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Genetic toxicity in vitro

Description of key information

2,2'(Octadec-9-enylimino)bisethanol CAS No 25307-17-9 was tested in a bacterial reverse mutation assay (Ames) with and without metabolic acitoivation (S9-mix) and showed no mutagenic activity. It was also found to be not mutagenic and not clastogenic in the mammalian cell gene mutation in the L5178Y TK +/- Mouse lymphoma assay and Chromosomal aberration test in human lymphocytes in-vitro, both with and with S9.

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP, Fully OECD 417 compliant
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Version / remarks:
Also OECD guideline No 471
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Target gene:
The five strains of Salmonella typhimurium (a): TA 1535, TA 1537, TA 98, TA 100 and TA 102. Each strain derived from Salmonella typhimurium LT 2 contains one mutation in the histidine operon, resulting in a requirement for histidine.
In addition, to increase their sensitivity to mutagenic items, further mutations have been added:
• the rfa mutation causes partial loss of the lipopolysaccharide barrier that coats the surface of
the bacteria and increases permeability to large molecules that do not penetrate the normal
bacteria cell wall,
• the uvrB mutation is a deletion of a gene coding for the DNA excision repair system, which
renders the bacteria unable to use this repair mechanism to remove the damaged DNA,
• the addition of the plasmid pKM 101 to strains TA 98, TA 100 and TA 102 enhances their
sensitivity of detection to some mutagens,
• in case of TA 102 strain, the histidine mutation is located on the multicopy plasmid pAQ1.
Species / strain / cell type:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA102
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix from Aroclor 1254 treated rats
Test concentrations with justification for top dose:
PRELIMINARY TOXICITY TEST
The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL. Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate.

MUTAGENICITY EXPERIMENTS
Since the test item was toxic in the preliminary test, the choice of the highest dose-level was
based on the level of toxicity, according to the criteria specified in the international guidelines.
Experiments without S9 mix:
The selected treatment-levels were:
⋅ 2.34, 4.69, 9.38, 18.75, 37.5 and 75 μg/plate, for all the strains in the first experiment,
⋅ 1.25, 2.5, 5, 10 and 20 μg/plate, for all the strains in the second experiment

Experiments with S9 mix:
The selected treatment-levels were:
⋅ 18.75, 37.5, 75, 150 and 300 μg/plate, for the TA 98, TA 1537 and TA 102 strains in the first experiment,
⋅ 9.38, 18.75, 37.5, 75 and 150 μg/plate, for the TA 100 and TA 1535 strains in the first experiment,
⋅ 6.25, 12.5, 25, 50 and 100 μg/plate, for all the strains in the second experiment


2.1.3 Positive controls
without S9 mix:
• 1 μg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,
• 50 μg/plate of 9-Aminoacridine (9AA): TA 1537 strain,
• 0.5 μg/plate of 2-Nitrofluorene (2NF): TA 98 strain,
• 0.5 μg/plate of Mitomycin C (MMC): TA 102 strain.
with S9 mix:
• 2 μg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,
• 10 μg/plate of 2-Anthramine (2AM): TA 102 strain.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO batch K33208450 435 (Merck Eurolab, Fontenay-Sous-Bois, France or distilled water for the Mitomycin C positive control.
- Justification for choice of solvent/vehicle: To achieve necessary solubility for test substance and the positive control substances.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 2-Anthramine
Remarks:
With S9 Mix
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Sodium Azide TA 1535 -TA100, 9-Aminoacridine TA 1537, 2-nitrofluorene TA 98, Mitomycin C TA102
Remarks:
Without S9 mix
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation) was used for the preliminary test and for the first experiment both without S9 mix and the first experiment with S9 mix.
preincubation; was used for the second experiment with S9.

The direct plate incorporation method was performed as follows: test item solution (0.05 mL),
S9 mix when required or phosphate buffer pH 7.4 (0.5 mL) and bacterial suspension (0.1 mL)
were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin
and maintained at 45°C). After rapid homogenization, the mixture was overlaid onto a Petri
plate containing minimum medium.

The preincubation method (b, c) was performed as follows: test item solution (0.05 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C, under shaking, before adding the overlay agar and pouring onto the surface of a minimum agar plate. After 48 to 72 hours of incubation at 37°C (see § 2.5), revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, Suffolk CB9 7 BN, UK).

Preliminary toxicity test

To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

Mutagenicity experiments

In two independent experiments, using three plates/dose-level, each strain was tested, with and without S9 mix, with:
• at least five dose-levels of the test item,
• the vehicle control,
• the appropriate positive control.
The sterility of the S9 mix was checked before the beginning and at the end of each experiment and was found to be satisfactory.
Evaluation criteria:
In each experiment, for each strain and for each experimental point, the number of revertants per plate was scored. The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

Acceptance criteria

This study is considered valid if the following criteria are fully met:
• the number of revertants in the vehicle controls is consistent with the historical data of the
testing facility
• the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with the historical data of the testing facility.

Evaluation criteria

A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.
Species / strain:
S. typhimurium, other: TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

RANGE-FINDING/SCREENING STUDIES:

The test item was freely soluble in the vehicle (DMSO) at 100 mg/mL.

 

Consequently, with a treatment volume of 50 μL/plate, the dose-levels were 10, 100, 500, 1000, 2500 and 5000 μg/plate. A marked precipitate was observed in the Petri plates when scoring the revertants at dose-levels ≥ 2500 μg/plate.

 

A moderate to marked toxicity was noted at dose-levels ≥ 100 μg/plate towards the three strains used without S9 mix and in the TA 100 strain with S9 mix and ≥ 500 μg/plate in the TA 98 and TA 102 strains with S9 mix

 

COMPARISON WITH HISTORICAL CONTROL DATA:

 

All values were within the historical control means for the vehicle and positive controls for each strain..

 

ADDITIONAL INFORMATION ON CYTOTOXICITY:

 

A marked toxicity was observed in all strains at dose-levels ≥ 150 μg/plate.

 

Conclusions:
Under our experimental conditions, the test item CECAJEL 210 (batch No. 9194) did not show any mutagenic activity in the bacterial reverse mutation test with Salmonella
Executive summary:

The objective of this study was to evaluate the potential of the test item CECAJEL 210

(batch No. 9194) to induce reverse mutation inSalmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

 

Methods

A preliminary toxicity test was performed to define the dose-levels of CECAJEL 210 to be used for the mutagenicity study. The test item was then tested in two independent experiments, with and without a metabolic activation system, the S9 mix, prepared from a liver microsomal fraction (S9 fraction) of rats induced with Aroclor 1254. Both experiments were performed according to the direct plate incorporation method except for the second test with S9 mix, which was performed according to the preincubation method (60 minutes, 37°C). Five strains of bacteriaSalmonella typhimurium: TA 1535, TA 1537, TA 98, TA 100 and TA 102 were used. Each strain was exposed to at least five dose-levels of the test item (three plates/dose-level). After 48 to 72 hours of incubation at 37°C, the revertant colonies were scored.

 

The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

 

The test item CECAJEL 210 was dissolved in dimethylsulfoxide (DMSO).

 

The dose-levels of the positive controls were as follows:

without S9 mix:

• 1 μg/plate of sodium azide (NaN3): TA 1535 and TA 100 strains,

• 50 μg/plate of 9-Aminoacridine (9AA): TA 1537 strain,

• 0.5 μg/plate of 2-Nitrofluorene (2NF): TA 98 strain,

• 0.5 μg/plate of Mitomycin C (MMC): TA 102 strain.

 

with S9 mix:

• 2 μg/plate of 2-Anthramine (2AM): TA 1535, TA 1537, TA 98 and TA 100 strains,

• 10 μg/plate of 2-Anthramine (2AM): TA 102 strain.

 

Results

The number of revertants for the vehicle and positive controls was as specified in the acceptance criteria. The study was therefore considered valid. Since the test item was toxic in the preliminary test, the choice of the highest dose-level was based on the level of toxicity, according to the criteria specified in the international guidelines.

 

Experiments without S9 mix:

The selected treatment-levels were:

2.34, 4.69, 9.38, 18.75, 37.5 and 75 μg/plate, for all the strains in the first experiment,

1.25, 2.5, 5, 10 and 20 μg/plate, for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. A moderate to marked toxicity was noted in all strains at dose-levels ≥ 10 μg/plate. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Experiments with S9 mix:

The selected treatment-levels were:

18.75, 37.5, 75, 150 and 300 μg/plate, for the TA 98, TA 1537 and TA 102 strains in the

first experiment,

9.38, 18.75, 37.5, 75 and 150 μg/plate, for the TA 100 and TA 1535 strains in the

first experiment,

6.25, 12.5, 25, 50 and 100 μg/plate, for all the strains in the second experiment.

 

No precipitate was observed in the Petri plates when scoring the revertants at any dose-level. A marked toxicity was observed in all strains at dose-levels ≥ 150 μg/plate. The test item did not induce any noteworthy increase in the number of revertants, in any of the five strains.

 

Conclusion

Under our experimental conditions, the test item CECAJEL 210 (batch No. 9194) did not show any mutagenic activity in the bacterial reverse mutation test withSalmonella

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 02 September 2009 and 25 November 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of the relevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Not applicable.
Species / strain / cell type:
lymphocytes: human
Details on mammalian cell type (if applicable):
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a volunteer who had been previously screened for suitability. The volunteer had not been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbitone and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9) (µg/ml)
4(20)-hour without S9 0*, 1.25*, 2.5*, 5*, 10*, 20, 30, MMC 0.4*
4(20)-hour with S9 0*, 2.5*, 5*, 10*, 20*, 30, 40, CP 5*

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9) (µg/ml)
24-hour without S9 0*, 1.25*, 2.5*, 5*, 10* 15, 20, MMC 0.2*
4(20)-hour with S9 0*, 2.5*, 5*, 10*, 20*, 30, 40, CP 5*

* Dose levels selected for metaphase analysis
MMC = Mitomycin C
CP = Cyclophosphamide
Vehicle / solvent:
The test material was dissolved in Dimethyl Sulphoxide (DMSO) as it formed a solution with the test material suitable for dosing.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
In the presence of S9 Migrated to IUCLID6: (CP)
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Dimethyl Sulphoxide (DMSO)
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
In the absence of S9 Migrated to IUCLID6: (MMC)
Details on test system and experimental conditions:
METHOD OF APPLICATION
-In medium

DURATION
- Preincubation period:
48 hrs

- Exposure duration:
Experiment 1 - 4 hrs with and without S9. Experiment 2 - 24 hrs without S9, 4 hrs with S9.

- Expression time (cells in growth medium):
20 hrs for 4 hrs exposure.

- Selection time (if incubation with a selection agent):
Not applicable.

- Fixation time (start of exposure up to fixation or harvest of cells):
24 hrs.


SELECTION AGENT (mutation assays):
No selection agent.

SPINDLE INHIBITOR (cytogenetic assays):
Demecolcine

STAIN (for cytogenetic assays):
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and coverslipped using mounting medium.


NUMBER OF REPLICATIONS:
Duplicate cultures


NUMBER OF CELLS EVALUATED:
100/culture


DETERMINATION OF CYTOTOXICITY
- Method:
mitotic index - A total of 2000 lymphocyte cell nuclei were counted and the number of cells in metaphase recorded and expressed as the mitotic index and as a percentage of the vehicle control value.

-Scoring of Chromosome Damage:
Where possible the first 100 consecutive well-spread metaphases from each culture were counted, where there were approximately 30 to 50% of cells with aberrations, slide evaluation was terminated at 50 cells. If the cell had 44-48 chromosomes, any gaps, breaks or rearrangements were noted according to the simplified system of Savage (1976) recommended in the 1983 UKEMS guidelines for mutagenicity testing . Cells with chromosome aberrations were reviewed as necessary by a senior cytogeneticist prior to decoding the slides.

OTHER EXAMINATIONS:
- Determination of polyploidy:
Frequency of polyploid cells


OTHER:
None.

Evaluation criteria:
A positive response was recorded for a particular treatment if the % cells with aberrations, excluding gaps, markedly exceeded that seen in the concurrent control, either with or without a clear dose-relationship. For modest increases in aberration frequency a dose response relationship is generally required and appropriate statistical tests may be applied in order to record a positive response.
Statistics:
The frequency of cells with aberrations excluding gaps and the frequency of polyploid cells was compared, where necessary, with the concurrent vehicle control value using Fisher's Exact test.
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Refer to information on results and tables
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
Results tables 1 to 8 are attached

Preliminary Toxicity Test
The dose range for the Preliminary Toxicity Test was 1.25 to 160 µg/ml. The maximum dose selected, 160 µg/ml, was based on the toxicity seen in
the Mouse Lymphoma Assay on the same test material (Harlan Laboratories Ltd Project No. 0142/0421). A precipitate of the test material was observed in the parallel blood-free cultures at the end of the exposure, at and above 40 µg/ml, in the 4(20)-hour with metabolic activation (S9) exposure group and the 24-hour continuous exposure group. In the 4(20)-hour without metabolic activation (S9) exposure group precipitate was seen at 160 µg/ml. Haemolysis was also seen in the blood cultures at the end of exposure in all three exposure groups at and above 80 µg/ml. Microscopic assessment of the slides prepared from the exposed cultures showed that metaphase cells were present up to 20 µg/ml in the 4(20) hour exposure groups in the
presence and absence of metabolic activation (S9). The maximum dose with metaphases present in the 24-hour continuous exposure was 10 µg/ml.
The mitotic index data are presented in the attached Table 1. The test material induced evidence of toxicity in all of the exposure groups.
The selection of the maximum dose level was based on toxicity and was 40 µg/ml and 30 µg/ml for the 4(20)-hour exposure groups with and without S9 respectively. For the continuous exposure group used in Experiment 2, the maximum dose selected was 20 µg/ml.
Chromosome Aberration Test - Experiment 1
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9) (µg/ml)
4(20)-hour without S9 0*, 1.25*, 2.5*, 5*, 10*, 20, 30, MMC 0.4*
4(20)-hour with S9 0*, 2.5*, 5*, 10*, 20*, 30, 40, CP 5*
The qualitative assessment of the slides determined that the toxicity was similar to that observed in the Preliminary Toxicity Test and that there were
metaphases suitable for scoring present at 20 µg/ml in the absence of metabolic activation (S9). In the presence of metabolic activation (S9) the
maximum dose level of the test material with metaphases suitable for scoring was 30 µg/ml. No precipitate of the test material was observed at the
end of exposure in either dose group.
The mitotic index data are given in attached Table 2. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was observed, and that 60% and 90% mitotic inhibition was achieved at 10 and 20 µg/ml respectively in the absence of S9. In the presence of S9 65% and 80% mitotic inhibition was achieved at 20 and 30 µg/ml respectively.
The maximum dose level selected for metaphase analysis was based on toxicity and was 10 µg/ml in the absence of S9 and 20 µg/ml in the presence of S9.
The chromosome aberration data are given in attached Table 4 and Table 5. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with aberrations either in the absence or presence of
metabolic activation.
The polyploid cell frequency data are given in attached Table 8. The test material did not induce a statistically significant increase in the numbers of
polyploid cells at any dose level in either of the exposure groups.

Chromosome Aberration Test - Experiment 2
The dose levels of the controls and the test material are given in the table below:
Group Final concentration of Bis (2-hydroxyethyl) oleyl amine (CAS Number 25307-17-9) (µg/ml)
24-hour without S9 0*, 1.25*, 2.5*, 5*, 10* 15, 20, MMC 0.2*
4(20)-hour with S9 0*, 2.5*, 5*, 10*, 20*, 30, 40, CP 5*
The qualitative assessment of the slides determined that there were metaphases suitable for scoring present at 20 µg/ml in the presence of S9. In the
absence of S9 the maximum test material dose level with metaphases suitable for scoring was 10 µg/ml. No precipitate of the test material was
observed at the end of exposure in either exposure group.
The mitotic index data are given in attached Table 3. They confirm the qualitative observations in that a dose-related inhibition of mitotic index was
observed, and that 28% mitotic inhibition was achieved at 10 µg/ml in the absence of S9. The higher dose level of 15 µg/ml had no metaphases for
scoring due to toxicity. In the presence of S9 76% mitotic inhibition was achieved at 20 µg/ml. Whilst the optimum 50% toxicity was not achieved or
exceeded in the absence of S9 because of the steepness of the toxicity curve, it was considered that the test material had been adequately tested.
The maximum dose level selected for metaphase analysis was the based on toxicity and was 10 and 20 µg/ml for the without and with S9 exposure
groups respectively.
The chromosome aberration data are given in attached Table 6 and Table 7. All of the vehicle control cultures had frequencies of cells with
chromosome aberrations within the expected range. The positive control materials induced statistically significant increases in the frequency of cells
with aberrations. The metabolic activation system was therefore shown to be functional and the test method itself was operating as expected.
The test material did not induce any statistically significant increases in the frequency of cells with chromosome aberrations either in the absence or
presence of metabolic activation.
The polyploid cell frequency data are given in attached Table 8. The test material did not induce a statistically significant increase in the numbers of
polyploid cells at any dose level in either of the exposure groups.

Remarks on result:
other: strain/cell type:
Remarks:
Migrated from field 'Test system'.

For the tables and figures of results mentioned above, please refer to the attached background material section for the following tables:

Table 1: Mitotic Index - Preliminary Toxicity Test

Table 2: Mitotic Index - Experiment 1

Table 3: Mitotic Index - Experiment 2

Table 4: Results of Chromosome Aberration Test - Experiment 1 Without Metabolic Activation (S9)

Table 5: Results of Chromosome Aberration Test - Experiment 1 With Metabolic Activation (S9)

Table 6: Results of Chromosome Aberration Test - Experiment 2 Without Metabolic Activation (S9)

Table 7: Results of Chromosome Aberration Test - Experiment 2 With Metabolic Activation (S9)

Table 8:  Mean Frequency of Polyploid Cells (%)

Conclusions:
Interpretation of results (migrated information):
negative

The test material did not induce a statistically significant increase in the frequency of cells with chromosome aberrations in either the absence or presence of a liver enzyme metabolising system in either of two separate experiments. The test material was therefore considered to be non-clastogenic to human lymphocytes in vitro.
Executive summary:

Introduction.  

This report describes the results of an in vitro study for the detection of structural chromosomal aberrations in cultured mammalian cells. It supplements microbial systems insofar as it identifies potential mutagens that produce chromosomal aberrations rather than gene mutations (Scottet al, 1990). The method used followed that described in the OECD Guidelines for Testing of Chemicals (1997) No. 473 "Genetic Toxicology: Chromosome Aberration Test" and Method B10 of Commission Regulation (EC) No. 440/2008 of 30 May 2008. The study design also meets the requirements of the UK Department of Health Guidelines for Testing of Chemicals for Mutagenicity.

Methods. 

Duplicate cultures of human lymphocytes, treated with the test material, were evaluated for chromosome aberrations at up to four dose levels, together with vehicle and positive controls. Four treatment conditions were used for the study, i.e. In Experiment 1, 4 hours in the presence of an induced rat liver homogenate metabolising system (S9), at a 2% final concentration with cell harvest after a 20-hour expression period and a 4 hour exposure in the absence of metabolic activation (S9) with a 20-hour expression period. In Experiment 2, the 4 hours exposure with addition of S9 was repeated (using a 1% final S9 concentration), whilst in the absence of metabolic activation the exposure time was increased to 24 hours.

Results. 

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

All the positive control materials induced statistically significant increases in the frequency of cells with aberrations indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any statistically significant increases in the frequency of cells with aberrations, in either of two separate experiments. The dose range included a dose level that induced at least 50% mitotic inhibition in the 4(20)-hour exposure groups. As a result of a steep toxicity curve less than optimum toxicity was achieved in the 24‑hour exposure group.

Conclusion. 

The test material was considered to be non-clastogenic to human lymphocytes in vitro.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The experimental phases of the study were performed between 08 September 2009 and 26 October 2009.
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study conducted to GLP and in compliance with agreed protocols, with no or minor deviations from standard test guidelines and/or minor methodological deficiencies, which do no effect the quality of therelevant results.
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
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):
Type and identity of media:
RPMI 1640 (R0)

Properly maintained:
Yes

Periodically checked for Mycoplasma contamination:
Yes

Periodically checked for karyotype stability:
No

Periodically "cleansed" against high spontaneous background:
Yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
phenobarbital and beta-naphthoflavone induced rat liver, S9
Test concentrations with justification for top dose:
The maximum dose level used was limited by test material induced toxicity.

Vehicle and positive controls were used in parallel with the test material. Solvent (DMSO) treatment groups were used as the vehicle controls. Ethylmethanesulphonate (EMS) Sigma batch 1419706 15108051 at 400 µg/ml and 150 µg/ml for Experiment 1 and Experiment 2 respectively, was used as the positive control in the absence of metabolic activation. Cyclophosphamide (CP) Acros batch A0164185 at 2 µg/ml was used as the positive control in the presence of metabolic activation.
Vehicle / solvent:
Vehicle used:
Vehicle (DMSO) treatment groups were used as the vehicle controls.


Justification for choice of vehicle:
Formed a solution suitable for dosing at the required concentration.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
With metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
Vehicle (DMSO) treatment groups were used as the vehicle controls.
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
Without metabolic activation
Details on test system and experimental conditions:
The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of 30 May 2008.

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4 hour exposure group in the presence of metabolic activation (1% S9) and a 24 hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.31 to 10 µg/ml in the absence of metabolic activation and 1.25 to 20 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.16 to 5 µg/ml in the absence of metabolic activation, and 1.25 to 20 µg/ml in the presence of metabolic activation.

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.
Evaluation criteria:
Please see "Any other information on materials and methods incl. tables" section.
Statistics:
Please see "Any other information on materials and methods incl. tables" section.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
non-mutagenic
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 of the test material used in the repeat of the preliminary toxicity test was 0.31 to 80 µg/ml for all three of the exposure groups.
In all three of the exposure groups there were marked dose related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test material when compared to the concurrent vehicle controls. The toxicity curve was steep in all three of the exposure groups. Precipitate of the test material was not observed at any of the dose levels. In the subsequent mutagenicity experiments the maximum dose was limited by toxicity.

Mutagenicity Test
A summary of the results from the Test is presented in attached Table 1.

Experiment 1

The results of the microtitre plate counts and their analysis are presented in attached Tables 2 to 7.

There was evidence of toxicity following exposure to the test material in both the absence and presence of metabolic activation, as indicated by the %RSG and RTG values. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred. Optimum levels of toxicity were achieved in the presence of metabolic activation (Table 6). Optimum levels of toxicity were not achieved in the absence of metabolic activation due to the very sharp onset of toxicity, despite using a very narrow dose interval. However, with no evidence of a response in either the absence or presence of metabolic activation in this experiment, or in the second experiment where the usual upper limit of acceptable toxicity was exceeded, and the subsequent dose level to the upper surviving dose level exhibiting an excessive level of toxicity, the test material was considered to have been adequately tested (Tables 3 and 6). The excessive toxicity observed at 10 µg/ml in the absence of metabolic activation, resulted in this dose not being plated for viability or 5-TFT resistance. Acceptable levels of toxicity were seen with both positive control substances (Table 3 and Table 6).

Neither of the vehicle control mutant frequency values were markedly outside the acceptable range of 50 to 200 x 10-6 viable cells. The value for the without metabolic activation vehicle control was the lower limit of the range. 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 (Tables 3 and 6).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 3 and 6). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 4 and 7.

Experiment 2

The results of the microtitre plate counts and their analysis are presented in attached Tables 8 to 13.

As was seen previously, there was evidence of a marked dose-related reduction in % RSG and RTG values in cultures dosed with the test material in the both the absence and presence of metabolic activation. There was no evidence of any significant reductions in viability (%V), therefore indicating that no residual toxicity had occurred in either the absence or presence of metabolic activation. Near optimum levels of toxicity were achieved in the presence of metabolic activation. Optimum levels of toxicity were not achieved in either the absence or presence of metabolic activation due to the sharp onset of toxicity, despite using a very narrow dose interval. However, a dose level that marginally exceeded the usual acceptable upper limit of toxicity was plated for viability and TFT resistance for each of the exposure groups (Tables 9 and 12). It was therefore considered that with no evidence of any response, in either the first or second experiment, using a dose range where optimum levels of toxicity were achieved, approached or exceeded, the test material had been adequately tested. The excessive toxicity observed at 5 µg/ml in the absence of metabolic activation, and at and above 17.5 µg/ml in the presence of metabolic activation, resulted in these doses not being plated for viability or 5-TFT resistance. Both positive controls induced acceptable levels of toxicity (Tables 9 and 12).

The 24-hour exposure without metabolic activation demonstrated that the extended time point had an effect on the toxicity of the test material. It was also noted that the lowering of the S9 concentration to 1% S9 in the second experiment resulted in greater levels of toxicity than those observed in the presence of 2% S9 in the first experiment.

Neither of the vehicle control mutant frequency values were outside the acceptable range of 50 to 200 x 10-6 viable cells. 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 (Tables 9 and 12).

The test material did not induce any statistically significant or dose related (linear-trend) increases in the mutant frequency x 10-6 per viable cell in either the absence or presence of metabolic activation (Tables 9 and 12). No precipitate of test material was observed at any of the dose levels.
The numbers of small and large colonies and their analysis are presented in Tables 10 and 13.
Remarks on result:
other: strain/cell type: Thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.
Remarks:
Migrated from field 'Test system'.

Please see Attached "Tables 1 to 10"

Due to the nature and quantity of tables it was not possible to insert them in this section.

Conclusions:
Interpretation of results (migrated information):
negative Non-mutagenic

The test material did not induce any toxicologically significant increases in the mutant frequency at the TK +/- locus in L5178Y cells and is therefore considered to be non mutagenic under the conditions of the test.
Executive summary:

Introduction. 

The study was conducted according to a method that was designed to assess the potential mutagenicity of the test material on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line. The method used meets the requirements of the OECD (476), Method B17 of Commission Regulation (EC) No. 440/2008 of30 May 2008.

Methods. 

Two independent experiments were performed. In Experiment 1, L5178Y TK +/- 3.7.2c mouse lymphoma cells (heterozygous at the thymidine kinase locus) were treated with the test material at eight dose levels, in duplicate, together with vehicle (solvent) and positive controls using 4-hour exposure groups both in the absence and presence of metabolic activation (2% S9). In Experiment 2, the cells were treated with the test material at eight dose levels using a 4‑hour exposure group in the presence of metabolic activation (1% S9) and a 24‑hour exposure group in the absence of metabolic activation.

The dose range of test material was selected following the results of a preliminary toxicity test. The dose range for Experiment 1 was 0.31 to 10 µg/ml in the absence of metabolic activation and 1.25 to 20 µg/ml in the presence of metabolic activation. The dose range for Experiment 2 was 0.16 to 5 µg/ml in the absence of metabolic activation, and 1.25 to 20 µg/ml in the presence of metabolic activation.

Results. 

The maximum dose level used was limited by test material induced toxicity. Precipitate of test material was not observed at any of the dose levels. The vehicle (solvent) controls had acceptable mutant frequency values that were within the normal range for the L5178Y cell line at the TK +/- locus. The positive control materials induced marked increases in the mutant frequency indicating the satisfactory performance of the test and of the activity of the metabolising system.

The test material did not induce any toxicologically significant dose-related increases in the mutant frequency at any dose level, either with or without metabolic activation, in either the first or the second experiment.

Conclusion. 

The test material was considered to be non-mutagenic to L5178Y cells under the conditions of the test.

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

Based on structure and mechanism of cytotoxicity, genototoxicity by 2,2'(Octadec-9-enylimino)bisethanol is not expected. In physiological circumstances, 2,2'(Octadec-9-enylimino)bisethanol has a cationic surfactant structure (cationic charged at physiological conditions) which leads to high adsorptive properties to negatively charged surfaces as cellular membranes. The lipophilic tail easily dissolves in the membranes, whereas the polar head causes disruption and leakage of the membranes leading to cell damage or lysis of the cell content. As a consequence, the whole molecule will not easily pass membrane structures. Noteworthy in this respect is that recent research shows that the log distribution coefficient for cationic surfactants between water and phospholipid are possibly several orders of magnitude higher than between water and oil.

Cytotoxicity through disruption of cell membrane will occur rather than absorption over the cell membrane into the cell and transfer to the nucleus to interact with DNA.

Additional information

2,2'(Octadec-9-enylimino)bisethanol CAS NO 25307-17-9 was tested in a bacterial reverse mutation assay (OECD 471) and was not mutagenic with or without S9 mix. It was also tested for mammalian cell gene mutation in the L5178Y TK +/- Mouse lymphoma assay and was not mutagenic. In addition it was tested in a Chromosomal aberration test in human lymphocytes in-vitro and was found to be not clastogenic. All tests were to current OECD/EU protocols carried out to GLP and with a clearly defined and described test substance. Based on this it can be concluded that 2,2'(Octadec-9-enylimino)bisethanol CAS NO 25307-17-9 would not be expected to have any genotoxic hazard to human health.

Justification for classification or non-classification

Based on the three negative in-vitro tests for genotoxicity 2,2'(Octadec-9-enylimino)bisethanol CAS No 25307-17-9 does not require classification as a mutagen according to the European Union CLP/GHS criteria.