rejoined
inaccurately, thus producing a mutated chromosome. Chromosome aberrations are generally evaluated in first post treatment mitoses. The majority of chemical mutagens induce aberration of the chromatid type, but chromosome type aberrations also occur.

Species / strain / cell type:

other: Human lymphocytes

Details on mammalian cell type (if applicable):

Blood samples were obtained from healthy donors not receiving medication. Blood cultures were set up in bulk within 24 hrs after collection in 75 cm² cell culture flasks.
- Type and identity of media: DMEM:F12 (Dulbecco's modified eagle medium/ Ham's F12 medium; mixture 1:1; Life Technologies GmbH, 76339 Eggenstein, Germany) containing 10 % FCS (fetal calf serum) provided by PAA Laboratories GmbH (35091 Cölbe, Germany), the antibiotic solution containing 10,000 U/mL penicillin and 10,000 µg/mL streptomycin (SEROMED, D-12247 Berlin). Additionally, the medium was supplemented with Phytohemagglutinin (PHA, final concentration 3 µg/mL, SEROMED), the anticoagulant heparin (25,000 U.S.P.-U/mL, NATTERMANN, 50829 Köln, Germany), and HEPES (final concentration 10 mM, Serva, 69115 Heidelberg, Germany).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/-naphthoflavone induced rat liver S9 was used as the metabolic activation system. The S9 was prepared from 8 - 12 weeks old male Wistar HanIbm rats.

Test concentrations with justification for top dose:

Experiment I
with metabolic activation: 11.9, 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL
without metabolic activation: 20.8, 36.4, 63.7, 111.4, 195.0, 341.2, 597.1, 1044.9, 1828.6 and 3200 µg/mL

Experiment II
with metabolic activation: 10.0, 20.0, 50.0, 125.0, 250.0, 500.0, 750.0, 1000.0, 1250.0 and 1500.0 µg/mL
without metabolic activation: 8.1, 14.2, 25.9, 43.5, 76.2, 133.3, 233.2, 408.2, 714.3 and 1250 µg/mL
Results from the rangefinding assay were used to determine the dose range to be used in the chromosomal aberrations assay.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol 0.5%
- Justification for choice of solvent/vehicle: solubility

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

880 (Experiment I) and 770 (Experiment II) µg/mL, positive control without metabolic activation

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:
Range-finder:
A preliminary cytotoxicity test was performed to determine the concentrations to be used in the mutagenicity assay. Cytotoxicity is characterized by the percentages of mitotic suppression in comparison to the controls by counting 1000 cells per culture in duplicate. The experimental conditions in this pre-test phase were identical to those required and described below for the mutagenicity assay. The pre-test phase was performed with 10 concentrations without S9 mix and 11 concentrations with S9 mix of the test substance and a solvent and positive control. All cell cultures were set up in duplicate. Exposure times were 4 h (with and without S9 mix). The preparation interval was 22 h after start of the exposure. Additional solvent control cultures (with and without S9 mix) were used in the presence of BrdU (5-bromodeoxyuridine; 6 µg/mL) to reassure the replication time of the cultured lymphocytes.

Dose Selection
The highest concentration used in the pre-test was chosen with regard to the current OECD Guideline for in vitro mammalian cytogenetic tests requesting for the top concentration clear toxicity with reduced mitotic indices below 50 % of control, and/or the occurrence of precipitation. In case of nontoxicity the maximum concentration should be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest, if formulability in an appropriate solvent is possible. With respect to the ability to formulate a homogeneous suspension of the test substance, 3200 µg/mL of test substance (approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous suspension in ethanol that was not applicable. Test substance concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test substance was observed before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.
Using reduced mitotic indices as an indicator for toxicity in Experiment I, toxic effects of about 50 % of control were observed after 4 h treatment with 597.1 µg/mL and above in the absence and presence of S9 mix. Considering these toxicity data, 1250 µg/mL (without S9 mix) and 1500 µg/mL (with S9 mix) were chosen as top concentrations in Experiment II. The cytogenetic evaluation of higher concentrations in the respective preparation interval (with and without S9 mix) was impossible due to strong test substance-induced toxic effects (low metaphase numbers, partially paralleled by poor metaphase quality and precipitation on the slides).

Exposure time 4 hours
The culture medium was replaced with serum-free medium (for treatment with S9 mix) or complete medium with 10 % FCS (v/v) (for treatment without S9 mix), containing the test substance.
For the treatment with metabolic activation 50 µL S9 mix per mL medium were used. Concurrent solvent and positive controls were performed. After 4 h the cells were spun down by gentle centrifugation for 5 minutes. The supernatant with the dissolved test substance was discarded and the cells were re-suspended in "saline G" solution (NaCl -8000 mg, KCl -400 mg, glucose x H2O -1100 mg, Na2HPO4 7H2O - 290 mg, KH2PO4 --150 mg). The washing procedure was repeated once as described. After washing the cells were re-suspended in complete culture medium and cultured until preparation.

Exposure time 22 hours (without S9 mix)
The culture medium was replaced with complete medium (with 10 % FCS) containing the test substance without S9 mix. The culture medium at continuous treatment was not changed until preparation of the cells. Concurrent solvent and positive controls were performed. All cultures were incubated at 37° C in a humidified atmosphere with 5.5 % CO2 (94.5 % air).

Preparation of the Cultures
Three hours before harvesting, colcemid was added to the cultures (final concentration 0.2 µg/mL). The cultures were harvested by centrifugation 22 h after beginning of treatment. The supernatant was discarded and the cells were re-suspended in approximately 5 mL hypotonic solution (0.0375 M KCl). The cell suspension was then allowed to stand at 37° C for 20 to 25 minutes. After removal of the hypotonic solution by centrifugation the cells were fixed with a mixture of methanol and glacial acetic acid (3 parts plus 1 part). At least two slides per experimental group were prepared by dropping the cell suspension onto a clean microscope slide. The cells for evaluation of cytogenetic damage were stained with Giemsa (MERCK, 64293 Darmstadt, Germany) or according to the Fluorescent plus Giemsa technique, respectively.

Analysis of Metaphase Cells
The slides were evaluated (according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik") using NIKON microscopes with 100 x oil immersion objectives. Breaks, fragments, deletions, exchanges and chromosomal disintegrations were recorded as structural chromosome aberrations. Gaps were recorded as well, but they were not included in the calculation of the aberration rates. At least 100 well spread metaphase plates per culture were scored for cytogenetic damage on coded slides, except for the positive control in Experiment II without metabolic activation, where only 50 metaphase plates were scored. Only metaphases with 46 +/-1 centromer regions were included in the analysis. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was determined. In addition, the number of polyploid cells in 250 metaphase cells (% polyploid metaphases) was scored.

Evaluation criteria:

A test substance is classified as non-mutagenic if:
- the number of induced structural chromosome aberrations in all evaluated dose groups is in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps).
- no significant increase of the number of structural chromosome aberrations is observed.

A test substance is classified as mutagenic if:
- the number of induced structural chromosome aberrations is not in the range of our historical control data (0.0 - 4.0 % aberrant cells, excluding gaps) and
- either a concentration-related or a significant increase of the number of structural chromosome aberrations is observed.

A test substance can be classified as aneugenic if:
- the number of induced numerical aberrations is not in the range of our historical control data (0.0 – 0.8 % polyploid cells).

Statistics:

Statistical significance was confirmed by means of the Fisher´s exact test (p < 0.05). However, both biological and statistical significance should be considered together. If the criteria for the test substance are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

With respect to the ability to formulate a homogeneous suspension of the test item, 3200 µg/mL of the test substance (approx. 4.8 mM) were applied as top concentration for treatment of the cultures in the pre-test. Doses over 3200 µg/mL led to an inhomogeneous suspension in ethanol that was not applicable. Test substance concentrations between 11.9 and 3200 µg/mL, and between 20.8 and 3200 µg/mL (with and without S9 mix, respectively) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, precipitation of the test item was observed before start of treatment at 36.4 µg/mL and above in the absence of S9 mix, and at 20.8 µg/mL and above in the presence of S9 mix. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance.


Polyploid metaphases:
Polyploid metaphases occured, but in both experiments, no biologically relevant increase in the rate of polyploid metaphases was found after treatment with the test item (0.0 – 0.6 %) as compared to the rates of the solvent controls (0.0 – 0.8 %).


POSITIVE CONTROLS
In both experiments, EMS (880 and 770 µg/mL, respectively) and CPA (37.5 µg/mL) were used as positive controls and showed distinct increases in cells with structural chromosome aberrations.

Summary of results of the chromosomal aberration study with PC-2007-140 (W 575 no solvent)

Exp.	Preparation interval	Test substance concentration in µg/mL	Polyploid cells in %	Mitotic indices in % of control	Incl. gaps	Aberrant cells in % excl. gaps	With exchanges
Exposure period 4 hrs without S9 mix
I	22 hrs	Solvent control	0.8	100.0	2.0	2.0	1.0
		Positive control	0.2	64.2	10.0	9.0^S	0.0
		20.8	0.2	73.4	3.0	2.0	0.0
		36.4P	0.4	82.6	2.5	1.5	0.5
		341.2P	0.2	61.3	2.0	1.5	0.0
		597.1P	0.4	50.5	0.5	0.5	0.0
Exposure period 22 hrs without S9 mix
II	22 hrs	Solvent control	0.4	100.0	2.0	2.0	0.0
		Positive#control	0.0	25.3	50.0	49.0^S	11.0
		43.5##	0.2	72.3	5.0	4.5	0.0
		76.2P	0.2	67.9	0.0	0.0	0.0
		133.3P	0.0	53.6	3.0	3.0	0.0
		233.2P	0.0	29.2	2.5	2.0	0.0
Exposure period 4 hrs with S9 mix
I	22 hrs	Solvent control	0.2	100.0	2.0	1.5	0.0
		Positive control	0.4	49.7	8.5	8.5^S	2.0
		11.9	0.0	84.7	1.0	1.0	0.0
		20.8P	0.4	88.9	1.5	1.0	0.5
		341.2P	0.6	65.3	0.0	0.0	0.0
		597.1P	0.4	73.4	2.5	2.0	0.0
II	22 hrs	Solvent control	0.0	100.0	2.0	1.0	0.0
		Positive control	0.0	34.6	22.0	20.0^S	1.0
		20.0	0.0	120.1	1.5	1.5	0.0
		50.0P##	0.0	111.0	3.5	3.3^S	0.0
		125.0P	0.0	110.2	1.5	1.5	0.0

 

P -Test substance precipitation was observed

S - Aberration frequency statistically significant higher than corresponding control values

# - Evaluation of 50 metaphases per culture

## - Evaluation of 200 metaphases per culture

Conclusions:

Based on the results of the read across study, similar absence of clastogenicity can be expected for the test substance in the chromosomal aberration study, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the clastogenic potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS' (active: 100%), in a chromosomal aberration assay, according to OECD Guideline 473, in compliance with GLP. In the assay, the human lymphocyte cultures were exposed to test substance, suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation. Test substance was tested up to cytotoxic or precipitating concentrations. The following experimental points were microscopically evaluated:11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation. In the absence of S9 mix, reduced mitotic indices of about or below 50% of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the cytogenicity endpoint. In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased, these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background. Under the study conditions, the read across substance was considered to be non-clastogenic in the chromosomal aberration study, with and without metabolic activation (Bohnenberger, 2009). Based on the results of the read across study, similar absence of clastogenicity can be expected for the test substance, 'di-C18-22 AAEMIM-MS'.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From April 09, 2018 to April 30, 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)

Version / remarks:

adopted 29 July 2016,

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

Thymidine kinase gene

Species / strain / cell type:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Group 1 (4-h without S9): 0.63, 1.25, 2.5, 5, 10 and 20 μg/mL
Group 2 (4-h with S9 (2%)): 0.63, 1.25, 2.5, 5, 10 and 20 μg/mL
Group 3 (24-h without S9): 1.25, 2.5, 5, 10, 20 and 30 μg/mL

The dose range of test substance used in the main test was selected following the results of a preliminary toxicity test.

The maximum dose levels in the mutagenicity test were limited by the onset of test substance precipitate in the 4 h exposure groups in both the absence and presence of metabolic activation, and a combination of test substnace induced toxicity and the onset of test substance precipitate in the 24 h exposure group in the absence of metabolic activation, as recommended by the OECD 490 guideline.

Vehicle / solvent:

Tetrahydrofuran (0.25%)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

THF

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

ethylmethanesulphonate

Details on test system and experimental conditions:

Cell Line
The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and
were frozen in liquid nitrogen at that time.

Cell Culture
The stocks of cells are stored in liquid nitrogen at approximately -196 °C. Cells were routinely cultured in RPMI 1640 medium with Glutamax-1 and HEPES buffer (20 mM) supplemented with Penicillin (100 units/mL), Streptomycin (100 μg/mL), Sodium pyruvate (1 mM), Amphotericin B (2.5 μg/mL) and 10% donor horse serum (giving R10 media) at 37 °C with 5% CO2 in air. The cells have a generation time of approximately 12 h and were subcultured accordingly. RPMI 1640 with 20% donor horse serum (R20), 10% donor horse serum (R10), and without serum (R0), are used during the course of the study. Master stocks of cells were tested and found to be free of mycoplasma.

Microsomal Enzyme Fraction
Lot No. PB/βNF S9 29/03/18 was used in this study, and was pre-prepared in-house (outside the confines of the study) following standard procedures. Prior to use, each batch of S9 is tested for its capability to activate known mutagens in the Ames test and a copy of the certificate of S9 efficacy is presented in Appendix 2. S9-mix was prepared by mixing S9, NADP (5 mM), G-6-P (5 mM), KCl (33 mM) and MgCl2 (8 mM) in R0. 20% S9-mix (i.e. 2% final concentration of S9) was added to the cultures of the preliminary toxicity test and mutagenicity test.

Test substance preparation: The test substance was a UVCB therefore the maximum proposed dose level in the solubility test was initially set at 5000 μg/mL, the maximum recommended dose level, and no correction for the purity of the test item was applied. The test substance was found to be insoluble in R0 medium at 50 and 25 mg/mL, dimethyl sulfoxide (DMSO) at 500, 250, and 125mg/mL, acetone at 500 and 250 mg/mL, and tetrahydrofuran (THF) at 500 mg/mL. The test substance formed a solution in THF at 250 mg/mL considered acceptable for dosing in the solubility checks. THF is toxic to L5178Y cells at dose volumes greater than 0.25% of the total culture volume. Therefore, the test substance was formulated at 250 mg/mL and dosed at 0.25% to give the maximum achievable dose level of 625 μg/mL. There was no significant change in pH when the test substance was dosed into media and the osmolality did not increase by more than 50 mOsm at the concentration levels investigated.

Control preparation
Vehicle and positive controls were used in parallel with the test substance in the Mutagenicity Test. Solvent (THF, Sigma-Aldrich batch STBH1613, purity >99.9%, expiry 07/11/22) exposure groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) (Sigma batch BCBS6100V, purity treated as 100%, expiry 17.11.22) at 400 μg/mL and 150 μg/mL, respectively, was used as the positive control in the 4-hour and 24-hour exposure groups in the absence of metabolic activation. Cyclophosphamide (Acros Organics batch A0373263, purity 97%, Expiry 22.02.19) at 1.5 μg/mL was used as the positive control in the presence of metabolic activation. The positive controls were formulated in DMSO.

Evaluation criteria:

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined, the increase in Mutation Frequency (MF) above the concurrent background exceeds the Global Evaluation Factor (GEF) and the increase is concentration related (e.g., using a trend test). The test chemical is then considered able to induce mutation in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly negative if, in all experimental conditions examined there is no concentration related response or, if there is an increase in MF, it does not exceed the GEF. The test chemical is then considered unable to induce mutations in this test system.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Remarks:

THF

Untreated negative controls validity:

not specified

Positive controls validity:

valid

 Preliminary cytotoxicity test

Dose (mg/mL)	% RSG (-S9) 4 h exposure	% RSG (+S9) 4 h exposure	% RSG (-S9) 24 h exposure
0	100	100	100
0.16	92	110	76
0.31	86	99	81
0.63	97	116	75
1.25	100	98	58
2.5	89	101	48
5	90	114	50
10	76	111	48
20	80	104	23
40	65	89	7

Summary of results

Concentration (µg/mL)			4 h -S9					Concentration (µg/mL)			4 h +S9
			%RSG	RTG	MF§						%RSG	RTG	MF§
0			100	1.00	140.03			0			100	1.00	127.77
0.63			96	0.99	146.25			0.63			102	1.02	132.90
1.25			98	0.93	142.67			1.25			105	0.95	156.03
2.5			99	0.97	136.15			2.5			108	0.98	130.01
5			105	1.08	152.20			5			74	0.74	102.60
10			93	0.97	152.91			10			68	0.71	104.53
20			92	0.93	137.90			20			60	0.60	121.28
		Ø	75					30		Ø	69
40		Ø	65					40		Ø	70
MF threshold for a positive response = 266.03								MF threshold for a positive response = 253.77
Positive control								Positive control
EMS								CP
400			74	0.48	1145.38			1.5			82	0.53	1230.16

Concentration (µg/mL)		24 h -S9
		%RSG	RTG	MF§
0		100	1.00	137.78
0.63	Ø	97
1.25		86	0.92	120.52
2.5		81	0.98	105.46
5		74	0.82	130.38
10		61	0.71	131.75
20		30	0.42	123.23
30		21	0.30	136.47
40	Ø	13
MF threshold for a positive response = 263.78
Positive control
EMS
150		71	0.58	911.84

Cell and 96-Well Plate Counts: Mutagenicity test (-S9) 4 h Exposure

Concentration (µg/mL)		Cell counts			Viability after day 2 2 cells/well				Resistant mutants after day 2 2000 cells/well
		0h	24h	48h
0	A B	6.60 7.39	7.15 7.62	10.22 10.13	81 76	72 79	80 78	79 73	20 22	21 20	17 19	23 15
0.63	A B	6.83 7.38	8.10 6.60	8.94 10.36	79 77	73 84			21 23	21 19
1.25	A B	7.28 7.43	7.55 7.12	9.46 9.63	78 79	70 77			18 14	21 24
2.5	A B	7.41 8.32	6.77 6.41	10.41 9.63	76 84	75 73			18 19	16 23
5	A B	7.83 7.24	7.76 7.53	9.84 9.25	83 76	76 78			21 22	22 22
10	A B	7.44 7.30	7.49 6.54	9.45 9.52	84 75	80 75			20 25	22 21
20	A B	7.94 7.54	6.51 6.36	9.69 9.75	80 76	72 82			17 18	25 18
30	A B	8.24 6.55	6.06 6.07	8.96 8.72	NP NP	NP NP			NP NP	NP NP
40	A B	7.79 6.74	4.68 5.04	9.69 9.62	NP NP	NP NP			NP NP	NP NP
Positive control EMS (µg/mL)
400	A B	7.57 8.05	6.11 5.81	8.76 7.84	67 60	61 63			67 69	49 85

Summary Analysis: Mutagenicity Test (-S9) 4 h Exposure 

Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
0		18.79	100	81.66	1.00	140.03
0.63		17.73	96	84.40	0.99	146.25
1.25		17.50	98	78.43	0.93	142.67
2.5		16.51	99	81.00	0.97	136.15
5		18.24	105	84.40	1.08	152.20
10		16.63	93	85.11	0.97	152.91
20		15.64	92	82.33	0.93	137.90
30	Ø	13.40	75
40	Ø	11.73	65
Positive control EMS
Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
400		12.37	74	53.01	0.48	1145.38

 

GEF =126, therefore MF threshold for a positive response = 26 

Large and Small ColoniesAnalysis: Mutagenicity Test (-S9) 4 h exposure

Concentration (µg/mL)		Viability after day 2				Small colonies after day 2				Large colonies after day 2
0	A B	81 76	72 79	80 78	79 73	10 10	9 11	7 10	10 7	10 12	12 9	10 9	13 8
0.63	A B	79 77	73 84			8 10	9 7			13 13	12 12
1.25	A B	78 79	70 77			6 3	10 12			12 11	11 12
2.5	A B	76 84	75 73			10 7	8 10			8 12	8 13
5	A B	83 76	76 78			11 8	10 6			10 14	12 16
10	A B	84 75	80 75			6 12	13 7			14 13	9 14
20	A B	80 76	72 82			7 7	14 6			10 11	11 12
400 EMS	A B	67 60	61 63			28 36	24 40			39 33	25 45

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		150	768	694	768	62.0	685	768	70.0	0.47
0.63		71	384	350	384	54.9	334	384	82.6	0.40
1.25		80	384	353	384	53.7	338	384	81.3	0.40
2.5		76	384	349	384	59.0	343	384	69.7	0.46
5		71	384	349	384	56.6	332	384	86.2	0.40
10		70	384	346	384	61.2	334	384	82.0	0.43
20		74	384	350	384	56.3	340	384	73.9	0.44
400 EMS		133	384	256	384	382.4	242	384	435.5	0.47

 

Cell and 96-Well Plate Counts: Mutagenicity Test (+S9) 4 h Exposure

Concentration (µg/mL)		Cell counts			Viability after day 2 2 cells/well				Resistant mutants after day 2 2000 cells/well
		0h	24h	48h
0	A B	8.37 8.28	6.35 6.15	9.40 9.36	82 79	79 82	85 80	81 75	14 21	22 18	21 21	21 21
0.63	A B	8.92 7.37	5.92 6.73	9.67 9.67	80 79	82 80			22 22	19 19
1.25	A B	9.12 8.57	6.06 5.87	9.60 9.86	73 82	74 81			23 23	19 22
2.5	A B	9.20 8.56	6.12 6.41	9.81 9.16	76 75	77 82			16 24	16 18
5	A B	8.40 8.25	5.85 6.48	6.67 7.42	84 74	80 83			14 17	13 21
10	A B	7.89 8.56	7.65 6.35	5.22 6.29	83 81	84 80			16 18	17 19
20	A B	7.75 7.49	6.25 6.47	5.94 6.14	82 79	81 78			15 22	15 23
30	A B	7.32 7.93	6.61 7.33	6.18 6.45	NP NP	NP NP			NP NP	NP NP
40	A B	7.99 8.31	6.49 5.31	7.23 6.94	NP NP	NP NP			NP NP	NP NP
Positive control CP (µg/mL)
1.5	A B	8.42 7.73	4.98 5.49	9.72 9.15	61 68	69 69			77 74	75 69

 

Summary Analysis: Mutagenicity Test (+S9) 4 h exposure

Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
0		14.66	100	90.77	1.00	127.77
0.63		15.29	102	90.38	1.02	132.90
1.25		14.51	105	82.33	0.95	156.03
2.5		14.86	108	82.33	0.98	130.01
5		10.86	74	90.38	0.74	102.60
10		10.07	68	96.26	0.71	104.53
20		9.60	60	89.59	0.60	121.28
30	Ø	11.00	69
40	Ø	10.45	70
Positive control CP
Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
1.5		12.35	82	59.42	0.53	1230.16

GEF =126, therefore MF threshold for a positive response = 253.77

 

Large and Small Colonies Analysis: Mutagenicity Test (+S9) 4 h Exposure

Concentration (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	82 79	79 82	85 80	81 75	7 9	6 10	9 8	9 8	7 12	16 8	12 13	12 13
0.63	A B	80 79	82 80			7 9	9 10			15 13	10 9
1.25	A B	73 82	74 81			9 10	7 10			14 13	12 12
2.5	A B	76 75	77 82			9 9	3 7			7 15	13 11
5	A B	84 74	80 83			4 8	5 7			10 9	8 14
10	A B	83 81	84 80			4 1	6 3			12 17	11 16
20	A B	82 79	81 78			7 8	6 9			8 14	9 14
1.5 CP	A B	61 68	69 69			49 48	47 42			28 26	28 27

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		125	768	702	768	49.5	675	768	71.1	0.42
0.63		63	384	349	384	52.9	337	384	72.2	0.43
1.25		74	384	348	384	59.8	333	384	86.5	0.41
2.5		74	384	356	384	46.0	338	384	77.5	0.38
5		63	384	360	384	35.7	343	384	62.5	0.37
10		56	384	370	384	19.3	328	384	81.9	0.20
20		64	384	354	384	45.4	339	384	69.6	0.40
1.5 CP		117	384	198	384	557.3	275	384	280.9	0.63

 

Cell and 96-Well Plate Counts: Mutagenicity Test (-S9) 24 h exposure

Concentration (µg/mL)		Cell counts			Viability after day 2 2 cells/well				Resistant mutants after day 2 2000 cells/well
		0h	24h	48h
0	A B	8.42 8.14	7.09 8.62	7.58 7.01	80 79	82 79	75 83	82 80	21 22	22 15	22 20	27 19
0.63	A B	8.69 8.61	6.42 6.35	8.15 7.88	NP NP	NP NP			NP NP	NP NP
1.25	A B	7.45 7.91	8.33 6.79	7.29 7.82	81 82	77 79			17 20	17 20
2.5	A B	7.16 8.17	7.61 6.02	7.54 8.35	83 87	82 80			22 17	16 18
5	A B	7.90 6.92	6.84 7.01	7.17 8.02	78 80	83 79			21 17	23 19
10	A B	6.64 6.68	7.17 7.43	7.57 7.31	75 81	75 81			19 17	22 18
20	A B	5.61 5.20	5.80 5.98	6.00 7.53	73 83	73 80			17 16	21 16
30	A B	4.84 5.41	4.89 4.77	6.98 6.14	74 80	70 80			21 17	17 19
40	A B	5.00 5.98	3.41 3.91	4.56 4.82	NP NP	NP NP			NP NP	NP NP
Positive control EMS (µg/mL)
150	A B	9.23 8.02	6.16 7.87	5.71 5.05	71 79	72 79			80 73	73 63

Summary Analysis: Mutagenicity Test (-S9) 24 h exposure

Concentration (µg/mL)		SG	%RSG	%V	RTG	MF
0		79.08	100	89.59	1.00	137.78
0.63	Ø	73.78	97
1.25		73.11	86	88.81	0.92	120.52
2.5		69.17	81	99.97	0.98	105.46
5		64.96	74	89.59	0.82	130.38
10		60.29	61	83.70	0.71	131.75
20		35.89	30	81.66	0.42	123.23
30		27.06	21	78.43	0.30	136.47
40	Ø	15.71	13
Positive control EMS
Concentration (µg/mL)		SG	%RSG	%V	RTG	MF§
150		54.25	71	76.59	0.58	911.84

 

GEF =126, therefore MF threshold for a positive response = 263.78

Large and Small Colonies Analysis: Mutagenicity Test (-S9) 24 h Exposure

Concentration (µg/mL)		Viability # after day 2				Small colonies # after day 2				Large colonies # after day 2
0	A B	80 79	82 79	75 83	82 80	9 10	7 8	10 8	12 10	12 12	15 7	12 12	15 9
1.25	A B	81 82	77 79			3 8	4 7			14 12	13 13
2.5	A B	83 87	82 80			10 9	9 3			12 8	7 15
5	A B	78 80	83 79			8 8	9 6			13 9	14 13
10	A B	75 81	75 81			7 7	10 6			12 10	12 12
20	A B	73 83	73 80			7 4	9 7			10 12	12 9
30	A B	74 80	70 80			7 9	8 9			14 8	9 10
150 EMS	A B	71 79	72 79			38 36	37 35			42 37	36 28

Mutation frequencies

Concentration (µg/mL)				Small colonies			Large colonies			Proportion small colony mutants
		Viable		Mutants			Mutants
		Yv	Nv	Ym	Nm	MF§	Ym	Nm	MF§
0		128	768	694	768	56.5	674	768	72.9	0.44
1.25		65	384	362	384	33.2	332	384	81.9	0.30
2.5		52	384	353	384	42.1	342	384	57.9	0.42
5		64	384	353	384	47.0	335	384	76.2	0.39
10		72	384	354	384	48.6	338	384	76.2	0.39
20		75	384	357	384	44.6	341	384	72.7	0.39
30		80	384	351	384	57.3	343	384	72.0	0.45
150 EMS		83	384	238	384	312.3	241	384	304.1	0.51

Conclusions:

Under the study conditions, the test substance was determined to be non-mutagenic in mouse lymphoma assay, with and without metabolic activation.

Executive summary:

An in vitro study was conducted to determine the genotoxic potential of the test substance, 'di-C18-22 AAEMIM-MS', using the thymidine kinase gene, according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y TK+/- cells. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at 6 dose levels (ranging from 0.63 to 20 μg/mL) in duplicate, together with vehicle (THF), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The dose range for the test substance in the main test was selected following the results of a preliminary toxicity test, where the tested doses ranged from 0.16 to 40 μg/mL in three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). The dose levels plated for viability and expression of mutant colonies in the preliminary test ranged from 0.16 to 40 μg/mL in three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). Precipitate of the test substance was observed at 40 µg/mL in the 4 h and 24 h exposure groups in the absence of metabolic activity, and at and above 20 µg/mL in the 4 h exposure group in the presence of metabolic activation, at the end of the exposure periods.Therefore, the maximum dose level used for the main test was limited by precipitate in the 4 h exposure groups, with and without S9, and combination of test substance induced toxicity and the onset of test substance precipitate in the 24 h exposure group, without S9. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test substance did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Under the study conditions, the test substance was considered not to induce mutations in the mouse lymphoma thymidine kinase locus assay, with and without metabolic activation (Envigo, 2018).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Bacterial reverse mutation assay (Ames test):

An in vitro study was conducted to determine the mutagenic potential of the test 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 test substance was determined to be non-mutagenic in Ames test, with or without metabolic activation (BioReliance, 2009).

 

Chromosomal aberration assay:

Study 1:

An in vitro study was conducted to determine clastogenic potential of a formulation containing 39% of the test substance, ‘C18 -22 AAEMIM-MS’, in a chromosomal aberration assay, according to OECD Guideline 473, in compliance with GLP. A preliminary toxicity test was performed to establish the dose range for the assay. In the preliminary toxicity assay, the maximum dose tested was 5000 µg/mL. Visible precipitate was observed in the treatment medium at dose level ≥ 15 µg/mL. Dose levels ≤ 5 µg/mL were soluble in treatment medium. Selection of dose levels for the chromosome aberration assay was based on cell growth inhibition relative to the solvent control. Substantial toxicity was observed at dose levels ≥ 50 µg/mL with and without S9, in the 4 h exposure group, and at dose level ≥ 15 µg/mL without S9, in the 20 h continuous exposure group. Based on these findings, the doses chosen for the main assay ranged from 2.5 to 50 µg/mL for both with and without S9, 4 h exposure groups, and ranged from 0.625 to 20 µg/mL for without S9, 20 h continuous exposure group. In the initial chromosome aberration assay, the cells were treated for 4 h and 20 h without S9 and for 4 h with S9. All cells were harvested 20 h after treatment initiation. Visible precipitate observed at dose ≥ 40µg/mL. Selection of doses for microscopic analysis was based on toxicity without S9, 4 and 20 h exposure groups. Due to lack of 50% toxicity, as measured by cell growth or mitotic inhibition, at any dose level in the with S9, 4 h exposure group, this portion of the chromosome aberration assay was repeated at dose levels 20, 30, 40, 50, 75, 100, 125 and 150 µg/mL. In the repeat chromosome aberration assay, the cells were treated for 4 h with S9. All cells were harvested 20 h after treatment initiation. Visible precipitate was observed in treatment medium at dose levels ≥ 40µg/mL. Dose levels ≤30 µg/mL was soluble in treatment medium. Selection of doses for microscopic analysis was based on toxicity in with S9 activated, 4 h exposure group. The percentage of cells with structural or numerical aberrations in test substance treated group was not significantly increased above that of the solvent control at any dose level. Under the study conditions, the substance was considered to be not clastogenic in Chinese Hamster Ovary (CHO-K1) cells, with and without metabolic activation (Bioreliance, 2004).

 

Study 2:

An in vitro study was conducted to determine the clastogenic potential of the read across substance, 'di-C16-18 and C18-unsatd. AAEMIM-MS' (active: 100%), in a chromosomal aberration assay, according to OECD Guideline 473, in compliance with GLP. In the assay, the human lymphocyte cultures were exposed to test substance, suspended in ethanol at concentrations between 11.9 - 3200 µg/mL with metabolic activation and 20.8 - 3200 µg/mL without metabolic activation. Test substance was tested up to cytotoxic or precipitating concentrations. The following experimental points were microscopically evaluated:11.9, 20.0, 20.8, 50.0, 125.0, 341.2, 597.1 µg/mL with metabolic activation and 20.8, 36.4, 43.5, 76.2, 133.3, 233.2, 341.2, 597.1 µg/mL without metabolic activation. In the absence of S9 mix, reduced mitotic indices of about or below 50% of control were observed at the highest evaluated concentrations. In the presence of S9 mix, concentrations showing clear cytotoxic effects were excluded from scoring for the cytogenicity endpoint. In Experiment II, in the absence of S9 mix, a single increase in chromosomal aberrations was observed, slightly exceeding the laboratory’s historical control data range, but since the value was not statistically significantly increased, these findings were considered as biologically irrelevant. A single statistically significant increase was observed in Experiment II, in the presence of S9 mix, but the value was clearly within the range of the laboratory’s historical control data and thus considered as being without biological relevance. Positive controls induced the appropriate response. There was no evidence of chromosome aberration induced over background. Under the study conditions, the read across substance was considered to be non-clastogenic in the chromosomal aberration study, with and without metabolic activation (Bohnenberger, 2009).

Mammalian cell gene mutation assay:

An in vitro study was conducted to determine the genotoxic potential of the test substance, 'di-C18-22 AAEMIM-MS', using the thymidine kinase gene, according to OECD Guideline 490, in compliance with GLP. This study was performed to investigate the potential of the test substance to induce mutations at the thymidine kinase locus (TK1) on chromosome 11 and/or structural chromosomal aberrations in mouse lymphoma L5178Y TK+/- cells. One main Mutagenicity Test was performed. In this main test, L5178Y TK +/- mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test substance at 6 dose levels (ranging from 0.63 to 20 μg/mL) in duplicate, together with vehicle (THF), and positive controls using 4 h exposure groups both in the absence and presence of metabolic activation (2% S9), and a 24 h exposure group in the absence of metabolic activation. The dose range for the test substance in the main test was selected following the results of a preliminary toxicity test, where the tested doses ranged from 0.16 to 40 μg/mL in three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). The dose levels plated for viability and expression of mutant colonies in the preliminary test ranged from 0.16 to 40 μg/mL in three exposure groups (i.e., 4 h with and without S9 and 24 h without S9). Precipitate of the test substance was observed at 40 µg/mL in the 4 h and 24 h exposure groups in the absence of metabolic activity, and at and above 20 µg/mL in the 4 h exposure group in the presence of metabolic activation, at the end of the exposure periods.Therefore, the maximum dose level used for the main test was limited by precipitate in the 4 h exposure groups, with and without S9, and combination of test substance induced toxicity and the onset of test substance precipitate in the 24 h exposure group, without S9. The vehicle control cultures had mutant frequency values that were acceptable for the L5178Y cell line at the TK +/- locus. The positive control substances induced marked increases in the mutant frequency, sufficient to indicate the satisfactory performance of the test and of the activity of the metabolizing system. The test substance did not induce any toxicologically significant increases in the mutant frequency at any of the dose levels in the main test, in any of the three exposure groups. Under the study conditions, the test substance was considered not to induce mutations in the mouse lymphoma thymidine kinase locus assay, with and without metabolic activation (Envigo, 2018).

Overall, based on the available study results from in vitro assays with the test as well as read across substance, the test substance, 'di-C18-22 AAEMIM-MS' is considered to be non-genotoxic.

Justification for classification or non-classification

Based on the available in vitro study results, the test substance, 'di-C18-22 AAEMIM-MS' does not warrant a genotoxicity classification, according to the EU CLP criteria (Regulation 1272/2008/EC).