2-Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- Experimental Ames test (OECD 471) on the substance itself "2 -Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide" (water and methanol free).

- Several experimental in vitro studies (Ames, MLA, HPRT and In Vitro Micronucleus test) on analogues.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

09-Apr-2012 to 26-apr-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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 / 650 µg/plate in DMSO for TA100

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 / 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 / 10 µg/plate in DMSO for WP2uvrA

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 / 5 µg/plate in saline for TA1535

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

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.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

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

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: 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 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

supporting study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis is based on “different compounds which have similar properties”.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- The source substance is identified as Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, also known as Sodium 3-[(2-carboxyethyl)(2-ethylhexyl)amino]propanoate (CAS no. 94441-92-6 | EC no. 305-318-6). It is a UVCB substance whose major constituent is sodium 2-ethylhexylimino-di-propionate. Minor constituents are sodium 2-ethylhexylimino-mono-propionate, unreacted acrylic acid and unreacted 2-ethylhexylamine.
- The target substance is identified as Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) (CAS no. 68610-44-6| EC no. 271-865-1). It is a UVCB substance whose major constituents are sodium 2-ethylhexylimino-mono-propionate and sodium 2-ethylhexylimino-di-propionate. Minor constituents are unreacted acrylic acid and unreacted 2-ethylhexylamine.

3. ANALOGUE APPROACH JUSTIFICATION
The target and source substances are essentially the same: both are UVCB substances composed of the exact same constituents with the exact same functional groups (i.e. carboxylic acid groups and secondary/tertiary amine groups). They are expected to have the same ADME profile and to share common mode of action and breakdown products. The target and source substances only differ in the overlapping ranges of their constituents, the content of sodium 2-ethylhexylimino-mono-propionate being especially higher in the target substance. This difference is expected to have no or very limited impact on the potency of effects exerted on exposed living organisms.

4. DATA MATRIX
Cf. read-across justification document attached in §13. Assessment reports.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

Tested up to the maximum recommended dose level (5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/plate)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/plate)

Vehicle controls validity:

valid

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

Remarks:

tested to the maximum rcommended dose level (5000 µg/plate)

Vehicle controls validity:

not valid

Positive controls validity:

not valid

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction was obtained from the liver of rats treated with Aroclor 1254 by the IP route. Each batch is validated by the Supplier for its ability to activate benzo(a)pyrene and 2-anthramine to mutagenic intermediates. It was preserved at -80°C.

Test concentrations with justification for top dose:

A preliminary test was conducted up to 5000 µg/plate (maximum dose level recommended by the OECD guideline).
No precipitate was observed. Moderate to marked cytotoxicity was noted without S9 mix at dose-levels ≥ 1000 µg/plate in the TA 98 and TA 102 strains and ≥ 2500 µg/plate in the TA 100 strain. No toxicity was noted with S9 mix.

On the basis of the results obtained in the preliminary test, the main experiments were conducted as following:
> Experiments without S9 mix:
156.3, 312.5, 625, 1250 and 2500 µg/plate, for TA 1535 and TA 100 strains in both experiments,
78.1, 156.3, 312.5, 625 and 1250 µg/plate, for the TA 1537, TA 98 and TA 102 strains in both experiments.
> Experiments with S9 mix:
312.5, 625, 1250, 2500 and 5000 µg/plate, for all the strains in both experiments.

Vehicle / solvent:

- Vehicle used: water
- Justification: freely soluble in the vehicle (water for injections) at 50 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA 1535 and TA 100, without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA 1537, without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

TA 98, without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

TA 102, without S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Anthramine

Remarks:

All the strains, with S9 Mix

Details on test system and experimental conditions:

METHOD OF APPLICATION:
The preliminary test and the first main experiment were conducted using the plate incorporation method.
The second main experiment was conducted using the preincubation method, incubation for 60 minutes at 37°C, under shaking).

EXPERIMENTAL DESIGN:
> In the preliminary test, 6 dose-levels (one plate/dose-level) were tested in 3 strains (TA 98, TA 100 and TA102) with and without S9 Mix.
> The main experiments were conducted using 3 plates/dose levels in the 5 strains (TA 98, TA 100, TA 1535, TA 1537 and TA 102), with and without S9 Mix. At least 5 dose-levls were tested in each experiment, with vehicle controls and positive controls.

EVALUATION OF THE RESULTS:
The number of revertants and the aspect of the bacterial lawn were scored per plates.

Evaluation criteria:

> Acceptance criteria:
This study is considered valid if the number of revertants in the vehicle controls and in the positive controls are consistent with the historical data of the testing facility.
> Evaluation criteria
A reproducible 2-fold increase (for the TA 98, TA 100 and TA 102 strains) or 3-fold increase (for the TA 1535 and TA 1537 strains) in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may also be taken into account in the evaluation of the data obtained.

Statistics:

The individual results and the mean number of revertants, with the corresponding standard deviation and ratio (mutants obtained in the presence of the test item/mutants obtained in the presence of the vehicle), are presented in tabular form.

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to marked toxicity at ≥ 1250 µg/plate in the TA 1535, TA 1537, TA 98 and TA 102 strains and at 2500 µg/plate in the TA 100 strain.

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 98, TA 100, TA 1535, TA 1537 and TA 102

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Moderate to marked toxicity in the second experiment (using preincubation method) at ≥ 2500 µg/plate in the TA 1535 and TA 1537 strains, and at 5000 µg/plate in the TA 98, TA 100 and TA 102 strains

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

The test item was tested according to the international guidelines (OECD 471) with Salmonella typhimurium strains, using both the plate incorporation and the preincubation methods, in the absence and in the presence of metabolic activation.
Under the experimental conditions, the test item did not show mutagenic activity.

Executive summary:

The objective of this study was to evaluate the potential of the test item to induce reverse mutation in Salmonella typhimurium. The study was performed according to the international guidelines (OECD 471, Commission Directive No. B13/14) and in compliance with the Principles of Good Laboratory Practice Regulations.

A preliminary toxicity test was performed to define the dose-levels to be used for the mutagenicity study.

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

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

The test item was dissolved in water for injections.

The test item was testd up to the maximum recommended dose level of 5000 µg/plate or up to the cytotoxicity, according to the strains and according to the presence or the absence of the metabolic activation.

The number of revertants for the vehicle and positive controls was in the acceptance criteria. The study was therefore considered valid.

No noteworthy increase in the number of revertants was noted in all the five strains.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study conducted according to current guidelines and under GLP.

Reason / purpose for cross-reference:

read-across: supporting information

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:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

X-linked hypoxanthine-guanine phosphoribosyl transferase (HGPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 mix from phenobarbital- and ß-naphthoflavone induced rats

Test concentrations with justification for top dose:

1st Experiment
without S9 mix
0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 µg/mL
with S9 mix
0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL
2nd Experiment
without S9 mix
0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 µg/mL
with S9 mix
0; 25.0; 50.0; 100.0; 200.0; 400.0; 600.0 µg/mL

Vehicle / solvent:

Due to the good solubility of the test substance in water, culture medium (Ham's F12) was used as most suitable vehicle.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Negative controls, with and without S9 mix, were treated with culture medium without test substance in parallel to the other treatment groups.

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Attachment period:20 - 24 hours
- Exposure duration:4 hour, removal of test substance by intense washing (4 hour exposure)
- Expression time (cells in growth medium):3 days (4-hour treatment)
- Selection time (if incubation with a selection agent):6 - 7 days
- Fixation time (start of exposure up to fixation or harvest of cells):10-11 days

SELECTION AGENT (mutation assays): Hypoxanthine-free Ham's F12 medium with 6-thioguanine (10 μg/mL), stable glutamine (200 mM), fetal calf serum (FCS)
STAIN (for cytogenetic assays):colonies were fixed with methanol, stained with Giemsa

NUMBER OF REPLICATIONS:Duplicate cultures were used for all experimental
groups.

NUMBER OF CELLS EVALUATED: Cloning efficiency 200 cells per dose group were seeded in 25 cm² flasks in duplicate using 5 mL. For selection of the mutants, six 75 cm2 flasks with 3x105 cells each from every treatment group, if possible, were seeded in 10 mL selection medium.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency

Evaluation criteria:

The cloning efficiency (CE, %) was calculated for each test group as follows:
CEabsolute = total number of colonies in the test group/total number of seeded cells in the test group x 100

The uncorrected mutant frequency (MFuncorr.) per 106 cells was calculated for each test group
as follows:
MFuncorr. = total number of mutant colonies / number of seeded cells x 106
MFcorr. = MFuncorr. / CE2 absolut x 100

A finding is assessed as positive if the following criteria are met:
• Increase in the corrected mutation frequencies (MFcorr.) both above the concurrent negative control values and our historical negative control data range (see Appendix 6).
• Evidence of the reproducibility of any increase in mutant frequencies.
• A statistically significant increase in mutant frequencies and the evidence of a doseresponse relationship.

The test substance is considered non-mutagenic according to the following criteria:
• The corrected mutation frequency (MFcorr.) in the dose groups is not statistically significantly increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 in highest tested concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In both experiments, in the presence and absence of S9 mix, after 4 hours treatment the morphology and attachment of the cells were adversely influenced in at least the highest applied concentration

Conclusions:

Interpretation of results: negative

In the absence and the presence of metabolic activation, DERIPHAT 160 C is not a mutagenic substance in the HPRT locus assay using CHO cells under the experimental conditions chosen.

Executive summary:

The substance test substance was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro according to OECD 476. Therefore two independent experiments were carried out, both with and without the addition of liver S9 mix from phenobarbital- and β-naphthoflavone induced rats (exogenous metabolic activation).

According to an initial range-finding cytotoxicity test for the determination of the experimental doses and taking into account the cytotoxicity actually found in the main experiments, the following doses were tested. Test groups printed in bold type were evaluated in this study:

1st and 2nd Experiment

without S9 mix

0; 12.5; 25.0; 50.0; 100.0; 200.0; 400.0 μg/mL

with S9 mix

0; 18.8; 37.5; 75.0; 150.0; 300.0; 600.0 μg/mL

Following attachment of the cells for 20-24 hours, cells were treated with the test substance for 4 hours in the absence and presence of metabolic activation. Subsequently, cells were cultured for 6-8 days and then selected in 6-thioguanine-containing medium for another week. Finally, the colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The negative controls gave mutant frequencies within the range expected for the CHO cellline.

Both positive control substances, EMS and DMBA, led to the expected increase in the frequencies of forward mutations.

In this study, in the 1st and 2nd Experiment, the highest concentrations evaluated for gene mutations were clearly cytotoxic in the absence and the presence of metabolic activation. Based on the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after the addition of a metabolizing system in two experiments performed independently of each other. Thus, under the experimental conditions of this study, the test substance is not mutagenic in the HPRT locus assay under in vitro conditions in CHO cells in the absence and the presence of metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

ANALOGUE APPROACH JUSTIFICATION:
See the read-across document

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with S9 in highest tested concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

Reference 6

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:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: colchicine: 0.1 µg/mL

Remarks:

without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 / 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:

Key result

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

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: human peripheral blood

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

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:
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 7

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis is based on “different compounds which have similar properties”.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- The source substance is identified as Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, also known as Sodium 3-[(2-carboxyethyl)(2-ethylhexyl)amino]propanoate (CAS no. 94441-92-6 | EC no. 305-318-6). It is a UVCB substance whose major constituent is sodium 2-ethylhexylimino-di-propionate. Minor constituents are sodium 2-ethylhexylimino-mono-propionate, unreacted acrylic acid and unreacted 2-ethylhexylamine.
- The target substance is identified as Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) (CAS no. 68610-44-6| EC no. 271-865-1). It is a UVCB substance whose major constituents are sodium 2-ethylhexylimino-mono-propionate and sodium 2-ethylhexylimino-di-propionate. Minor constituents are unreacted acrylic acid and unreacted 2-ethylhexylamine.

3. ANALOGUE APPROACH JUSTIFICATION
The target and source substances are essentially the same: both are UVCB substances composed of the exact same constituents with the exact same functional groups (i.e. carboxylic acid groups and secondary/tertiary amine groups). They are expected to have the same ADME profile and to share common mode of action and breakdown products. The target and source substances only differ in the overlapping ranges of their constituents, the content of sodium 2-ethylhexylimino-mono-propionate being especially higher in the target substance. This difference is expected to have no or very limited impact on the potency of effects exerted on exposed living organisms.

4. DATA MATRIX
Cf. read-across justification document attached in §13. Assessment reports.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Cytotoxicity from 3330 µg/mL and up to 5000 µg/mL

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

without

Genotoxicity:

ambiguous

Remarks:

The increase was only observed at the intermediate dose of 1281 µg/ml. No increase was seen below and above, up to 5000 µg/ml.

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

lymphocytes: huamn primary lymphocytes

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Long treatment (ca. 24 hours)

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

read-across: supporting information

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:

in vitro mammalian cell gene mutation tests using the thymidine kinase gene

Target gene:

thymidine kinase (TK) locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Key result

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.

Reference 9

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across hypothesis is based on “different compounds which have similar properties”.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
- The source substance is identified as Sodium N-(2-carboxyethyl)-N-(2-ethylhexyl)-β-alaninate, also known as Sodium 3-[(2-carboxyethyl)(2-ethylhexyl)amino]propanoate (CAS no. 94441-92-6 | EC no. 305-318-6). It is a UVCB substance whose major constituent is sodium 2-ethylhexylimino-di-propionate. Minor constituents are sodium 2-ethylhexylimino-mono-propionate, unreacted acrylic acid and unreacted 2-ethylhexylamine.
- The target substance is identified as Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide (methanol free) (CAS no. 68610-44-6| EC no. 271-865-1). It is a UVCB substance whose major constituents are sodium 2-ethylhexylimino-mono-propionate and sodium 2-ethylhexylimino-di-propionate. Minor constituents are unreacted acrylic acid and unreacted 2-ethylhexylamine.

3. ANALOGUE APPROACH JUSTIFICATION
The target and source substances are essentially the same: both are UVCB substances composed of the exact same constituents with the exact same functional groups (i.e. carboxylic acid groups and secondary/tertiary amine groups). They are expected to have the same ADME profile and to share common mode of action and breakdown products. The target and source substances only differ in the overlapping ranges of their constituents, the content of sodium 2-ethylhexylimino-mono-propionate being especially higher in the target substance. This difference is expected to have no or very limited impact on the potency of effects exerted on exposed living organisms.

4. DATA MATRIX
Cf. read-across justification document attached in §13. Assessment reports.

Reason / purpose for cross-reference:

read-across source

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested up to the maximum recommended dose level (5000 µg/mL)

Vehicle controls validity:

valid

Positive controls validity:

valid

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Based on an In Vitro test available for the substance itself (Ames test) or several In Vitro tests on analogue substances (Ames test, MLA, HPRT, Micronuclues test...), the substance "2 -Propenoic acid, methyl ester, reaction products with 2-ethyl-1-hexanamine and sodium hydroxide" (water and methanol free) is considered not to induce mutagenic effects.