Sodium 3-(2-propyn-1-yloxy)-1-propanesulfonate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-06-23 - 1993-07-05 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Justification for type of information:

see target record

Cross-reference

Reason / purpose for cross-reference:

read-across source

Remarks:

target record

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Study period:

1993-06-23 - 1993-07-05 (experimental phase)

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Well-documented study according to OECD 471 with minor deviations: performed on suitable read-across substance and only four strains of S. typhimurium (TA1535, TA1537, TA98, TA100) were used, data on E.coli WP2 strains or S. typhimurium TA102 are lacking. However, since these strains were mainly included in the recent version of OECD 471 because the four formerly only recommended S. typhimurium strains TA1535, TA1537, TA98 and TA100 may not detect certain oxidising mutagens, cross-linking agents and hydrazines, and this mode of action is not likely to occur based on the chemical structure of the test item, this restriction is considered to be negligible.

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The rational for the analogue approach is the high structural similarity between the source and the target substance. Propargyl 3-sulfopropyl ether, potassium salt, and Propargyl 3-sulfopropyl ether, sodium salt, are structurally identical except the inorganic counterion, potassium resp. sodium. This difference is considered very minor as both cations are ubiquitously present in the body fluids, and the organic moieties are identical containing three functional groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the alkine, ether and sulfo group.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)

Source Chemical: Propargyl 3-sulfopropyl ether, potassium salt, EC 618-959-4, CAS 93637-00-4, SMILES Code C#CCOCCCS(=O)(=O)[O-].[K+], molecular formula C6H9O4KS, Mol. Weight 216.2994 g/mol

Target Chemical: Propargyl 3-sulfopropyl ether, sodium salt, EC 608-454-7, CAS 30290-53-0, SMILES Code C#CCOCCCS(=O)(=O)[O-].[Na+], molecular formula C6H9O4NaS, Mol. Weight 200.19 g/mol

Both substances do not contain impurities to an extent which is expected to alter the outcome of the experimental results or read-across approach.

3. ANALOGUE APPROACH JUSTIFICATION
According to REACH Annex XI, chapter 1.5, “Substances whose physicochemical, toxicological and ecotoxicological properties are likely to be similar or follow a regular pattern as a result of structural similarity may be considered as a group, or "category" of substances.”… “The similarities may be based on:
1) a common functional group;
2) the common precursors and/or the likelihood of common breakdown products via physical and biological processes, which result in structurally similar chemicals…”.
Hence, Propargyl 3-sulfopropyl ether, sodium salt was analyzed regarding these criteria in the order as stated above:
1) Propargyl 3-sulfopropyl ether, sodium salt, is an organic salt with a sodium cation as inorganic counterion. The inorganic cation sodium (Na+) is widely distributed throughout the body and a normal constituent in the electrolyte system of vertebrates. Hence, it suggests itself to predominantly focus on the organic anion and regard it unchanged as a first step. So, the complete organic cation shall serve as a ‘functional group’ in this case. Further analogues can therefore be easily found by exchanging the inorganic counterion into a similar one of a similar size and low or no intrinsic toxic properties. Obvious here are e.g. potassium, hydrogen or ammonium.
2) Due to the ionic structure of all above mentioned salts, they all dissociate readily into the respective ions when getting into contact with water, which can be scientifically concluded. Propargyl 3-sulfopropyl ether, sodium salt, is distributed as a 50% aqueous solution and hence very soluble in water; the registered substance containing water is fully miscible in water. A similar behaviour can be assumed for POPS-K. In consequence, both substances can be reasonably expected to be present completely dissociated in the body fluids predominantly consisting of water. So, the organic moiety is identical in both substances and can be regarded as common breakdown product according to the Regulation. The substances structurally only differ in their inorganic cation, which can be considered as a very minor difference as both cations are ubiquitously present in the body fluids.
The data matrix displays exemplarily the chlorides of the inorganic counterions in question, sodium and potassium. Both salts show mild to moderate irritating effects, data available on POPS-K indicate very minor irritating effects not sufficient for classification. In general, the observed effects can be considered as rather consistent given the magnitude of effects, ionic structure of the cations, the content of the cations in the actual source and target chemical and the available data quality.
In both RTECS and GESTIS Substance Database of the German IFA providing various information on hazardous substances at the workplace, no information is given that NaCl or KCl are sensitizing which is comprehensible out of the following reasons: both sodium and potassium are ubiquitously present in the body and no information is given on autoimmune diseases associated with these ions. Further, these cations are not capable to act as (pre-)haptene or allergen. Immune responses are associated with proteins, and those ion are neither a protein nor capable of binding on them or modify them in a manner that the immune system is capable of recognizing them. Hence, a immune response could maximally be caused by the organic anion, which is identical in both source and target substance.
With regard to acute toxicity, also here possible differences may only arise from the cation. As displayed in the data matrix, potassium is in general of higher toxicity compared to sodium. Hence, a read-across is unlikely to underestimate the actual hazard of the registered substance, and more likely to overestimate it. Hence, read-across does not pose a potential risk and can be justified.

According to the RTECS database, for both NaCl and KCl, there are positive effects noted in various assays related to mutagenicity. According to the GESTIS database however, „There are no indications that NaCl has any mutagenic effects. NaCl solutions of very low concentrations have been used as solvents for test substances in a variety of mutagenicity tests (because of their inactivity). Positive reactions found in isolated cases on cultivated mammalian cells or in microorganisms were probably caused by osmotic effects and are not attributable to mutagenicity. There are no indications that NaCl has any carcinogenic effects.“ (http://gestis-en.itrust.de/nxt/gateway.dll/gestis_en/000000.xml?f=templates$fn=default.htm$vid=gestiseng:sdbeng$3.0). For KCl, that information is not given, but expectable, as also potassium is contained in cell culturing media, and the same osmotic effects in higher concentrations are expectable. Summarizing, there is no indication given that the exchange of the cation (Na+ or K+) would result in a different outcome of gene mutation testing in bacteria, hence, read-across is justified.
An obvious difference is that the potassium salt may be isolated as solid, whereas the sodium salt undergoes slight changes during isolation, can hence not be isolated as such and so the water must be considered as stabilizer in its identification. However, when being dissolved resp. diluted in the body fluids predominantly consisting of water, this difference can be neglected.

4. DATA MATRIX
There is not sufficient data on both complete, non-dissociated substances available to allow a direct comparison. Further, QSAR estimation revealed identical phys.-chem. properties, as e.g. for EpiSuite (US EPA) estimations, the inorganic ion is not regarded. However, as stated above, both organic salts immediately dissociate into the respective ions. Hence, the toxicity of the more relevant organic anion, Propargyl 3-sulfopropyl ether, does not need to be regarded for depicting possible differences or similarities, as it is identical in both molecules, and it is sufficient to compare the different cations only. Exemplarily, sodium and potassium chloride are compared, data is derived from RTECS (http://ccinfoweb.ccohs.ca/rtecs/search.html)

For the table, please refer to the attached justification

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD guideline 471 (May 26, 1983)
Salmonella / microsome test as described by Ames et al. (1973a, 1975) and Maron and Ames (1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, protected from light

Target gene:

his-

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Dr. W. Göggelmann, GSF München/Neuherberg and received on August 24,1989.
- Suitability of cells: 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).
All tester strains were routinely checked for crystal violet sensitivity (deep rough character), UV sensitivity (uvrB) and ampicillin resistance (pKM101).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

8, 40, 200, 1 000 and 5 000 µg/plate, as required by the guideline; no bacteriotoxic effects were observed at any concentrations in the pre-test up to 10 000 µg/plate

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-Nitro-1,2-phenylene diamine; 2-Aminoanthracene

Remarks:

The positive controls sodium azide, 9-aminoacridine hydrochloride and 4-nitro-1,2-phenylene diamine were used without S 9 mix, whereas the positive control 2-aminoanthracene was used with S 9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his minimal agar

NUMBER OF REPLICATIONS: Three plates per strain and concentration, two independent experiments

DETERMINATION OF CYTOTOXICITY
The number of colonies, the number of revertants and the background growth were determined as a measure of the toxicity of the test item.

Evaluation criteria:

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 has revealed a sufficient bacterial density in the suspension.

Assessment of mutagenicity and bacteriotoxicity
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:

Presentation and evaluation of data
The results are compiled in tabular form. 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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not exceeded
- Precipitation: none stated

RANGE-FINDING/SCREENING STUDIES:
No mutagenic and bacteriotoxic effects were observed at any concentrations. The total number of viable cells, the number of revertants and the background growth were in the range of biological variability.

Conclusions:

The study was conducted according to OECD 471 of 1983 under GLP with minor deficiencies compared to the recent guideline and is sufficiently documented. Positive and negative controls gave the appropriate results. Hence, the available study is sufficiently reliable to assess the genotoxic potential of propargyl-3-sulfopropyl ether, potassium salt, towards bacteria. The evaluation of the individual dose groups, with respect to the parameters relevant for assessment (dose effect and doubling), showed no biologically relevant mutagenic effect of POPS at concentrations ranging from 8 to 5 000 µg/plate.

Executive summary:

The test article POPS was investigated in the Salmonella/microsome test for point mutations using four Salmonella typhimurium LT2 mutants in an OECD 471 (1983) study under GLP. These tester strains were the histidine auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100.

Concentrations ranging from 10 to 10 000 µg per plate were employed in the preliminary toxicity test. The solvent was H2O.

No bacteriotoxic effects were observed. The total number of viable cells, the count of revertants and the background growth were within the range of biological variability.

On the basis of this result, the first main test (4% S9) employed the following concentrations: 8, 40, 200, 1 000 and 5 000 µg/plate.

No evidence of biologically significant bacteriotoxic or mutagenic activity of POPS was found. Neither with, nor without S9 mix was a biologically relevant or dose-related increase of the reversion rates observed, when compared to the negative control (solvent).

The same concentrations were used in the second main test as in the first main test. The concentration of S9 was increased to 10% S9.

Results with regard to absence of bacteriotoxic and mutagenic effect could be confirmed by the second main test.

The sensitivity of the test system was demonstrated by the marked mutagenic effects exerted on each strain by 2 of the following 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 POPS caused no mutagenic effect at concentrations ranging from 8 to 5 000 µg/plate.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

OECD guideline 471 (May 26, 1983)
Salmonella / microsome test as described by Ames et al. (1973a, 1975) and Maron and Ames (1983)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

potassium 3-(prop-2-yn-1-yloxy)propane-1-sulfonate

EC Number:

618-959-4

Cas Number:

93637-00-4

Molecular formula:

C6H9O4KS

IUPAC Name:

potassium 3-(prop-2-yn-1-yloxy)propane-1-sulfonate

Test material form:

solid: particulate/powder

Remarks:

yellowish

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: ambient, protected from light

Method

Target gene:

his-

Species / strain

Species / strain / cell type:

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

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: The strains were obtained from Dr. W. Göggelmann, GSF München/Neuherberg and received on August 24,1989.
- Suitability of cells: 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).
All tester strains were routinely checked for crystal violet sensitivity (deep rough character), UV sensitivity (uvrB) and ampicillin resistance (pKM101).

Metabolic activation:

with and without

Metabolic activation system:

Aroclor 1254 induced rat liver S9

Test concentrations with justification for top dose:

8, 40, 200, 1 000 and 5 000 µg/plate, as required by the guideline; no bacteriotoxic effects were observed at any concentrations in the pre-test up to 10 000 µg/plate

Vehicle / solvent:

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

Controls

Untreated negative controls:

yes

Remarks:

solvent control

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: 4-Nitro-1,2-phenylene diamine; 2-Aminoanthracene

Remarks:

The positive controls sodium azide, 9-aminoacridine hydrochloride and 4-nitro-1,2-phenylene diamine were used without S 9 mix, whereas the positive control 2-aminoanthracene was used with S 9 mix.

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48h

SELECTION AGENT (mutation assays): his minimal agar

NUMBER OF REPLICATIONS: Three plates per strain and concentration, two independent experiments

DETERMINATION OF CYTOTOXICITY
The number of colonies, the number of revertants and the background growth were determined as a measure of the toxicity of the test item.

Evaluation criteria:

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 has revealed a sufficient bacterial density in the suspension.

Assessment of mutagenicity and bacteriotoxicity
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:

Presentation and evaluation of data
The results are compiled in tabular form. 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.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

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

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: not exceeded
- Precipitation: none stated

RANGE-FINDING/SCREENING STUDIES:
No mutagenic and bacteriotoxic effects were observed at any concentrations. The total number of viable cells, the number of revertants and the background growth were in the range of biological variability.

Applicant's summary and conclusion

Conclusions:

The study was conducted according to OECD 471 of 1983 under GLP with minor deficiencies compared to the recent guideline and is sufficiently documented. Positive and negative controls gave the appropriate results. Hence, the available study is sufficiently reliable to assess the genotoxic potential of propargyl-3-sulfopropyl ether, potassium salt, towards bacteria. The evaluation of the individual dose groups, with respect to the parameters relevant for assessment (dose effect and doubling), showed no biologically relevant mutagenic effect of POPS at concentrations ranging from 8 to 5 000 µg/plate.

Executive summary:

The test article POPS was investigated in the Salmonella/microsome test for point mutations using four Salmonella typhimurium LT2 mutants in an OECD 471 (1983) study under GLP. These tester strains were the histidine auxotrophic strains TA 1535, TA 1537, TA 98 and TA 100.

Concentrations ranging from 10 to 10 000 µg per plate were employed in the preliminary toxicity test. The solvent was H2O.

No bacteriotoxic effects were observed. The total number of viable cells, the count of revertants and the background growth were within the range of biological variability.

On the basis of this result, the first main test (4% S9) employed the following concentrations: 8, 40, 200, 1 000 and 5 000 µg/plate.

No evidence of biologically significant bacteriotoxic or mutagenic activity of POPS was found. Neither with, nor without S9 mix was a biologically relevant or dose-related increase of the reversion rates observed, when compared to the negative control (solvent).

The same concentrations were used in the second main test as in the first main test. The concentration of S9 was increased to 10% S9.

Results with regard to absence of bacteriotoxic and mutagenic effect could be confirmed by the second main test.

The sensitivity of the test system was demonstrated by the marked mutagenic effects exerted on each strain by 2 of the following 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 POPS caused no mutagenic effect at concentrations ranging from 8 to 5 000 µg/plate.