1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C8-10 (even numbered) acyl derivs., hydroxides, inner salts

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: C8-18 AAPB and Formamidopropylbetaine
- In vitro mammalian chromosome aberration test with Chinese hamster lung fibroblasts (met. act.: with and without) (OECD Guideline 473, GLP, RL1); read across: Formamidopropylbetaine

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

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:

dated May 30, 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

adopted July 21, 1997

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

his-operon (Salmonella strains); trp (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: uvrB- (Salmonella); uvrA- (E. coli)

Metabolic activation:

with and without

Metabolic activation system:

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: 33, 100, 333, 1000, 2500 and 5000 μg/plate
(all concentrations adjusted to purity)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionized water
- Justification for choice of solvent/vehicle: due to 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

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene; with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: Experiment I: in agar (plate incorporation); Experiment II: preincubation

DURATION
- Preincubation period: 60 minutes. at 37°C (only Experiment II)
- Exposure duration: least 48 hours at 37 °C in the dark

NUMBER OF REPLICATIONS: triplicates

DETERMINATION OF CYTOTOXICITY
- Method: background growth; reduction in the number of revertants below the indication factor of 0.5

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:

According to the OECD guideline 471, a statistical analysis of the data is not mandatory.

Key result

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

Remarks:

only in strain TA 100 toxic effects were observed at 5000 μg/plate without S9 mix in experiment I and with and without S9 mix in experiment II

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment I without S9 mix, the data in the solvent control of strain TA 100 were slightly above our historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Conclusions:

C8-10 Alkylamidopropyl betaine was evaluated in the bacterial reverse mutation assay (Ames test) using Salmonella typhimurium tester strains TA98, TA100, TA1535, and TA1537 and Escherichia coli tester strain WP2 uvrA in the presence and absence of rat liver S9 mix. Under the conditions of the study, the test substance was negative for mutagenic potential.

Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to C8-10 Alkylamidopropyl betaine (36% a.i. in aqueous solution) in deionized water at concentrations of 0 (control), 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in the first experiment (plate incorporation assay) and 0 (control), 33, 100, 333, 1000, 2500 and 5000 μg/plate in the second experiment (preincubation assay) in the presence and absence of mammalian metabolic activation (rat liver S9 mix).. All concentrations were adjusted to purity.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were absent in nearly all strains, only in strain TA 100 toxic effects were observed at 5000μg/plate without S9 mix in experiment I and with and without S9 mix in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

In experiment I without S9 mix, the data in the solvent control of strain TA 100 were slightly above the historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

 

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C8-10 Alkylamidopropyl betaine did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08. Jun. 2005 - 19. Oct. 2005

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

The V79 (ATCC, CCL-93) cells are stored over liquid nitrogen (vapour phase) in the cell bank of BSL BIOSERVICE, as large stock cultures allowing the repeated use of the same cell culture batch in experiments. Routine checking of mycoplasma infections was carried out before freezing.

Metabolic activation:

with and without

Metabolic activation system:

S9 liver microsomal fraction, prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw).

Test concentrations with justification for top dose:

Pre-test toxicity: 0.0075, 0.015, 0.03, 0.06, 0.125, 0.25, 0.5, 1, 2.5 and 5 µL/mL
Experiment 1: 0.25, 0.5, 1,2.5 and 5 µL/mL (with and without S9); 1, 2.5 and 5 µL/mL selected for microscopic evaluation
Experiment 2: 0.125, 0.25, 0.5, 1, 2.5 and 5 µL/mL (without S9); 1, 2.5 and 5 µL/mL selected for microscopic evaluation
1, 2, 3, 4 and 5 µL/mL (with S9); 2, 3, 4 and 5 µL/mL selected for microscopic evaluation

Vehicle / solvent:

cell culture medium (MEM medium)

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: without metabolic activation; 15 µg/mL (3 hours treatment), 5 µg/mL (24 h treatment)

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: with metabolic activation

Details on test system and experimental conditions:

Seeding of the Cultures:
Three or four days old stock cultures (in exponential growth) with higher than 50% confluency were trypsinised at 37 DC for 5 min. by adding a trypsine solution in Ca-Mg-free PBS. The enzymatic treatment was stopped with complete culture medium. A single cell suspension was prepared. The
trypsine concentration for all subculturing steps was 0.2%. The cells were rinsed with Ca-Mg-free PBS prior to the trypsine treatment. The cells were seeded into Quadriperm dishes which contain microscopic slides (at least 2 chambers per dish and test group). Into each chamber 1E04 - 5E04 cells were seeded with regard to preparation interval. The medium was minimum essential medium supplemented with 10% FCS.
Treatment
Experiment I: Short time exposure: Two days after seeding of the cells, the culture medium was replaced with serum-free medium containing the test item and 50 µL/mL S9 mix (with metabolic activation). Additional negative and positive controls were performed with and without metabolic activation. 4 h after treatment the cultures were washed twice with PBS and cultured in complete medium for the remaining culture time.
Experiment II: Short time exposure (with metabolic activation), long time exposure (without metabolic activation): The treatment with metabolic activation was performed as described above for experiment 1. In the experiment without metabolic activation, two days after seeding the cells were incubated with the test item in complete medium (MEM with 10 % FCS) for 20 h. The ce11s were prepared at the end of the incubation. Additional negative and positive controls were tested. All cultures are incubated at 37 °C in a humidified atmosphere with 5.0 % CO2 (95.0 % air).
Preparation of the Cultures:
17.5 h (4 hand 20 h treatment) and 25.5 h (28 h) after the start of the treatment Colcemid® was added to the cultures (0.2 µg/mL culture medium).2.5 h later, the cells were treated on the slides in the chambers with hypotonic solution (0.4 % KCl) for 20 min at 37 °C. After incubation in the hypotonic solution the cells were fixed with 3 + 1 methanol + glacial acetic acid. All the steps were carried out on precision hot plates. After fixation the cells were stained with Giemsa.
Analysis of Metaphase Cells
All slides, including those of posItIve and negative controls were independently coded before microscopic analysis. Evaluation of the cultures was performed [according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik" (4)] using microscopes with 100 x oil immersion objectives. As structural chromosomal aberrations breaks, fragments, deletions, exchanges and chIOmosomal disintegration were recorded. Gaps were recorded as well but not included in the calculation of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally. At least 200 well spread metaphases per concentration and negative/positive controls were scored for cytogenetic damage. The cells scored contained 22 ± 1 centromeres. To describe a cytotoxic effect the mitotic index (% cells in mitosis) was detelmined by counting
the number of mitotic cells in 1000 cells. Additionally the number of polyploid cells was scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

Evaluation criteria:

There are several criteria for detennining a positive result: a clear and dose-related increase in the number of cells with aberrations, a biologically relevant response for at least one of the dose groups, which is higher than the laboratory negative control range (up to 4.5% aberrant cells).
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary. However, for the interpretation of the data, both biological and though evaluated statistical significance should be
considered together.
A test item is considered to be negative if there is no biologically relevant increase in the percentages of aberrant cells above concurrent control levels, at any dose group. Although most experiments will give clearly positive or negative results, in some cases the data set will preclude making a definitive judgement about the activity of the test substance.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Experiment 1:

Dose Group	Concentration [µL/mL]	Treatment time	Fixation interval	Mean % aberrant cells
				incl. gaps	excl. gaps
without metabolic activation
C	0	4 h	20 h	2.0	0.5
3	1	4 h	20 h	3.5	2.0
4	2.5	4 h	20 h	2.0	1.0
5	5	4 h	20 h	3.5	1.5
EMS	900 µg/mL	4 h	20 h	10.5	8.0
with metabolic activation
C	0	4 h	20 h	3.0	1.5
3	1	4 h	20 h	2.5	1.0
4	2.5	4 h	20 h	3.5	3.5
5	5	4 h	20 h	4.5	1.5
CPA	0.83 µg/mL	4 h	20 h	10.0	9.0

200 cells evaluated for each concentration

C: Negative Control (culture medium)

EMS: Ethylmethanesulfonate (positive control without metabolic activation)

CPA: Cyclophosphamide) (positive control with metabolic activation)

No precipitation of the test item was noted in all dose groups evaluated

 

 

Experiment 2:

Dose Group	Concentration [µL/mL]	Treatment time	Fixation interval	Mean % aberrant cells
				incl. gaps	excl. gaps
without metabolic activation
C	0	20 h	20 h	3.0	0.5
4	1	20 h	20 h	3.0	1.5
5	2.5	20 h	20 h	2.0	1.0
6	5	20 h	20 h	5.5	3.0
EMS	400 µg/mL	20 h	20 h	12.0	9.0
with metabolic activation
C	0	4 h	20 h	4.0	1.5
2	2	4 h	20 h	5.0	2.5
3	3	4 h	20 h	6.5	3.5
4	4	4 h	20 h	3.8	1.8
5	5	4 h	20 h	6.0	3.0
CPA	0.83 µg/mL	4 h	20 h	11.5	10.5

200 cells evaluated for each concentration

C: Negative Control (culture medium)

EMS: Ethylmethanesulfonate (positive control without metabolic activation)

CPA: Cyclophosphamide) (positive control with metabolic activation)

No precipitation of the test item was noted in all dose groups evaluated

Conclusions:

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item Formamidopropyldimethylbetaine (38.2% a.i.) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item is considered to be non-clastogenic.

Executive summary:

The test item Formamidopropyldimethylbetaine (38.2% a.i.) was investigated for a possible potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation with S9 homogenate. The selection of the concentrations used in experiment I and II based on data from the solubility test and the pre-experiment according to the guidelines.

In experiment I with and without metabolic activation 5 µL/mL was selected as highest dose group for the microscopic analysis of chromosomal aberrations. In experiment II with and without metabolic activation 5 µL/mL was selected as highest dose group. The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. At least 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for microscope analysis: Experiment I: with and without metabolic activation: 1, 2.5 and 5 µL/mL Experiment II: without metabolic activation: 1, 2.5 and 5 µL/mL with metabolic activation: 2, 3, 4 and 5 µL/mL.

Precipitation: The test item was diluted in culture medium (MEM medium). No precipitation of the test item was noted in all dose groups evaluated.

Toxicity: In experiments I and II no toxic effects of the item (indicated by a decrease of the rel. mitotic index below 70 %) were noted in all dose groups evaluated with and without metabolic activation.

Clastogenicity: In experiment I without metabolic activation the aberration rate of the negative control (0.5 %) was within the historical control data of the negative control (0.0 % - 4.0 %). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 2.0 % (1 µL/mL) , 1.0 % (2.5 µL/mL) and 1.5 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment I with metabolic activation the number of aberrant cells noted for the negative control (l.5 %) was within the historical control data of the negative control (0.0 % - 4.5 %). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 1 % (1 µL/mL) , 3.5 % (2.5 µL/mL) and 1.5 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment II without metabolic activation the aberration rate of the negative control (0.5 %) was within the historical control data of the negative control (0.0 % - 4.0 %,). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 1.5 % (1 µL/mL) , 1 % (2.5 µL/mL) and 3 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control. In experiment II with metabolic activation the aberration rate of the negative control (1.5 %) was within the historical control data of the negative control (0.0 % - 4.5 %). The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. The mean values noted were 2.5 % (2 µL/mL), 3.5 % (3 µL/mL), 1.8 % (4 µL/mL) and 3 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control. Polyploid cells show the occurrence of polyploid metaphases. No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item. EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal abfmation. In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, Formamidopropyldimethylbetaine did not induce structural

chromosomal aberrations in the V79 Chinese hamster cell line. Therefore,

Formamidopropyldimethylbetaine is considered to be non-clastogenic.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03-Oct-2011 to 08-Nov-2011

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

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

L5178Y mouse lymphoma cells are used because they are sensitive indicators of mutagenic activity of a broad range of chemical classes.
The TK mutational system is able to detect base pair alterations, frame shift mutations, small deletions, and clastogenic effects.
Cells deficient in thymidine kinase (TK) due to the forward mutation (TK+/- to TK-/-) are resistant to the cytotoxic effects of the pyrimidine analogue trifluorothymidine (TFT). TK deficient cells cannot incorporate the analogue into its phosphorylated derivative (nucleotide); the nucleotides needed
for cellular metabolism are obtained solely from de novo synthesis. In the presence of TK, TFT is converted into nucleotides, which are lethal to the cells. Thus, cells, which will survive in culture medium containing TFT, are mutated, either spontaneously or by the action of the test substance, giving rise to a TK deficient phenotype. Furthermore, by applying the TFT-selection procedure it is possible to discriminate between the two different classes of TFT-resistant mutants (small and large colonies). The large colonies are believed to be the result of mutants with single gene mutations (substitutions, deletions of base-pairs) affecting the TK gene. The small colonies are believed to be the result of chromosomal damage to the TK and adjacent genes.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media:

-basic medium: RPMI 1640 Hepes buffered medium containing penicillin (50 U/mL )/streptomycin (50 μg/mL), 1 mM sodium pyruvate and 2 mM L-glutamin
-growth medium (R10): basic medium + 10% (v/v) heat-inactivated (>= 30 min 56°C) horse serum
exposure media:
-for 3 hour exposure (R5):Cells were exposed to the test substance in basic medium supplemented with 5% (v/v) heat-inactivated horse serum;
-for 24 hour exposure (R10):Cells were exposed to the test substance in basic medium supplemented with 10% (v/v) heat-inactivated horse serum
-selective medium: basic medium + 20% (v/v) heat-inactivated horse serum and 5 μg/mL trifluorothymidine (TFT)
-non-selective medium: basic medium + 20% (v/v) heat-inactivated horse serum

- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 microsomal fractions from rat liver

Test concentrations with justification for top dose:

concentrations were corrected for purity

dose range finding test:
without and with 8% (v/v) metabolic activation: 33, 100, 333, 1000, 1882 µg/mL

experiment 1:
without and with 8% (v/v) metabolic activation 1, 3.3, 10, 33, 100, 333, 1000, 1882 µg/mL

experiment 2:
without and with 12% (v/v) metabolic activation 1, 3.3, 10, 33, 100, 333, 1000, 1882 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: RPMI 1640
- Justification for choice of solvent/vehicle: substance did not precipitate in exposure medium

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: with metabolic activation; 7.5 µg/mL

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

other: without metobolic activation; 15 µg/mL (3 hours treatment), 5 µg/mL (24 h treatment)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 3 h, 24 h
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days (cloning efficiency), 12 days (mutant frequency)
- Fixation time (start of exposure up to fixation or harvest of cells): 15 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: solvent controls – duplicates; test substance and positive controls – single/concentration. Two independent
experiments, both in the absence and presence of S9-mix.

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

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

OTHER EXAMINATIONS:
- Other: small (chromosomal damage) and large (single gene mutations) colonies

OTHER:

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria:
a) The absolute cloning efficiency of the solvent controls (CEday2) is between 65 and 120%. An acceptable number of surviving cells (1E06) could be analysed
for expression of the TK mutation.
b) The spontaneous mutation frequency in the solvent control is ≥ 50 per 106 survivors and ≤ 170 per 1E06 survivors.
c) The growth rate (GR) over the 2-day expression period for the negative controls should be between 8 and 32 (3 hours treatment) and between 32-180
(24 hours treatment).
d) The mutation frequency of MMS should not be below 500 per 1E06 survivors, and for CP not below 700 per 1E06 survivors.

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:

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: 7.29 at 1882 µg/mL (=highest concentration tested) compared to 7.28 in the solvent control
- Effects of osmolality: 0.310 Osm/kg at 1882 µg/mL compared to 0.288 Osm/kg in the solvent control
- Precipitation: no precipitation up to 1882 µg/mL

RANGE-FINDING/SCREENING STUDIES:
3 h and 24 h treatment without S9-mix: 33, 100, 333, 1000 and 1882 μg/mL
3 h treatment with 8% (v/v) S9-mix: 33, 100, 333, 1000 and 1882 μg/mL

In the absence and presence of S9-mix, no toxicity in the relative suspension growth was observed up to and including the highest test substance concentration of 1882 μg/mL compared to the suspension growth of the solvent controls.


EXPERIMENT 1
3 h treatment without and with 8% (v/v) S9 mix: 1, 3.3, 10, 33, 100, 333, 1000 and 1882 μg/mL exposure medium

No severe toxicity was observed at all dose levels in the absence and presence of S9-mix.
No significant increase in the mutation frequency at the TK locus was observed after treatment with the test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the test substance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.

EXPERIMENT 2
3 h treatment, 12% (v/v) S9-mix: 1, 3.3, 10, 33, 100, 333, 1000 and 1882 μg/mL exposure medium
24 h treatment, without metabolic activation: 1, 3.3, 10, 33, 100, 333, 1000 and 1882 μg/mL exposure medium
No severe toxicity was observed at all dose levels in the absence and presence of S9-mix.
No significant increase in the mutation frequency at the TK locus was observed after treatment with test substance either in the absence or in the presence of S9-mix. The numbers of small and large colonies in the testsubstance treated cultures were comparable to the numbers of small and large colonies of the solvent controls.


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 except the mutation frequency of two of the solvent control cultures controls: first experiment; in the presence of S9-mix and second experiment, in the absence of S9-mix (both slightly below historical minimum), but experiment is considered valid.

ADDITIONAL INFORMATION ON CYTOTOXICITY.
No toxicity was observed up to and including the highest tested dose level in both experiments in the absence and presence of S9-mix.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Conclusions:

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

Executive summary:

In a mammalian cell gene mutation assay detecting forward mutations at the thymidine-kinase (TK) locus mouse lymphoma L5178Y cells cultured in vitro were exposed to Formamidopropyldimethylbetain (37 %) at concentrations of 1, 3.3, 10, 33, 100, 333, 1000 and 1882 μg/mL in the presence and absence of mammalian metabolic activation (S9-mix). Formamidopropyldimethylbetain was tested up to the limit concentration of 0.01 M (= 1882 µg/mL). The test was performed in 2 independent experiments: 

Experiment 1:

Without and with 8% (v/v) metabolic activation, 3 h treatment

Experiment 2:

Without metabolic activation, 24 h treatment

With 12% (v/v) metabolic activation, 3 h treatment

 

The numbers of small and large colonies in the treated cultures of both experiments were comparable to the numbers of small and large colonies of the solvent controls. Formamidopropyldimethylbetain did not induce a significant increase in the mutation frequency in the presence or absence of mammalian metabolic activation. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix and exposure period.

 

The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background. This study is classified as acceptable.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009-10-21 to 2009-12-14

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)

Version / remarks:

1997

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine Kinase Locus

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Additional strain / cell type characteristics:

other: Clone 3.7.2C

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9

Test concentrations with justification for top dose:

Pre-Experiment: 39.1; 78.1; 156.3; 312.5; 625; 1250; 2500; 5000 µg/mL

Experiment I:
without S9 mix (4 hours treatment): 2.4; 4.9; 9.8; 19.5; 39.0; 58.5; 78.0 µg/mL; evaluated: 2.4; 4.9; 9.8; 39 µg/mL
with S9 mix (4 hours treatment): 4.9; 9.8; 19.5; 39.0; 78.0; 117.0; 156.0 µg/mL; evaluated: 4.9; 9.8; 19.5; 39.0; 78.0 µg/mL

Experiment II:
without metabolic activation (24 hours treatment): 2.5; 5.0; 10.0; 20.0; 40.0; 50.0; 60.0 µg/mL; evaluated: 10; 20; 40; 50; 60 µg/mL
with metabolic activation (4 hours treatment): 10.0; 20.0; 40.0; 80.0; 100.0; 110.0; 120.0 µg/mL; evaluated: 40, 80, 100, 110, 120 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: deionised water (10 %)
- Justification for choice of solvent/vehicle: solubility properties

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

deionised water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Details on test system and experimental conditions:

METHOD OF APPLICATION:

DURATION
- Exposure duration: Experiment I: 4 hours with and without metabolic activation
Experiment II: 24 hours without metabolic activation, 4 hours with metabolic activation
- Expression time (cells in growth medium): 48 hours
- Selection time (if incubation with a selection agent): 10 to 15 days

SELECTION AGENT (mutation assays): RPMI 1640 medium by addition of 5 µg/mL TFT (Trifluorothymidine)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: approx. 4000 cells per well

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

A test item is classified as mutagenic if the induced mutation frequency reproducibly exceeds a threshold of 126 colonies per 1E6 cells above the corresponding solvent control.
A relevant increase of the mutation frequency should be dose-dependent.
A mutagenic response is considered to be reproducible if it occurs in both parallel cultures.
However, in the evaluation of the test results the historical variability of the mutation rates in the solvent controls of this study are taken into consideration.
Results of test groups are generally rejected if the relative total growth is less than 10 % of the vehicle control unless the exception criteria specified by the IWGT recommendations are fulfilled.
Whenever a test item is considered mutagenic according to the above mentioned criteria, the ratio of small versus large colonies is used to differentiate point mutations from clastogenic effects. If the increase of the mutation frequency is accompanied by a reproducible and dose dependent shift in the ratio of small versus large colonies clastogenic effects are indicated.

Statistics:

Linear regression analysis (least squares) using SYSTAT11 (SYSTAT Software, Inc., 501, Canal Boulevard, Suite C, Richmond, CA 94804, USA)

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not effected
- Effects of osmolality: not increased
- Precipitation: No
- Other confounding effects: None

RANGE-FINDING/SCREENING STUDIES:
The highest concentration used in the pre-test was chosen with regard to the solubility of the test item in deionised water. Test item concentrations between 39.1 and 5000 µg/mL were used to evaluate toxicity in the presence (4 h treatment) and absence (4 h and 24 h treatment) of metabolic activation.
Strong toxic effects were observed at 78.1 µg/mL and above in the absence of metabolic activation (4 h treatment) and at 312.5 µg/mL and above in the presence of metabolic activation. Following continuous treatment (24 hours) toxic effects as described above occurred already at the lowest concentration of 39.1 µg/mL and above.
The test medium was checked for precipitation at the end of each treatment period (4 or 24 hours) before the test item was removed. No precipitation occurred up to the maximum concentration with and without metabolic activation at both treatment intervals.
The dose range of the main experiments was limited by toxicity of the test item. The individual concentrations were generally spaced by a factor of 2.0. A closer spacing was used in the upper concentration range to cover toxic effects more closely. A very narrow dose spacing was used in the second experiment with and without metabolic activation to cover the recommended cytotoxic range of approximately 10-20% relative total growth.

COMPARISON WITH HISTORICAL CONTROL DATA:
In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.


ADDITIONAL INFORMATION ON CYTOTOXICITY:
Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.
The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

Summary Table

			relative	mutant		relative	mutant
	conc. µg	S9	total	colonies/		total	colonies/
	per mL	mix	growth	10^6cells	threshold	growth	10^6cells	threshold
Column	1	2	3	4	5	6	7	8
Experiment I / 4 h treatment			culture I			culture II
Solv. control with water		-	100.0	147	273	100.0	141	267
Pos. control with MMS	19.5	-	32.4	424	273	23.7	403	267
Test item	2.4	-	131.7	122	273	67.5	209	267
Test item	4.9	-	122.7	134	273	129.3	139	267
Test item	9.8	-	103.9	137	273	126.0	104	267
Test item	19.5	-	43.7	275	273	96.9	96	267
Test item	39.0	-	31.1	187	273	39.3	181	267
Test item	58.5	-	culture was not continued^#			culture was not continued^#
Test item	78.0	-	culture was not continued^#			culture was not continued^#
Solv. control with water		+	100.0	130	256	100.0	141	267
Pos. control with CPA	3.0	+	61.5	232	256	90.0	250	267
Pos. control with CPA	4.5	+	44.8	320	256	54.6	348	267
Test item	4.9	+	66.2	164	256	137.6	99	267
Test item	9.8	+	104.3	140	256	134.1	146	267
Test item	19.5	+	57.7	202	256	125.6	134	267
Test item	39.0	+	111.4	105	256	147.5	83	267
Test item	78.0	+	43.5	149	256	108.2	154	267
Test item	117.0	+	culture was not continued^#			culture was not continued^#
Test item	156.0	+	culture was not continued^#			culture was not continued^#
Experiment II / 24 h treatment			culture I			culture II
Solv. control with water		-	100.0	158	284	100.0	153	279
Pos. control with MMS	13.0	-	19.2	569	284	25.0	573	279
Test item	2.5	-	culture was not continued^##			culture was not continued^##
Test item	5.0	-	culture was not continued^##			culture was not continued^##
Test item	10.0	-	65.9	217	284	78.7	264	279
Test item	20.0	-	44.3	208	284	71.1	230	279
Test item	40.0	-	33.1	173	284	45.8	208	279
Test item	50.0	-	38.5	182	284	29.8	214	279
Test item	60.0	-	20.7	209	284	16.2	241	279
Experiment II / 4 h treatment			culture I			culture II
Solv. control with water		+	100.0	164	290	100.0	145	271
Pos. control with CPA	3.0	+	52.2	355	290	25.0	312	271
Pos. control with CPA	4.5	+	23.6	385	290	23.3	407	271
Test item	10.0	+	culture was not continued^##			culture was not continued^##
Test item	20.0	+	culture was not continued^##			culture was not continued^##
Test item	40.0	+	75.5	171	290	102.9	189	271
Test item	80.0	+	40.2	210	290	61.6	159	271
Test item	100.0	+	47.1	133	290	49.0	149	271
Test item	110.0	+	32.6	183	290	42.0	167	271
Test item	120.0	+	22.0	203	290	29.0	201	271

Threshold = number of mutant colonies per 10⁶cells of each solvent control plus 126

#      culture was not continued due to exceedingly severe cytotoxic effects
##    culture was not continued since a minimum of only four analysable concentrations is required

Conclusions:

The test item did not induce mutations in the mouse lymphoma thymidine kinase locus assay using the cell line L5178Y in the absence and presence of metabolic activation.

Executive summary:

The study was performed to investigate the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix:                     2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL
with S9 mix:                         4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix:                                10; 20; 40; 50; and 60 µg/mL

with S9 mix:                               40; 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50 % in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % RTG was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5 % in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT^â11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups.

In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 1E6 cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 1E6 cells. The solvent controls remained within the range of the historical data.

Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for Test Guidelines Ninth Addendum to the OECD Guidelines for the Testing of Chemicals, February 1998, adopted July 21, 1997, Guideline No. 476 "In vitro Mammalian Cell Gene Mutation Test“ and Commission Regulation (EC) No. 440/2008 B.17: ”Mutagenicity –In vitro Mammalian Cell Gene Mutation Test“, dated May 30, 2008 for in vitro mutagenicity (mammalian forward gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- in vivo, mouse micronucleus assay (similar to OECD Guideline 474, GLP, RL2), intraperitoneal, 100 mg/kg bw; read-across: C8-18 and C18 unsatd. AAPB

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-01-13 to 1987-02-13

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Mammalian erythrocyte micronucleus test as described by described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975), the later released OECD Guideline 474 (1983) is based to a great extend on this publication.

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

other: OF1 (I. O. P. S. Caw)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: no data
- Weight at study initiation: no data
- Assigned to test groups randomly: animals were not formally randomized
- Fasting period before study: no data
- Housing: 2 (preliminary study) or 5 (main study) in plastic cages, on dust free bedding
- Diet: ad libitum, commercial pelleted diet UAR - A04C-R
- Water: ad libitum from plastic bottles
- Acclimation period: minimum 24 h


ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C +/- 1.5 °C
- Humidity: relative humidity 55 +/- 15%
- Air changes): minimum of 8 air changes per hour
- Photoperiod: 12 h light / 12 h dark cycle

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s)/solvent(s) used: sterile distilled water. The pure compound and all dilutions were kept at 4°C during the test.

Details on exposure:

Preliminary study:
- No. of animals per dose: 2 males/2 females
- Frequency of treatment: two times; 24 h between injections
- Doses: 100, 200, 500, 1000, 2000 mg/kg bw i.p.
- Time of sacrifice: 6 hours after second injection
- Observation time points: 15 min after first administration, then periodically up to 30 hours.
- Type of observations: clinical signs, mortality
- Tolerated doses: 100 - 500 mg/kg bw

Main study
- No. of animals per dose: 5 males/5 females
- Frequency of treatment: two times; 24 h between injections
- Doses: 20, 200 mg/kg bw i.p., injected volume: each 10 ml/kg bw
- Positive control: cyclophosphamide 100 mg/kg bw i.p., injected volume: each 10 ml/kg bw
- Negative control: sterile distilled water, injected volume 10 ml/kg bw
- Time of sacrifice: 6 hours after second injection by cervical dislocation
- Number of samples: two smears of bone marrow/each animal
- EXAMINATIONS: Number of cells with micronuclei / 1000 polychromatic erythrocytes (PCE/NCE ratio: not given)

Duration of treatment / exposure:

30 hours

Frequency of treatment:

Two times, 24 h between the two injections

Post exposure period:

30 h after first injection
6 h after second injection

Dose / conc.:

20 other: mg/kg bw /injection (two injections at an interval of 24 h)

Remarks:

nominal conc.

Dose / conc.:

200 other: mg/kg bw /injection (two injections at an interval of 24 h)

Remarks:

nominal conc.

No. of animals per sex per dose:

Preliminary study: 2 males, 2 females
Main study: 5 males, 5 females

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide, 5 males and 5 females
- Doses / concentrations: 100 mg/kg bw

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
preliminary study (details see "any other information on materials and methods")
In this preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of >/= 100 mg/kg bw/day. At doses >/= 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields): As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment.

DETAILS ON SMEAR PREPARATION: The bone marrow was extracted from the femurs using foetal calf serum centrifuged and put into suspension again. For each animal, two smears were prepared, air-dried and stained with freshly filtered May Grunwald and Giemsa staining.

Statistics:

Student´s test
Exact bilateral comparison test

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

in preliminary dose finding study lethal at doses >/= 1000 mg/kg bw

Vehicle controls validity:

valid

Positive controls validity:

valid

Preliminary study:

Mortality:1000 mg/kg bw: 4/4 animals died within 30 hours after first administration 2000 mg/kg bw: 4/4 animals died within 4 hours after first administration

Clinical signs:
>= 100 mg/kg bw: piloerection
>= 500 mg/kg bw: ptosis
1000 mg/kg bw: loss of righting reflex

Main study:
Mean numbers of micronucleated erythrocytes/1000 polychromatic erythrocytes:
- 20 mg/kg bw: 0.8, Sd dev 0.7 (males), 1.6 Sd dev 1.5 (females)
- 200 mg/kg bw: 1.2 Sd dev 1.0 (males), 1.0 Sd dev 1.3 (females)
- Negative control (vehicle sterile distilled water): 1.4 Sd dev 0.8 (males), 1.8 Sd dev 2.2 (females)
- Positive control (cyclophosphamide): 67.8 Sd dev 11.9 (males), 44.8 Sd dev 7.7 (females)

Conclusions:

No significant increase in the number of micronucleus-bearing erythrocytes was observed following two intraperitoneal administration of Coco AAPB (a.i.: 30 % ). The results for each treated group were comparable with those obtained for the negative control group. The results obtained with cyclophosphamide positive control (100 mg/kg) are significantly positive. Test article Coco AAPB induced no mutagenic effect in the mouse.

Executive summary:

In a OF1 (I.O.P.S. Caw) mouse bone marrow micronucleus assay, performed as described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975), 5 male and 5 female animals were treated i. p. with Coco AAPB (30 % a.i). The test method by W. Schmidt is almost equivalent to the procedure described by OECD guideline 474. In a preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of >/= 100 mg/kg bw/day. At doses >/= 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

The mean number of micronucleated erythrocytes/1000 polychromatic erythrocytes in males and female mice at 20 and 200 mg/kg bw/day were unaffected compared to the negative controls. The administration of 100 mg cyclophosphamide/kg bw serving as the positive control led to clearly elevated numbers of micronucleated erythrocytes.

It can be concluded, that Coco AAPB (30 % a.i) induced no clastogenic effect in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OECD 474 for in vivo cytogenetic mutagenicity data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

For the target substance C8-10 Alkylamidopropyl betaine, an in vitro gene mutation study in bacteria (Ames Test) is available. Further reliable, relevant and adequate data is available from in vitro gene mutation studies in bacteria conducted with the closely related source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine.

In vitro gene mutation tests in mammalian cells (mouse lymphoma L5178Y cells) were conducted with the source substances C8-18 AAPB and Formamidopropylbetaine. An in vitro mammalian chromosome aberration test in Chinese hamster lung fibroblasts (V79) is available with the source substance Formamidopropylbetaine. An in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test) was conducted with the source substance C8-18 and C18 unsatd. AAPB.

All tests were consistently negative. There is no evidence for a genotoxic potential of the target and source substances.

A justification for read-across is given below.

 

In vitro data

Ames tests

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997) and EU method B.13/14 (30 May 2008), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium and Escherichia coli WP2 uvrA were exposed to C8-10 Alkylamidopropyl betaine (36% a.i. in aqueous solution) in deionized water at concentrations of 0 (control), 3, 10, 33, 100, 333, 1000, 2500 and 5000 μg/plate in the first experiment (plate incorporation assay) and 0 (control), 33, 100, 333, 1000, 2500 and 5000 μg/plate in the second experiment (preincubation assay) in the presence and absence of mammalian metabolic activation (rat liver S9 mix).. All concentrations were adjusted to purity.

The plates incubated with the test item showed normal background growth up to 5000 μg/plate with and without S9 mix in all strains used.

Toxic effects, evident as a reduction in the number of revertants (below the indication factor of 0.5), were absent in nearly all strains, only in strain TA 100 toxic effects were observed at 5000μg/plate without S9 mix in experiment I and with and without S9 mix in experiment II.

No substantial increase in revertant colony numbers of any of the five tester strains was observed following treatment with the test item 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.

In experiment I without S9 mix, the data in the solvent control of strain TA 100 were slightly above the laboratory's historical control range. Since this deviation is rather small, this effect is considered to be based upon biologically irrelevant fluctuations in the number of colonies.

Appropriate reference mutagens were used as positive controls. They showed a distinct increase in induced revertant colonies.

In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, C8-10 Alkylamidopropyl betaine did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used.

 

Similar negative results were obtained with the source substances C8-18 and C18 unsatd. AAPB and Formamidopropylbetaine:

In a reverse gene mutation assay in bacteria according to EU Method B.14 (Version Commission Directive 92/69/EEC), strains TA1535, TA 1537, TA 1538, TA 98 and TA 100 of S. typhimurium were exposed to C8-18 and C18 unsatd. AAPB. The test was performed with concentrations up to and including cytotoxic concentrations in the absence and the presence of mammalian metabolic activation

No evidence of biologically significant mutagenic activity of the test item was found in the presence and absence of metabolic activation, up to and including its cytotoxic limit. The positive controls induced the appropriate responses in the corresponding strains and activity of metabolizing system was confirmed.

There was no evidence of induced mutant colonies over background.

 

The potential of Formamidopropylbetaine to induce mutations was investigated in a reverse gene mutation assay in bacteria according to OECD Guideline 471. The assay was performed in two independent experiments. Both took place as a plate incorporation test, using the Salmonella typhimurium strains T A 1535, TA 1537, TA 98, TA 100, TA 102. All experiments were in the absence and in the presence of a metabolic activation by an Aroclor 1254 induced rat liver post mitochondrial fraction (S9).

Experiment 1: 62, 185, 556, 1667, 5000 µg/plate

No mutagenic effects, evident as an elevation of the number of revertant colonies or toxic effects, evident as a reduction of the number of colonies occurred with or without metabolic activation with the test substance concentrations. Due to this, the concentrations of the second test were chosen as follows:

Experiment 2: 1000, 2000, 3000, 4000, 5000 µg/plate

No mutagenic or toxic effects occurred. Negative (solvent) and positive control treatments were included for all strains in both experiments. All mean numbers of revertant colonies on negative control plates fell within acceptable ranges and were significantly elevated by positive control treatments.

Based on the results of this study it is concluded that Formamidopropylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay.

 

Mammalian cell gene mutation assays

In a mammalian cell gene mutation assay according to OECD Guideline 476 (1997) and EU Method B.17 (2008), the potential of C8 -18 AAPB to induce mutations at the mouse lymphoma thymidine kinase locus using the cell line L5178Y was tested.

The assay was performed in two independent experiments, using two parallel cultures each. The first main experiment was performed with and without liver microsomal activation and a treatment period of 4 h. The second experiment was performed with a treatment period of 24 hours in the absence and 4 hours in the presence of metabolic activation.

The main experiments were evaluated at the following concentrations:

Experiment I:

without S9 mix: 2.4; 4.9; 9.8; 19.5; and 39.0 µg/mL

with S9 mix: 4.9; 9.8; 19.5; 39.0; and 78.0 µg/mL

Experiment II:

without S9 mix: 10; 20; 40; 50; and 60 µg/mL

with S9 mix: 40: 80; 100; 110; and 120 µg/mL

Relevant cytotoxic effects indicated by a relative total growth of less than 50% in both parallel cultures were observed in the absence of metabolic activation at 39 µg/mL in experiment I following 4 hour treatment and at 40 µg/mL and above in experiment II following 24 hours treatment. In the presence of metabolic activation toxic effects as described above occurred at 100 µg/mL and above in experiment II. No reproducible cytotoxic effects were noted in the first experiment with metabolic activation. The recommended toxic range of approximately 10-20 % relative total growth was covered in the second experiment with and without metabolic activation.

The isolated minor reduction of the relative total growth to 43.5% in the first culture of experiment I with metabolic activation was not considered a real toxic effect since no comparable reduction was observed in the parallel culture under identical conditions.

No substantial and reproducible dose dependent increase of the mutation frequency was observed with and without metabolic activation. The mutation frequency did not reproducibly reach or exceed the threshold of 126 above the mutation frequency of the corresponding solvent control in any of the experimental parts. An isolated increase exceeding the threshold was noted in the first culture of experiment I without metabolic activation at 19.5 µg/mL. However, this increase was judged as irrelevant fluctuation since it was not reproduced in the parallel culture under identical experimental conditions. Furthermore, the increase was not dose dependent as indicated by the lacking statistical significance. In experiment II the mutant frequency exceeded the range of the historical solvent control data at several test points without metabolic activation (both cultures) and at one test point with metabolic activation (culture I). However, the threshold described above was not reached at any test point of the second experiment and no dose dependent increase was indicated by statistical analysis.

A linear regression analysis (least squares) was performed to assess a possible dose dependent increase of mutant frequencies using SYSTAT11statistics software. No significant dose dependent trend of the mutation frequency indicated by a probability value of <0.05 was determined in all experimental groups. In this study the range of the solvent controls was from 130 up to 164 mutant colonies per 106cells; the range of the groups treated with the test item was from 83 up to 275 mutant colonies per 106cells. The solvent controls remained within the range of the historical data. Methylmethanesulfonate (19.5 µg/mL in experiment I and 13.0 µg/mL in experiment II) and cyclophosphamide (3.0 µg/mL and 4.5 µg/mL in both main experiments) were used as positive controls and showed a distinct increase in induced total mutant colonies at acceptable levels of toxicity with at least one of the concentrations of the controls.

There was no concentration related positive response of induced mutant colonies over background.

 

In a mammalian cell gene mutation assay detecting forward mutations at the thymidine-kinase (TK) locus mouse lymphoma L5178Y cells cultured in vitro were exposed to Formamidopropylbetaine (37 %) at concentrations of 1, 3.3, 10, 33, 100, 333, 1000 and 1882 μg/mL in the presence and absence of mammalian metabolic activation (S9-mix). Formamidopropyldimethylbetain was tested up to the limit concentration of 0.01 M (= 1882 µg/mL). The test was performed in 2 independent experiments: 

Experiment 1:

Without and with 8% (v/v) metabolic activation, 3 h treatment

Experiment 2:

Without metabolic activation, 24 h treatment

With 12% (v/v) metabolic activation, 3 h treatment

The numbers of small and large colonies in the treated cultures of both experiments were comparable to the numbers of small and large colonies of the solvent controls. Formamidopropylbetaine did not induce a significant increase in the mutation frequency in the presence or absence of mammalian metabolic activation. This result was confirmed in an independent repeat experiment with modifications in the concentration of the S9-mix and exposure period.

The positive controls did induce the appropriate response. There was no evidence of induced mutant colonies over background.

 

Chromosome aberration test

Formamidopropylbetaine (38.2% a.i.) was investigated for a possible potential to induce structural chromosomal aberrations in V79 cells of the Chinese hamster in vitro in the absence and presence of metabolic activation with S9 homogenate. The selection of the concentrations used in experiment I and II based on data from the solubility test and the pre-experiment according to the guidelines.

In experiment I with and without metabolic activation 5 µL/mL was selected as highest dose group for the microscopic analysis of chromosomal aberrations. In experiment II with and without metabolic activation 5 µL/mL was selected as highest dose group. The chromosomes were prepared 20 h after start of treatment with the test item. The treatment intervals were 4 h with and without metabolic activation (experiment I) and 4 h with and 20 h without metabolic activation (experiment II). Two parallel cultures were set up. At least 100 metaphases per culture were scored for structural chromosomal aberrations. The following concentrations were evaluated for microscope analysis: Experiment I: with and without metabolic activation: 1, 2.5 and 5 µL/mL Experiment II: without metabolic activation: 1, 2.5 and 5 µL/mL with metabolic activation: 2, 3, 4 and 5 µL/mL.

Precipitation: The test item was diluted in culture medium (MEM medium). No precipitation of the test item was noted in all dose groups evaluated.

Toxicity: In experiments I and II no toxic effects of the item (indicated by a decrease of the rel. mitotic index below 70 %) were noted in all dose groups evaluated with and without metabolic activation.

Clastogenicity: In experiment I without metabolic activation the aberration rate of the negative control (0.5 %) was within the historical control data of the negative control (0.0 % - 4.0 %). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 2.0 % (1 µL/mL) , 1.0 % (2.5 µL/mL) and 1.5 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment I with metabolic activation the number of aberrant cells noted for the negative control (l.5 %) was within the historical control data of the negative control (0.0 % - 4.5 %). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 1 % (1 µL/mL) , 3.5 % (2.5 µL/mL) and 1.5 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control.

In experiment II without metabolic activation the aberration rate of the negative control (0.5 %) was within the historical control data of the negative control (0.0 % - 4.0 %,). The number of aberrant cells found after treatment with the test item was within the historical control data range of the negative control. The mean values noted were 1.5 % (1 µL/mL) , 1 % (2.5 µL/mL) and 3 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control. In experiment II with metabolic activation the aberration rate of the negative control (1.5 %) was within the historical control data of the negative control (0.0 % - 4.5 %). The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control. The mean values noted were 2.5 % (2 µL/mL), 3.5 % (3 µL/mL), 1.8 % (4 µL/mL) and 3 % (5 µL/mL). The number of aberrant cells found in the groups treated with the test item did not show a biologically relevant increase as compared to the corresponding negative control. Polyploid cells show the occurrence of polyploid metaphases. No biologically relevant increase in the frequencies of polyploid cells was found after treatment with the test item. EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases in cells with structural chromosomal abfmation. In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, Formamidopropylbetaine did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, Formamidopropylbetaine is considered to be non-clastogenic.

 

In vivo data

The cytogenetic activity of the AAPBs was tested in a OF1 (I. O. P. S. Caw) mouse bone marrow micronucleus assay, performed as described by W. Schmid - The Micronucleus test, Mutation Research, 31, 9-15 (1975). 5 male and 5 female animals were treated i. p. with C8-18 and C18 unsatd. AAPB (30 % a. i). The test method by W. Schmidt is almost equivalent to the procedure described by OECD guideline 474. In a preliminary study the test animals were administered twice (in a 24 hours interval) each 100, 200, 500, 1000 and 2000 mg/kg bw/day by intraperitoneal injection. Clinical signs and mortality were observed up to 30 hours after the first administration. Clinical signs like piloerection and ptosis were seen at doses of ≥ 100 mg/kg bw/day. At doses ≥ 1000 mg/kg bw/day the mice died within 30 and 4 hours after the first administration. The tolerated doses were in the range of 100 to 500 mg/kg bw/day. Therefore, the dose of 200 mg/kg bw/day was selected as the high dose and 20 mg/kg bw/day (10 % of the high dose) as the low dose. As the test substance was applied twice with a 24 h interval (although only one timepoint was chosen for sacrifice), the result of the sacrifice 6h later may be regarded as a result of a 30h and a 6h treatment. The dose level of 200 mg/kg bw/day (corresponding to 60 mg active substance/kg bw/day) is considered to be sufficiently high based on the effects found in the preliminary study and due to the highly irritating properties of the compound.

The mean number of micronucleated erythrocytes/1000 polychromatic erythrocytes in males and female mice at 20 and 200 mg/kg bw/day were unaffected compared to the negative controls. The administration of 100 mg cyclophosphamide/kg bw serving as the positive control led to clearly elevated numbers of micronucleated erythrocytes.

It can be concluded, that C8-18 and C18 unsatd. AAPB (30 % a. i) induced no clastogenic effect in this in vivo cytogenicity study on mice at dose levels of 20 and 200 mg/kg bw/day.

Conclusion

Reliable, relevant and adequate data on the genetic toxicity of the target substance and the source closely related source substances is available from in vitro gene mutation studies in bacteria (Ames Tests), in vitro gene mutation studies in mammalian cells (L5178Y/ TK Mouse Lymphoma assay), an in vitro chromosome aberration assay and one in vivo cytogenicity study (in vivo Mammalian Erythrocyte Micronucleus Test).

As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the substances.

In conclusion there is no evidence for genotoxic properties for the target substance C8-10 Alkylamidopropyl betaine.

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. The target and source substances contain the same functional groups. Thus a common mode of action can be assumed.

The only deviation within this group of substances is a variety in their carbon chain length, which obviously does not have a relevant impact on genetic toxicity as demonstrated by the available data on the target and source substances.

 

a. Structural similarity and functional groups

The target substance C8-10 Alkylamidopropyl betaine is a UVCB substance manufactured from fatty acids (C8 and C10) and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

The source substances C8-18 AAPB and C8-18 and C18 unsatd. AAPB are UVCB substances manufactured from natural fatty acids or oils with N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate. As their origin is from natural sources, the used fatty acids may have a mixed slightly varying composition with an even numbered chain length from C8 to C18. Unsaturated C18 amounts may be included.

The source substance Formamidopropylbetaine is a monoconstituent substance manufactured from formic acid and N, N-dimethylpropylenediamine (DMAPA) and further reacted with sodium monochloroacetate.

 

b. Differences

Differences in chemical and other intrinsic properties of the target and source substances could potentially arise from the following facts:

-Different amounts of different carbon chain lengths (carbon chain length distribution):

Higher amounts of higher chain lengths and corresponding lower amounts of lower chain length lead to a rising average lipophilicity as can be seen from the increasing log Kow from Formamidopropylbetaine (log Kow: -3.3), C8-10 Alkylamidopropyl betaine (log Kow: 2.2), C12 AAPB (log Kow: 3.54), C8-18 AAPB (log Kow: 4.23).

However, based on the available toxicological data it is demonstrated, that this read-across approach is nevertheless reliable.

 

- Different amounts of unsaturated fatty ester moieties:

The source substance C8-18 and C18 unsatd. AAPB contains considerable amounts of unsaturated C18 chains, which represents a worst case with respect to some toxicological endpoints, mainly local effects (e.g. irritation, sensitisation) and reactivity. But in general, variability in the fatty acid moiety is not expected to be relevant to the intrinsic genetic toxicity of the substances.  

The provided structural similarities and impurity profiles support the proposed read-across hypothesis with high confidence.

 

Comparison of genotoxicity data

 

Endpoints	Target substance	Source substances
	C8-10 Alkylamidopropyl betaine	C8-18 and C18 unsatd. AAPB	C8-18 AAPB	Formamidopropylbetaine
Genotoxicity   in vitro Gene mutation in bacteria	key_Genetic toxicity in vitro: 73772-45-9 / 73772-46-0_8.4.1_Evonik_2014_OECD471   OECD TG 471, S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2 Metabolic activation: with and without cytotoxicity: only in strain TA 100 at 5000 μg/plate; vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 1 (reliable without restrictions), GLP	Sup_RA_Genetic toxicity in vitro: 147170-44-3_8.4.1_Zschimmer_1996_EEC 92_69 EU Method B.13/14 S. typhimurium TA 1535, TA 100, TA 1537, TA 1538 and TA 98 Metabolic activation: with and without cytotoxicity: first evidence of toxicity at 10000 µg/plate with and without S9; vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 2 (reliable with restrictions), no GLP	No data	sup_RA_Genetic toxicity in vitro: 120128-90-7_8.4.1_Evonik_2005_OECD471   OECD TG 471, gene mutation: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102 Metabolic activation: with and without cytotoxicity: no; vehicle controls valid: yes; negative controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 1 (reliable without restrictions), GLP
Gene mutation in mammalian cells	No data, read-across	No data	Key_RA_Genetic toxicity in vitro: 97862-59-4_8.4.3_MoLy_Evonik_2010_OECD-476 OECD TG 476, mouse lymphoma L5178Y cells Metabolic activation: with and without cytotoxicity: yes; vehicle controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 1 (reliable without restriction), GLP	key_RA_Genetic toxicity in vitro: 120128-90-7_8.4.3_Evonik_2012_OECD476   OECD TG 476, mouse lymphoma L5178Y cells Metabolic activation: with and without cytotoxicity: no; vehicle controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 1 (reliable without restriction), GLP
Chromosome aberration	No data, read-across	No data	No data	key_RA_Genetic toxicity in vitro: 120128-90-7_8.4.2_Evonik_2005_OECD473   OECD TG 473, Chinese hamster lung fibroblasts (V79) Metabolic activation: with and without cytotoxicity: no; vehicle controls valid: yes; positive controls valid: yes   Genotoxicity: negative   Reliability: 1 (reliable without restriction), GLP
Genotoxicity in vivo	No data, read-across	Key_Genetic toxicity in vivo: 61789-40-0_8.4.4_Goldschmidt_France_1987_micronucleus test in vivo Study result type: experimental result similar to OECD TG 474,mouse, intraperitoneal,100 mg/kg bw   toxicity: yes (in preliminary dose finding study lethal at doses >/= 1000 mg/kg bw); vehicle controls valid: yes; positive controls valid: yes Genotoxicity negative   Reliability: 2 (reliable with restrictions), GLP	No data	No data

 

The target substance C8-10 Alkylamidopropyl betaine  was not mutagenic in the Ames test. Cytotoxicity was only observed in strain TA 100 at the limit concentration of 5000 μg/plate. Also the source substances Formamidopropylbetaine and C8-18 and C18 unsatd. AAPB were not mutagenic in several reverse gene mutation assays in bacteria and showed little or no signs of cytotoxicity.

Based on comparable results in the Ames test as well as on structural similarities, it is concluded that the outcome of themutation assay in mammalian cells as well as the chromosome aberration assays conducted with the source substances Formamidopropylbetaine, C8-18 AAPB and C8-18 and C18 unsatd. AAPB is also relevant for the target substance C8-10 Alkylamidopropyl betaine.

 

In a mammalian cell gene mutation assay according to OECD Guideline 476 the source substances Formamidopropylbetaine and C8-18 AAPB induced no concentration related positive response of induced mutant colonies over background when tested up limit or cytotoxic concentrations.

The source substance Formamidopropylbetaine did not induce chromosome aberrations in a chromosome aberration assay according to OECD Guideline 473 when tested up to limit concentrations.

The source substance C8-18 and C18 unsatd. AAPB was not clastogenic in a mouse bone marrow micronucleus assay.

 

Quality of the experimental data of the analogues:

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

The key studies were conducted according to EU Method B.13/14/OECD TG 471, OECD TG 476, OECD TG 473 or similar to OECD TG 474 and were reliable (RL1) or reliable with restrictions (RL2).

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

As fatty acids independently from their chain length and degree on unsaturation are generally considered to be not genotoxic, a variability in the fatty acid moiety is not expected to have any influence on the genotoxic activity of the substances.

Based on the comparable negative outcome of the Ames test for the target and source substances as well as on structural similarities of the target and the source substances as presented above and in more detail in the general justification for read across, it can be concluded that the available data on mammalian cell gene mutation and clastogenicity from the source substances Formamidopropylbetaine, C8-18 AAPB and C8-18 and C18 unsatd. AAPB are also valid for the target substance C8-10 Alkylamidopropyl betaine.

There was no evidence of mutagenic or clastogenic intrinsic properties in any of the performed studies

Justification for classification or non-classification

Based on the available data, C8-10 Alkylamidopropyl betaine  does not need to be classified for mutagenicity according to regulation (EC) 1272/2008. Thus, no labelling is required.