N-(2-hydroxyethyl)-N-[2-[(1-oxooctyl)amino]ethyl]-β-alanine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

All available tests were consistently negative:

- Ames test with S. typhimurium TA 98, TA 100, TA 1535, TA 1537, E coli WP2 uvrA (met. act.: with and without) (OECD TG 471, GLP, RL1)

- In vitro mammalian chromosome aberration test with Chinese hamster lung fibroblasts (met. act.: with and without) (OECD Guideline 473, GLP, RL1)

- Mammalian cell gene mutation assay with mouse lymphoma L5178Y cells (TK) (met. act.: with and without) (OECD Guideline 476 and GLP); read across: Amphoacetates C8-18

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:

2007-03-20 to 2007-03-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

June 8, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus (Salmonella typhimurium strains) and tryptophan locus (E. coli strain)

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

other: additional rfa, gal, chl, bio and uvrB mutations in S. typhimurium strains and uvrA mutation in E.coli strain

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
First test: 3, 10, 33, 100, 333, 1000, 3330, 5000 µg/plate
Second test: 100, 333, 1000, 3330, 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

2-nitrofluorene

sodium azide

methylmethanesulfonate

other: (without metabolic activation)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 h

NUMBER OF REPLICATIONS: 3 replicates, 2 independent repeats

DETERMINATION OF CYTOTOXICITY
- Method: reduction of the bacterial background lawn, increase in the size of the microcolonies, reduction of revertant colonies

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 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 (mutagenic) in the test if:
a) The total number of revertants in tester strain TA 100 is greater than two (2) times the concurrent control, or the total number of revertants in tester strains TA 1535, TA 1537, TA98 or WP2uvrA is greater than three (3) times the concurrent control.
b) In case a positive response will be repeated, the positive response should be reproducible in at least one independently repeated experiment.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Statistics:

No formal hypothesis testing was done.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks:

With the exception of tester strain TA 100 in the absence of S9-mix, where a moderate reduction of the revertant colonies compared to the solvent control was observed at the highest tested concentration.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES:
- Toxicity: No reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants were observed. With the exception of tester strain TA 100 in the absence of S9-mix, where a moderate reduction of the revertant colonies compared to the solvent control was observed at the highest tested concentration.
- Mutagenicity: In the dose range finding test, no increase in the number of revertants was observed upon treatment with the test substance under all conditions tested.

COMPARISON WITH HISTORICAL CONTROL DATA:
negative and strain-specific positive control values were within laboratory historical control data ranges

ADDITIONAL INFORMATION ON CYTOTOXICITY (DEFINITIVE TEST):
In tester strain TA 100 in the absence of S9-mix, a moderate reduction of the revertant colonies was observed at the highest tested concentration, however the reduction was not below the historical control data range. In the presence of S9-mix, fluctuations in the number of revertant colonies just below or around the laboratory historical control data range were observed. However, since no dose-relationship was observed, the reductions are not considered to be caused by toxicity of the test substance.
In all other tester strains both in the first and second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

Conclusions:

Based on the results of this study it is concluded that Amphopropionate C8 (50%) is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (July, 1997) and EU Method B. 13/14 (June, 2000) strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed in two independent experiments to Amphopropionate C8 (50 %) at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (first test) and 100, 333, 1000, 3330, 5000 µg/plate (second test) in the absence and presence of mammalian metabolic activation using the plate incorporation method.

 

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of the test item on the plates was not observed at the start or at the end of the incubation period in both tester strains.

In tester strain TA 100 in the absence of S9-mix, a moderate reduction of the revertant colonies was observed at the highest tested concentration, however the reduction was not below the historical control data range. In all other tester strains both in the first and second mutation assay, there was no reduction of the bacterial background lawn and no biologically relevant decrease in the number of revertants at any of the concentrations tested in all tester strains in the absence and presence of S9-mix.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C8 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C8 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.  This study is classified as acceptable. It satisfies the requirements for Test Guideline OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007-04-04 to 2007-06-01

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

June 8, 2000

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral lymphocytes

Details on mammalian cell type (if applicable):

Blood collected from healthy adult, non-smoker, male volunteers (average age: 40 years)
- Type and identity of media: RPMI 1640 medium (lnvitrogen Corporation), supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum (lnvitrogen Corporation), L-glutamine (2 mM) (Merck), penicillin/streptomycin (50 U/ml and 50 µg/mL respectively) (lnvitrogen Corporation) and 30 U/mL heparin (Sigma).

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9-mix

Test concentrations with justification for top dose:

Dose range finding test: 33, 100, 333, 1000 and 3020 µg/mL with and without S9-mix (3 h, 24 h an 48 h exposure time, 24 h and 48 h fixation)
1st cytogenetic assay: 333, 1000 and 3020 µg/mL with and without S9-mix (3 h exposure time, 24 h fixation)
2nd cytogenetic assay: 300, 1000 and 3020 µg/mL with S9-mix (3 h exposure time, 48 h fixation)
25, 100, 300, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (48 h exposure time, 48 h fixation)
300, 750, 1000, 1500, 1750 and 2000 µg/mL without S9-mix (24 h exposure time, 24 h fixation)
Cytogenetic assay 2A: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (24 h exposure time, 48 h fixation).

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 medium (lnvitrogen Corporation, Breda, The Netherlands).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h, 24 h, and 48 h
- Expression time (cells in growth medium): 20-22 h for the 3 h exposure and 24 fixation time; 44-46 h for the 3 h exposure and the 48 h fixation time; cells that were treated for 24 h and 48 h were fixed immediately after 24 h and 48
- Fixation time (start of exposure up to fixation or harvest of cells): 24 h / 48 h

SPINDLE INHIBITOR (cytogenetic assays): colchicine (0.5 µg/mL), during the last 2.5 to 3 h of the culture period
STAIN (for cytogenetic assays): 5% (v/v) Giemsa solution

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture (In case the number of aberrant cells, gaps excluded, was ≥ 25 in 50 metaphases, no more metaphases were examined.)

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations in the absence of a clear dose-response relationship.
A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant increase in the number of cells with chromosome aberrations.

Statistics:

Chi-square test, one-sided, p < 0.05

Species / strain:

lymphocytes: human peripheral lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

without metabolic activation after 24 and 48 h exposure at the highest evaluated concentrations

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH/osmolality: pH and osmolarity of 3020 pg/mL: 7.30 and 296 mOsm/kg / solvent control: 7.41 and 285 mOsm/kg
- Evaporation from medium: no
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
The number of cells with chromosome aberrations, polyploid cells and cells with endoreduplicated chromosomes in the solvent control cultures was within the laboratory historical control data range. The positive control chemicals (MMC-C and CP) both produced statistically significant increases in the frequency of aberrant cells.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
Clearly reduced mitotic indices could be observed without metabolic activation after 24 and 48 h exposure at the highest evaluated concentrations.

Conclusions:

In conclusion, it can be stated that under the experimental conditions reported, the test item Amphopropionate C8 is not clastogenic in human lymphocytes in vitro, when tested up to cytotoxic or the recommended limit concentration of 0.01 M (corresponding to 3020 µg/mL).

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, June 2000, peripheral human lymphocyte cultures were exposed to Amphopropionate C8 (50.6% a.i.) at the following concentrations:

1st cytogenetic assay: 333, 1000 and 3020 µg/mL with and without S9-mix (3h exposure time, 24 fixation)

2nd cytogenetic assay: 300, 1000 and 3020 µg/mL with S9-mix (3h exposure time, 48 h fixation)

25, 100, 300, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (48 h exposure time, 48 h fixation)

300, 750, 1000, 1500, 1750 and 2000 µg/mL culture medium without S9-mix (24 h exposure time, 24 h fixation)

Cytogenetic assay 2A: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (24 h exposure time, 48 h fixation).

The highest treatment concentration in this study, 3020 µg/mL (0.01 M) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 473.

Clearly reduced mitotic indices could be observed without metabolic activation after 24 and 48 h exposure at the highest evaluated concentrations.

The positive and negative controls induced appropriate responses.

Both in the absence and presence of metabolic activation, Amphopropionate C8 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of Amphopropionate C8 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

There was no evidence of structural chromosome aberrations induced over background.

 

Reference 3

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
This read-across is based on the hypothesis that source and target substances have similar toxicological properties because
• they are manufactured from similar or identical precursors under similar conditions
• they share structural similarities with common functional groups: tertiary amines, amides, fatty acid chains with differing C-chain length, and short chain carboxylic acids (acetic / propenoic) of comparable length

Therefore, read-across from the existing studies on the source substances is considered as an appropriate adaptation to the standard information requirements of the REACH Regulation for the target substance, in accordance with the provisions of Annex XI, 1.5 of the REACH Regulation.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
See general justification for read-across attached to IUCLID chapter 13.

3. ANALOGUE APPROACH JUSTIFICATION
See general justification for read-across attached to IUCLID chapter 13.

4. DATA MATRIX
See general justification for read-across attached to IUCLID chapter 13.

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Reason / purpose for cross-reference:

read-across: supporting information

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Interpretation of results: negative with and without metabolic activation

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

For the assessment of the genotoxic potential of Amphopropionate C8 a reverse gene mutation assay in bacteria as well as a chromosome aberration assay are available, which were conducted on the substance itself. Furthermore, an in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) conducted with the closely related substance Amphoacetates C8-18 is available. A justification for read-across is given below.

 

Bacterial reverse mutation assays

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (July, 1997) and EU Method B. 13/14 (June, 2000) strains of S. typhimurium (TA 1535, TA 1537, TA 98 and TA 100) and E. coli (WP2 uvr A) were exposed in two independent experiments to Amphopropionate C8 (50 %) at concentrations of 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (first test) and 100, 333, 1000, 3330, 5000 µg/plate (second test) in the absence and presence of mammalian metabolic activation using the plate incorporation method.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C8 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C8 is not mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay. 

 

Similar results were obtained with the source substance Amphoacetates C8-18:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 strains TA 1535, TA 1537, TA 98 and TA 100 of S. typhimurium) were exposed to Amphoacetates C8-18 at concentrations of 1.3, 6.4, 32, 160 and 800 µg/plate in the absence and presence of mammalian metabolic activation using the plate incorporation method.

Negative up to cytotoxic concentration (0.4 µL/plate) with and without metabolic activation in the Salmonella typhimurium reverse mutation assay, in strain TA100 only. For the other strains, no cytotoxicity and no increase in the number of revertants were observed at the highest dose tested (0.4 μL/plate). However, the highest recommended dose was not reached for these strains.

 

Mammalian cell cytogenetics assays

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473, adopted 21 July 1997 and EU Method B.10, June 2000, peripheral human lymphocyte cultures were exposed to Amphopropionate C8 (50.6% a.i.) at the following concentrations:

1st cytogenetic assay: 333, 1000 and 3020 µg/mL with and without S9-mix (3h exposure time, 24 fixation)

2nd cytogenetic assay: 300, 1000 and 3020 µg/mL with S9-mix (3h exposure time, 48 h fixation)

25, 100, 300, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (48 h exposure time, 48 h fixation)

300, 750, 1000, 1500, 1750 and 2000 µg/mL culture medium without S9-mix (24 h exposure time, 24 h fixation)

Cytogenetic assay 2A: 50, 100, 250, 500, 750, 1000, 1250 and 1500 µg/mL without S9-mix (24 h exposure time, 48 h fixation).

The highest treatment concentration in this study, 3020 µg/mL (0.01 M) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 473.

Clearly reduced mitotic indices could be observed without metabolic activation after 24 and 48 h exposure at the highest evaluated concentrations.

The positive and negative controls induced appropriate responses.

Both in the absence and presence of metabolic activation, Amphopropionate C8 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in two independent experiments. No effects of Amphopropionate C8 on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix.

There was no evidence of structural chromosome aberrations induced over background.

 

Supporting data are also available for the source substance Amphoacetates C8-18:

In a mammalian cell cytogenetics assay (chromosome aberrations) according to OECD guideline 473,

human peripheral blood lymphocytes were exposed to Amphoacetates C8-18 at the following concentrations:

First cytogenetic test (doses selected for analysis):

Without and with S9-mix, 3 h exposure time, 24 h fixation time: 100, 450 and 500 µg/ mL

Second cytogenetic test (doses selected for analysis):

Without S9-mix, 24 hr exposure; 24 hr fixation: 100, 200 and 300 µg/ mL

Without S9-mix, 48 hr exposure; 48 hr fixation: 10, 200 and 240 µg/ mL

With S9-mix, 3 hr exposure; 48 hr fixation: 100, 300 and 400 µg/ mL

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

Amphoacetates C8-18 did not induce a statistically significant or biologically relevant increase in the number of cells with chromosome aberrations in the absence and presence of S9-mix, in either of the two independently repeated experiments.

Finally, it is concluded that this test is valid and that Amphoacetates C8-18 is not clastogenic in human lymphocytes under the experimental conditions described in this report. Amphoacetates C8-18 may have the potential to disturb mitotic processes.

 

Mammalian cell gene mutation assay

In a mammalian cell gene mutation assay according to OECD guideline 476 (TK assay), L5178Y mouse lymphoma cells cultured in vitro were exposed to Amphoacetates C8-18 at the following concentrations:

Experiment 1 (doses selected for measurement of mutation frequency):

Without S9-mix, 3 hours treatment: 0.1, 1, 5, 10, 33, 50, 75 and 100 µg/mL

With (8% (v/v) S9-mix, 3 hours treatment: 33, 100, 125, 150, 175, 200 and 225 µg/mL

Experiment 2 (doses selected for measurement of mutation frequency):

Without S9-mix, 24 hours treatment: 0.08, 0.8, 4, 8, 17, 25, 34 and 42 µg/mL

With (12% (v/v) S9-mix, 3 hours treatment: 4, 42, 84, 125, 146, 168, 210 and 230 μg/mL

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range. Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, Amphoacetates C8-18 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in a repeat experiment with modifications in the duration of treatment time.

In the presence of S9-mix, Amphoacetates C8-18 did not induce a significant increase in the mutation frequency in the first experiment. This result was confirmed in an independent experiment with modifications of the S9-mix concentration added for metabolic activation.

In conclusion, Amphoacetates C8-18 is not mutagenic in the TK mutation test system under the experimental conditions described in the report.

 

Supporting data with acrylic acid:

Acrylic acid did not induce gene mutations in Salmonella or CHO cells (HPRT locus) but was

clearly positive in the mouse lymphoma assay and in thein vitrochromosomal aberration test.

Since in the mouse lymphoma assay small colonies were induced preferentially, the mutagenic potential of acrylic acid seems to be limited to clastogenicity.In vivo, acrylic acid did not induce mutagenic effects in either rat bone marrow cells or mouse germ cells after oral administration. Based on the results, acrylic acid is not considered to be mutagenicin vivo(EU RAR, 2002).

 

There was no evidence of mutagenic or clastogenic intrinsic properties in any of the performed studies. There are no data gaps for the endpoint genotoxicity. No human information is available for this endpoint. However, there is no reason to believe that these results would not be applicable to humans.

 

Justification for read-across

For details on substance identity and detailed toxicological profiles, please refer also to the general justification for read-across given at the beginning of the CSR and attached as pdf document to IUCLID section 13.

This read-across approach is justified based on structural similarities as well as on a similar toxicological profile. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

 

Structural similarity and functional groups

The target substanceAmphopropionate C8is manufactured from capric acid andaminoethylethanolamie (AEEA) to form 1-(2-Hydroxyethyl)-2-Heptylimidazoline. Excess AEEA is removed from the reaction mixture by distillation at elevated temperature. In a further step2-propenoic acid is added to formAmphopropionate C8. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

The source substanceAmphoacetates C8-C18is manufactured from1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and chloroacetic acid in the presence of sodium hydroxide. The molar relation between 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C7-C17 odd-numbered, C17-unsatd. alkyl) derivs. and chloroacetic acid ranges from 1:1 to 1:2. As by-product, hydrochloric acid is formed during the reaction, that is neutralized with adding more sodium hydroxide.

 

Differences

Chain length:

The target substance Amphopropionate C8 contains C8 chains, whereas the major C chain in the target substance is C12.In general the absorption declines withincreasingalkyl chain length (Ramirez et al. 2001). Thus, the target substance may be bioavailable to a slightly higher degree.

 

Degree of unsaturation:

In contrast to the target substance Amphopropionate C8, the source substance Amphoacetates C8-C18 contains some amounts of unsaturated C18 chains.

An increase in the degree of unsaturation may lead to a slightly higher irritation potential (HERA, 2002; Stillman, 1975; Aungst, 1989). Apart from that, fatty acids irrespective of their degree of unsaturation are in general non-toxic. Irritation studies are available for the target substance itself, thus, for other endpoints,this difference in composition is of no toxicological relevance.

 

Propionate vs. acetate functions:

The target substance Amphopropionate C8 contains propionate functions, whereas thesource substance Amphoacetates C8-C18 contains acetate functions. The shorter acetate chains might lead to slightly higher absorption.

 

Presence of residual acrylic acid:

The target substanceAmphopropionate C8 may contain some small amounts of residual acrylic acid, in contrast to the source substance Amphoacetates C8-C18.

However, supporting data from acrylic acid demonstrate that this difference in composition is of no toxicological relevance for human health endpoints.

 

Comparison of genotoxicity data

 

	Target substance	Source substance
Endpoint	Amphopropionate C8	Amphoacetates C8-C18
Genetic toxicity, Ames test	sup_RA_Genetic toxicity in vitro_64265-45-8_8.4.1_Evonik_2007_OECD471   OECD TG 471, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2, with and without met.act. (S9 mix) Cytotoxicity: no, but tested up to limit concentrations Genotoxicity: negative   1 (reliable without restriction), GLP	sup_RA_Genetic toxicity in vitro_931-291-0_8.4.1_Evonik_1994_OECD471   OECD TG 471, S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100, with and without met.act. (S9 mix) Cytotoxicity: yes Genotoxicity: negative   2 (reliable with restrictions), GLP
		sup_RA_Genetic toxicity in vitro_931-291-0_8.4.1_Evonik_1991_OECD471   OECD TG 471, S. typhimurium TA 1535, TA 1537, TA 98 and TA 100, with and without met.act. (S9 mix) Cytotoxicity: yes Genotoxicity: negative   2 (reliable with restrictions), GLP
Genetic toxicity, mammalian cell gene mutation assay	No data; read-across	key_RA_Genetic toxicity in vitro_8.4.3_931-291-0_Rhodia_2010_OECD476   OECD TG 476, mouse lymphoma L5178Y cells, with and without met.act. (S9 mix) Cytotoxicity: yes Genotoxicity: negative   1 (reliable without restriction), GLP
Genetic toxicity, chromosome aberration	key_RA_Genetic toxicity in vitro_64265-45-8_8.4.2_Evonik_2007_OECD473   OECD TG 473, human peripheral lymphocytes, with and without met.act. (S9 mix) Cytotoxicity: yes Genotoxicity: negative   1 (reliable without restriction), GLP	sup_RA_Genetic toxicity in vitro_8.4.2_931-291-0_Rhodia_2010_OECD473   OECD TG 473, human peripheral lymphocytes, with and without met.act. (S9 mix) Cytotoxicity: yes Genotoxicity: negative   1 (reliable without restriction), GLP

 

For the assessment of the genotoxic potential of Amphopropionate C8 a reverse gene mutation assay in bacteria as well as mammalian cell gene mutation assay is available conducted with the target substance itself.

Furthermore, an in vitro gene mutation study in mammalian cells (L5178Y/ TK Mouse Lymphoma assay) conducted with the closely related substance Amphoacetates C8-18 is available. All available tests were consistently negative.

 

Supporting data are available for Acrylic acid, which did not induce gene mutations in Salmonella or CHO cells (HPRT locus) but was clearly positive in the mouse lymphoma assay and in thein vitrochromosomal aberration test. Since in the mouse lymphoma assay small colonies were induced preferentially, the mutagenic potential of acrylic acid seems to be limited to clastogenicity.In vivo, acrylic acid did not induce mutagenic effects in either rat bone marrow cells or mouse germ cells after oral administration. Based on the results, acrylic acid is not considered to be mutagenicin vivo(EU RAR, 2002).

 

Quality of the experimental data of the analogues:

The available data are adequate and sufficiently reliable to justify the read-across approach.

The studies were conducted according to OECD Guidelines 471, 476 and 473, respectively, and are reliable or reliable with restrictions (RL1-2).

The test materials used in the respective studies represent the source substance as described in the hypothesis in terms of substance identity and minor constituents.

Overall, the study results are adequate for the purpose of classification and labelling and risk assessment.

 

Conclusion

The structural similarities between the source and the target substances presented above and in more detail in the general justification for read-across support the read-across hypothesis. Adequate and reliable scientific information indicates that the source and target substances have similar toxicity profiles.

The negative outcome of the complete testing battery of in vitro genotoxicity tests for the source substance Amphoacetates C8-C18 are also valid for the target substance Amphopropionate C8.The negative results from the bacterial reverse mutation assay and the in vitro chromosome aberration assay, which are available for the source substance as well as for the target substance, further justify this read-across approach.

Thus, no classification for genotoxic properties is required. 

 

References

Aungst, 1989. Structure/Effect Studies of Fatty Acid Isomers as Skin Penetration Enhancers and Skin Irritants. Pharmaceutical Research, March 1989, Volume 6, Issue 3, pp 244-247

 

EU RAR, 2002:European Union, Risk Assessment Report: Acrylic acid, CAS No: 79-10-7, Risk Assessment. European Union Risk Assessment Report, 1st Priority List, Vol. 2

 

HERA, 2002: Fatty Acid Salts – Human Health Risk Assessment

 

Ramírez M, Amate L,Gil A. Absorption and distribution of dietary fatty acids from different sources. Early Human Development 2001 Nov;65 Suppl:S95-S101

 

Stillman et al., 1975. Relative irritancy of free fatty acids of different chain length. Contact Dermatitis. 1975;1(2):65-9.

Justification for classification or non-classification

Based on reliable, relevant and adequate data, Amphopropionate C8 is considered to be not mutagenic and not clastogenic. According to Regulation EC No 1272/2008 no classification and labelling for mutagenicity is required.