trisodium 3-[{3-[bis(2-carboxylatoethyl)amino]propyl}(C12-18-(even numbered) and C18-(unsaturated) alkyl)amino]propanoate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

There is test data for β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt in the Ames bacterial reverse mutation assay OECD guideline 471 and in the In-vitro micronucleus assay in human lymphocytes according to OECD Guideline 487. There is also an L5178Y TK +/- Mouse lymphoma assay to OECD Guideline 490, available for the read across substanceCoco iminodiglycinate registered as Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS No 2098351-38-1). This is a close structural analogue. A mouse lymphoma test onβ-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt is planned but it will not be completed until July due to capacity issues at the CRO.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 November 2017 - 10 December 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

21 July 1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

31 May 2008

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

Species / strain / cell type:

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

Species / strain / cell type:

E. coli WP2

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix induced by Aroclor 1254 (500 mg/kg bw)

Test concentrations with justification for top dose:

Dose-range finding test (without and with S9; tester strains TA100 and WP2uvrA): 1.7, 5.4, 17, 52, 164, 512, 1600 and 5000 µg/plate (reported as part of experiment 1)
First experiment (without S9; tester strains TA1535, TA1537 and TA98): 17, 52, 164, 512, 1600 and 2500 μg/plate
First experiment (with S9; tester strains TA1535, TA1537 and TA98): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (without S9, tester strains TA1537, TA98 and TA100): 17, 52, 164, 512, 1600 and 2500 μg/plate
Second experiment (without S9, tester strains TA1535 and WP2uvrA): 17, 52, 164, 512, 1600 and 5000 μg/plate
Second experiment (with S9, all tester strains): 17, 52, 164, 512, 1600 and 5000 μg/plate

Vehicle / solvent:

- Vehicle used: Milli-Q water
- Justification for choice of vehicle: a previously performed solubility test showed that the test item was dissolved in Milli-Q water.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: ICR-191, 2-aminoanthracene

Remarks:

For details on positive control substances, see table 1

Details on test system and experimental conditions:

Two individual experiments were performed. The dose range-finding study with tester strains TA100 and TA1537 was reported as part of the first experiment. The first experiment was a direct plate assay. The second experiment was a pre-incubation assay and was performed to obtain more information about the possible mutagenicity of the test item.

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period (second experiment): 30 ± 2 minutes at 70 rpm at 37 ± 1°C
- Exposure duration: 48 ± 4 h (in the dark at 37.0 ± 1.0 °C)

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain.

METHODS: The following solutions were successively added to 3 mL molten top agar: 0.1 mL of a fresh bacterial culture (10^9 cells/ml) of one of the tester strains, 0.1 mL of a dilution of the test item in Milli-Q water or control solution and either 0.5 mL S9-mix (in case of activation assays) or 0.5 mL 0.1 M phosphate buffer (in case of non-activation assays). The ingredients were mixed on a Vortex and the content of the top agar tube was poured onto a selective agar plate. After solidification of the top agar, the plates were inverted and incubated in the dark at 37.0 ± 1.0°C for 48 ± 4 h. In the pre-incubation assay (second experiment) this procedure was preceeded by pre-incubation of the solutions.

DETERMINATION OF CYTOTOXICITY
- Method: the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.
- Other: precipitation of the test item was recorded

COLONY COUNTING:
- The revertant colonies were counted automatically with the Sorcerer Colony Counter. Plates with sufficient test item precipitate to interfere with automated colony counting were counted manually. Evidence of test item precipitate on the plates and the condition of the bacterial background lawn were evaluated when considered necessary, macroscopically and/or microscopically by using a dissecting microscope.

Evaluation criteria:

DECISION CRITERIA:
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is not greater than two times the concurrent vehicle control, and the total number of revertants in tester strains TA1535, TA1537 or TA98 is not greater than three times the concurrent vehicle control.
b) The negative response should be reproducible in at least one follow-up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in the tester strain TA100 or WP2uvrA is greater than two times the concurrent vehicle control, or the total number of revertants in tester strains TA1535, TA1537, TA98 is greater than three times the concurrent vehicle control.
b) In case a follow up experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one follow up experiment.

In addition to the criteria stated above, any increase in the total number of revertants should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

ACCEPTABILITY CRITERIA:
a) The vehicle control and positive control plates from each tester strain (with or without S9-mix) must exhibit a characteristic number of revertant colonies when compared against relevant historical control data generated at Charles River Den Bosch.
b) The selected dose-range should include a clearly toxic concentration or should exhibit limited solubility as demonstrated by the preliminary toxicity range-finding test or should extend to 5 mg/plate.
c) No more than 5% of the plates are lost through contamination or some other unforeseen event. If the results are considered invalid due to contamination, the experiment will be repeated.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 512 (without S9) and at and above 1600 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above1600 µg/plate (without S9) and at 5000 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1600 µg/plate (without S9) and at 5000 µg/plate (with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at and above 1600 µg/plate (without and with S9)

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- In both experiments, in all tester strains in the absence and in the presence of S9-mix, no increase in the number of revertants was observed upon treatment with the test item under all conditions tested.
- In both experiments, precipitation of the test item on the plates was not observed at the start end the end of the incubation period in any tester strain.

Table 2 Historical Control Data of the Solvent Control

	TA1535		TA1537		TA98		TA100		WP2uvrA
S9-mix	-	+	-	+	-	+	-	+	-	+
Range	3 – 29	3 - 27	3 – 20	3 – 23	8 - 41	8 - 55	63 – 176	54 - 160	10 – 59	9 - 69
Mean	10	11	6	7	16	23	108	107	25	32
SD	3	4	2	3	5	7	19	20	7	8
n	2356	2336	2264	2235	2319	2360	2341	2336	2075	2078

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Table 3 Historical Control Data of the Positive Control Items

	TA1535		TA1537		TA98
S9-mix	-	+	-	+	-	+
Range	125 – 1248	73 – 1206	55 – 1353	54 – 1051	365 – 1995	250 – 1977
Mean	846	219	787	353	1406	887
SD	146	119	345	162	258	349
n	2348	2229	2003	2234	2200	2276

	TA100		WP2uvrA
S9-mix	-	+	-	+
Range	439 – 1848	408 - 2651	93 – 1951	111 - 1359
Mean	901	1232	1094	437
SD	168	343	477	149
n	2335	2327	2021	2085

SD = Standard deviation

n = Number of observations

Historical control data from experiments performed between Oct 2015 and Oct 2017. 

Conclusions:

In an AMES test, performed according to OECD guideline 471 and GLP principles, Sodium cocopropylenediamine propionate was found not to be mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay, with or without metabolic activation.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 November 2017 - 04 February 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Blood was collected from healthy adult, non-smoking volunteers
- Suitability of cells: Recommended in the international OECD guideline.
- Age of volunteers and Average Geneteration Time (AGT):
Dose-range finding study: age 31, AGT = 13.8 h
First cytogenetic assay: age 22, AGT = 13.2 h
Cytogenetic assay 1A: age 31, AGT = 13.2 h
Second cytogenetic assay: age 31, AGT = 15.2 h

- Blood sample collection: Blood samples were collected by venipuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.

MEDIA USED
- Culture medium: Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) fetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/ml and 50 µg/ml respectively) and 30 U/ml heparin.
- Lymphocyte cultures: Whole blood (0.4 ml) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/ml) phytohaemagglutinin was added.

Cytokinesis block (if used):

Cytochalasine B (5 μg/ml) for 24 hours (1.5 times normal cell cycle)

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose-range finding test: 15.6, 31.3, 62.5, 125, 250 and 500 µg/mL
Based on the results of the dose-range finding test the following dose levels were selected for the first cytogenetic assay.
First assay (without S9): 25, 125, 250, 300, 350, 400 and 450 µg/mL
First assay (with S9): 25, 250, 500, 600, 700 and 800 µg/mL
Second assay (without S9): 50, 100, 200, 250, 300, 350 and 400 µg/mL

Dose levels selected for scoring:
First assay (without S9): 125, 350 and 450 µg/mL
First assay (with S9): 25, 500 and 600 µg/mL
Second assay (without S9): 50, 100 and 200 µg/mL

Vehicle / solvent:

- Vehicle used: RPMI culture medium
- Justification for choice of vehicle: A solubility test was performed based on visual assessment where the test item was dissolved in RPMI 1640 medium.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

other: Colchicine

Details on test system and experimental conditions:

Two individual cytogenetic assays were performed: a first assay in the absence and in the presence of S9 for an exposure time of 3 hours (without cytokinesis-block) and a second assay, to confirm the results of the first assay, in the absence of S9 for an exposure time of 24 hours (with cytokinesis-block).

METHOD OF APPLICATION: in medium

DURATION
- Culture period of lymphocytes: 46 ± 2 hours
- Exposure duration: 3 hours (first assay) and 24 hours (second assay)
- Harvest time: 27 hours (first assay) and 24 hours (second assay)

ENVIRONMENTAL CONDITIONS:
- Humidity: set to maintain 80 - 100% (actual range 34 - 89%), containing 5.0 ± 0.5% CO2 in air
- Temperature: set to maintain 37.0 ± 1.0°C (actual range 34.9 - 37.2°C)

NUMBER OF REPLICATIONS:2

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
To harvest the cells, cell cultures were centrifuged (5 min, 365 g) and the supernatant was removed. Cells in the remaining cell pellet were re-suspended in 1% Pluronic F68. After centrifugation (5 min, 250 g), the cells in the remaining pellet were swollen by hypotonic 0.56% (w/v) potassium chloride (Merck) solution. Immediately after, ethano:acetic acid fixative (3:1 v/v) was added. Cells were collected by centrifugation (5 min, 250 g) and cells in the pellet were fixated carefully with 3 changes of ethanol:acetic acid fixative (3:1 v/v).
Fixed cells were dropped onto cleaned slides, which were immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue. The slides were marked with the Charles River Den Bosch study identification number and group number. At least two slides were prepared per culture. Slides were allowed to dry and thereafter stained for 10 - 30 min with 5% (v/v) Giemsa (Merck) solution in Sörensen buffer pH 6.8. Thereafter slides were rinsed in water and allowed to dry. The dry slides were automatically embedded and mounted with a coverslip in an automated cover slipper.

NUMBER OF CELLS EVALUATED: At least 1000 binucleated cells per culture and at least 1000 mononucleated cells per culture were scored for micronuclei.

METHOD OF SCORING: by light microscope

CRITERIA FOR SCORING: 
The following criteria for scoring of binucleated cells were used:
• Main nuclei that were separate and of approximately equal size.
• Main nuclei that touch and even overlap as long as nuclear boundaries are able to be distinguished.
• Main nuclei that were linked by nucleoplasmic bridges.
The following cells were not scored:
• Trinucleated, quadranucleated, or multinucleated cells.
• Cells where main nuclei were undergoing apoptosis (because micronuclei may be gone already or may be caused by apoptotic process).
The following criteria for scoring micronuclei were adapted from Fenech, 1996:
• The diameter of micronuclei should be less than one-third of the main nucleus.
• Micronuclei should be separate from or marginally overlap with the main nucleus as long as there is clear identification of the nuclear boundary.
• Micronuclei should have similar staining as the main nucleus.

DETERMINATION OF CYTOTOXICITY
- Method: cytotoxicity was determined by calculating the Cytokinesis-Block Proliferation Index (CBPI):
%cytostasis = 100-100 [(CNPIt-1)/(CBPIc-1)]
where t=test item or control treatment culture and c=vehicle control culture
CBPI = [(no. mononucleate cells)+(2x no. binucleate cells)+(3x no. multinucleate cells)]/total no. of cells

Rationale for test conditions:

Test conditions were based on OECD guideline.

Evaluation criteria:

EVALUATION CRITERIA:
A test item is considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if all of the following criteria are met:
- At least one of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
- The increase is dose-related in at least one experimental condition when evaluated with a Cochran Armitage trend test.
- Any of the results are outside the 95% control limits of the historical control data range.

A test item is considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
- None of the test concentrations exhibits a statistically significant (Chi-square test, one-sided, p < 0.05) increase compared with the concurrent negative control.
- There is no concentration-related increase when evaluated with a Cochran Armitage trend test.
- All results are inside the 95% control limits of the negative historical control data range.


ACCEPTABILITY CRITERIA:
- The concurrent negative control data are considered acceptable when they are within the 95% control limits of the distribution of the historical negative control database.
- The concurrent positive controls should induce responses that are compatible with those generated in the historical positive control database.
- The positive control item colchicine induces a statistically significant increase in the number of mononucleated cells with micronuclei and the positive control items MMC-C and CP induces a statistically significant increase in the number of binucleated cells with micronuclei. The positive control data will be analyzed by the Chi-square test (one-sided, p < 0.05).

Statistics:

Graphpad Prism version 4.03 (Graphpad Software, San Diego, USA) was used for statistical analysis of the data.

Key result

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

Additional information on results:

- Precipitation: in the dose-range finding test, at a concentration of 500 µg/mL, no precipitation in the culture medium was observed.
- pH (at a concentration of 5000 µg/mL): 7.7 (7.8 in the solvent control)
- Osmolarity (at a concentration of 5000 µg/mL): 301 mOsm/kg (294 mOsm/kg in the solvent control)

In both experiments, both in the absence and presence of S9-mix, the test item did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.

ACCEPTABILITY
- The number of mono- and binucleated cells with micronuclei found in the solvent control was within the 95% control limits of the distribution of the historical negative control database
- The positive control chemicals, mitomycin C and cyclophosphamide both produced a statistically significant increase in the number of binucleated cells with micronuclei. The positive control chemicals colchicine and MMC-C produced a statistically significant increase in the number of mono- and binucleated cells with micronuclei. In addition CP also showed a statistically significant increase in the number of binucleated cells with micronuclei. The number of mono- and binucleated cells with micronuclei found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.

Conclusions:

The outcome of an in vitro micronucleus test, performed according to OECD guideline 487 and GLP principles, showed that Sodium cocopropylenediamine propionate is not clastogenic or aneugenic in human lymphocytes under the experimental conditions described in the report.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 April 2018 - 29 May 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

29 July 2016

Deviations:

no

GLP compliance:

yes

Type of assay:

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

Specific details on test material used for the study:

Stability at higher temperatures: yes, maximum temperature: ~70°C
Appearance: clear amber liquid
Concentration: 29.7% aqueous solution
Storage conditions: at room temperature

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: L5178Y/TK+/- -3.7.2C mouse lymphoma cells from American Type Culture Collection, (ATCC, Manassas, USA), (2001)
- Suitability of cells: recommended test system in international guidelines

MEDIA USED
- Type and identity of media including CO2 concentration if applicable:
* Basic medium: RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/mL and 50 μg/mL, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin
* Growth medium: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Exposure medium: 3 hr exposure: basic medium, supplemented with 5% (v/v) heat-inactivated horse serum (=R5 medium); 24 hr exposure: basic medium, supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
* Selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium) and 5 μg/ml trifluorothymidine
* Non-selective medium: basic medium supplemented with 20% (v/v) heat-inactivated horse serum (total amount of serum = 20%, R20-medium)
- Properly maintained: yes, stock cultures of the cells were stored in the freezer (-150°C).
- Periodically checked for Mycoplasma contamination: yes
- Periodically 'cleansed' against high spontaneous background: yes, prior to testing

Metabolic activation:

with and without

Metabolic activation system:

Rat liver microsomal enzymes (S9 homogenate) from male Sprague Dawley rats that have been dosed orally with a suspension of phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Dose-range finding test, 3 hr and 24 hr exposure (with and without S9-mix): 313, 625, 1250, 2500 and 5000 μg/mL
Additional dose-range finding test, 3 hr and 24 hr exposure (with and without S9-mix): 9.4, 19, 38, 75, 150 and 300 μg/mL

First mutagenicity test:
Without S9-mix: 0.94, 1.9, 3.8, 7.5, 15, 30, 60, 70, 80, 90 and 100 μg/mL exposure medium.
With S9-mix: 3.8, 7.5, 15, 30, 70, 150, 175, 200, 225, 250, 275 and 300 μg/mL exposure medium.

Second mutagenicity test (without S9-mix): 0.63, 1.25, 2.5, 5, 10, 15, 20, 25, 30, 35 and 40 µg/mL exposure medium.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 (exposure medium (R5) Hepes buffered medium
- Justification for choice of solvent/vehicle: the test item formed a clear solution in the vehicle which was also the medium used.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): below 1 x 10^6 cells/mL for the 3 hour exposure period and 1.25 x 10^5 cells/mL for the 24 hour exposure period.

DURATION
- Cleansing period: Prior to dose-range finding and mutagenicity testing, the mouse lymphoma cells were grown for 1 day in R10-medium containing 10^-4 M hypoxanthine, 2 x 10^-7 M aminopterine and 1.6 x 10^-5 M thymidine (HAT-medium) to reduce the amount of spontaneous mutants, followed by a recovery period of 2 days on R10-medium containing hypoxanthine and thymidine only. After this period cells were returned to R10-medium for at least 1 day before starting the experiment.
- Exposure duration: 3 hours (experiment 1; with and without S9-mix); 24 hours (experiment 2; without S9-mix).
- Expression time: 2 days in which at least 4 x 10^6 cells were subcultured every day
- Selection time (if incubation with a selection agent): 11 or 12 days

ENVIRONMENTAL CONDITIONS (set to maintain):
All incubations were carried out in a humid atmosphere (80 - 100%, actual range 63 - 99%) containing 5.0 ± 0.5% CO2 in air in the dark at 37.0 ± 1.0°C (actual range 34.9 - 37.5°C).

SELECTION AGENT: TFT

STAIN: 0.5 mg/ml 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)

NUMBER OF REPLICATIONS:
- test concentrations: 1
- positive control: 1
- solvent control: 2

NUMBER OF CELLS EVALUATED: 9.6 x 10^5 cells per concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth (relative suspension growth x relative cloning efficiency/100)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWGT as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.
- A test item is considered positive (mutagenic) in the mutation assay if it induces a mutation frequency of more than the mutation frequency in the controls + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.
- A test item is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.
- A test item is considered negative (not mutagenic) in the mutation assay if: none of the tested concentrations reaches a mutation frequency of the mutation frequency in the controls + 126.

ACCEPTABILITY CRITERIA:
a) The absolute cloning efficiency of the solvent controls is between 65 and 120% in order to have an acceptable number of surviving cells analyzed for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors.
c) The suspension growth over the 2-day expression period for the solvent controls should be between 8 and 32 for the 3 hour treatment, and between 32 and 180 for the 24 hour treatment.
d) The positive control should demonstrate an absolute increase in the total mutation frequency, that is, an increase above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6. At least 40% of the IMF should be reflected in the small colony MF. And/or, the positive control has an increase in the small colony MF of at least 150 x 10^-6 above that seen in the concurrent solvent control (a small colony IMF of 150 x 10^-6).

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

See 'additional information on results' for details

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Solubility: the test item did not precipitate in the exposure medium up to and including the concentration of 5000 μg/mL. Since testing up to 5000 μg/mL is recommended in the guidelines, this concentration was used as the highest test item concentration in the dose-range finding test.
- pH (at 5000 μg/mL): 7.42, compared to 7.51 in the solvent control
- Osmolarity (at 5000 μg/mL): 0.306 OSm/kg, compared to 0.299 Osm/kg in the solvent control

RANGE-FINDING/SCREENING STUDIES:
Dose-range finding study 1: Both in the absence and presence of S9-mix, hardly any or no cell survival was observed at all test item concentrations tested. An additional dose range finding test was performed with a test item concentration range of 9.4 to 300 µg/mL in the absence and presence of S9-mix with a 3 hour treatment period.

Dose-range finding study 2:
- After 3 hours of treatment, in the absence of S9-mix, the relative suspension growth was 22% at the test item concentration of 75 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test item concentrations of 150 μg/mL and above.
- After 3 hours of treatment, in the presence of S9-mix, the relative suspension growth was 46% at the test item concentration of 150 μg/mL compared to the relative suspension growth of the solvent control. Hardly any cell survival was observed at the test item concentration of 300 μg/mL.
- After 24 hours of treatment, the relative suspension growth was 5% at the test item concentration of 38 μg/mL compared to the relative suspension growth of the solvent control. Hardly any or no cell survival was observed at test item concentrations of 75 μg/mL and above.

FIRST MUTAGENICITY TEST:
- Toxicity: in the absence of S9, the dose levels of 0.94 to 30 μg/mL showed no cytotoxicity and 80 to 100 μg/mL showed similar cytotoxicity. In the presence of S9 the dose levels of 200 to 300 μg/mL were too toxic for further testing. Dose levels selected to measure mutation frequencies at the TK-locus were: 0.94, 1.9, 7.5, 30, 60, 70, 80 and 100 μg/mL exposure medium (without S9-mix) and 3.8, 7.5, 15, 30, 70, 150 and 175 μg/mL exposure medium (with S9-mix).
- In the absence of S9-mix, the relative total growth of the highest test item concentration was 26% compared to the total growth of the solvent controls. In the presence of S9-mix, the relative total growth of the highest test item concentration was 17% compared to the total growth of the solvent controls.
- No biologically relevant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

SECOND MUTAGENICITY TEST:
- Toxicity: The dose levels of 0.63 to 10 μg/mL showed no cytotoxicity. The dose levels of 35 and 40 μg/mL wre too toxic for further testing. The dose levels selected to measure mutation frequencies at the TK-locus were: 1.25, 2.5, 5, 10, 15, 20, 25 and 30 µg/mL exposure medium.
- The relative total growth of the highest test item was 16% compared to the total growth of the solvent controls.
- No significant increase in the mutation frequency at the TK locus was observed after treatment with the test item. The numbers of small and large colonies in the test item treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

HISTORICAL CONTROL DATA: see table 1 and table 2

ACCEPTABILITY:
- The mutation frequency found in the solvent control cultures was within the acceptability criteria of this assay and within the 95% control limits of the distribution of the historical negative control database. Except in the first experiment in the presence of S9-mix in which the mutation frequency of one of the solvent control cultures was not within the range of the acceptability criteria. However, the mutation frequency of one of the solvent control cultures (187 per 10^6 survivors) in the absence of S9-mix was recorded to be outside the range of ≥ 50 per 10^6 survivors and ≤ 170 per 10^6 survivors. According to the OECD guideline 490, a single solvent control group is sufficient. Furthermore, the mutation frequency of the other solvent control was within the acceptability criteria, the mutation frequency was just above the upper limit and none of the tested concentrations reached a mutation frequency of MF(controls) + 126. Therefore the determination of the mutagenicity of the test item with only one solvent control had no effect on the results of the study.

- Positive control chemicals both produced significant increases in the mutation frequency and the mutation frequency found in the positive control cultures was within the 95% control limits of the distribution of the historical positive control database.
- The suspension growth over the two-day expression period for cultures treated with exposure medium was between 15 and 17 (3 hour treatment) and 89 and 91 (24 hour treatment).

- In the second mutation experiment, the increase in the small colony MF was not met. The induced MF reflected in the small colony IMF was not 40% and no increase in the small colony MF of at least 150 x 10^-6 was observed. However the positive control was above the spontaneous background MF (an induced MF (IMF)) of at least 300 x 10^-6 and a three-fold increase in small colonies was observed, this deviation had no effect on the results of the study.

Table 1 Historical control data of the solvent control

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	96	92	96
SD	29	30	29
n	268	248	285
Upper control limit (95% control limits)	160	152	160
Lower control limit (95% control limits)	32	31	32

SD = Standard deviation

n = Number of observations

Table 2 Historical data for the positive control

	Mutation frequency per 10^6survivors
	- S9-mix		+ S9-mix
	3 hour treatment	24 hour treatment	3 hour treatment
Mean	808	684	1669
SD	239	206	843
n	136	124	146
Upper control limit (95% control limits)	1465	1222	4196
Lower control limit (95% control limits)	152	146	-859

SD = Standard deviation

n = Number of observations

Distribution historical positive control data from experiments performed between November 2014 and November 2017. 

Conclusions:

Based on the results of an in vitro mammalian cell gene mutation test, performed according to OECD guideline 490 and GLP principles, Sodium cocopropylenediamine propionate is not mutagenic in the TK mutation test system under the experimental conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

No mode of action / human relevance framework is required as all three in-vitro studies were negative. There is no concern for potential for mutagenic, clastogenic or aneugenic potential β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt.

Additional information

There is a fully CLP compliant Mouse Lymphoma, mammalian cell gene mutation assay OECD 490 for the close structural analogueCoco iminodiglycinate registered as Amines, N-C12-18-alkyltrimethylenedi-, reaction products with chloroacetic acid, sodium salts (CAS No 2098351-38-1the difference being that it has ethyl side chain groups rather than propyl side chain groups in β-Alanine, N-(2-carboxyethyl)-N-[3-[(2-carboxyethyl)amino]propyl]-, N-C12-18-alkyl derivs., trisodium salt, but is otherwise the same being based on the same diamine.

Justification for classification or non-classification

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