C16-18 (evennumbered, C18 unsaturated) alkyl bis(2-hydroxyethyl) amine oxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There are two in-vitro genotoxicity studies available for Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides, an in-vitro micronucleus test in human lymphocytes and an L5178 TK +/- Mouse lymphoma assay. Both studies were carried out to the current OECD guidelines to GLP. In addition there is an Ames test used for read across from the source substance 2,2’-(octadec-9-enylimino)diethanol CAS No 93962-62-0.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

19 March 1990 - 3 April 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed under GLP and accoding to OECD/EC guidelines. In the current study the guidelines was not updated yet and this test does not contains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. These strains are preferred to detect cross-linking mutagens.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Qualifier:

according to guideline

Guideline:

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

Principles of method if other than guideline:

In the current study the guidelines was not updated yet and this test does not contains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. These strains are preferred to detect cross-linking mutagens.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes involved in histidine synthesis.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see remarks section

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

preliminary toxicity test (with and without S9-mix): 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
experiment 1 without S9-mix: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
experiment 1 with S9-mix: 1.0, 3.3, 10.0, 33.3, 100.0 µg/plate
experiment 2 without S9-mix: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
experiment 2 with S9-mix: 1.0, 3.3, 10.0, 33.3, 100.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given, common solvent for this type of test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimtehylsulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

other: see remarks section for details

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: each dose is tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: all colonies were counted.

DETERMINATION OF CYTOTOXICITY
- Method: other: Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates.

Evaluation criteria:

An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range
for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be a t least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding test with strain
TA100 or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing has been done.
A test substance was considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or
without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance was considered positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9-mix 33.3µg/plate gives a moderate reduction and elimination at 100µg/plate. WithS9-mix with S9-mix 100µg/plate gives a moderate reduction and elimination at 333µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: not observed
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES: The survival of the TA100culture i s determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. In the absence of S9-mix the survival of strain TA100 is moderately reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. In the presence o f S9-mix the survival of strain TA100 is moderately reduced at a test substance concentration of 100 µg/plate and eliminated at and above 333 µg/plate. Based on these data, the test substance was tested up to a concentration of 33.3 µg/plate i n the absence of S9-mix and up to 100 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100) therefore from the results of this study it can be concluded that AROMOX O/12 is not mutagenic in the ames test.

Executive summary:

The test item was tested in the Ames Salmonella/microsome plate test up to 33.3 µg/plate in the absence o f S9 -mix and up to 100 µg/plate in the presence of S9 -mix. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test item can, therefore, be considered as not mutagenic in this test system.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

19 March 1990 - 3 April 1990

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed under GLP and accoding to OECD/EC guidelines. In the current study the guidelines was not updated yet and this test does not contains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. These strains are preferred to detect cross-linking mutagens.

Justification for type of information:

There are two in-vitro genotoxicity studies available for Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides, an in-vitro micronucleus test in human lymphocytes and an L5178 TK +/- Mouse lymphoma assay. Both studies were carried out to the current OECD guidelines to GLP. In addition there is an Ames test used for read across from the source substance 2,2’-(octadec-9-enylimino)diethanol CAS No 93962-62-0. The higher degree of unsaturation in the 2,2’-(octadec-9-enylimino)diethanol is expected to result in higher reactivity from the increased double bonds in the unsaturated C18 in the substance which at 80% is significantly higher than in the target substance Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides which has only 35% unsaturated C18. Due to this higher reactivity 2,2’-(octadec-9-enylimino)diethanol would be more likely to react with DNA than Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides so read across to this substance will not underestimate the potential for mutagenicity in the Ames test.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

In the current study the guidelines was not updated yet and this test does not contains: E. coli WP2 uvrA, or E. coli WP2 uvrA (pKM101), or S. typhimurium TA102. These strains are preferred to detect cross-linking mutagens.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Genes involved in histidine synthesis.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

other: see remarks section

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

preliminary toxicity test (with and without S9-mix): 1.0, 3.3, 10.0, 33.3, 100, 333, 1000, 3330, 5000 µg/plate
experiment 1 without S9-mix: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
experiment 1 with S9-mix: 1.0, 3.3, 10.0, 33.3, 100.0 µg/plate
experiment 2 without S9-mix: 0.33, 1.0, 3.3, 10.0, 33.3 µg/plate
experiment 2 with S9-mix: 1.0, 3.3, 10.0, 33.3, 100.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: not given, common solvent for this type of test.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

dimtehylsulfoxide (DMSO)

Positive controls:

yes

Positive control substance:

other: see remarks section for details

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: each dose is tested in triplicate in each strain.

NUMBER OF CELLS EVALUATED: all colonies were counted.

DETERMINATION OF CYTOTOXICITY
- Method: other: Selection of an adequate range of doses was based on a preliminary toxicity test with strain TA100, both with and without S9-mix. Nine concentrations have been tested in duplicate for toxicity. The highest concentration of test article used in the subsequent mutagenesis assay was that which gave a reduced survival on the non-selective plates.

Evaluation criteria:

An Ames test was considered acceptable if it met the following criteria:
a) The negative control data (number of spontaneous revertants per plate) should reasonably fall within the laboratory background historical range
for each tester strain.
b) The positive control chemicals should produce responses in all tester strains which also reasonably fall within the laboratory historical range documented for each positive control substance. Furthermore, the mean plate count should be a t least two times the concurrent vehicle control group mean.
c) The selected dose range should include a clearly toxic concentration as demonstrated by the preliminary toxicity range-finding test with strain
TA100 or should extend to 5 mg/plate.

Statistics:

No formal hypothesis testing has been done.
A test substance was considered negative (not mutagenic) in the Ames test if:
a) The total number of revertants in any tester strain at any concentration was not greater than two times the solvent control value, with or
without metabolic activation.
b) The negative response should be reproducible in at least one independently repeated experiment.
A test substance was considered positive (mutagenic) in the Ames test if:
a) It induced at least a 2-fold, dose related increase in the number of revertants with respect to the number induced by the solvent control in any of the tester strains, either with or without metabolic activation. However, any mean plate count of less than 20 was considered to be not significant. If the test substance showed in the first test only a positive response at one or two concentrations, the assay was repeated with doses just below and exceeding those showing positive effects in the first test.
b) The positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other extenuating factors might enter into the final evaluation decision.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9-mix 33.3µg/plate gives a moderate reduction and elimination at 100µg/plate. WithS9-mix with S9-mix 100µg/plate gives a moderate reduction and elimination at 333µg/plate.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: not observed
- Other confounding effects: no data


RANGE-FINDING/SCREENING STUDIES: The survival of the TA100culture i s determined by comparing the number of colonies on the plates containing the test substance with those on the solvent control plate. In the absence of S9-mix the survival of strain TA100 is moderately reduced at a test substance concentration of 33.3 µg/plate and eliminated at and above 100 µg/plate. In the presence o f S9-mix the survival of strain TA100 is moderately reduced at a test substance concentration of 100 µg/plate and eliminated at and above 333 µg/plate. Based on these data, the test substance was tested up to a concentration of 33.3 µg/plate i n the absence of S9-mix and up to 100 µg/plate in the presence of S9-mix.

COMPARISON WITH HISTORICAL CONTROL DATA: The negative and strain-specific positive control values fell within laboratory background historical ranges indicating that the test conditions were optimal and that the metabolic activation system functioned properly.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative

The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100) therefore from the results of this study it can be concluded that AROMOX O/12 is not mutagenic in the ames test.

Executive summary:

The test item was tested in the Ames Salmonella/microsome plate test up to 33.3 µg/plate in the absence o f S9 -mix and up to 100 µg/plate in the presence of S9 -mix. The test substance did not induce a dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535; TA1537; TA98 and TA100). These results were confirmed in an independently repeated experiment. The test item can, therefore, be considered as not mutagenic in this test system.

Reference 3

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 03 August 2017. Experimental completion date 02 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

Identification: Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6
Alternative Name: Aromox T/12
Physical state/Appearance: Extremely pale yellow paste
Batch: 1548134
Purity: 50.66%
Expiry Date: 17 March 2018
Storage Conditions: Room temperature in the dark

The test item was considered to be a UVCB and was supplied as 50.66 % in a paste in diethylene glycol and water. Concentrations were adjusted for the active ingredient.

Target gene:

Not applicable

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:
Preliminary Toxicity Test: female, aged 30 years
Main Experiment: female, aged 21 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital/B-naphthoflavone induced rat liver S9

Test concentrations with justification for top dose:

Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL. Due to formulation difficulties and the necessity of using acetone as the vehicle the maximum achievable dose level was 2500 μg/mL.

Main Experiment:
4-hour exposure group (-S9): 0, 10, 20, 40, 80, 100, 120, 160 μg/mL
4-hour exposure group (+S9): 0, 10, 20, 40, 80, 100, 120, 160 μg/mL
24-hour exposure group (-S9):0, 10, 20 ,30, 40, 50, 60, 80 μg/mL

The selection of the maximum dose level for the Main Experiment was based on toxicity

Vehicle / solvent:

Acetone

The test item was insoluble in culture media at 25 mg/mL and dimethyl sulphoxide (DMSO) at 250 mg/mL but was soluble in acetone at 500 mg/mL in solubility checks performed in-house.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 μg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine (DC)

Remarks:

Absence of S9-mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 0.05 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 0.05 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.
24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9.9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 0.05 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 9.77 to 2500 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 100, 120 and 160 μg/mL.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 10, 20, 40, 80, 100, 120 and 160 μg/mL.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest. The dose range of test item used was 10, 20, 30, 40, 50, 60 and 80 μg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.
Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.
When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations. The Study Director may make a judgement based on experience and the biological relevance of the data and any justification for acceptance of the data will be included in the report. Scoring additional cells (where appropriate) or performing a repeat experiment possibly using modified experimental conditions (e.g. concentration spacing, other metabolic activation conditions (i.e. S9 concentration or S9 origin)) could be useful.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Species / strain:

primary culture, other: whole blood

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:

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250 and 2500 μg/mL. The maximum dose was the maximum achievable dose level.
A precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure, at and above 625 μg/mL, in the 4-hour exposure group in the absence of S9 and at and above 312.5 μg/mL in the 4-hour exposure group in the presence of S9 and in the 24-hour continuous exposure group.
Hemolysis was observed following exposure to the test item at and above 156.25 μg/mL in all three exposure groups. Hemolysis is an indication of a toxic response to the erythrocytes and not indicative of any genotoxic response to the lymphocytes.
A reduced cell pellet was also noted at the end of the exposure period, at and above 312.5 μg/mL in all three exposure groups demonstrating overall toxicity to the cells.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to 78.13 μg/mL in the 4-hour exposure groups and at and above 39.06 μg/mL in the 24-hour exposure group. The test item demonstrated a very sharp toxicity curve. In the 4-hour exposure groups there were no binucleate cells present at and above 156.25 μg/mL and only modest toxicity demonstrated at 78.13 μg/mL. In the 24-hour exposure group near optimum toxicity was demonstrated with 45% cytostasis at 39.06 μg/mL and no binucleate cells present at and above 78.13 μg/mL.
The selection of the maximum dose level for the Main Experiment was based on toxicity and was 160 μg/mL for the 4-hour exposure groups and was 80 μg/mL for the 24-hour exposure group.

Micronucleus Test – Main Experiment
The qualitative assessment of the slides determined that there was a small increase in toxicity compared to the preliminary toxicity test and that there were binucleate cells suitable for scoring up to 40 μg/mL in all three exposure groups.
The CBPI data for the short exposure groups and for the 24-hour exposure group are given in Table 2 and Table 3, respectively. The 4-hour exposure groups demonstrated a very sharp toxicity curve between 40 μg/mL and 80 μg/mL with modest toxicity being demonstrated at 40 μg/mL and complete toxicity at 80 μg/mL with no cells available for scoring. The 4-hour exposure in the presence of S9 achieved toxicity approaching optimum with 34% cytostasis at 40 μg/mL. The 24-hour exposure group achieved 33%, 45% and 69% cytostasis at 20, 30 and 40 μg/mL, respectively. The maximum dose level selected for analysis of binucleate cells was based on toxicity and was 40 μg/mL for all three exposure groups.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

Micronucleus Test – Main Experiment

The dose levels of the controls and the test item are given in the table below:

Exposure Group	Final concentration of test itemTallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6(µg/mL)
4-hour without S9	0*, 10*, 20*, 40*, 80, 100, 120, 160, MMC0.2*
4-hour with S9 (2%)	0*, 10, 20*, 40*, 80, 100, 120, 160, CP5*
24-hour without S9	0*, 10*, 20*, 30*, 40*, 50, 60, 80,DC0.075*

* = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC = Mitomycin C

CP = Cyclophosphamide

DC = Demecolcine

DISCUSSION

Although the 4-hour exposure group of the main test only achieved very modest toxicity at 40 μg/mL with 17% cytostasis, the 24-hour exposure group in the absence of S9 was scored at a concentration which exceeded optimum toxicity and therefore it is considered that the test item has been adequately tested in the absence of S9.

The 4-hour exposure group in the presence of S9 achieved 34% cytostasis at 40 μg/mL which is marginally less than optimum. However, since the toxicity curve is so steep and the higher dose level of 80 μg/mL had no cells suitable for scoring it is considered unlikely that optimum toxicity would be achieved and therefore the test item is considered to be adequately tested in the presence of S9.

In the 24-hour exposure in the absence of S9 the dose range included a dose level which achieved marginally greater than optimum toxicity and in the presence of S9 the dose range included a dose level which achieved toxicity approaching optimum. The test item was therefore considered to be adequately tested in the absence and presence of S9.

The test item was markedly toxic with a steep toxicity curve which made achieving optimum toxicity difficult. There was a small increase in toxicity between the preliminary toxicity test and the main experiment which may have been due to slight differences in the formulations or from the use of a different blood donor. These small variations between experiments and the steep toxicity curve are considered to make further experiments of little value and unlikely to yield improved results.

Conclusions:

The test item, Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

Introduction

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

Methods

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at up to four dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2% final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be limited by toxicity. The dose levels selected for the Main Test were as follows:

Exposure Group	Final concentration of test itemTallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6(µg/mL)
4-hour without S9	10, 20, 40, 80, 100, 120, 160
4-hour with S9 (2%)	10, 20, 40, 80, 100, 120, 160
24-hour without S9	10, 20, 30, 40, 50, 60, 80

Results

All vehicle (acetone) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

The test item did not induce any statistically significant increases in the frequency of cells with micronuclei. The test item was markedly toxic with a steep toxicity curve which made achieving optimum toxicity difficult. In the 24-hour exposure in the absence of S9 the dose range included a dose level which achieved marginally greater than optimum toxicity and in the presence of S9 the dose range included a dose level which achieved toxicity approaching optimum.

Conclusion

The test item, Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6 was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 29 August 2017 Experimental Completion Date: 10 October 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: in vitro mammalian cell gene mutation tests using the thymidine kinase gene (migrated information)

Specific details on test material used for the study:

Identification: Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6
Alternative Name: Aromox T/12
Physical state/Appearance: Extremely pale yellow paste
Batch: 1548134
Expiry Date: 27 March 2018
Storage Conditions: Room temperature in the dark


The test item was considered to be a UVCB* and was supplied as 50.66 % in a paste in diethylene glycol and water. Concentrations were adjusted for the active ingredient.

Target gene:

thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

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 hours and were sub-cultured accordingly. RPMI 1640 with 20% donor horse serum (R20) 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.


Cell Cleansing
The TK +/- heterozygote cells grown in suspension spontaneously mutate at a low but significant rate. Before the stocks of cells were frozen they were cleansed of homozygous (TK -/-) mutants by culturing in THMG medium for 24 hours. This medium contained Thymidine (9 µg/mL), Hypoxanthine (15 µg/mL), Methotrexate (0.3 µg/mL) and Glycine (22.5 µg/mL). For the following 24 hours the cells were cultured in THG medium (i.e. THMG without Methotrexate) before being returned to R10 medium.

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fraction

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 9.77, 19.53, 39.06, 78.13, 156.25, 312.5, 625, 1250, 2500 µg/mL

Main Experiment: 4 hours, with and without S9 mix: 0, 2.5, 5, 10, 20, 30, 40, 50 amd 60 µg/mL (+S9 also 70 and 80 µg/mL)

Main Experiment: 24 hours, without S9-mix: 0, 0.31, 0.63, 1.25, 2.5, 5, 10, 15 and 20 µg/mL

Vehicle / solvent:

Acetone

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Acetone

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

Preliminary Toxicity Test
A preliminary toxicity test was performed on cell cultures at 5 x 105 cells/mL, using a 4 hour exposure period both with and without metabolic activation (S9), and at 1.5 x 105 cells/mL using a 24-hour exposure period without S9. The dose range used in the preliminary toxicity test was 9.77 to 2500 µg/mL for all three of the exposure groups. Following the exposure period the cells were washed twice with R10, resuspended in R20 medium, counted using a Coulter counter and then serially diluted to 2 x 105 cells/mL.

The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured after 24 hours by counting and diluting to 2 x 105 cells/mL. After a further 24 hours the cultures were counted and then discarded. The cell counts were then used to calculate Suspension Growth (SG) values. The SG values were then adjusted to account for immediate post Concentration toxicity, and a comparison of each Concentration SG value to the concurrent vehicle control performed to give a percentage Relative Suspension Growth (%RSG) value.

Results from the preliminary toxicity test were used to set the test item dose levels for the mutagenicity experiments. Maximum dose levels were selected using the following criteria:

i) For non-toxic test items the upper test item concentrations will be 10 mM, 2 mg/mL or 2 µL/mL whichever is the lowest. When the test item is a substance of unknown or variable composition (UVCB) the upper dose level may need to be higher and the maximum concentration will be 5 mg/mL.

ii) Precipitating dose levels will not be tested beyond the onset of precipitation regardless of the presence of toxicity beyond this point.

iii) In the absence of precipitate and if toxicity occurs, the highest concentration should lower the Relative Total Growth (RTG) to approximately 10 to 20 % of survival. This optimum upper level of toxicity was confirmed by an IWGT meeting in New Orleans, USA (Moore et al., 2002).

Main Experiment
Several days before starting the experiment, an exponentially growing stock culture of cells was set up so as to provide an excess of cells on the morning of the experiment. For the 4-hour exposure groups both with and without metabolic activation, the cells were counted and processed to give 1 x 106 cells/mL in 10 mL aliquots in R10 medium in sterile plastic universals. In the 24-hour exposure in absence of metabolic activation the cells were processed to give 0.3 x 106 cells/mL in 10 mL cultures established in 25 cm2 tissue culture flasks The Concentrations were performed in duplicate (A + B), at up to ten dose levels of the test item (2.5 to 60 µg/mL for the 4-hour exposure group in the absence of metabolic activation, 2.5 to 80 µg/mL for the 4-hour exposure group in the presence of metabolic activation, and 0.31 to 20 µg/mL for the 24-hour exposure group in the absence of metabolic activation). To each universal was added 2 mL of S9-mix (2%) if required, 0.1 mL of the Concentration dilutions, (0.15 or 0.2 mL for the positive control) and sufficient R0 medium to bring the total volume to 20 mL (R10 is used for the 24-hour exposure group). The Concentration vessels were incubated at 37 °C for 4 or 24 hours with continuous shaking using an orbital shaker within an incubated hood.


Measurement of Survival, Viability and Mutant Frequency
At the end of the Concentration period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/mL. The cultures were incubated at 37 °C with 5% CO2 in air and sub-cultured every 24 hours for the expression period of two days, by counting and dilution to 2 x 105 cells/mL, unless the mean cell count was less than 3 x 105 cells/mL in which case all the cells were maintained.

On Day 2 of the experiment, the cells were counted, diluted to 104 cells/mL and plated for mutant frequency (2000 cells/well) in selective medium containing 4 µg/mL 5 trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/mL and plated (2 cells/well) for viability (%V) in non-selective medium.

The daily cell counts were used to obtain a Relative Suspension Growth (%RSG) value that gives an indication of post Concentration toxicity during the expression period as a comparison to the vehicle control, and when combined with the Viability (%V) data a Relative Total Growth (RTG) value.

Plate Scoring
Microtitre plates were scored using a magnifying mirror box after ten to fourteen days’ incubation at 37 °C with 5% CO2 in air. The number of positive wells (wells with colonies) was recorded together with the total number of scorable wells (normally 96 per plate). The numbers of small and large colonies seen in the TFT mutation plates were also recorded (Cole et al., 1990). Colonies are scored manually by eye using qualitative judgement. Large colonies are defined as those that cover approximately ¼ to ¾ of the surface of the well and are generally no more than one or two cells thick. In general, all colonies less than 25% of the average area of the large colonies are scored as small colonies. Small colonies are normally observed to be more than two cells thick. To assist the scoring of the TFT mutant colonies 0.025 mL of thiazolyl blue tetrazolium bromide (MTT) solution, 2.5 mg/mL in phosphate buffered saline (PBS), was added to each well of the mutation plates. The plates were incubated for approximately two to three hours. MTT is a vital stain that is taken up by viable cells and metabolized to give a brown/black color, thus aiding the visualization of the mutant colonies, particularly the small colonies.

Evaluation criteria:

Please see "Any other information on materials and methods incl. tables"

Key result

Species / strain:

mouse lymphoma L5178Y cells

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:

Preliminary Cytotoxicity Test
In all three of the exposure groups, there was evidence of marked reductions in the relative suspension growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The onset of toxicity was very sharp in all three of the exposure groups indicating that achieving optimum levels of toxicity would be very difficult. Precipitate of the test item was observed at and above 625 µg/mL in the 4-hour and 24-hour exposure groups in the absence of metabolic activation, and at and above 312.5 µg/mL in the 4-hour exposure group in the presence of metabolic activation. In the subsequent mutagenicity test the maximum dose levels were limited by test item-induced toxicity.

Mutagenicity Test
4-hour exposure in the absence and presence of metabolic activation
As was seen previously, there was evidence of marked toxicity following exposure to the test item in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was also evidence of a modest reduction in viability (%V) in the absence of metabolic activation, therefore indicating that residual toxicity had occurred. Based on the %RSG and RTG values observed, optimum levels of toxicity were achieved in the absence of metabolic activation. Whilst optimum levels of toxicity were not achieved in the presence of metabolic activation due to the steep toxicity curve of the test item, despite using a narrow dose interval, a dose level that exceeded the usual upper limit of acceptable toxicity was plated for viability and 5-TFT resistance as sufficient cells were available at the time of plating. The excessive toxicity observed at and above 40 µg/mL in the absence of metabolic activation, and at and above 70 µg/mL in the presence of metabolic activation, resulted in these dose levels not being plated for viability or 5-TFT resistance. The toxicity observed at 30 µg/mL in the absence of metabolic activation exceeded the upper acceptable limit of 90%, therefore, this dose was excluded from the statistical analysis. Acceptable levels of toxicity were seen with both positive control substances.

The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. Both of the positive controls produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily and that the metabolic activation system was functional.

The test item did not induce any toxicologically increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels (including the dose levels that achieved or exceeded optimum levels of toxicity), in either the absence or presence of metabolic activation.

Precipitate of the test item was observed at and above 70 µg/mL in the presence of metabolic activation at the end of the exposure period.

24-hour exposure in the absence of metabolic activation
As was seen previously, there was evidence of marked toxicity following exposure to the test item as indicated by the %RSG and RTG values (Table 9). There was no evidence of marked reductions in viability (%V), therefore indicating that residual toxicity had not occurred in this exposure group. Based on the RTG and / or %RSG values observed, optimum levels of toxicity were considered to have been achieved. The excessive toxicity observed at and above 15 µg/mL resulted in these dose levels not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with the positive control substance (Table 9).

The 24-hour exposure without metabolic activation group, demonstrated that the extended time point had a marked effect on the toxicity of the test item. The vehicle control cultures had mutant frequency values that were considered acceptable for the L5178Y cell line at the TK +/- locus. The positive control produced marked increases in the mutant frequency per viable cell indicating that the test system was operating satisfactorily.

The test item did not induce any toxicologically increases in the mutant frequency x 10-6 per viable cell, at any of the dose levels, including the dose level that achieved optimum levels of toxicity.

No precipitate of the test item was observed at any of the dose levels at the end of the exposure period.

Summary of Results

Main Experiment

Concentration (µg/mL)		4-Hours-S9					Concentration (µg/mL)		4-Hours+S9
		%RSG	RTG	MF§					%RSG	RTG	MF§
0		100	1.00	178.99			0		100	1.00	179.29
2.5		91	1.03	202.12			2.5	Ø	93
5		97	1.09	183.43			5		94	1.12	140.40
10		76	0.72	249.82			10		89	1.16	121.80
20		21	0.21	255.81			20		74	0.82	158.33
30	X	2	0.02	479.83			30		23	0.28	154.75
40	Ø	0					40		60	0.83	133.55
50	Ø	0					50		40	0.48	168.90
60	Ø	0					60	X	2	0.04	149.12
							70	Ø	1
							80	Ø	0
MF threshold for a positive response = 304.99							MF threshold for a positive response = 305.29
Positive control							Positive control
EMS							CP
400		90	0.73	1576.18			1.5		81	0.78	714.89

               

Concentration (µg/mL)		24-Hours-S9
		%RSG	RTG	MF§
0		100	1.00	206.84
0.31		89	1.29	140.89
0.63		80	0.83	212.78
1.25		76	0.81	194.37
2.5		50	0.67	192.18
5		33	0.48	207.94
10		12	0.33	199.95
15	Ø	1
20	Ø	0
MF threshold for a positive response = 332.84
Positive control
EMS
150		62	0.39	1248.88

Conclusions:

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Executive summary:

Introduction

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method was designed to be compatible with the OECD Guidelines for Testing of Chemicals No 490 "In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene" adopted 29 July 2016, Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008, the US EPA OPPTS 870.5300 Guideline, and in alignment with the Japanese MITI/MHW guidelines for testing of new chemical substances.

 

Methods…….

One main experiment was performed. In the main experiment, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test item at up to ten dose levels, in duplicate, together with vehicle (acetone) and positive controls using 4‑hour exposure groups both in the absence and presence of metabolic activation (2% S9) and a 24-hour exposure in the absence of metabolic activation.

 

The dose range of test item used in the main test was selected following the results of a preliminary toxicity test. The dose levels plated out for viability and expression of mutant colonies were as follows:

 

Main Experiment

Group	Concentration of Tallow bis(2-hydroxyethyl)amineoxide CAS No 61791-46-6: (µg/mL) plated for mutant frequency
4-hour without S9	2.5, 5, 10, 20, 30, 40
4-hour with S9 (2%)	5, 10, 20, 30, 40, 50, 60
24-hour without S9	0.31, 0.63, 1.25, 2.5, 5, 10

 

 Results……..

The maximum dose levels used in the main test were limited by test item-induced toxicity. Precipitate of the test item was observed at and above 70 µg/mL in the 4-hour exposure group in the presence of metabolic activation. The vehicle (acetone) controls had acceptable mutant frequency values that were within the acceptable range for the L5178Y cell line at the TK +/- locus. The positive control items induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolizing system.

 

The test item 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.

Conclusion

The test item did not induce any increases in the mutant frequency at the TK +/- locus in L5178Y cells that exceeded the GEF, consequently it is considered to be non-mutagenic in this assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No mode of action / human relevance framework is required as all three in-vitro studies were negative. There is no concern for potential for mutagenic, clastogenic or aneugenic potential for Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides,

Additional information

There are two in-vitro genotoxicity studies available forEthanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides, an in-vitro micronucleus test in human lymphocytes and an L5178 TK +/- Mouse lymphoma assay. Both studies were carried out to the current OECD guidelines to GLP. In addition there is an Ames test used for read across from the source substance 2,2’-(octadec-9-enylimino)diethanol CAS No 93962-62-0. The higher degree of unsaturation in the 2,2’-(octadec-9-enylimino)diethanol is expected to result in higher reactivity from the increased double bonds in the unsaturated C18 in the substance which at 80% is significantly higher than in the target substance Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides which has only 35% unsaturated C18. Due to this higher reactivity 2,2’-(octadec-9-enylimino)diethanol would be more likely to react with DNA than Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides so read across to this substance will not underestimate the potential for mutagenicity in the Ames test.

Justification for classification or non-classification

The clear negative results in the Ames test, in-vitro micronucleus test in human lymphocytes and L5178 TK +/- Mouse lymphoma assay but with and without S9 metabolic activation means there are no concerns for any mutagenic, clastogenic or aneugenic effects from Ethanol, 2,2-iminobis-N-tallow alkyl derivatives, N-oxides. No in-vivo testing is required and no classification is required.