Reaction products of 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-17 and C17 unsatd. alkyl) derivs. and sodium hydroxide and 2-propenoic acid

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)

- 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

- In vitro mammalian chromosome aberration test with Chinese hamster lung fibroblasts (met. act.: with and without) (OECD Guideline 473, GLP, RL1); read across: Amphopropionate C8 / 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:

2015-07-16 to 2015-07-31

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:

May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

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

Details on mammalian cell type (if applicable):

Regular checking of the properties of the Salmonella typhimurium and Escherichia coli strains regarding the membrane permeability, ampicillin resistance; UV sensitivity, and amino acid requirement as well as normal spontaneous mutation rates is performed

Additional strain / cell type characteristics:

other: Salmonella strains: rfa-, uvrB-; E. coli strain: uvrA-

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 Mix

Test concentrations with justification for top dose:

Pre-Experiment/Experiment I: 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate
Experiment II: 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water
- Justification for choice of solvent/vehicle: solubility properties and relative nontoxicity to the bacteria

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

sodium azide

methylmethanesulfonate

other: 4-nitro-o-phenylene-diamine (without metabolic activation); 2-aminoanthracene (with metabolic activation)

Details on test system and experimental conditions:

METHOD OF APPLICATION:plate incorporation (experiment I) and preincubation (experiment II)

DURATION
- Preincubation period: 60 min (only experiment II)
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): all Salmonella typhimurium strains: histidine; E. coli strain: tryptophane

NUMBER OF REPLICATES: 3

DETERMINATION OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants; reduction of the bacterial background lawn

Evaluation criteria:

A test item is considered as a mutagen if a biologically relevant increase in the number of revertants exceeding the threshold of twice (strains TA 98, TA 100, and WP2 uvrA) or thrice (strains TA 1535 and TA 1537) the colony count of the corresponding solvent control is observed.A dose dependent increase is considered biologically relevant if the threshold is exceeded at more than one concentration.
An increase exceeding the threshold at only one concentration is judged as biologically relevant if reproduced in an independent second experiment.
A dose dependent increase in the number of revertant colonies below the threshold is regarded as an indication of a mutagenic potential if reproduced in an independent second experiment.
However, whenever the colony counts remain within the historical range of negative and solvent controls such an increase is not considered biologically relevant.

Statistics:

not mandatory

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:

cytotoxicity

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: To evaluate the toxicity of the test item a pre-experiment was performed with all strains used. Eight concentrations were tested for toxicity and mutation induction with each 3 plates. The pre-experiment is reported as main experiment I, since the following criteria are met: Evaluable plates (>0 colonies) at five concentrations or more in all strains used.

COMPARISON WITH HISTORICAL CONTROL DATA: In experiment I the number of colonies did not quite reach the lower limit of the laboratory's historical control data in the negative control of strain TA 1535 with metabolic activation. Since this deviation is rather small, this effect is judged to be based upon statistical fluctuations and has no detrimental impact on the outcome of the study.

ADDITIONAL INFORMATION ON CYTOTOXICITY:

The plates incubated with the test item showed reduced background growth at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000-5000	1000-5000	1000-2500	2500
TA 1537	1000-5000	1000-5000	333-2500	1000-2500
TA 98	1000-5000	1000-5000	333-2500	1000-2500
TA 100	333-5000	333-5000	333-2500	1000-2500
WP2 uvrA	1000-5000	1000-5000	2500	2500

Toxic effects, evident as a reduction in the number of revertants (below the induction factor of 0.5), were observed at the following concentrations (µg/plate):

Strain	Experiment I		Experiment II
	without S9 mix	with S9 mix	without S9 mix	with S9 mix
TA 1535	1000-5000	2500-5000	1000-2500	2500
TA 1537	1000-5000	2500-5000	1000-2500	2500
TA 98	1000-5000	1000-5000	333-2500	1000-2500
TA 100	33; 333-5000	333-5000	33-2500	333-2500
WP2 uvrA	2500-5000	2500-5000	2500	2500

Conclusions:

During the described mutagenicity test and under the experimental conditions reported, no substantial increase in revertant colony numbers of any of the five tester strains was observed at any concentration level, neither in the presence nor absence of metabolic activation (S9 mix). There was also no tendency of higher mutation rates with increasing concentrations in the range below the generally acknowledged border of biological relevance. Therefore, Amphopropionate C12-18 is considered to be non-mutagenic in this Salmonella typhimurium and 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 (2008) 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 C12 -18 (ca. 40% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation method and at concentrations of 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate using the reincubation method both in the absence and presence of mammalian metabolic activation.

 

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of Amphopropionate C12 -18 did not occur up to the highest investigated dose. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C12 -18 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C12 -18 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:

experimental study

Adequacy of study:

key study

Study period:

07-May-2010 to 13-Jul-2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Principles of method if other than guideline:

The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

Species strain
- Type and identity of media:
-RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

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, 3 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL
Without S9-mix, 24 hours treatment: 100, 333, 1000, 3330 and 5000 µg/mL and 0.3, 1, 3, 10 and 33 µg/mL
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
(additional concentrations were not evaluated as there was a sufficient number of analysable concentrations and toxicity at higher doses)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640 culture medium
- Justification for choice of solvent/vehicle: Test compound was soluble in RPMI 1640 culture medium and this has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 to 12 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

The global evaluation factor (GEF) has been defined by the IWTG as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.

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

Additional information on results:

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

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 100 µg/mL and higher in the absence of S9, 3 hours treatment; at dose levels of 333 µg/mL and higher in the presence of S9, 3 hours treatment; at dose levels of 100 µg/mL and higher in the absence of S9, 24 hours treatment

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
In the absence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 84 and 90% compared to the total growth of the solvent controls after the 3 and 24 hours treatment period, respectively.

In the presence of S9-mix, the relative total growth of the highest test substance concentration was reduced by 89 and 92% compared to the total growth of the solvent controls after the 3 hours treatment period in the first and second experiment, respectively.

In the first experiment in the presence of S9-mix (3h treatment), there were only 7 analysable concentrations obtained, due to toxicity. However these conditions were repeated in the second experiment where 8 analysable concentrations were evaluated. This slight deviation was not considered to alter the study integrity.

In the second experiment in the presence of S9-mix, the recommended toxic range of approximately 10-20% RTG was not covered in the conditions with S9-mix. But the RTG was 8%, and a mutation frequency similar to that of the next inferior dose within a very narrow dose range, and not more than 2-fold compared to the negative control. The test conditions were similar to those of the first experiment where an appropriate RTG range was obtained, except for the metabolic activation concentration which was higher in experiment 2.

Conclusions:

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.

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 C12 -18 a reverse gene mutation assay in bacteria 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 as well as mammalian cell gene mutation assays conducted with the closely related substances Amphopropionate C8 and Amphoacetates C8-18 are 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 (2008) 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 C12 -18 (ca. 40% a.i.) at concentrations of 3, 10, 33, 100, 333, 1000, 2500 and 5000 µg/plate using the plate incorporation method and at concentrations of 0.3, 1, 3, 10, 33, 100, 333, 1000 and 2500 µg/plate using the reincubation method both in the absence and presence of mammalian metabolic activation.

The positive controls induced the appropriate responses in the corresponding strains and metabolic activation was confirmed. Precipitation of Amphopropionate C12 -18 did not occur up to the highest investigated dose. Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), occurred in all strains with and without metabolic activation.

In both mutation assays, no increase in the number of revertants over background was observed upon treatment with Amphopropinate C12 -18 under all conditions tested.

Based on the results of this study it is concluded that Amphopropionate C12 -18 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.

 

Supporting data are available for the closely related source substances Amphopropionate C8 and Amphoacetates C8-18:

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

 

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 uL/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 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.

 

Mammalian cell cytogenetics assa

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.

 

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 the in vitro chromosomal 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 mutagenic in 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 substance Amphopropionates C12-18 is manufactured from fatty acids (C12-18, C18unsatd.) and aminoethylethanolamie (AEEA) to form 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17 odd-numbered, C17unsatd. alkyl) derivs. This is further reacted with 2-propenoic acid in the presence of sodium hydroxide (alternatively, sodium 2-propenoate can be used) and water. The molar relation between 1H-Imidazole-1-ethanol, 4,5-dihydro-, 2-(C11-C17, C17unsatd. alkyl) derivs. and 2-propenoic acid is somewhat below 1:1. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

The source substance Amphopropionate C8 is manufactured from capric acid and aminoethylethanolamine (AEEA) to form 1-(2-Hydroxyethyl)-2-Heptylimidazoline. Excess AEEA is removed from the reaction mixture by distillation at elevated temperature. In a further step 2-propenoic acid is added to form Amphopropionate C8. Most of the excess 2-propenoic acid is stripped off by distillation. However, a small amount remains in the aqueous solution.

 

The source substance Amphoacetates C8 -C18 is manufactured from 1H-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 source substance Amphopropionate C8 contains shorter C chains, whereas the major C chain in the target substance is C12.

In general the absorption declines with increasing alkyl chain length (Ramirez et al. 2001). Therefore the source substances with the shorter alkyl chain lengths are assumed to represent a worst-case scenario due to higher absorption rates than the target substance.

 

Degree of unsaturation:

In contrast to the source substance Amphopropionate C8, the target substance Amphopropionates C12-18 as well as the source substance Amphoacetates C8-C18 contain 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 Amphopropionates C12-18 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 substance Amphopropionates C12-18 as well as the source substance Amphopropionate 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 are available to demonstrate that this difference in composition is of no toxicological relevance.

 

Comparison of genotoxicity data

 

	Target substance	Source substances
Endpoint	Amphopropionates C12-18	Amphopropionate C8	Amphoacetates C8-C18
Genetic toxicity, Ames test	key_Genetic toxicity in vitro_93820-52-1_8.4.1_Evonik_2015_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: yes Genotoxicity: negative   1 (reliable without restriction), GLP	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	No data, read-across	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 C12 -18 a reverse gene mutation assay in bacteria 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 as well as mammalian cell gene mutation assays conducted with the closely related substances Amphopropionate C8 and Amphoacetates C8-18 are available. All available tests were consistently negative.

 

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 the in vitro chromosomal 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 mutagenic in vivo.

Thus, the small residual amounts of acrylic acid, that may be present in the target substance, are of no toxicological relevance.

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 results from the bacterial reverse mutation assay, which is available for the source substances as well as for the target substance, further justify this read-across approach.

The negative outcome of the complete testing battery of in vitro genotoxicity tests for the source substances Amphopropionate C8 and Amphoacetates C8-C18 are also valid for the target substance Amphopropionates C12 -18. 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, Amphopropionates C12 -18 is considered to be not mutagenic and not clastogenic. According to Regulation EC No 1272/2008 no classification and labelling for mutagenicity is required.