Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In conclusion the full set of genotoxicity tests required by the REACH regulation is negative.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01. Dec. 2004 - 23. Dec. 2004
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
Metabolic activation:
with and without
Metabolic activation system:
Mammalian liver post-mitochondrial fraction (S9)
Test concentrations with justification for top dose:
Experiment 1: 62, 185, 556, 1667, 5000 µg/plate
Experiment 2: 1000, 2000, 3000, 4000, 5000 µg/plate
Vehicle / solvent:
demineralized water
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: without S9: Sodium azide, 9-Aminoacridine, 2-Nitrofluorene, Mitomycin C (MMC); with S9: 2-Aminoanthracene, Benzo[a]pyrene
Details on test system and experimental conditions:
Bacteria : Salmonella typhimurium
Strains : TA 1535, TA 1537, TA 98, TA 100, TA102
Date of receipt : 21 st September 1995, 3rd March 1998, 9th August 2002
Supplier : Fa. Boehringer Ingelheim Pharma GmbH & Co. KG, former Dr. Karl Thomae GmbH, Dept. of Exp. Pathology and Toxicology, 88397 Biberach an der Riß, Germany. Bruce Ames Laboratory, Department of Molecular and Cell Biology, 401 Barker Hall, Berkeley, California 94720-0001.
Storage : The Salmonella strain cultures are stored as stock cultures in ampoules with nutrient broth + 9% DMSO (Merck, 1.02950.0500) in liquid nitrogen.
Metabolic Activation : Mammalian liver post-mitochondrial fraction (S9)
Species : Rat
Enzyme-inducing agent : Aroclor 1254
Supplier : Molecular Toxicology, INC., Annapolis, USA

Bacteria were grown overnight for 10 hours in nutrient broth at 37°C / 110 rpm. For inoculation stock cultures, stored at -196°C (Salmonella typhimurium strains) were used. They had been checked for strain characteristics of rfa-character, UVrB-deletion, resistance to ampicillin and tetracycline. Checks were carried out according to Maron and Ames (3). All treatments were performed at the end of the incubation period.

The test was performed by direct plate incorporation method. The mutagenicity experiments with his- Salmonella typhimurium were performed with
minor modifications of the method described by Ames et al. (3). Modifications were as follows:
2 ml Top agar, 100 µL test substance, 100 µL bacterial suspension and 500 µL S9-Mix (or 500 µL phosphate buffer) were mixed in a test tube and poured on the surface of the Minimal-Glucose-Agar plates. For each strain and dose level three plates were used.
Each experiment contained positive controls to check the activity of the metabolizing system and the mutagenicity of the bacteria in triplicate as well as sixfold negative controls to check the spontaneous reversion rate of each used bacteria strain. After solidification the plates were incubated upside down for at least 48 hours at 3r C in the dark.
Evaluation criteria:
A test substance producing no biologically relevant positive response at anyone of the test points is considered to be non-mutagenic in this system. A biologically relevant response is described as follows: If the number of revertants is at least twice the spontaneous reversion rate in one of the
strains and if there is a concentration related, increasing number of revertants over the range tested.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102
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 [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with 5 different strains of Salmonella typhimurium

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

Without S9

Positive control

548±42

> 1000

313±13

633±119

> 1000

Negative control

20±2

20±2

19±2

61±3

264±38

62

25±8

29±4

24±5

54±3

230±10

185

18±´4

28±5

24±8

52±5

248±27

556

26±3

22±3

19±4

56±7

296±58

1667

26±7

26±9

26±6

61±2

291±97

5000

21±2

27±7

17±4

45±6

266±22

 

With S9 *

Positive control

369±86

287±34

840±163

> 1000

> 1000

Negative control

23±6

32±3

27±4

72±12

338±48

62

23±7

29±4

24±2

81±10

432±72

185

21±3

35±10

33±6

76±7

370±61

556

23±3

33±5

40±4

85±6

351±43

1667

26±1

32±3

33±2

82±4

360±56

5000

23±2

24±3

24±4

82±6

312±51

T=Toxicity

P=Precipitation

C=Contamination

M=Mean

SD=Standard deviation

 


 

Experiment 2:  

 

Dose [µg/plate]

Mean number of revertant colonies/3 replicates (±S.D.) with 5 different strains of Salmonella typhimurium

 

TA 1535

TA 1537

TA 98

TA 100

TA 102

 

Without S9

Positive control

430±62

> 1000

328±2

883±62

> 1000

Solvent control

18±4

31±4

24±4

53±6

286±29

1000

16±2

26±1

22±3

62±5

285±37

2000

14±4

28±4

22±2

55±10

267±63

3000

19±4

28±3

24±2

50±13

345±14

4000

17±4

32±9

22±4

59±3

253±13

5000

20±5

26±4

22±0

50±6

329±64

 

With S9 *

Positive control

237±30

632±87

784±142

> 1000

> 1000

Solvent control

15±2

31±4

28±10

72±6

315±49

1000

18±5

32±5

22±8

54±8

391±26

2000

17±4

35±3

28±3

64±4

387±32

3000

21±6

26±7

22±2

71±9

381±53

4000

17±3

23±2

27±5

61±6

371±65

5000

19±4

30±3

30±8

65±9

323±54

T=Toxicity

P=Precipitation

C=Contamination

M=Mean

SD=Standard deviation

Conclusions:
Interpretation of results (migrated information):
negative

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

The purpose of the study was to investigate the potential of the test substance Formamidopropyldimethylbetaine to induce mutations using the bacterial reverse mutation assay.

The study was conducted in accordance with the OECD guidelines for testing of chemicals No. 471 "Bacterial reverse mutation test" (1997) and with the procedure described in the Commission Directive 2000/32/EC (2000).

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). The test substance and the positive controls were triple tested, the negative controls sixfold. Demineralized water (A. dem.) was used as vehicle for all dilutions of the test substance.

Five concentrations were tested in each experiment, using 0.1 ml for each plate. The test substance was tested in the following concentrations:

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 Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
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:
other: 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:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 1640 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
Migrated to IUCLID6: with metabolic activation; 7.5 µg/mL
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
Migrated to IUCLID6: 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 (106) 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 106 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 106 survivors, and for CP not below 700 per 106 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:
Interpretation of results (migrated information):
negative

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.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
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:
other: GLP 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:
other: without S9: EMS - Ethylmethanesulfonate; with S9: CPA - Cyclophosphamide
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 1 x10 4 - 5 x 10 4 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 OEeD 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:
Interpretation of results (migrated information):
negative

In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item UB 2740 50 % (Formamidopropyldimethylbetaine) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item UB 2740 50% (Formamidopropyldimethylbetaine) is considered to be non-clastogenic.
Executive summary:
The test item UB 2740 50% (Formamidopropyldimethylbetaine) 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, the test item UB 2740 50% (Formamidopropyldimethylbetaine)

did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item UB 2740 50%

(Formamidopropyldimethylbetaine) is considered to be non-clastogenic.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

In vitro - Ames test:

The purpose of the study was to investigate the potential of the test substance Formamidopropyldimethylbetaine to induce mutations using the bacterial reverse mutation assay.

The study was conducted in accordance with the OECD guidelines for testing of chemicals No. 471 "Bacterial reverse mutation test" (1997) and with the procedure described in the Commission Directive 2000/32/EC (2000).

The assay was performed in two independent experiments. Both took place as a plate incorporation test, using the Salmonella typhimurium strains TA 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). The test substance and the positive controls were triple tested, the negative controls sixfold. Demineralized water (A. dem.) was used as vehicle for all dilutions of the test substance.

Five concentrations were tested in each experiment, using 0.1 mL for each plate. The test substance was tested in the following concentrations:

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 Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay.

In vitro – mammalian cell gene mutation assay

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 Formamidopropyldimethylbetaine (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). The substance 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. Formamidopropyldimethylbetaine 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.

 

In conclusion there was no evidence of induced mutant colonies over background.

 

In vitro - Chromosome aberration test:

The test item UB 2740 50% (Formamidopropyldimethylbetaine) 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 aberration. In conclusion, it can be stated that during the described in vitro chromosomal aberration test and under the experimental conditions reported, the test item UB 2740 50% (Formamidopropyldimethylbetaine) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item UB 2740 50%(Formamidopropyldimethylbetaine) is considered to be non-clastogenic.

 


Justification for selection of genetic toxicity endpoint
Data from three GLP compliant guideline studies with reliability 1. The full set of genotoxicity tests required by the REACH regulation is negative.

Short description of key information:
Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay and the mammalian cell gene mutation assay using mouse lymphoma L5178Y cells. In an in vitro chromosomal aberration test, the test item UB 2740 50% (Formamidopropyldimethylbetaine) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, Formamidopropyldimethylbetaine is considered to be non-clastogenic.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Formamidopropyldimethylbetaine is not mutagenic in the Salmonella typhimurium reverse mutation assay and the mammalian cell gene mutation assay using mouse lymphoma L5178Y cells. In an in vitro chromosomal aberration test, the test item UB 2740 50% (Formamidopropyldimethylbetaine) did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, Formamidopropyldimethylbetaine is considered to be non-clastogenic.

In conclusion the full set of genotoxicity tests required by the REACH regulation is negative. According to Directive 67/548/EEC as well as GHS Regulation EC No 1272/2008 no classification and labelling for mutagenic toxicity is necessary.