N-(2-carboxyethyl)-N-octyl-β-alanine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

An analogue substance was determined to be not genotoxic.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read across to sodium lauriminodipropionate Study performed to modern guidelines with independant repeat assays over two time points.

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test

GLP compliance:

yes

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S-9

Test concentrations with justification for top dose:

0.21, 0.28, 0.38 ug/ml in the 8 hour non-activated study
0.28, 0.38, 0.50 ug/ml in the 12 hour non-activated study
0.48, 0.63, 0.84 ug/ml in the 8 hour S-9 study
0.63, 0.84, 1.13 ug/ml in the 12 hour S-9 study

Vehicle / solvent:

Water

Positive controls:

yes

Positive control substance:

triethylenemelamine

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Cytotoxicity / choice of top concentrations:

cytotoxicity

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

Negative.

Executive summary:

Read across is considered valid as this same read-across is used in US EPA review of this class of substance.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Full GLP report to current guidelines, 2013 Read-across justified in US EPA review of this class of substances

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Assessment of the potential mutagenicity of the test item on the thymidine kinase, TK +/-, locus of the L5178Y mouse lymphoma cell line.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

The L5178Y TK+/- 3.7.2c mouse lymphoma cell line was obtained from Dr. J. Cole of the MRC Cell Mutation Unit at the University of Sussex, Brighton, UK. The cells were originally obtained from Dr. D. Clive of Burroughs Wellcome (USA) in October 1978 and were frozen in liquid nitrogen at that time.

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/-naphthoflavone

Test concentrations with justification for top dose:

The dose range used in the preliminary toxicity test was 19.53 to 5000 μg/ml for all three of the exposure groups.
Experiment 1 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (19.53 to 625 μg/ml in both the absence and presence of metabolic activation)
Experiment 2 treatments were performed in duplicate (A + B), both with and without metabolic activation (S9-mix) at eight dose levels of the test item (5 to 160 μg/ml in the absence of metabolic activation, and 100 to 450 μg/ml in the presence of metabolic activation),

Vehicle / solvent:

Water

Untreated negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

The treatment vessels were incubated at 37°C for 4 hours with continuous shaking using an orbital shaker within an incubated hood.
At the end of the treatment period, for each experiment, the cells were washed twice using R10 medium then resuspended in R20 medium at a cell density of 2 x 105 cells/ml. The cultures were incubated at 37 °C with 5% CO2 in air and subcultured every 24 hours for the expression period of two days by counting and diluting to 2 x 105 cells/ml.
On Day 2 of the experiment, the cells were counted, diluted to 104 cells/ml and plated for mutant frequency (2000 cells/well) in selective medium containing 4 μg/ml 5-trifluorothymidine (TFT) in 96-well microtitre plates. Cells were also diluted to 10 cells/ml and plated (2 cells/well) for viability (%V) in non-selective medium.

Evaluation criteria:

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

Statistics:

Small significant increases designated by the UKEMS statistical package will be reviewed using the above criteria, and may be disregarded at the Study Director's discretion.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

not applicable

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In all three of the exposure groups there was evidence of marked dose-related reductions in the Relative Suspension Growth (%RSG) of cells treated with the test item when compared to the concurrent vehicle controls. The toxicity was very steep in all three exposure groups.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

The steep toxicity curve observed proved that it would be hard to achieve optimum toxicity. The excessive toxicity observed at and above 468.75 μg/ml in the absence of metabolic activation and at 625 μg/ml in the presence of metabolic activation resulted in these dose levels not being plated for viability or 5-TFT resistance. In the presence of metabolic activation a dose level (468.75 μg/ml) was later excluded from statistical analysis as the RTG value also fell below the acceptable level of toxicity.

Conclusions:

Interpretation of results (migrated information):
negative with metabolic activation
negative without metabolic activation

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

Executive summary:

Valid study, but difficulties in finding optimum concentrations for test due to sharp cut-off in toxicity. This is considered common with surface active agents that will disrupt membranes at criticial concentrations.

Reference 3

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:

09-Apr-2012 to 26-apr-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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 uvr A

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:

Experiment 1
Preliminary test (without and with S9) TA100 and WP2uvrA: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Main study: TA1535, TA1537 and TA98:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Water
- Justification for choice of solvent/vehicle:
Test compound was stable and soluble in water and water has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9

Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9

Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9

Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

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

NUMBER OF CELLS EVALUATED: 10E8 per plate

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 EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat 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 independently repeated experiment.

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

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

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

RANGE-FINDING/SCREENING STUDIES:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

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

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No toxicity or mutagenicity was observed up to and including the top dose of 5000 µg/plate

Conclusions:

Interpretation of results (migrated information):
negative

Sodium N-(2-carboxylethyl)-N-(2-ethylhexyl)-ß-alaninate is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay

Executive summary:

Sodium N-(2-carboxylethyl)-N-(2-ethylhexyl)-ß-alaninate did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of S9-metabolic activation. These results were confirmed in an independently repeated experiment.

 

In this study, the negative and strain-specific positive control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Reference 4

Endpoint:

in vitro cytogenicity / micronucleus study

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12-Apr-2012 to 28-Jan-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Species / strain / cell type:

lymphocytes: human peripheral blood

Details on mammalian cell type (if applicable):

Type and identity of media:
Blood samples
Blood samples were collected by venapuncture 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.

- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal 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.

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:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24 exposure; 24 hr fixation: 100, 333, 1000, 3330 and 5000 µg/mL
First cytogenetic test:
Without and with S9-mix, 3hr exposure; 27 hr fixation: 1000, 3330 and 5000 µg/mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 4000 and 5000 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Culture medium
- Justification for choice of solvent/vehicle:
Test compound was stable in water and soluble in culture medium. Culture medium has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: mitomycin CMMC-C 0.25 and 0.38 µg/mL for a 3 hours exposure period and 0.15 and 0.225 µg/mL for a 24 hours exposure period

Remarks:

without S9

Positive control substance:

other: colchicine: 0.1 µg/mL

Remarks:

without S9

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: 15 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration:
Short-term treatment
Without and with S9-mix: 3 hr treatment, 24 hr recovery/harvest time
Continuous treatment
Without S9-mix: 24 hr treatment/harvest time

ARREST OF CELL DIVISION: 5 µg/mL Cytochalasine B
STAIN: Giemsa

NUMBER OF REPLICATIONS: duplicates

NUMBER OF CELLS EVALUATED: 1000/culture (mono- and binucleated cells)

DETERMINATION OF CYTOTOXICITY
- The cytostasis/cytotoxicity was determined using the cytokinesis-block proliferation index (CPBI index)

Evaluation criteria:

A test substance was considered positive (clastogenic or aneugenic) in the in vitro micronucleus test if:
a) It induces a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono or binucleated cells with micronuclei.
b) A statistically significant and biologically relevant increase is observed in the number of mono or binucleated cells with micronuclei in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic or aneugenic) in the in vitro micronucleus test if:
a) none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of mono and binucleated cells with micronuclei.
b) The number of mono and binucleated cells with micronuclei was within the laboratory historical control data range.

Statistics:

The incidence of micronucleated cells (cells with one or more micronuclei) for each exposure group was compared to that of the solvent control using Chi-square statistics:

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

only after 3 hours exposure period

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH:
Solvent control: 8.06
5000 µg/ml: 8.22
- Effects of osmolality: No
Solvent control: 0.281 mOsm/kg
5000 µg/ml: 0.293 mOsm/kg

- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/mL

RANGE-FINDING/SCREENING STUDIES:
- No toxicity was observed up to and including the highest tested dose of 5000 µg/ml in the absence and presence of S9, 3 hr treatment/27 hr fixation; at dose levels of 3330 and 5000 µg/ml in the absence of S9 for the continuous treatment of 24 hr.


COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- No severe toxicity was observed up to and including the highest tested dose level in both experiments

Conclusions:

Interpretation of results (migrated information):
ambiguous without metabolic activation (only after 3 hours exposure time)
negative with metabolic activation

Finally, it is concluded that this test is valid and that Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate is not clastogenic or aneugenic in human lymphocytes in the presence of S9-mix up to a concentration of 5000 µg/ml.

In addition Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate is not clastogenic or aneugenic in human lymphocytes after prolonged exposure period up to a dose level of 2000 µg/ml.

Although Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate induces the formation of micronuclei in human lymphocytes after 3 hours exposure time in the absence of S9 metabolic activation, this was only observed at an intermediate concentration of 1281 µg/ml. Concentrations below and above, up to 5000 µg/ml, did not show an increase in the number of micronucleated cells. Therefore the biological relevance of this increase is doubtful and the test results are considered equivocal.

Executive summary:

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 chemical colchicine produced a statistically significant increase in the number of mononucleated cells with micronuclei. In addition colchicine also showed a statistically significant increase in the number of binucleated cells with micronuclei in the first cytogenetic assay. It was therefore concluded that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

In the first cytogenetic assay, in the presence of S9-mix, at the 3 hours exposure time, Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei. However, in the absence of S9-mix, Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate induced a statistically significant increase in the number of binucleated cells with micronuclei at an intermediate concentration of 1281 µg/ml a.i.. Although this increase is not dose dependent, the number of binucleated cells with micronuclei is above the historical control data range. Since it was only observed at an intermediate concentration, the biological relevance of this increase is doubtful.  In the second cytogenetic assay with a 24 hours continuous exposure time, Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate did not induce a statistically significant or biologically relevant increase in the number of mono- and binucleated cells with micronuclei.  

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Justification for type of information:

See Section 13 for read-across justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

Preliminary results from a GLP mouse lymphoma assay (OECD 476) show no mutagenicity with or without S9 mix in concentrations up to 5000 µg/ml, or to a cytotoxicity level of 81%. The dossier will be updated as soon as the test report is available.

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity level of 81%

Executive summary:

This study on Sodium N-(2 -carboxyethyl)-N-(2 -ethylhexyl)-β-alaninate, CAS No 94441 -92 -6, is performed under GLP and is being finalized right now.

The preliminary results from this study shows that Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate is not mutagenic in the mouse lymphoma L5178Y test system with or without S9 mix. This was tested in concentrations up to 5000 µg/ml, or to a cytotoxicity level of 81%. The dossier will be updated as soon as the test report is available.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6,will betested for genetic toxicity in three in vitro tests. Both the two available tests showed no biological relevant evidence on genetic toxicity. This is also supported by the preliminary results from the third test which is being finalized at this moment.

The bacterial reverse mutation assay (Ames test), reliability rating 1, showed that the substance did not cause any substantial increase in revertant colony numbers of any of the five tested strains at any dose level, either in the presence or absence of metabolic activation (S-9 mix). No evidence of mutagenic potential was seen in this study.

 

In the in vitro test on micronucleus assay in human lymphocytes, reliability rating 1,Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6,was not clastogenic or aneugenic, in presence of metabolic activation by S9 mix or after long time exposure without metabolica activation by S9 mix.The increased incidence of formation of micronuclei in one of the middle doses after the shortest exposure time of 3 hours without S9 metabolic activation is not considered to have any biological relevance since the lower and higher doses were negative. It is therefore not considered to be required from a legal, scientific or animal welfare point of view to perform anyin vivomicronucleus test.

The third genetic toxicity study, the mouse lymphoma assay, is being finalized at the moment. This study is performed according to OECD Guideline 476 and GLP. The preliminary results indicates thatSodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6,do not induce mutations in the mouse lymphoma thymidine kinase locus (TK+/-), but the dossier will be updated as soon as the test report is available.

Based on the available data, it can be concluded that Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, would not be expected to have any genotoxic hazard to human health.

Justification for selection of genetic toxicity endpoint

No specific study has been selected as the conclusion that the substance is not genotoxic/mutagenic is based on the two negative studies available, the Ames test and the in vitro micronucleus assay in human lymphocytes, and the preliminary results from the in vitro Mouse Lymphoma study which is being finalized at this moment.

Short description of key information:

Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, has been tested in three in-vitro genotoxicty studies.

The first study available on Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, is the bacterial reverse mutation assay (Ames test) compliant to OECD Guideline 471 and performed under GLP. The reliability rating of the test is 1 and no evidence of mutagenic potential was reported in this study.

The second available study on genetic toxicity is the in vitro micronucleus assay in human lymphocytes, (OECD Guideline 487), which is GLP compliant and has reliability rating 1. No biological relevant evidence of clastogenic or aneugenic effects was seen in this study.

No indications on clastogenic or aneugenic effects were seen in the presence of S9-mix up to the highest tested concentration of 5000 μg/ml, or in absence of S9-mix after prolonged exposure period up to a dose level of 2000 μg/ml. After the shortest duration of exposure, 3 hours, and in absence of S9 mix one of the middle dose groups showed significantly increased number of micronulclei in human lymphocytes. The concentrations below and above, up to 5000 µg/ml did not show any increased number of micronucleated cells. The biological relevance of this increase is questionable and the overall test result is considered to be negative.

The third genetic toxicity study on Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, is the mouse lymphoma assay according to OECD Guideline 476. This study is performed under GLP and is being finalized right now. The preliminary results from this study show that Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-ß-alaninate is not mutagenic in the mouse lymphoma L5178Y test system with or without S9 mix. This was tested in concentrations up to 5000 µg/ml, or to a cytotoxicity level of 81%. The dossier will be updated as soon as the test report is available.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the lack of biological relevant findings in the two available in-vitro tests for genotoxicity, Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, CAS No 94441-92-6, 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.