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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 has not been specifically tested for genetic toxicity, but there is test data on 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol CAS No 1218787-30-4 and 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 (tested as CAS No 61791-44-4) which were both tested in the Ames bacterial reverse mutation assay.  There is also data which can be used for read across for another structurally related substance with a very similar carbon chain length distribution, 2,2'(Octadec-9-enylimino)bisethanol CAS No 25307-17-9 which was tested in two additional in-vitro genotoxicty studies to current protocol and carried out to GLP with well-defined test substance.  Both 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 and 2,2'(Octadec-9-enylimino)bisethanol CAS No 25307-17-9 are more unsaturated and therefore more reactive than 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2, so they are considered as a worst case for possible genetic toxicology. 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol CAS No 1218787-30-4 has an equivelent level of saturation and a comparible alkyl chain lenght as 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 and is thought to be a highl comparable molecule on the basis of structural similarity. Addtionally, 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 was shown to be negative in Derek Nexus QSAR for genotoxicity for both S. typhimurium and E. coli strains.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
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:
guideline study
Justification for type of information:
See section 13.2 for the read-across justification.
Reason / purpose for cross-reference:
read-across source
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:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
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:
guideline study
Justification for type of information:
See section 13.2 for the read-across justification.
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
reference to other study
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 bacteria
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 04 Feb 2021 to 15 Jun 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
See section 13.2 for the read-across justification.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
Point mutations which involve substitution, addition or deletion of one or a few DNA base pairs.
All Salmonella: rfa, uvrB genes
E. coli: trp, uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A pKM 101
Additional strain / cell type characteristics:
other: The strain is deficient in the DNA nucleotide excision repair system.
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9: liver, rat
- method of preparation of S9 mix: according to Ames et al.
- final culture medium final concentrations in the S9 mix of:
8 mM MgCl2
33 mM KCl
5 mM glucose-6-phosphate
4 mM NADP
This solution was mixed with the liver 9000 x g supernatant fluid in the following proportion:
co-factor solution 9.5 parts
liver preparation 0.5 parts
- quality controls of S9 (e.g., enzymatic activity, sterility, metabolic capability)
a) Alkoxyresorufin-O-dealkylase activities
b) Test for the presence of adventitious agents
c) Promutagen activation (including biological activity in the Salmonella typhimurium assay
using 2-aminoanthracene and benzo[a]pyrene)
Test concentrations with justification for top dose:
Tester strains of TA98 and TA100 were subjected to the test item in the pre-experiment at the following concentrations: 3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate.

The test item concentrations to be applied in the main experiments were chosen according to the
results of the pre-experiment. 5000 µg/plate was selected as the
maximum concentration. The concentration range covered two logarithmic decades. Two independent
experiments were performed at the following concentrations:
Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA1535 and TA1537 (without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100 and E. coli WP2 uvrA (pKM101), TA1535 and TA1537
(with metabolic activation))
Experiment II:
0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(with metabolic activation))
Vehicle / solvent:
Negative controls (A. dest., Eurofins Munich, Lot No. 210219, 210319, 210409) and solvent controls (DMSO, AppliChem Lot No. 0001926479) were treated in the same way as all dose groups.

The S9 mix substitution buffer was used in the study as a replacement for S9 mix, without metabolic
activation (-S9).
Phosphate-buffer (0.2 M) contains per litre of purified water:
0.2 M NaH2PO4 x H2O 120 mL
0.2 M Na2HPO4 880 mL
The two solutions were mixed and the pH was adjusted to 7.4. Sterilisation was performed for 20 min
at 121 °C in an autoclave.
This 0.2 M phosphate-buffer was mixed with 0.15 M KCl solution (sterile) in the following proportion:
0.2 M phosphate-buffer 9.5 parts
0.15 M KCl solution 0.5 parts
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
2.5 µg/plate for TA98, TA100, TA1535 and TA1537; 10 µg/plate for E. coli WP2 uvrA (pKM101); with metabolic activation for all
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
E. coli WP2 uvrA (pKM101); without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other:
Remarks:
TA98, TA1537; Without metabolic activation
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
sodium azide
Remarks:
TA100, TA1535; Without metabolic activation
Details on test system and experimental conditions:
NUMBER OF REPLICATIONS:
- Replicatoins: In triplicate
- Number of independent experiments: 2

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding (if applicable): 109 cells/mL
- Test substance dissolved in DMSO and diluted prior to treatment

TREATMENT AND HARVEST SCHEDULE:
- Preincubation period, if applicable: For the pre-incubation method 100 µL of the test item-preparation is pre-incubated with the tester strains (100 µL) and sterile buffer or the metabolic activation system (500 µL) for 60 min at 37 °C prior to adding the overlay agar (2000 µL) and pouring onto the surface of a minimal agar plate.
- Exposure duration/duration of treatment: After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark

MUTAGENICITY:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.


- CRITERIA OF VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020)):
- S9 + S9
STRAIN|- S9 min | - S9 max | + S9 min | + S9 max
TA98 14 61 15 60
TA100 44 143 60 154
TA1535 5 35 4 37
TA1537 3 35 5 41
E. coli WP2 uvrA (pKM101) 110 315 142 381
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of revertant rates over the control plate
- at least five different concentrations of each tester strain are analysable.
Evaluation criteria:
MUTAGENICITY:
- The Mutation Factor is calculated by dividing the mean value of the revertant counts by the mean values of the solvent control (the exact and not the rounded values are used for calculation).
A test item is considered as mutagenic if:- a clear and dose-related increase in the number of revertants occurs and/or- a biologically relevant positive response for at least one of the dose groups occurs in at least one tester strain with or without metabolic activation.
A biologically relevant increase is described as follows:- if in tester strains TA98, TA100 and E. coli WP2 uvrA (pKM101) the number of reversions is at least twice as high- if in tester strains TA1535 and TA1537 the number of reversions is at least three times higher as compared to the reversion rate of the solvent control
METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Cytotoxicity can be detected by a clearing or rather diminution of the background lawn (indicated as "N" or “B”, respectively in the result tables) or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control.

- CRITERIA OF VALIDITY:
A test is considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to ampicillin (TA98, TA100, E. coli WP2 uvrA (pKM101))
- the negative control plates (A. dest.) with and without S9 mix are within the following ranges (mean values of the spontaneous reversion frequency are within the historical control data range (2017 – 2019 for all tester strains, except for E. coli WP2 uvrA (pKM101). For this tester strain the period was December 2019 to May 2020)):
- S9 + S9
STRAIN|- S9 min | - S9 max | + S9 min | + S9 max
TA98 14 61 15 60
TA100 44 143 60 154
TA1535 5 35 4 37
TA1537 3 35 5 41
E. coli WP2 uvrA (pKM101) 110 315 142 381
- corresponding background growth on both negative control and test plates is observed.
- the positive controls show a distinct enhancement of
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
No known confounding factors were present in the test substance.

RANGE-FINDING/SCREENING STUDIES (if applicable):
pre-experiment at the following concentrations:
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate

STUDY RESULTS
- Concurrent vehicle negative and positive control data
All controls resulted in values within acceptable limits.
For all test methods and criteria for data analysis and interpretation:
- No concentration-response relationship is presented
- Statistical analysis; According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

Ames test:
- Signs of toxicity: cyto toxicity observed with increasing concentration both with and without S9 mix
- Mean number of revertant colonies per plate and standard deviation: Both values are presented in the study for all strains used. An additional Mutation Factor has been calculated by dividing the mean revertants of the test item with the mean revertants of the vehicle control. Cytotoxicity may result in decrease in revertants seen with increasing concentration.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)


Positive Historical Control without S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Substance 4-NOPD NaN3 NaN3 4-NOPD MMS
Conc./plate 10 µg 10 µg 10 µg 40 µg 1 µL
Mean 443.2 614.2 826.2 123.3 1702.7
SD 168.7 208.2 362.2 49.4 551.4
Min 89 166 28 33 831
Max 2013 2493 1863 570 3391
RSD [%] 38.1 33.9 43.8 40 32.4
n 985 1034 935 934 89

Negative Historical Control without S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Mean 28.4 95.4 15.6 15.6 181.5
SD 8 16.3 5.9 6.1 42.7
Min 14 44 5 3 110
Max 61 143 35 35 315
RSD [%] 28.2 17 37.8 39.2 23.5
n 976 1023 926 927 87

Positive Historical Control with S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Substance 2-AA 2-AA 2-AA 2-AA 2-AA
Conc./plate 2.5 µg 2.5 µg 2.5 µg 2.5 µg 10 µg
Mean 1305.9 1054.5 185.7 187.3 708.4
SD 792.7 611 130.1 104 153.5
Min 70 119 19 23 424
Max 3609 2920 1856 1476 1138
RSD [%] 60.7 57.9 70.1 55.5 21.7
n 976 1026 929 929 89

Negative Historical Control with S9
TA98 TA100 TA1535 TA1537 WP2 uvrA (pKM101)
Mean 29.9 92.7 14.1 16.2 219.4
SD 7.3 14 5.3 6.3 48.6
Min 15 60 4 5 142
Max 60 154 37 41 381
RSD [%] 24.5 15.1 37.5 38.8 22.2
n 974 1020 923 924 87

Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I, precipitation was noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). In experiment II, precipitation was observed at concentrations of 1000 µg/plate (without and with metabolic activation). The observed precipitation did not interfere with the scoring and thus it did not impact the results.

Conclusions:
In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, CAS 1218787-30-4 did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used.
Therefore, CAS 1218787-30-4 is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

 


The test item, CAS 1218787-30-4, was investigated for its potential to induce gene mutations according to the plate incorporation test (experiment I) and the pre-incubation test (experiment II) using Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and tester strain E. coli WP2 uvrA (pKM101).
In two independent experiments several concentrations of the test item were used. Each assay was conducted with and without metabolic activation. The concentrations, including the controls, were tested in triplicate. The following concentrations of the test item were prepared and used in the experiments:


 



Experiment I:
3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA1535 and TA1537 (without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100 and E. coli WP2 uvrA (pKM101), TA1535 and TA1537
(with metabolic activation))


 



Experiment II:
0.316, 1.0, 3.16, 10.0, 31.6, 100, 316 and 1000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(without metabolic activation))
3.16, 10.0, 31.6, 100, 316, 1000, 2500 and 5000 µg/plate
(TA98, TA100, TA1535, TA1537 and E. coli WP2 uvrA (pKM101)
(with metabolic activation))


 


Precipitation of the test item was observed in all tester strains used in experiment I and II. In experiment I, precipitation was noted at concentrations of 316 µg/plate and higher (with and without metabolic activation). In experiment II, precipitation was observed at concentrations of 1000 µg/plate (without and with metabolic activation). The observed precipitation did not interfere with the scoring; thus it did not impact the results.


 


The microbial contamination observed in a few plates in experiment I at concentrations of
5000 µg/plate did not affect the quality or integrity of the results as the microbial contamination could be clearly distinguished from the Salmonella revertants and thus did not affect the evaluation.


 


Toxic effects of the test item were noted in all tester strains evaluated in experiment I and II.


 


In experiment I toxic effects of the test item were observed in tester strains TA98 and TA100 at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). Toxic effects of the test item were also noted in tester strain TA1535 concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate (with metabolic activation). Toxic effects of the test item were also observed in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 316 µg/plate and higher (without metabolic activation) and at concentrations of 2500 µg/plate and higher (with metabolic activation).


 



In experiment II toxic effects of the test item were noted in tester strain TA98 at concentrations of 3.16 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation). In tester strain TA100, toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of1000 µg/plate and higher (with metabolic activation). Toxic effects of the test item were also observed in tester strain TA1535 at concentrations of 10.0 µg/plate and higher (without metabolic activation)
and at concentrations of 1000 µg/plate and higher (with metabolic activation). In tester strain TA1537, toxic effects of the test item were noted at concentrations of 31.6 µg/plate and higher (without metabolic activation) and at concentrations of 316 µg/plate and higher (with metabolic activation).


 


Toxic effects of the test item were also observed in tester strain E. coli WP2 uvrA (pKM101) at concentrations of 100 µg/plate and higher (without metabolic activation) and at concentrations of 1000 µg/plate and higher (with metabolic activation). The reduction in the number of revertants down to a mutation factor of ≤ 0.5 found in tester strain TA1537 at a concentration of 3.16 µg/plate (without metabolic activation) was regarded as not biologically relevant due to lack of a dose-response relationship and lack of concomitant clearing of the background lawn.


 


No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with the test item at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II. In addition all validity crierion were met.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vitro:


 


2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2, has not been specifically tested but there is test data on 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol CAS No 1218787-30-4 and 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 which were tested in a bacterial reverse mutation assay (Ames) and were found not mutagenic with or without S9 mix. Also 2,2'(Octadec-9-enylimino)bisethanol CAS No 25307-17-9 a structurally similar substance was 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, which was tested in two additional in-vitro genotoxicty studies to current protocol and carried out to GLP with well-defined test substance. Both these read across substances are more unsaturated and therefore more reactive than 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2, so they are considered as a worst case for possible genetic toxicology. Based on this it can be concluded that, 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 would not be expected to have any genotoxic hazard to human health. Based on this there are no requirements for in-vivo genotoxicity testing.


 


Justification for selection of genetic toxicity endpoint


2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 has not been specifically tested for genetic toxicity, but there is test data on 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol CAS No 1218787-30-4 and 2,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 (tested as CAS No 61791-44-4) which were tested in the Ames bacterial reverse mutation assay. 2,2'-(C16-18 (evennumbered) alkyl imino) diethanol CAS No 1218787-30-4 is highly comparable to the target substance based off structural similarity including functional groups, appropriate alkyl chain length and saturation level. ,2’-(C16-18 (even numbered, C18 unsaturated) alkyl imino) diethanol CAS No 1218787-32-6 is more unsaturated and therefore more reactive than 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2, so it is considered as a worst case for possible genetic toxicology. Addtionally, 2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2 was shown to be negative in Derek Nexus QSAR for genotoxicity for both S. typhimurium and E. coli strains and was considered inactive for genotoxicity.

Justification for classification or non-classification

2,2’-(Octadecylimino)bisethanol CAS No 10213-78-2, has not been specifically tested but there is test data on three similar substances which are considered to be worst case for read across and very unlikely to underestimate any genotoxicity of this substance. All four in-vitro genotoxicity tests were consistently negative and therefore it is conclude that 2,2’-(Octadecylimino)bisethanol does not require classification as a mutagen according to the European Union CLP/GHS criteria. No in-vivo genotoxicty testing is required.