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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The key in-vitro genotoxicity studies for this endpoint addressing both bacterial mutagenicity and mammalian mutagenicity were all negative within the whole range of the amphoteric, glycinate category. In order to strengthen the read across within the substance group, the genotoxic profile of all four substances were evaluated using the ToxTracker screening tool. ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. No genotoxic or oxidative stress-inducing properties were detected for the amphoteric glycinate substances in this screening test. Also the QSAR toolbox, ACD/ToxSuite model, TOPKAT model, Derek tool and VEGA platform give no indications on mutagenic or carcinogenic properties for the data showing results with valid reliability index.

The only available in-vitro mammalian clastogenicity test (OECD 473) is performed on the smallest substance within the group (shortest alky chain, lowest number of amine residues and carboxymethylated groups). The test report on the Human Lymphocyte Cytogenetics study conclude that Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) is clastogenic based on a statistically significant increase on the mean percent aberrant cells at the top dose in Experiment II, 24 hour treatment without S9 metabolic activation. There was also a larger increase seen in Experiment I at 50µg/mL after 4 hour exposure without metabolic activation. However in this case the result was not statistically significant as the control value was at the top of the historical range. Also there was marked cytotoxicity with the relative mitotic index of 47% and the report concluded that the increase was cytotoxicity related. 

The statistical significance seen in Experiment II at 50µg/mL was due to the control mean percent aberrant cells being at the bottom of the historical control range at 0.3% a factor of ten lower than at 4 hours. There was also cytotoxicity, but to a lesser degree than seen at 4 hours, with the relative mitotic index 58%. The higher relative mitotic index corresponded to a lower mean percentage of aberrant cells at 4.7% which is only just above the top of the historical control range of 4.2%. This compares to the mean percentage of 5.3% after 4 hours of treatment without S9 metabolic activation. Overall the increase in mean percent of aberrant cells seen in Experiment II at 50µg/mL can be considered most likely due to cytotoxicity rather than clastogenicity. Also there were clearly no effects on the mean percent aberrant cells even in Experiment I with 4 hour exposure in the presence of S9 metabolic activation at 100µg/mL in the absence of significant cytotoxicity. The lack of effects with metabolic activation indicates that there is little possibility of a systemic clastogenic effect in experimental animals. 

As part of the weight of evidence there is also data for related substance Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) which has been tested in the in-vitro micronucleus assay also in human lymphocytes. This substance is closely structurally related to Coco iminodiglycinate, (Amines, N-C12-18 -alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), the difference being that it contains propyl side chain groups rather than ethyl groups, but is otherwise the same being based on the same diamine. While it has not been included in the group for other end points there is no reason to believe that this difference in structure could have any influence on possible clastogenic potential. In the in-vitro micronucleus test on(β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) there was no evidence of clastogenic or aneugenic activity at concentrations of 450µg/mL after 3 hour exposure without S9 metabolic activation, 600 µg/mL after 3 hour exposure with S9 metabolic activation and 200 µg/mL after 24 hour exposure without S9 metabolic activation.

Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) has also been tested in the L5178Y TK +/- Mouse Lymphoma Assay. No clastogenic effect was induced by the test item, neither with nor without S9 metabolic activation, at concentrations up to 0.025mg/mL without S9 metabolic activation and 0.1mg/mL with S9 metabolic activation. None of the concentrations, with or without S9 metabolic activation, induced a frequency of small colonies >40%. Evidently the substance did not induce any clastogenic effect. The highest concentrations resulted in reduction of the relative total growth, with values of 11.1% without S9 metabolic activation and 17.4% with S9 metabolic activation, so they were cytotoxic levels.

The lack of any clastogenicity or anugenicity for the structurally related ( β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) at significantly higher concentrations in the in-vitro micronucleus assay together with a lack of any evidence of clastogenicity in theL5178Y TK +/- Mouse Lymphoma Assay with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), gives significant support to a weight of evidence assessment of clastogenicity. It supports a view that the apparent clastogenicity seen with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) in the in-vitro human lymphocyte chromosome aberrations study only after 24 hours exposure in the absence of S9 metabolic activation was not true clastogenicity but rather due to the cytotoxicity present at that concentration. The genotoxic profiles of all four substances have also been evaluated using the ToxTracker screening tool. ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. No genotoxic or oxidative stress-inducing properties were detected for any of the amphoteric glycinate substances in this screening test. Also the QSAR toolbox, ACD/ToxSuite model, TOPKAT model, Derek tool and VEGA platform give no indications on mutagenic or carcinogenic properties for the data showing results with valid reliability index.

The overall conclusion is that Coco iminodiglycinate, (Amines, N-C12 -18 -alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351 -38 -1) is not mutagenic, clastogenic or genotoxic.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-05-31 to 2016-06-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
bacterial reverse mutation assay
Target gene:
His
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
rat S9 liver microsomal fraction
Test concentrations with justification for top dose:
0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate
A correction factor of 3.31 was applied to consider the active amphoteric ingredient (without sodium salt) of the test item.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: A. dest.
Untreated negative controls:
yes
Remarks:
A. dest., Eurofins Lot No. 160322, 160510
Negative solvent / vehicle controls:
yes
Remarks:
A. dest., Eurofins Lot No. 160322, 160510
Positive controls:
yes
Positive control substance:
other:
Remarks:
Positive control without metabolic activation: sodium azide for tester strains TA 100, TA 1535; 4-nitro-o-phenylene-diamine for TA 98, TA 1537; methylmethanesulfonate for TA 102; with metabolic activation: 2-aminoanthracene for all strains
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
-For the plate incorporation method the following materials were mixed in a test tube and poured over the surface of a minimal agar plate:
100 µL Test solution at each dose level, solvent control, negative control or reference mutagen solution (positive control),
500 µL S9 mix (for testing with metabolic activation) or S9 mix substitution buffer (for testing without metabolic activation),
100 µL Bacteria suspension (cf. Preparation of Bacteria, pre-culture of the strain),
2000 µL Overlay agar.
After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.


- Preincubation period: 60 min; 100 µL of the test item preparation was 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: at least 48 h; After solidification the plates were inverted and incubated at 37 °C for at least 48 h in the dark.



NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used. (In a one case only two plates were evaluated)


DETERMINATION OF CYTOTOXICITY
- Method: clearing or rather diminution of the background lawn or a reduction in the number of revertants down to a mutation factor of approximately ≤ 0.5 in relation to the solvent control

Evaluation criteria:
The Mutation Factor is calculated by dividing the mean value of the revertant counts through 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 TA 98, TA 100 and TA 102 the number of reversions is at least twice as high
- if in tester strains TA 1535 and TA 1537 the number of reversions is at least three times higher
than the reversion rate of the solvent control.
Statistics:
According to 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.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiment I and II
Additional information on results:
No precipitation of the test item was observed in any tester strain used in experiment I and II (with and without metabolic activation).
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 strain TA 98 at a concentration of 5.0 µL/plate (with and without metabolic activation). In tester strain TA 100 toxic effects of the test item were noted at concentrations of 1.0 µL/plate and higher (without metabolic activation) and at a concentration of 5.0 µL/plate (with metabolic activation). In tester strain TA 1537 toxic effects of the test item were observed at concentrations of 2.5 µL/plate and higher (without metabolic activation) and at a concentration of 5.0 µL/plate (with metabolic activation).
In experiment II toxic effects of the test item were noted in tester strain TA 98 at concentrations of 1.0 µL/plate and higher (without metabolic activation). In tester strains TA 100, TA 1535 and TA 1537 toxic effects of the test item were seen at concentrations of 1.0 µL/plate and higher (without metabolic activation) and at a concentration of 5.0 µL/plate (with metabolic activation). In tester strain TA 102 toxic effects of the test item were observed at a concentration of 5.0 µL/plate (without metabolic activation).
No biologically relevant increases in revertant colony numbers of any of the five tester strains were observed following treatment with Coco iminodiglycinate at any concentration level, neither in the presence nor absence of metabolic activation in experiment I and II.
All criteria of validity were met
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.
Conclusions:
Interpretation of results (migrated information): negative

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) did not cause gene mutations by base pair changes or frameshifts in the genome of the tester strains used. Therefore, Coco iminodiglycinate is considered to be non-mutagenic in this bacterial reverse mutation assay.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA 98, TA 100, TA 1535, TA 1537 and TA 102 of Salmonella typhimurium were exposed to Coco iminodiglycinate at concentrations of 0.0100, 0.0316, 0.100, 0.316, 1.0, 2.5 and 5.0 µL/plate (experiment I and II), in the presence and absence of mammalian metabolic activation according to the plate incorporation method (experiment I) and the pre-incubation method (experiment II). Coco iminodiglycinate (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) was tested up to the limit concentration of 5.0 µL/plate in all tester strains used.

The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

 

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:
2016-05-03 to 2016-11-18
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany
Type of assay:
in vitro mammalian chromosome aberration test
Specific details on test material used for the study:
Name: Coco iminodiglycinate
Chemical Name: active ingredient: Sodium cocopropylenediamine glycinate
CAS No.: active ingredient: 97659-51-3
Batch No.: 1259678
Molecular Weight: 482.5 g/mol
Physical State: liquid
Colour: yellow
Density: 1.14 g/cm3
pH Value: 8.7 as 20% solution
Active Components: 30.2% Coco iminodiglycinate
59.2% water
10.6% NaCl
Expiry Date: 16 December 2016
Storage Conditions: room temperature
Species / strain / cell type:
lymphocytes: Human
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 1640
- Properly maintained: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 Wistar phenobarbital and β-naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-Experiment
5, 10, 25, 50, 100, 250, 500, 1000, 1500 and 2000 µg/mL

The following concentrations were selected in the main experiments for the microscopic analysis:
Experiment I
Without metabolic activation, 4 h treatment, 24 h preparation interval:
10, 25 and 50 µg/mL
With metabolic activation, 4 h treatment, 24 h preparation interval:
25, 50 and 100 µg/mL
Experiment II
Without metabolic activation, 24 h treatment, 24 h preparation interval:
10, 25 and 50 µg/mL

Vehicle / solvent:
- Vehicle(s)/solvent(s) used: The test item was prepared/dissolved in cell culture medium (RPMI).
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the cells and the S9 activity.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
RPMI
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Remarks:
without metabolic activation (400 and 600 µg/mL)
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
RPMI
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with metabolic activation (5.0 µg/mL)
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in cell culture medium (RPMI)

TREATMENT TIME:
4 hours (Experiment I with and without metabolic activation)
24 hours (Experiment II without metabolic activation)

FIXATION INTERVAL: 24 hours (Experiment I and II with and without metabolic activation)
NUMBER OF REPLICATIONS: 2 independent experiments
NUMBER OF CELLS SCORED: 300 per concentration (150 cells per culture). An Exception in experiment I without metabolic activation was the positive control EMS (1st culture 150 cells, 2nd culture 100 cells) and with metabolic activation the positive control CPA (1st culture 25 cells, 2nd culture 75 cells). An exception in experiment II was the positive control EMS (50 cells per culture).

DETERMINATION OF CYTOTOXICITY
- Method: Mitotic index; Proliferation Index BrdU

Evaluation criteria:
There are several criteria for determining a positive result:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data

Statistics:
A statistical evaluation was used as an aid for interpretation of the results.
Statistical significance at the 5% level (p < 0.05) was evaluated by the Fischer´s exact test. The p value was used as a limit in judging for significance levels in comparison with the corresponding negative control.
The chi² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations.
Key result
Species / strain:
lymphocytes: Human
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Different intepretation of the data if possible. See Applicant's summary and conclusion.
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
No precipitation of the test item was noted without and with metabolic activation in all dose groups evaluated in experiment I and II.

Summary: Experiment I, without and with metabolic activation

 

Dose Group

Concen-tration [µg/mL]

Relative Mitotic Index [%]

Prolife-ration
Index

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

without

4 h treatment,

24 h preparation interval

C

0

100

1.36

4.0

3.0

0.0% - 3.0% aberrant cells, excl. gaps

-

-

1

10

94

/

4.3

2.7

-

-

2

25

73

/

3.3

2.0

-

-

3

50

47

1.29

6.7

5.3

-

-

EMS

600

64

/

12.0

10.8

-

+

 

 

 

with

4 h treatment, 24 h preparation interval

C

0

100

1.11

4.7

4.0

0.0% - 3.7% aberrant cells, excl. gaps

-

-

2

25

88

/

6.7

4.0

-

-

3

50

90

/

4.0

2.7

-

-

4

100

100

1.04

5.0

3.7

-

-

CPA

5.0

49

/

22.0

20.0

-

+

The mitotic index was determined in 1000 cells per culture of each test group.

The relative values of the mitotic index are related to the negative controls.

C:       Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

CPA:  Cyclophosphamide

a:        - without precipitation. + with precipitation

b:           statistical significant increase compared to negative controls (Fisher’s exact test, p< 0.05),
+: significant; -not significant

Summary: Experiment II without metabolic activation

 

Dose Group

Concen-tration [µg/mL]

Relative Mitotic Index [%]

Prolife-ration
Index

Mean %

Aberrant Cells

Historical Laboratory Negative Control Range

Precipi-tationa

Statistical Signifi-canceb

incl. Gaps

excl. Gaps

without

24 h treatment,

24 h preparation interval

C

0

100

1.39

1.3

0.3

0.0% - 4.2% aberrant cells, excl. gaps

-

-

3

10

40

/

2.7

1.0

-

-

4

25

81

/

3.3

2.0

-

-

5

50

58

1.11

5.7

4.7

-

+

EMS

400

27

/

25.0

24.0

-

+

The mitotic index was determined in 1000 cells per culture of each test group.

The relative values of the mitotic index are related to the negative controls.

 

C:       Negative Control (Culture Medium)

EMS: Ethylmethanesulfonate

a:        - without precipitation. + with precipitation

b:        statistical significant increase compared to negative controls (Fishers exact test, p< 0.05),
+: significant; -not significant

Conclusions:
The test report on the Human Lymphocyte Cytogenetics study conclude that Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) is clastogenic based on a statistically significant increase on the mean percent aberrant cells at the top dose in Experiment II, 24 hour treatment without S9 metabolic activation. There was also a larger increase seen in Experiment I at 50µg/mL after 4 hour exposure without metabolic activation. However in this case it was not statistically significant as the control value was at the top of the historical range. Also there was marked cytotoxicity with the relative mitotic index of 47% and the report concluded that the increase was cytotoxicity related.
The statistical significance seen in Experiment II at 50µg/mL was due to the control mean percent aberrant cells being at the bottom of the historical control range at 0.3% a factor of ten lower than at 4 hours. There was also cytotoxicity, but to a lesser degree than seen at 4 hours, with the relative mitotic index 58%. The higher relative mitotic index corresponded to a lower mean percentage of aberrant cells at 4.7% which is only just above the top of the historical control range of 4.2%. This compares to the mean percentage of 5.3% after 4 hours of treatment without S9 metabolic activation. Overall the increase in mean percent of aberrant cells seen in Experiment II at 50µg/mL can be considered most likely due to cytotoxicity rather than clastogenicity. Also there were clearly no effects on the mean percent aberrant cells even in Experiment I with 4 hour exposure in the presence of S9 metabolic activation at 100µg/mL in the absence of significant cytotoxicity. The lack of effects with metabolic activation indicates that there is little possibility of a systemic clastogenic effect in experimental animals.
As part of the weight of evidence there is also data for related substance Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) which has been tested in the in-vitro micronucleus assay also in human lymphocytes. This substance is closely structurally related to Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), the difference being that it contains propyl side chain groups rather than ethyl groups, but is otherwise the same being based on the same diamine. While it has not been included in the group for other end points there is no reason to believe that this difference in structure could have any influence on possible clastogenic potential. In the in-vitro micronucleus test on (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) there was no evidence of clastogenic or aneugenic activity at concentrations of 450µg/mL after 3 hour exposure without S9 metabolic activation, 600 µg/mL after 3 hour exposure with S9 metabolic activation and 200 µg/mL after 24 hour exposure without S9 metabolic activation.
Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1)has also been tested in the L5178Y TK +/- Mouse Lymphoma Assay. No clastogenic effect was induced by the test item, neither with nor without S9 metabolic activation, at concentrations up to 0.025mg/mL without S9 metabolic activation and 0.1mg/mL with S9 metabolic activation. None of the concentrations, with or without S9 metabolic activation, induced a frequency of small colonies >40%. Evidently the substance did not induce any clastogenic effect). The highest concentrations resulted in reduction of the relative total growth, with values of 11.1% without S9 metabolic activation and 17.4% with S9 metabolic activation, so they were cytotoxic levels.
The lack of any clastogenicity or anugenicity for the structurally related (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) at significantly higher concentrations in the in-vitro micronucleus assay together with a lack of any evidence of clastogenicity in the L5178Y TK +/- Mouse Lymphoma Assay with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), gives significant support to a weight of evidence assessment of clastogenicity. It supports a view that the apparent clastogenicity seen with Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1)in the in-vitro human lymphocyte chromosome aberrations study only after 24 hours exposure in the absence of S9 metabolic activation was not true clastogenicity but rather due to the cytotoxicity present at that concentration. The genotoxic profiles of all four substances have also been evaluated using the ToxTracker screening tool. ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. No genotoxic or oxidative stress-inducing properties were detected for any of the amphoteric glycinate substances in this screening test. Also the QSAR toolbox, ACD/ToxSuite model, TOPKAT model, Derek tool and VEGA platform give no indications on mutagenic or carcinogenic properties for the data showing results with valid reliability index.

The overall conclusion is that Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) is not clastogenic.
Executive summary:

A chromosome aberration assay was carried out in order to investigate a possible potential of Coco iminodiglycinate to induce structural chromosome aberrations in human lymphocytes.The metaphases were prepared 24 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation (experiment I) and 24 h without metabolic activation (experiment II). Duplicate cultures were set up. Per culture 150 metaphases were scored for structural chromosomal aberrations.

The test item was dissolved in cell culture medium and diluted prior to treatment.

The following concentrations were evaluated:

Experiment I

Without metabolic activation, 4 h treatment, 24 h preparation interval: 10, 25 and 50 µg/mL

With metabolic activation, 4 h treatment, 24 h preparation interval: 25, 50 and 100 µg/mL

Experiment II

Without metabolic activation, 24 h treatment, 24 h preparation interval: 10, 25 and 50 µg/mL

No precipitation of the test item was noted without and with metabolic activation in all dose groups evaluated in experiment I and II.

In experiment I without metabolic activation, toxic effects (decrease below 70% rel. mitotic index) were seen at a concentration of 50 µg/mL and higher. With metabolic activation, cytotoxicity was observed at a concentration of 250 µg/mL. In experiment II without metabolic activation, a biologically relevant decrease of the relative mitotic index was noted at the concentration of 50 µg/mL and higher.

No reduction of the proliferation index was indicated in experiment I with and without metabolic activation. In experiment II a biologically relevant decreases of the proliferation index was observed, thus indicating a cell cycle delay.

In experiment I without metabolic activation the aberration rates were within the historical negative control data, except for the highest concentration evaluated. This induction seemed to be cytotoxicity related as the mitotic index of the concentration 50 µg/mL was reduced to 47%. With metabolic activation, the aberration rates of the negative control and the concentration 25 µg/mL were slightly shifted to a higher degree of chromosomal aberrations regarding the historical control range of the negative control . Given that the higher test substance concentrations were within the control data, the marginal induction was not regarded as biologically relevant.

In experiment II without metabolic activation the aberration rates were within the historical negative control data, except for the highest concentration of 50 µg/mL.

In the experiments I and II without and withmetabolic activation no biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item as compared to the controls.

The Fisher´s exact test was performed to verify the results in the experiment. A statistical significance (p < 0.05) was only present at the concentration 50 µg/mL in experiment II.

The chi² Test for trend was performed to test whether there was a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in experiment I without and with metabolic activation. In experiment II, a dose-related increase in chromosome damage was determined.

EMS (400 and 600 µg/mL) and CPA (5.0 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

There was evidence of chromosome aberration induced over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 473 for in vitro cytogenetic mutagenicity data.

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:
2016-05-31 - 2016-06-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline Study
Qualifier:
according to
Guideline:
other: OECD Guidline 490 (In vitro Mammalian Cell Gene Muattaion Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test
Deviations:
no
Qualifier:
according to
Guideline:
other: IWGT Recommendations
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Germany)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI
- Properly maintained: Yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Liver S9 of Wistar Phenobarbital and ß-Naphthoflavone-induced rat liver S9 mix
Test concentrations with justification for top dose:
Pre-experiment without and with metabolic activation: 0.025, 0.05, 0.10, 0.25, 0.5, 1.0, 1.5 and 2 mg/mL

Main Experiment
without metabolic activation: 0.0005, 0.0010, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.025 mg/mL
with metabolic activation: 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1 mg/mL





Vehicle / solvent:
RPMI cell culture medium was used as solvent (RPMI + 5% HS).
Untreated negative controls:
yes
Negative solvent / vehicle controls:
no
Positive controls:
yes
Positive control substance:
other: benzo(a)pyrene 3.5 µg/mL
Positive control substance:
other: ethylmethanesulfonate 300 µg/mL
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: 10 µg/mL
Details on test system and experimental conditions:
METHOD OF APPLICATION: suspended in medium
DURATION: 4 h (short-term exposure)
Expression time (cells in growth medium): 48 h
Selection time (if incubation with selection agent): about 14 days

SELECTION AGENT ( mutation assay) 5 µg/mL trifluorothymidine
NUMBER OF REPLICATIONS: one experiments with single exposure; cells were seeded in 4 plates and evaluated
NUMBER OF CELLS SEEDED: 2000 cells per well
DETERMINATION OF CYTOTOXICITY: relative total growth (RTG)
Evaluation criteria:
The test item is considered mutagenic if following criteria are met:
-The induced mutant frequency meets or exceeds the Global Evaluation factor (GEF) of 126 per 106 cells
- A dose-dependent increase in mutant frequency is detected.

Besides, combined with a positive effect in the mutant frequency, an increased occurrence of small colonies (≥40% of total colonies) is an indication for potential clastogenic effects and/or chromosomal aberrations.
According to the OECD guideline, the biological relevance is considered first for the interpretation of results. Statistical methods might be used as an aid in evaluation the test result.
A test item is considered to be negative if the induced mutant frequency is below the GEF and the trend test is negative.

Statistics:
The non-parametric Mann-Whitney test is applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative control.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
Interpretation of results: negative

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) is considered to be non-mutagenic in the in vitro mammalian cell gene mutation assay (thymidine kinase locus) in mouse lymphoma L5178Y cells.
Executive summary:

The test item Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) was assessed for its potential to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y. The selection of the concentrations used in the main experiment was based on data from the pre-experiment. In the main experiment without metabolic activation 0.025 mg/mL and with metabolic activation 0.1 mg/mL were selected as the highest concentrations. The experiment without and with metabolic activation was performed as a 4 h short-term exposure assay.

The test item Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1) was investigated at the following concentrations:

without metabolic activation:

0.0005, 0.0010, 0.0025, 0.005, 0.01, 0.015, 0.02 and 0.025 mg/mL

and with metabolic activation:

0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09 and 0.1 mg/mL

No precipitation of the test item was noted in the experiment.

Growth inhibition was observed in the main experiment without and with metabolic activation.

In the main experiment without metabolic activation the relative total growth (RTG) was 11.1% for the highest concentration (0.025 mg/mL) evaluated. The highest concentration evaluated with metabolic activation was 0.1 mg/mL with a RTG of 17.4%. In the main experiment no biologically relevant increase of mutants was found after treatment with the test item (without and with metabolic activation).The Global Evaluation Factor (GEF; defined as the mean of the negative/vehicle mutant frequency plus one standard deviation; data gathered from ten laboratories) was not exceeded by the induced mutant frequency at any concentration.

No dose-response relationship was observed. Additionally, in the main experiment colony sizing showed no clastogenic effects induced by the test item under the experimental conditions (without and with metabolic activation).

EMS, MMS and B[a]P were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. Additionally, MMS and B[a]P significantly increased the number of small colonies, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
As part of the weight of evidence there is also data on the related substance Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) which has been tested in the in-vitro micronucleus assay also in human lymphocytes. This substance is closely structurally related to Coco iminodiglycinate, (Amines, N-C12-18 -alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), the difference being that it contains propyl side chain groups rather than ethyl groups, but is otherwise the same being based on the same diamine.
Reason / purpose:
read-across source
Species / strain:
lymphocytes: peripheral human lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The outcome of an in-vitro micronucleus test, performed according to OECD guideline 487 and GLP principles, showed that Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the report.

The in-vitro micronucleus data for Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) is used as supporting data on the genetic toxicity endpoint for the Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1).
 
This substances are closely structurally related, the difference being that Sodium cocopropylenediamine propionate (β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salts with CAS no 2136366-30-6) contains propyl side chain groups rather than ethyl groups, but is otherwise the same being based on the same diamine. While it has not been included in the group for other end points there is no reason to believe that this difference in structure could have any influence on possible clastogenic potential.
Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
ToxTracker assay
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Between 11/08/2016 and 17/08/2016
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
ToxTracker is a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. The differential induction of the GFP reporters as well as cytotoxicity of the tested compounds is determined by flow cytometry. The selected compounds were analysed in the absence and presence of an S9 rat liver extract-based metabolising system. Exposure to control compounds is included in each test to determine technical performance and reproducibility of the ToxTracker assay. Quantitative data analysis is done using ToxPlot software.
The study was not performed under GLP, however general principles to conduct proper scientifically correct in vitro experiments were adhered to.
Qualifier:
no guideline available
Principles of method if other than guideline:
The ToxTracker assay is a panel of six validated GFP-based mouse embryonic stem (mES) reporter cell lines that can be used to identify the biological reactivity and potential carcinogenic properties of newly developed compounds in a single test. ToxTracker is a mammalian stem cell-based assay that monitors activation of specific cellular signalling pathways for detection of the biological reactivity of compounds. In contrast to the cancer-derived cell lines that are currently used for in vitro genotoxicity testing, stem cells are genetically stable and proficient in all cellular pathways required for accurate detection of potentially carcinogenic properties of compounds. Extensive whole-genome transcription profiling has led to identification of a panel of biomarker genes that are preferentially activated upon exposure to different classes of carcinogens and toxicants. (1) To allow easy assessment of the activation status of these biomarker genes, the system uses generated green fluorescent (GFP) mES reporter cell lines. These reporters were created using bacterial artificial chromosomes (BAC) that contain the complete biomarker gene including promoter and regulatory elements ensuring physiological regulation of the GFP reporters following transfection into stem cells. (2)
ToxTracker consists of a panel of six different mES GFP reporter cell lines representing four distinct biological responses that are associated with carcinogenesis, i.e. general cellular stress, DNA damage, oxidative stress and the unfolded protein response. (3)

(1) Hendriks, G., Atallah, M., Raamsman, M., Morolli, B., van der Putten, H., Jaadar, H., Tijdens, I., Esveldt-van Lange, R., Mullenders, L., van de Water, B., et al. (2011). Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment. Mutat Res 709-710, 49–59.

(2) Hendriks, G., Atallah, M., Morolli, B., Calléja, F., Ras-Verloop, N., Huijskens, I., Raamsman, M., van de Water, B., and Vrieling, H. (2012). The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals. Toxicol. Sci. 125, 285–298.

(3) Hendriks G, Derr RS, Misovic B, Morolli B, Calléja FMGR, Vrieling H. (2016). The Extended ToxTracker Assay Discriminates Between Induction of DNA Damage, Oxidative Stress, and Protein Misfolding. Toxicol Sci. 150, 190–203.
GLP compliance:
no
Remarks:
This screening study was not performed under GLP, however general principles to conduct proper scientifically correct in vitro experiments were adhered to.
Type of assay:
other: ToxTracker assay
Target gene:
Biological damage: Cellular pathway: Biomarker gene:

DNA damage ATR/Chk1 DNA damage signaling Bscl2
NF-kB signaling Rtkn

Oxidative stress Nrf2 antioxidant response Srxn1
Nrf2-independent Blvrb

Protein damage Unfolded protein response Ddit3

Cellular stress p53 signaling Btg2
Species / strain / cell type:
other: mammalian cell line
Remarks:
GFP-based mouse embryonic stem (mES) reporter cell line
Details on mammalian cell type (if applicable):
Extensive whole-genome transcription profiling has led to identification of a panel of biomarker genes that are preferentially activated upon exposure to different
classes of carcinogens and toxicants. (1) To allow easy assessment of the activation status of these biomarker genes, green fluorescent (GFP) mES reporter cell lines have been generated. These reporters were created using bacterial artificial chromosomes (BAC) that contain the complete biomarker gene including promoter and regulatory elements ensuring physiological regulation of the GFP reporters following transfection into stem cells. (2)

(1) Hendriks, G., Atallah, M., Raamsman, M., Morolli, B., van der Putten, H., Jaadar, H., Tijdens, I., Esveldt-van Lange, R., Mullenders, L., van de Water, B., et al. (2011). Sensitive DsRed fluorescence-based reporter cell systems for genotoxicity and oxidative stress assessment. Mutat Res 709-710, 49–59.

(2) Hendriks, G., Atallah, M., Morolli, B., Calléja, F., Ras-Verloop, N., Huijskens, I., Raamsman, M., van de Water, B., and Vrieling, H. (2012). The ToxTracker assay: novel GFP reporter systems that provide mechanistic insight into the genotoxic properties of chemicals. Toxicol. Sci. 125, 285–298.
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S9 liver extract from alachlor-induced rats (Moltox)
Test concentrations with justification for top dose:
The highest compound concentration will induce significant cytotoxicity (50-70%). The appropriate dose range for the ToxTracker analysis is determined in a extensive dose range finding. For the dose range finding, as well as the ToxTracker analysis a maximum concentration of 10 mM or 2 mg/ml is used, in line with the advised maximum concentration in the current guidelines for in vitro genotoxicity testing.

Doses used for testing were 0, 0.001, 0.002, 0.004, 0.008, 0.016% Amines, N-C12-18-alkyltrimethylenedi-, N-(carboxymethyl) derivs., sodium salts (CAS no 2098351-38-1) without S9.
Doses used for testing were 0, 0.004, 0.008, 0.016, 0.031, 0.063% Amines, N-C12-18-alkyltrimethylenedi-, N-(carboxymethyl) derivs., sodium salts (CAS no 2098351-38-1) with S9.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Remarks:
DMSO as solvent
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
other:
Remarks:
Without S9: Cisplatin = positive control DNA damage. Dimethyl maleate = positive control oxidative stress. Tunicamycin = positive control protein damage. With S9: Cisplatin = positive control DNA damage Aflatoxin = positive control metabolic activation
Details on test system and experimental conditions:
The ToxTracker assay requires only standard cell culture facilities and a low-end flow cytometer. The ToxTracker reporter cells are maintained by culturing them in gelatin-coated dishes in the presence of irradiated primary mouse embryonic fibroblasts (MEFs). During chemical exposures and reporter analysis the ToxTracker cells are cultured in the absence of fibroblasts in Buffalo rat liver (BRL)-conditioned mES cell culture medium.

For chemical testing, the six independent mES reporter cell lines are seeded in gelatin-coated 96-well cell culture plates in 200 μl BRL-conditioned ES cell medium (40.000 cells per well). 24 h after seeding the cells in the 96-well plates, medium is aspirated and fresh BRL-conditioned ES cell medium containing 10% foetal calf serum and the diluted chemicals is added to the cells. For each tested compound, five concentrations are tested in 2-fold dilutions. The highest compound concentration will induce significant cytotoxicity (50-70%). The appropriate dose range for the ToxTracker analysis is determined in a extensive dose range finding. For the dose range finding, as well as the ToxTracker analysis a maximum concentration of 10 mM or 2 mg/ml is used, in line with the advised maximum concentration in the current guidelines for in vitro genotoxicity testing. In case concentrations are limited by solubility or the occurrence of precipitation in the culture medium, the maximum soluble concentration will be used in the assay.

For the dose range finding, wild type mES cells are exposed to 20 different concentrations of the test substances. Cytotoxicity is estimated by cell count after 24 h exposure using a flow cytometer and is expressed as percentage of viable cells after 24 h. exposure compared to unexposed controls. Compounds are diluted in DMSO, water or PBS (see Table 3). For cytotoxicity assessment in the ToxTracker assay, the relative cell survival for the six different reporter cell lines is averaged.

GFP reporter induction is always compared to a vehicle control treatment. DMSO concentration is similar in all wells for a particular compound and never exceeds 1%. All compounds have been tested in at least three completely independent experiments. Positive reference treatments with cisplatin (DNA damage), diethyl maleate (oxidative stress) and tunicamycin (unfolded protein response) are included in all experiments. Metabolic activation is included in the ToxTracker assay by addition of S9 liver extract from alachlor-induced rats (Moltox). Cells are exposed to five concentrations of the test compounds in the presence of S9 and required co-factors (RegenSysA+B, Moltox) for 3 h. After washing, cells are incubated for 24 h in fresh BRLconditioned ES cell medium.

Induction of the GFP reporters is determined after 24 h exposure using a Guava easyCyte 8HT flow cytometer (Millipore). Only GFP expression in intact single cells is determined. Mean GFP fluorescence and cell concentrations in each well is measured, which is used for cytotoxicity assessment. In case auto-fluorescence of the test substances was observed in the dose range finding, wild type mES cells were exposed to the samples at the same concentrations as used in the ToxTracker. The mean fluorescence caused by the compound was then subtracted from the ToxTracker results of the respective compound.
Evaluation criteria:
Induction of the six reporter genes after treatment with test substance up to cytotoxic concentrations.

Validity of the assay was confirmed using exposure to the reference compounds specific for the pathways evaluated. Diethyl maleate (DEM) induced primarily the oxidative stress related reporters Srxn1 and Blvrb, tunicamycin induced the unfolded/misfolded protein stress response (Ddit3), and cisplatin showed induction of the DNA damage response (Bscl2, Rtkn) and p53-mediated cellular stress (Btg2).

Activation of the different GFP reporters by the control compounds is fully compliant with historical data thereby confirming the technical validity of the performed tests.
Statistics:
A statistical evaluation of the results is not regarded as necessary.
Species / strain:
other: mammalian cell line
Remarks:
GFP-based mouse embryonic stem (mES) reporter cell line
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
experiemnt I and II (with and without S9)
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
All four tested substances within the ampoteric, glycinate substance group induced significant levels of cytotoxicity (>50%) at a concentrations of 0.02% or lower when tested in the absence of S9 liver extract. None of the four tested substances showed increased cytotoxicity when cells were exposed in presence of S9 rat liver extract, indication that the substances did not require metabolic activation to become biological reactive. The 50% cytotoxicity level for the tested substances in presence of S9 varied between 0.04 and 0.07%. The reduced cytotoxicity when tested in the presence of S9 which is likely caused by a reduced exposure time of 3 h compared to 24 h for exposures in absence of S9. The six ToxTracker reporter cell lines showed a comparable cytotoxic response to the test compounds. For this reason, the cell survival graphs in the GFP induction figures show the average cytotoxicity of the six different cell lines.

None of the four tested ampoteric, glycinate substances induced the Bscl2-GFP or Rtkn-GFP DNA damage reporters when tested in the absence of a metabolising system. Activation of the Bscl2-GFP reporter is associated with induction of promutagenic DNA lesions that interfered with DNA replication. Activation of the Rtkn-GFP genotoxicity reporters correlates with induction of DNA strand breaks. Also when tested in the presence of a metabolising system, non of the ampoteric, glycinate substances activated the genotoxicity reporters in the ToxTracker assay. Together, no indication for genotoxic properties was found for the four tested ampoteric, glycinate substances.

None of the four tested substances induced significant levels of cellular oxidative stress when exposed in absence of a metabolising system, even at high cytotoxic concentrations. Oxidative stress is established by the Srxn1- GFP and Blvrb-GFP reporters. When tested in the presence of S9, only the biggest substance (longest alkyl-unsaturated, highest number amine and carboxyl groups) with CAS no 2060541-49-1 slightly activated the Srxn1-GFP oxidative stress reporter. Srxn1 activation is directly controlled by the Nrf2 anti-oxidant response pathway. However, the induction levels were >10-fold lower compared to the positive control Diethyl maleate. No oxidative stress by the other three substances was detected when exposed in the presence of S9.

In the absence of a metabolising system, all the amphoteic, glysincate substances but the biggest one with CAS no 2060541-49-1 significantly induced the Ddit3-GFP reporter, that is associated with protein damage and the unfolded protein response. Activation of the unfolded protein response was already observed at low cytotoxic concentrations. When tested in the presence of a metabolising system, induction of protein damage was reduced, which is generally correlated to the S9 exposure Protocol. After the 3 h exposure of cells in presence of compound and S9, cells were washed and allowed to recover up to 24 h. During this recovery time, cells are able to repair the cellular damage and will down-regulate the unfolded protein response. Only the smallest amphoteric, glycinate substance with CAS no 2098351-38-1 also activated the protein damage response in the presence of S9.

All criteria of validity were met.
Remarks on result:
other:
Remarks:
No genotoxicity nor indiction of oxidative stress was observed in the ToxTracker assay in any of the tested concentration in the absence or presence of a metabolising system.
Conclusions:
Interpretation of results: negative

In conclusion, it can be stated that in the described genotoxicity test and under the experimental conditions reported, the following can be concluded for Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS no 2098351-38-1):

• Strong cytotoxicity (>50%) was observed at a concentration of 0.02%
• No genotoxicity was observed in the ToxTracker assay at the highest tested concentration in the absence or presence of a metabolising system.
• No induction of oxidative stress was observed in the ToxTracker assay at the highest tested concentration in the absence or presence of a metabolising system.
• Strong induction of the unfolded protein response was observed, to comparable levels as the positive control.
• No increased reactivity was observed in the presence of a metabolising system.

Therefore, Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS no 2098351-38-1) is considered to be non-mutagenic in this ToxTracker screening test.
Executive summary:

ToxTracker is a screening study using a panel of mammalian stem cell lines that contain different fluorescent reporters for induction of DNA damage, oxidative stress and protein damage. All four substances within the amphoteric, glycinate substance group were tested in this assay up to cytotoxic levels with and without S9 rat liver extract-based metabolising system. The positive controls induced the appropriate responses. No genotoxic or oxidative stress-inducing properties were detected for any of the amphoteric, glycinate substances. All but the biggest substance (longest alkyl-unsaturated, highest number amine and carboxyl groups) with CAS no 2060541-49-1 induced significant levels of protein damage, already a low cytotoxic concentrations. There was no indication that the toxicity of the tested substances was increased due to metabolic activation in the liver.

 

This study is classified as acceptable weight of evidence to support the negative genotoxicity profile of the amphoteric, glycinate substances.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

Based on the general negative in vitro profile for genotoxicity Coco iminodiglycinate, (Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts with CAS no 2098351-38-1), does not require classification as a mutagen according to Regulation (EC) No 1272/2008 of the European Parliament and of the Council of 16 December 2008.