Potassium 3-sulphopropyl methacrylate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Genetic toxicity:

- in vitro: Gene mutation (Bacterial reverse mutation test / Ames test), S. typhimurium TA1535, TA 1537, TA 98, TA100, up to 5000 µg/plate with and without metabolic activation (OECD 471, GLP): negative ± S9

- in vitro: Gene mutation (Bacterial reverse mutation test / Ames test), S. typhimurium TA1535, TA 1537, TA 98, TA100, TA 1538, up to 5000 µg/plate with and without metabolic activation (OECD 471, GLP): negative ± S9

- in vitro: chromosome aberration, V79 cells, up to 5000 µg/plate with and without metabolic activation (OECD 473, GLP): negative ± S9

- in vitro: Gene mutation in mammalian cells (HPRT assay), V79 cells, up to 5000 µg/plate with and without metabolic activation (OECD 476, GLP): negative ± S9

Link to relevant study records

Reference

Reference 1

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:

25 January - 31 January 1983

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

other: OECD Guideline Draft No. 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(strains: Salmonella typhimurium TA1535, TA 1537, TA 98, TA100 and TA 1538 were used)

Qualifier:

according to guideline

Guideline:

other: EU Method B.14 (Draft) , (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

yes

Remarks:

(strains: Salmonella typhimurium TA1535, TA 1537, TA 98, TA100 and TA 1538 were used)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his-

Species / strain / cell type:

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

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Liver microsomal fraction (S9 mix), prepared from the livers of male Wistar rats treated with Aroclor 1254

Test concentrations with justification for top dose:

1.58, 5, 15.8, 50, 158, 500, 1580 and 5000 µg/plate with and without metabolic activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: distilled water
- On the day of the experiment, the test material was dissolved in distilled water at the highest investigated dose. The other doses were dilutions from this stock solution with the solvent in half-log intervals.

Untreated negative controls:

other: solvent control (distilled water)

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

methylmethanesulfonate

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Preincubation period: The bacteria were grown overnight in a shaking water bath for 16 hours at 37°C in 2.5% Nutrient Broth No. 2. After centrifugation, the bacteria were resuspended to a concentration of approximately 1 x 10E8 to 2 x 10E9 cells per millilitre in 0.16% Nutrient Broth and 0.5% Sodium chloride. The concentration of germs was controlled photometrically and determined in an experimental test with Histidine-rich potassium chloride solution on selective agar plates.

DETERMINATION OF CYTOTOXICITY
- To estimate the toxicity of the test material prototrophic bacteria (spontaneous revertants of TA 1537 = RTA) were added as an internal standard of to the selective agar plates together with TA 1537. The difference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test material was compared to the number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100 % survival rate.
Additionally, the toxicity of the test material can be evidenced in the mutagenicity assay, by a reduction in the number of revertants or by a clearing of the background lawn.

Evaluation criteria:

A material is identified as a mutagen in this test system if there is a reproducible demonstration of a dose effect relation with a 2-fold increase in the number of revertants over the controls in at least one strain . With the strain TA 100 a 1.5-fold increase is the criterion for a positive result .

Statistics:

The difference in the number of colonies on plates with and without added prototrophic bacteria at each dose level of the test material was compared to the number of colonies obtained with the negative control. The ratio of the two values was expressed as relative survival rate with the negative control at a 100 % survival rate.
Additionally, the toxicity of the test material can be evidenced in the mutagenicity assay, by a reduction in the number of revertants or by a clearing of the background lawn.

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(No relevant increase of the revertant colony numbers was obtained in Salmonella typhimurium strain used at all dose levels tested in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings.)

Cytotoxicity / choice of top concentrations:

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

Remarks:

(In the toxicity experiment with 3-Sulfopropyl-methacrylsauereester (SPM), neither quantitative nor qualitative evidence of a toxic effect was observed.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

(No relevant increase of the revertant colony numbers was obtained in Salmonella typhimurium strain used at all dose levels tested in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings.)

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Remarks:

(In the toxicity experiment with 3-Sulfopropyl-methacrylsauereester (SPM), neither quantitative nor qualitative evidence of a toxic effect was observed.)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the current study, the following results have been compiled:

GERM CONCENTRATIONS :
The concentrations of Salmonella typhimurium used in this assay were as follows:
TA 98 = 9.8 x 10 exp. 8 cells/ml
TA 100 = 5.7 x 10 exp. 8 cells/ml
TA 1535 = 1.5 x 10 exp. 9 cells/ml
TA 1537 = 9.9 x 10 exp. 8 cells/ml
TA 1530 = 1.3 x 10 exp. 9 cells/ml
in the experiment without metabolic activation,

TA 98 = 1.5 x 10 exp. 9 cells/ml
TA 100 = 7.4 x 10 exp. 8 cells/ml
TA 1535 = 1.3 x 10 exp. 9 cells/ml
TA 1537 = 1.5 x 10 exp. 9 cells/ml
TA 1538 = 1.3 x 10 exp. 9 cells/ml
in the experiment with metabolic activation.

Control plates with the used solvent (negative control) showed numbers of spontaneous revertant colonies per plate which were within the normal range of those cited in the literature .
Control plates with reference mutagens (positive controls) showed a distinct increase of the revertant colonies with the tester strains. This confirmes the reversion properties of each strain. The positive results of the mutagens 2-Amino-anthracene and Benzo(a)pyrene indicate that the metabolizing system was functioning.
The aseptic control showed no contamination for the S-9 mix.

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The study was performed according to the OECD Guideline 471 with deviations (strain TA 1538 instead of TA 102) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not induce significant increases in the frequency of revertant colonies in any of the bacterial strains. The test material was considered to be non-mutagenic under the conditions of the test.

Executive summary:

The test substance 3-Sulfopropyl-methacrylsauereester (SPM) was investigated according to OECD TG471 for its potential to cause gene mutations in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538). Under the conditions of this experiment 3-Sulfopropyl-methacrylsauereester (SPM) was found to cause no toxic effects. Up to the highest investigated dose, no relevant increase of the revertant colony numbers was obtained in any Salmonella typhimurium strain in comparison with the corresponding controls. The presence of microsomal activation did not influence these findings. In conclusion, it can be stated that during the described mutagenicity test and under the experimental conditions reported, the test material caused neither base-pair substitutions, nor frameshift mutations. Therefore the test results with 3-Sulfopropyl-methacrylsauereester (SPM) revealed no indication of gene mutagenic activity.

Reference 2

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:

07 May - 14 July 1992

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP - guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

(strains: no E.coli WP2 strains used)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

his operon (Salmonella strains)

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

Description of tester strains
To demonstrate point mutagenic effects the histidine auxotrophic mutants of Salmonella typhimurium LT2 were used. These strains were selected specifically for this test. To identify the two basic classes of point mutations, i.e. base pair substitutions and frameshift mutations, several strains were used which cover both types. Salmonella typhimurium TA 1535 and TA 1537 were selected by Ames et al. (1973b), whereas the Salmonella typhimurium TA 100 and TA 98 were developed by McCann et al. (1975).
Genotypes of bacterial strains widely used in reverse mutation assays
Amino acid marker Other relevant mutations Plasmid
Strain Histidine mutation Type of mutation Main DNAtarget Cell wall DNA-repair

TA1535 hisG46 base pair substitution GC rfa uvrB- no
TA 100 hisG46 base pair substitution GC rfa uvrB- pKM101
TA 1537 hisC3076 frameshift GC rfa uvrB- no
TA 98 hisD3052 frameshift GC rfa uvrB- pKM101

All strains contain GC base pairs at the site of the histidine mutation and are therefore selective for agents which react predominantly with these bases. The permeability of the cell wall for large molecules increases in the presence of the rfa mutation. The uvrB- strains are defective in excision repair, making them more sensitive both to the mutagenic and lethal effects of a wide variety of mutagens, since these strains cannot excise DNA adducts. The plasmid pKM101 carries a gene (muc+) which participates in "SOS" DNA-repair, a repair pathway which is induced by DNA damage and which confers an increased resistance to the lethal effects of many mutagens, at the expense of increased mutability. Bacteria carrying pKM101 are therefore more mutable than 0(1%4101- strains and have a higher spontaneous mutation rate. This plasmid also carries an ampicillin resistance gene, a useful marker which allows pKM101+ strains to grow on ampicillin plates.
Origin of strains:
The strains were obtained from Dr. W. Göggelmann, GSF München/Neuherberg and received at IBR Hannover on August 24,1989.
Production of stock cultures:
Permanent master cultures of the tester strains were stored in liquid nitrogen. In addition, master cultures on nutrient agar plates were stored at 4° C. Ampicillin was added to plates used for the R-factor strains, TA 98 and TA 100. All tester strains were routinely checked for crystal violet sensitivity (deep rough character), UV sensitivity (uvrB) and ampicillin resistance (pKM101). For each test and strain, frozen permanents were transferred to 30 ml of nutrient broth. The samples were incubated overnight in a shaking waterbath at 37°C for 16 hours.

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented fraction (S9 mix), prepared from the livers of Wistar rats treated with Aroclor 1254

Test concentrations with justification for top dose:

Preliminary range-finding test : 10, 32, 100, 320, 1000, 3200 and 10000 µg/plate (with TA 100)
Main tests : 8, 40, 200, 1000 and 5000 μg/plate (Main test 1 : 4% S9 ; Main test 2 : 10% S9) with and without metabolic activation

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

(solvent without test article)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-nitro-1,2-phenylene-diamine, 2-aminoanthracene (see 'Any other information on results incl. tables' for details).

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

The test was performed according to Maron and Ames (1983) and to OECD guideline 471 (May 26, 1983). In order to select the doses for the main test, a preliminary range finding was conducted with seven concentrations of the test article (10, 32, 100, 320, 1000, 3200 and 10000µg/plate). In each case, 0.1 ml of test article solution, 0.1 ml of TA 100 bacterial suspension (overnight culture, about 10E9 cells/ml), 0.5 ml of buffer and 2.0 ml of top agar were mixed and emptied onto petri dishes. In a parallel experiment, 0.1 ml of a bacterial suspension diluted to 10E-6 and 0.1 ml of test article solution were plated onto nutrient broth agar plates. The number of colonies, the number of revertants and the background growth were determined as a measure of the toxicity of SPM.
The following concentrations were therefore chosen for the main tests: 8, 40, 200, 1000 and 5000 µg/plate. The SPM sample was dissolved in H2O.
The main test was performed as follows:
0.1 ml of each test article solution, 0.1 ml of bacterial suspension, 0.5 ml of S 9 mix or buffer and 2.0 ml of top agar were mixed and poured onto petri dishes containing solid agar. The petri dishes were incubated at 37°C for 48 hours and the revertant colonies were counted. Three plates per strain and concentration, both with and without S 9 mix, were used for the mutant count. For the negative (solvent without test article ) and the positive control three plates each were used. The bacterial suspensions used were obtained from 16-hour cultures in nutrient broth which had been incubated at 37°C and shaken at 90 rpm. These suspensions were used for the determination of mutant counts. The number of viable cells was established concomitantly when determining the titer. After incubation for 48 hours at 37°C, the colonies were counted by an ARTEK COUNTER MODEL 982B. Automatic counts were validated by manual counting at regular intervals.
The following criteria were used for the acceptance of an assay:
-The negative controls had to be within the expected range as defined by published data (Maron and Ames 1983).
- The positive controls had to show sufficient effects as defined by the laboratory's experience.
-The titer determination had to reveal a sufficient bacterial density in the suspension
The test was repeated in an independent experiment.

Evaluation criteria:

A reproducible and dose-related increase of mutant counts for at least one strain is considered positive . For TA 98 and TA 1535 a twofold increase of revertants compared to the negative controls should be reached , whereas for TA 1537 a threefold increase should be attained . For TA 100 a 1,5-fold titer increase is regarded as an indication of potential mutagenicity . Otherwise the results are considered to be negative .
The criterion for a biologically significant bacteriotoxic effect is a reduction in the number of colonies/plate or revertants/plate or in background growth by more than 50% relative to the respective negative control .

Statistics:

Individual values (number of revertants per plate) , mean values and standard deviations , and revertant quotients are presented for each strain and concentration . The quotients were calculated by dividing the mean value for the number of revertants in each test group with the mean number of revertants in the solvent control group . Calculations were performed with BIOSYS SOFTWARE AMES-TEST III .

Key result

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Preliminary Test :
No mutagenic and bacteriotoxic effects were observed at any concentration . The total number of viable cells , the number of revertants and the background growth were in the normal range .

Main Test 1 (4% S9) :
No mutagenic effect could be observed. Compared to negative controls (solvent), neither a biologically relevant nor a dose-related increase of reversion rates was found. This observation was irrespective of the presence of S9 mix .

Main Test 2 (10% S9) :
Also with this changed test conditions the results with regard to lack of mutagenicity and bacteriotoxicity were confirmed in the second main test .

Remarks on result:

other: all strains/cell types tested

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The study was performed according to the OECD Guideline 471 with deviations (no E.coli WP2 strains was tested) according to the principles of the good laboratory practice and therefore considered to be of high quality (reliability Klimisch 2). The vehicle and the positive control substances fulfilled validity criteria of the test system. In conclusion , the results indicate that SPM caused no mutagenic effects at concentrations ranging from 8 to 5000 µg/plate in the four tester strains TA 1535, TA 1537, TA 98 and TA 100. .

Executive summary:

The test substance Methacrylsäure-(3-sulfopropyl)-ester, K-salt (SPM) was investigated according to OECD TG471 in the Salmonella/microsome test for point mutations using four Salmonella typhimurium LT2 mutants. These tester strains were the histidine auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100. Concentrations ranging from 10 to 10000 µg per plate were employed in the preliminary toxicity test. The solvent was H2O.

No bacteriotoxic effects were caused at concentrations ranging from 10 to 10000 µg/plate. The total number of viable cells, the number of revertants and the background growth were within the normal range. On the basis of this result, the main tests employed the following concentrations: 8, 40, 200, 1 000 and 5 000 µg/plate. No evidence of mutagenic activity of SPM was found. Neither with nor without S 9 mix was a biologically relevant or dose-related increase in reversion rate observed in comparison with the negative control (solvent). The sensitivity of the test system was demonstrated by the marked mutagenic effects exerted by the positive controls 9-aminoacridine hydrochloride, sodium azide, 4-nitro-1,2-phenylene diamine and 2-aminoanthracene.

In summary, it may be concluded that the test article SPM caused no mutagenic effect at concentrations ranging from 8 to 5000 µg/plate.

Reference 3

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:

29 May - 23 August 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 473 and under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes

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):

- Type and identity of media:
Medium A(for cultivation) :
*Minimum Essential Medium (MEM), Gibco
*10% Fetal Calf Serum (FCS), Biochrom
*2 mM L-Glutamin, Biochrom
*100 U/ml Penicillin, 100 µg/ml Streptomycin, Biochrom
*1% Non Essential Amino Acid, Gibco
Medium B (for treatment of test article and reference substances) :
-Medium A without FCS

- Properly maintained: yes ( Incubation of the cells was always performed at 37°C in a CO2-incubator (5% CO2)).

- Periodically checked for karyotype stability: yes, the karyotype of each analysed metaphase was determined

- Periodically checked for Mycoplasma contamination: yes, free of mycoplasma contamination (last periodic check on 20.05.1992 by ELISA)

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mix (obtained from Cytotest Cell Research GmbH & Co. KG. Lot No. 060792; Protein content : 31.6 mg/ml; 3 mg Glu-6-phosphate / ml S-9 Mix (10 mM); 4 mg NADP / ml S-9 Mix (5 mM). The S-9 Mix is added to yield a final concentration of 4%.

Test concentrations with justification for top dose:

Range-finding-test: 19.5 , 39 , 78 , 156 , 312 , 625 , 1250 , 2500 and 5000 µg/ml (with and without activation)

Main test:
- First test : 0.1 , 0.5 , 1.0 , 2.5 , 5 mg/ml (in 17 h assay) ; 2.5 and 5 mg/ml (in 24 h assay) with and without activation
- Second test : 1.0 , 2.5 , 5 mg/ml with and without activation

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Test material was dissolved in medium B (see at "Any other information on materials and methods incl. tables")

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

Cells treated with medium B were used as control

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION: various incubation periods

STAIN (for cytogenetic assays): Fluoresence/Giemsa technique

RANGE-FINDING TEST
In order to determine the sensitivity of the test and the cytotoxicity of the test article a range-finding cytotoxicity test was performed. V79 cells were seeded at a density of 1.0 E6 cells/flask and incubated at 37°C with 5% CO2 for 24 h in medium A. Subsequently, cells were incubated for 5 h in the presence of the following concentrations of the test article SPM: 5000; 2500; 1250; 625; 312; 156; 78; 39 and 19.5 µg/ml medium B with and without metabolic activation (S9-mix). Cells treated with medium B were used as negative control. The cells were then washed, trypsinized, and counted. The cell suspensions were adjusted to 1.0 E3 cells/ml and 200 cells were plated out in 5 ml culture medium in triplicate on petridishes with a diameter of 6 cm. Subsequently the cells were incubated for one week in medium A. After this time, the dishes were rinsed with Hank's solution and the colonies were fixed with methanol. The colonies were then stained with Giemsa solution and counted.
The respective data are presented in Table I.

MAIN-TEST
General description
day 1:
Cell cultivation with a defined cell concentration for 24 h at 37°C / 5 % CO2.
day 2:
a) Treatment of cells (4 slides) with test article or controls for 5 hours in medium B. Subsequently, replacement of medium B by medium A.
b) Treatment of one slide of the above with BrdU, to determine post-treatment mitosis.
day 3:
Treatment of cells with colcemid (0.2 µg/ml) two hours prior to termination of the test ( 17 and 24 h, respectively in a first test and 17 h in the second test).
Treatment of cells with hypotonic solution (KCl).
Fixation of cells in methyl alcohol/acetic acid.
Slides were dried at room temperature for 48 h.
day 5:
a) Giemsa staining of cells treated with test article or controls.
b) Fluorescence/Giemsa technique staining of cells treated with test article or controls and BrdU.

Specific test description :
Depending on the duration of the test (17, and 24 h), cells were seeded one day prior to treatment in medium A on glass slides. Cell densities were 3.3 E4 cells / ml. Incubation was performed in Quadriperm dishes (Heraeus) at 37°C / 5% CO2 over a period of 24 hours. The cells were then treated with different concentrations of the test article diluted in medium B, with and without S-9 Mix over a period of 5 hours.
All doses of the test article were tested at least in triplicates, i.e. two slides for evaluation of chromosomal aberrations and one slide of each concentration of the test article and of the positive and negative controls being treated with BrdU. The slides exposed to BrdU allowed differentiation of first, second and third post-treatment mitosis. Treatment with BrdU was performed in the first test only.
The following test concentrations of SPM were chosen in the first main test with and without S-9: 5; 2.5; 1.0; 0.5 and 0.1 mg/ml (17 h) and 5 and 2.5 mg/ml (24 h). In the second test the concentrations of 5; 2.5 and 1 mg/ml were used.
Untreated cells were used as negative controls and MMS- (without S-9) and cyclophosphamide (with S-9) -treated cells as positive controls.
After the 5h treatment, cells were washed twice with buffer 1, and medium B was replaced by medium A. The BrdU-treatmend of one slide of each group was continued. Two hours prior to termination of the test (17 and 24 h, respectively) cells were treated with 0.2 µg,/m1 colcemid. For both tests, cells were treated with 0.4% KCl and fixed in methyl alcohol/acetic acid. Slides were dried at room temperature for 48 h.
The slides treated with BrdU were moistened with buffer 3 and conditioned with ultraviolet light (UV) at 254 nm for 30 min. Afterwards the slides were incubated in concentrated buffer 2 (2 x SSC) for 30 minutes at 59 ± 1°C.
All slides were stained in 5 % Giemsa solution for 10 minutes at room temperature, dried overnight and analysed by microscope.
For determination of the type of post-treatment mitosis 50 metaphases were evaluated on the BrdUtreated slides of each test concentration in the first test. For evaluation of the mitotic index 1000 cells were analysed (Tables II-III).
Prior to the determination of chromosomal aberrations, slides were coded by a number combination providing at least two slides for each test point. Chromosomal aberrations were determined by microscopic analysis of 100 metaphases, containing 22 ± 2 centromeres.
In cases of reduced survival and/or reduced metaphase numbers, e.g. due to cytotoxic effects of the positive controls, a lower number of cells or metaphases was evaluated for determining posttreatment mitosis or chromosomal aberrations. The karyotype of each analysed metaphase was determined and numerical aberrations were recorded.
Whenever polyploid metaphases were found, they were recorded individually. Polyploid cells were not considered in the evaluation of chromosomal aberrations.

Evaluation criteria:

For determination of the type of post-treatment mitosis 50 metaphases were evaluated on the BrdUtreated slides of each test concentration in the first test. For evaluation of the mitotic index 1000 cells were analysed.
Prior to the determination of chromosomal aberrations, slides were coded by a number combination providing at least two slides for each test point. Chromosomal aberrations were determined by microscopic analysis of 100 metaphases, containing 22 ± 2 centromeres.
In cases of reduced survival and/or reduced metaphase numbers, e.g. due to cytotoxic effects of the positive controls, a lower number of cells or metaphases was evaluated for determining posttreatment mitosis or chromosomal aberrations. The karyotype of each analysed metaphase was determined and numerical aberrations were recorded. Whenever polyploid metaphases were found, they were recorded individually. Polyploid cells were not considered in the evaluation of chromosomal aberrations.

Key result

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

Additional information on results:

During the first main test, post-treatment mitosis was analysed using cells treated with BrdU which were incubated either in the presence or absence of S-9 mix. Evaluation of these slides revealed that after 17 h all cells treated with SPM as well as the negative control and the cells treated with either MMS or CPA as positive controls remained in their first post-treatment mitosis (Table II). After 24h, half or the vast majority of cells treated with SPM were at their second mitosis (Table III).

The evaluation of the mitotic index revealed no reduced survival of cells after 17h and 24h irrespective of the presence of S9-mix compared to the negative control (Table II and III).

Due to these results (vast majority of cells at their first mitosis, no reduced survival of cells) the second main test, with and without S9-mix, was performed with an incubation period of 17h.

The slides were further evaluated for chromosomal aberrations. The total number of cells with aberrations and the types of aberrations were determined. The results on the types of aberrations are presented in table IV and V. In table IVa and Va results are reported as percentages of aberrant cells.

In both experiments no remarkable increase of aberrant cells was observed for any test concentration of SPM at any harvest time either in the presence or absence of S-9. In addition all results were found within the spontaneous variability range observed in previous studies in our laboratory (e.g. maximum mean values for chromosomal aberrations observed in the negative control).

The positive controls (MMS/CPA) induced a remarkable increase of chromosomal aberrations compared to the negative controls and were within the usual variability ranges (e.g. maximum mean numbers of cells with aberrations excl. gaps at 17h p.a.: 7.5% (MMS) in the absence of S-9 mix and 11.5% (CPA) in the presence of S-9 mix).
No dose-dependent increase of polyploid cells was observed in the course of analysing metaphases for structural aberrations.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The study was performed according to the OECD Guideline 473 without deviations and considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The positive controls (MMS/CPA) induced a remarkable increase of chromosomal aberrations compared to the negative controls and were within the usual variability ranges (e.g. maximum mean numbers of cells with aberrations excl. gaps at 17h p.a.: 7.5% (MMS) in the absence of S-9 mix and 11.5% (CPA) in the presence of S-9 mix). No dose-dependent increase of polyploid cells was observed in the course of analysing metaphases for structural aberrations. In conclusion, the results of all experiments indicate that the test article SPM had no significant effect on the frequency of structural chromosomal aberrations in vitro under the test conditions described in this study report.

Executive summary:

The clastogenic potential of SPM was evaluated in a chromosome aberration test in vitro. The cytogenetic assay is performed by using cells of the established Chinese hamster cell line V79, a widely used culture system for in vitro mutagenicity testing. Cell cultures are exposed to the test article both with and without exogenous metabolic activation. Treated cells are arrested in a metaphase-like stage of mitosis and chromosome preparations are made. Preparations are stained and metaphase cells are analyzed for chromosomal aberrations at their first post-treatment mitosis. During the first main test, post-treatment mitosis was analysed using cells treated with BrdU which were incubated either in the presence or absence of S-9 mix. Evaluation of these slides revealed that after 17 h all cells treated with SPM as well as the negative control and the cells treated with either MMS or CPA as positive controls remained in their first post-treatment mitosis. After 24h, half or the vast majority of cells treated with SPM were at their second mitosis. The evaluation of the mitotic Index revealed no reduced survival of cells after 17h and 24h irrespective of the presence of S9 -mix compared to the negative control. Due to these results (vast majority of cells at their first mitosis, no reduced survival of cells) the second main test, with and without S9 -mix, was performed with an incubation period of 17h. The slides were further evaluated for chromosomal aberrations. The total number of cells with aberrations and the types of aberrations were determined. In both experiments no remarkable increase of aberrant cells was observed for any test concentration of SPM at any harvest time either in the presence or absence of S-9. In addition all results were found within the spontaneous variability range observed in previous studies in the testing laboratory (e.g. maximum mean values for chromosomal aberrations observed in the negative control). The positive controls (MMS/CPA) induced a remarkable increase of chromosomal aberrations compared to the negative controls and were within the usual variability ranges (e.g. maximum mean numbers of cells with aberrations excl. gaps at 17h p.a.: 7.5% (MMS) in the absence of S-9 mix and 11.5% (CPA) in the presence of S-9 mix). No dose-dependent increase of polyploid cells was observed in the course of analysing metaphases for structural aberrations.

In conclusion, the results of all experiments indicate that the test article SPM had no significant effect on the frequency of structural chromosomal aberrations in vitro under the test conditions described in this study report.

Reference 4

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:

08 May - 10 June 1992

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study is performed according to OECD Guideline 476 and under GLP conditions. No relevant deviations reported.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

HPRT-locus

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- V79 from chinese hamster obtained from ICN-Flow

- Type and identity of media:
Culture medium:
Minimal essential medium with Earles salts (MEM) + 10 % (v/v) fetal calf serum (FCS)
+1 % (v/v) non-essential amino acids (NEAA)
+1 % (v/v) penicillin / streptomycin
+2 mM L-glutamine
Incubation medium: Culture medium without FCS
Selection medium: Culture medium with 6-thioguanine (10 µg/ml)

- Periodically checked for Mycoplasma contamination: yes, checked for mycoplasma contamination on 19.05.92 (tested by ELISA)

Metabolic activation:

with and without

Metabolic activation system:

S-9 Mix: obtained from CCR Darmstadt(+5 mM Na2-NADP + 10 mM glucose-6-phosphate; 4 % (v/v) final concentration in incubation medium)

Test concentrations with justification for top dose:

Range-finding-test : 19.5 , 39 , 78 , 156 , 312 , 625 , 1250 , 2500 and 5000 µg/ml (with and without activation)

Main test : 156 , 312 , 625 , 1250 , 2500 and 5000 µg/ml (with and without activation)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: Solutions made up in incubation medium

Untreated negative controls:

yes

Remarks:

untreated cells

Negative solvent / vehicle controls:

yes

Remarks:

cells treated with incubation medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

9,10-dimethylbenzanthracene

other: methanesulfonic acid ethyl ester

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

STAIN (for cytogenetic assays): The colonies were stained with Giemsa solution

RANGE-FINDING TEST
In order to determine the cytotoxic potential of the test article, a range-finding test with and without exogenous metabolic activation (S-9-mix) was performed. Eight different test concentrations within the solubility limits of the test article were used: 5000; 2500; 1250; 625; 312; 156; 78; 39 and 19.5 µg/ml.
The V79 cells were seeded in a density of 1.0 E6 cells / flask and incubated for 24 h in culture medium. Afterwards, for a period of five hours, the cells were exposed to the test article or to the controls which were diluted in incubation medium. The cells were then washed, trypsinized, and counted. The cell suspensions were adjusted to 1.0 E3 cells / ml and 200 cells were plated out in 5 ml culture medium in triplicate on petridishes with a diameter of 6 cm. Subsequently the cells were incubated for one week in culture medium. After this time, the dishes were rinsed with Hank's solution and the colonies were fixed with methanol. The colonies were then stained with Giemsa solution and counted.

MAIN-TEST
In the main test, cell cultures were exposed for five hours, with and without exogenous metabolic activation to the six highest concentrations of the test article used in the rangefinding test : 5000; 2500; 1250; 625; 312 and 156 µg/ml. This was done in the same manner as described above. In addition, two positive controls, one with metabolic activation (9,10-dimethy1-1,2-benzanthracene) and one without (methanesulfonic acid ethyl ester), furthermore a triplicate negative control (untreated cells) were tested. In addition to the above described procedure, 1.0 E6 cells / flask were cultivated in cultivation medium for one week to allow stable phenotypic expression of the induced mutation at the HPRT-locus. Afterwards the cells were subcultivated and 1.0 E6 cells were seeded in each of four petridishes containing 10 ml selection medium (10 cm in diameter). The plate were incubated for an additional week. During this time only the cells with a functional inactive HPRT locus will survive and form a colony. Afterwards the colonies were fixed, stained and counted.

Evaluation criteria:

For the cytotoxicity tests mean colony number and standard deviation were calculated for each concentration tested. The relative and the absolute cloning efficiency (CE) was determined by the number of the colonies in the petri dishes. The relative CE describes the cloning efficiency of the treated cells in relation to the cloning efficiency of the untreated cells (negative controls) and is a parameter for the survival of the cells. The absolute CE is needed for the calculation of the mutant frequency (MF). It was calculated by the following formulas:

relative CE = mean of the colony number/mean value of the colony number of all negative controls * 100

absolute CE = mean of the colony number/seeded cell number * 100

The number of mutant colonies in the selective medium is adjusted relative to the number of colonies in non-selective medium to derive the mutant frequency (MF). This value described the number of mutant per million clonable cells.

MF = (mean of mutated colony number * 1.0 E6 cells/absolute CE * number of seeded cells) * 100

Statistics:

For the statistical evaluation of the results the following procedure was chosen. All MFvalues were tested for significance in the t-test, which was used because of homogenous group variance. Only those results, which were significantly different (limit of significance p = 0.05) compared to the highest negative controls are reported .

Key result

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

Additional information on results:

In the range-finding test with and without S-9 no significant cytotoxicity of the test article was measured in the range of 19.5 to 5000 µg/ml compared to the negative control. In the main tests with or without metabolic activation (S-9) no significant increase of the mutation frequency was observed compared to the highest negative control. In both tests performed, the positive controls induced a significant response under the chosen conditions.

Conclusions:

Interpretation of results:
negative with and without metabolic activation

The study was performed according to the OECD Guideline 476 without deviations and according to the principles of the good laboratory practice and therefore considered to be of the highest quality (reliability Klimisch 1). The vehicle and the positive control substances fulfilled validity criteria of the test system. The test material did not induce gene mutations at the HPRT locus in V79 cells. The test material was considered to be non-mutagenic under the conditions of the test.

Executive summary:

Methacrylsaure-(3-sulfopropyl)ester, K-Salz (SPM) was investigated using the gene mutation assay in Chinese hamster V79 cells (V79 / HPRT) for its ability to induce gene mutations. In the range-finding test with and without S-9 no significant cytotoxicity of the test article was measured in the range of 19.5 to 5000 µg/ml compared to the negative control. Based on these results 5 mg/ml was chosen as the highest concentration for the main test.

When SPM was tested in the main tests with or without metabolic activation (S-9) no significant increase of the mutation frequency was observed compared to the highest negative control. The statistical analysis of the MF values (MF: mutant frequency) was done by using the t-test.

In both tests performed, the positive controls induced a significant response under the chosen conditions.

In conclusion, the results of all experiments indicate that the test article SPM has no biologically significant effect on the mutation frequency in the HPRT-locus under the test conditions described in this report.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

There are several data available, all assessed with at least Klimisch 2, sufficiently covering all required endpoints, i.e. gene mutation in both bacteria and mammalian cells, and chromosome aberrations (micronucleus induction) in mammalian cells. Those in vitro genetic toxicity tests are designed, i.a. via addition of additional enzymes (metabolic activation; Aroclor 1254 induced rat liver S9), to mimic the exposure of humans to a potential genotoxic agent very close. They are accepted surrogates for human data, and no indication is given that the obtained results are not relevant for humans / risk assessment. Hence, the database is of good quality, sufficient to exclude that any risk with regard to genotoxic effects may arise for humans from 2-Propenoic acid, 2-methyl-,3-sulfopropylester, potassium salt.

All available tests gave consistently negative results, which makes a mode of action analysis impossible, as no conclusions from any events can be drawn.

Justification for classification or non-classification

All available test results for gene mutations or chromosome aberrations are consistently negative, and no need for classification as mutagen or directly genotoxic carcinogen was identified, and hence, as toxicokinetic data indicate a body-wide distribution including germ cells, no need for classification as germ cell mutagen is evident from the given data.

According to Regulation 1272/2008, “This hazard class (Germ cell mutagenicity) is primarily concerned with substances that may cause mutations in the germ cells of humans that can be transmitted to the progeny. However, the results from mutagenicity or genotoxicity tests in vitro and in mammalian somatic and germ cells in vivo are also considered in classifying substances and mixtures within this hazard class.” Further, “Substances which are positive in in vitro mammalian mutagenicity assays, and which also show chemical structure activity relationship to known germ cell mutagens, shall be considered for classification as Category 2 mutagens.”

This is not the case here, and so 2-Propenoic acid, 2-methyl-,3-sulfopropylester, potassium salt does not need to be classified as germ cell mutagen.