Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information
1. 1983, Ames test, TA98, TA100, TA1535, TA1537 and TA1538, not mutagenic 2. 2012, In vitro micronucleus test, not mutagenic 3. Prediction using TOXTREE (v.2.5.0), Chemservice S.A., 2011
Link to relevant study records
Reference
Endpoint:
in vitro cytogenicity / micronucleus study
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Qualifier:
according to guideline
Guideline:
other: OECD 487 - In Vitro Micronucleus Test
Deviations:
yes
Remarks:
expression phase and harvest time were slightly modified - this deviations are not considered to influence the outcome of the study
GLP compliance:
yes (incl. QA statement)
Remarks:
Hess. Ministerium für Umwelt, Energie, Landwirtschaft udn Verbraucherschutz, Wiesbaden, Germany
Type of assay:
in vitro mammalian cell micronucleus test
Species / strain / cell type:
lymphocytes: human lymphocytes
Details on mammalian cell type (if applicable):
Blood samples were obtained from healthy, non-smoking donors not receiving medication. For this study, blood was collected from a female donor (23 years old) for the first experiment and from a 33 year-old female donor for Experiment II. All donors had a previously established low incidence of micronuclei in their peripheral blood lymphocytes. Blood samples were drawn by venous puncture and collected in heparinized tubes. The tubes were sent to Harlan CCR to initiate cell cultures within 24 hrs after blood collection. If necessary, the blood was stored before use at 4 °C.
Metabolic activation:
with and without
Metabolic activation system:
liver S9 mix from phenobarbital/beta-naphthoflavone treated male rats
Test concentrations with justification for top dose:
657.3 µg/mL, 1150.3 µg/mL and 2013 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: deionised water, the final concentration of deinonised water in the culture medium was 10 % (v/v)
- Justification for choice of solvent/vehicle: The solvent was chosen due to its solubility properties and its relative non-toxicity to the cell cultures. The solubility of the test item in the solvent is approximately 1500 g/L.
Negative solvent / vehicle controls:
yes
Remarks:
Concurrent solvent controls (culture medium with 10 % deionised water) (local tap water deionised at Harlan CCR)).
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: demecolcin
Remarks:
Dilutions of stock solutions were prepared on the day of the experiment. Stability of Demecolcin, Mitomycin C and CPA in solution is unknown but a mutagenic response in the expected range is sufficient biological evidence of chemical stability.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 40 hours
- Exposure duration: 4 and 20 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B
STAIN (for cytogenetic assays): Giemsa

NUMBER OF CELLS EVALUATED: at least 1000 binuecleate cells per culture

DETERMINATION OF CYTOTOXICITY
- Method: other: To describe a cytotoxic effect the CBPI (cytokinesis-block proliferation index) was determined in approximately 500 cells per culture and cytotoxicity is expressed as % cytostasis. A CBPI of 1 (all cells are mononucleate) is equivalent to 100 % cytostasis (7).
Evaluation criteria:
The micronucleus assay is considered acceptable if it meets the following criteria:
a) The number of micronuclei found in the negative and solvent controls falls within the range of the laboratory historical control data.
b) The positive control substances should produce significant increases in the number of cells with micronuclei.

Evaluation of Results

A test item can be classified as non-mutagenic if:
- the number of micronucleated cells in all evaluated dose groups is in the range of the laboratory historical control data and/or
- no statistically significant or concentration-related increase in the number of micronucleated cells is observed.

A test item can be classified as mutagenic if:
- the number of micronucleated cells is not in the range of the historical laboratory control data and
- either a concentration-related increase of micronucleated cells in three test groups or a statistically significant increase of the number of micronucleated cells is observed.

Statistics:
Statistical significance was confirmed by means of the Chi square test. However, both biological and statistical significance should be considered together. If the criteria for the test item mentioned above are not clearly met, the classification with regard to the historical data and the biological relevance is discussed and/or a confirmatory experiment is performed.
Species / strain:
lymphocytes: human lymphocytes
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
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: No relevant influence on osmolarity or pH value was observed.
- Effects of osmolality: No relevant influence on osmolarity or pH value was observed.
- Water solubility: 1500 g/L
- Precipitation: no precipitation of the test item in the pre-test nor in the actual experiments

RANGE-FINDING/SCREENING STUDIES:
With respect to the molecular weight of the test item, 2013.0 µg/mL of 1-(3-sulphonatopropyl) pyridinium (approx. 10 mM) was applied as top concentration for treatment of the cultures in the pre-test. Test item concentrations between 13.1 and 2013.0 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. In the pre-test on toxicity, no precipitation of the test item was observed at the end of treatment. Since the cultures fulfilled the requirements for cytogenetic evaluation, this preliminary test was designated Experiment I.

COMPARISON WITH HISTORICAL CONTROL DATA: yes, no confounding factors observed
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

The test item 1-(3-sulphonatopropyl)pyridinium, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro in the absence and presence of metabolic activation by S9 mix. The occurrence of micronuclei in interphase cells provides an indirect but easy and rapid measure of chromosomal damage and aneugenicity. Micronuclei arise from chromosomal fragments or whole chromosomes. Micronuclei rarely occur spontaneously but are inducible by clastogens or agents affecting the spindle apparatus.

Two independent experiments were performed. In Experiment I, the exposure period was 4 hours with and without S9 mix. In Experiment II, the exposure periods were 4 hours with S9 mix and 20 hours without S9 mix. The cells were prepared 40 hours after start of treatment with the test item.

In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenetic damage on coded slides. To determine a cytotoxic effect the CBPI was determined in approximately 500 cells per culture and cytotoxicity is described as % cytostasis.

The highest treatment concentration in this study, 2013.0 µg/mL (approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the OECD Guideline 487 for the in vitro mammalian cell micronucleus test.

No precipitation of the test item in the culture medium was observed at the end of treatment. No relevant influence on osmolarity or pH value was observed.

In this study, at both preparation intervals, in the absence as well as in the presence of S9 mix, no biologically relevant cytotoxicity indicated by a clearly CBPI expressed as cytostasis could be observed (Table 4 and 5).

In both experiments, in the absence and presence of S9 mix, no biologically relevant increase in the number of cells carrying micronuclei was observed (see Table 6, 7, 8 and 9). The micronucleus rates of the cells after treatment with the test item (0.10 - 0.75 % micronucleated cells) were close to the range of the solvent control values (0.15 - 0.35 % micronucleated cells) and within the range of the laboratory historical control data. However, statistically significant increases were observed in Experiment I and II in the presence of S9 mix after treatment with 2013.0 µg/mL (0.70, 0.75 % micronucleated cells, respectively). The values are clearly within the range of the historical control data (0.20 - 1.70 % micronucleated cells) and therefore regarded as biologically irrelevant.

In both experiments, either Demecolcin (75.0 ng/mL), MMC (2.0 µg/mL) or CPA (10.0 or 15.0 µg/mL) were used as positive controls and showed distinct increases in cells with micronuclei.

In conclusion, it can be stated that under the experimental conditions reported, the test item 1-(3-sulphonatopropyl)pyridinium did not induce micronuclei in human lymphocytesin vitrowhen tested up to the highest required concentration.

Table 3a: Summary of results of the in vitro micronucleus test in human lymphocytes with 1-(3-sulphonatopropyl)pyridinium

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs without S9 mix

I

40 hrs

Negative control

1.86

 

0.45

 

 

Solvent control1

1.84

 

0.30

 

 

Positive control2

1.67

22.4

 9.20S

 

 

657.3

1.81

4.2

0.30

 

 

1150.3

1.82

2.6

0.30

 

 

2013.0

1.73

13.6

0.20

Exposure period 20 hrs without S9 mix

II

40 hrs

Negative control

1.92

 

0.25 / 0.40***

 

 

Solvent control1

1.92

 

0.35

 

 

Positive control3

1.71

23.2

2.60S/3.00S***

 

 

657.3

1.89

2.7

0.30

 

 

1150.3

1.82

10.5

0.15

 

 

2013.0

1.87

5.0

0.20

* For the positive control groups, the relative values are related to the negative controls; for the test item treatment groups are related to the solvent controls.

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

***  The number of micronucleated cells was determined in a sample of 2000 mononucleated cells

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

1     Deionised water 10.0 % (v/v)
2
           MMC           2.0 µg/mL

3           Demecolcin75.0 ng/mL

Table 3b: continued Summary of results of the in vitro micronucleus test in human lymphocytes with 1-(3-sulphonatopropyl)pyridinium

Exp.

Preparation

Test item

Proliferation

Cytostasis

Micronucleated

 

interval

concentration

index

in %*

cells

 

 

in µg/mL

CBPI

 

in %**

Exposure period 4 hrs with S9 mix

I

40 hrs

Negative control

1.69

 

0.30

 

 

Solvent control1

1.78

 

0.15

 

 

Positive control2

1.37

46.2

 2.40S

 

 

657.3

1.69

10.9

0.45

 

 

1150.3

1.72

8.2

0.50

 

 

2013.0

1.77

0.8

 0.70S

II

40 hrs

Negative control

1.57

 

0.40

 

 

Solvent control1

1.60

 

0.25

 

 

Positive control3

1.48

16.5

 2.80S

 

 

657.3

1.56

6.2

0.10

 

 

1150.3

1.60

0.3

0.30

 

 

2013.0

1.57

4.8

 0.75S

*     For the positive control groups, the relative values are related to the negative controls;
for the test item treatment groups the values are related to the solvent controls

**    The number of micronucleated cells was determined in a sample of 2000 binucleated cells

S     The number of micronucleated cells is statistically significantly higher than corresponding control values

1     Deionised water 10.0 % (v/v)
2
           CPA  15.0 µg/mL

3           CPA  10.0 µg/mL

Table 4: Cytotoxicity of 1-(3-sulphonatopropyl)pyridinium to the cultures of human lymphocytes (experiment 1).

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

4 hrs

40 hrs

1.86

---

Solvent control

4 hrs

40 hrs

1.84

---

13.1

4 hrs

40 hrs

n.d.

n.d.

22.9

4 hrs

40 hrs

n.d.

n.d.

40.0

4 hrs

40 hrs

n.d.

n.d.

70.1

4 hrs

40 hrs

n.d.

n.d.

122.6

4 hrs

40 hrs

1.79

5.9

214.6

4 hrs

40 hrs

1.77

8.6

375.6

4 hrs

40 hrs

1.75

11.1

657.3

4 hrs

40 hrs

1.81

4.2

1150.3

4 hrs

40 hrs

1.82

2.6

2013.0

4 hrs

40 hrs

1.73

13.6

With S9 mix

Negative control

4 hrs

40 hrs

1.69

---

Solvent control

4 hrs

40 hrs

1.78

---

13.1

4 hrs

40 hrs

n.d.

n.d.

22.9

4 hrs

40 hrs

n.d.

n.d.

40.0

4 hrs

40 hrs

n.d.

n.d.

70.1

4 hrs

40 hrs

n.d.

n.d.

122.6

4 hrs

40 hrs

1.72

7.2

214.6

4 hrs

40 hrs

1.72

8.0

375.6

4 hrs

40 hrs

1.76

2.1

657.3

4 hrs

40 hrs

1.69

10.9

1150.3

4 hrs

40 hrs

1.72

8.2

2013.0

4 hrs

40 hrs

1.77

0.8

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*
  Mean value of two cultures
n.d. Not determined
Table 5: Cytotoxicity of 1-(3-sulphonatopropyl)pyridinium to the cultures of human lymphocytes (experiment 2).

Concentration
(µg/mL)

Exposure time

Preparation interval

CBPI
per 500 cells*

Cytostasis (%)

Without S9 mix

Negative control

20 hrs

40 hrs

1.92

---

Solvent control

20 hrs

40 hrs

1.92

---

13.1

20 hrs

40 hrs

n.d.

n.d.

22.9

20 hrs

40 hrs

n.d.

n.d.

40.0

20 hrs

40 hrs

n.d.

n.d.

70.1

20 hrs

40 hrs

n.d.

n.d.

122.6

20 hrs

40 hrs

1.80

12.4

214.6

20 hrs

40 hrs

1.87

5.2

375.6

20 hrs

40 hrs

1.85

7.3

657.3

20 hrs

40 hrs

1.89

2.7

1150.3

20 hrs

40 hrs

1.82

10.5

2013.0

20 hrs

40 hrs

1.87

5.0

With S9 mix

Negative control

4 hrs

40 hrs

1.57

---

Solvent control

4 hrs

40 hrs

1.60

---

214.6

4 hrs

40 hrs

1.57

4.5

375.6

4 hrs

40 hrs

1.63

n.c.

657.3

4 hrs

40 hrs

1.56

6.2

1150.3

4 hrs

40 hrs

1.60

0.3

2013.0

4 hrs

40 hrs

1.57

4.8

Experimental groups evaluated for cytogenetic damage are shown in bold characters
*     Mean value of two cultures
n.d.Not determined
n.c. Not calculated as the CBPI is equal or higher than the solvent control value

Conclusions:
Interpretation of results (migrated information):
negative without metabolic activation
negative with metabolic activation

The study was performed according to the OECD Guideline 487 with deviations (expression phase and harvest time were slightly modified - these deviations are not considered to influence the outcome of the study) and is considered to be of the highest quality (reliability Klimisch 1). The vehicle water and the positive control substances fulfilled validity criteria of the test system. The positive controls mitomycin C, demecolcin and cyclophosphamide induced micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. None of the cultures treated with 1-(3-sulphonatopropyl)pyridinium in the absence and in the presence of S9 mix showed biologically relevant or statistically significant increased numbers of micronuclei. Based on this test, 1-(3-sulphonatopropyl)pyridinium is considered not to be non-mutagenic in human lymphocytes.
Executive summary:

The test item 1-(3-sulphonatopropyl)pyridinium, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro (Bohnenberger, 2012). Cultured human lymphcytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2 without S9 -mix (exposure period 20 hrs, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2013.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487. In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration. In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, statistically significant increases were observed in Experiment I and II in the presence of S9 mix after treatment with 2013.0 µg/mL (0.70, 0.75 % micronucleated cells, respectively). The values are clearly within the range of the historical control data (0.20 - 1.70 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 1-(3-sulphonatopropyl)pyridinium is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

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

Additional information

Additional information from genetic toxicity in vitro:

Genetic toxicity - gene mutation:

The test substance 1-(3 -sulfopropyl)-pyridinium-betain (PPS) was investigated for its potential to cause gene mutation in Salmonella typhimurium strains (TA98, TA100, TA1537, TA1535 and TA1538) (Guenard, 1983). The study was performed according to the OECD Guideline 471 with deviations (Salmonella 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. Under the conditions of this experiment 1-(3-sulfopropyl)-pyridinium-betain (PPS) 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 1-(3 -sulfopropyl)-pyridinium-betain (PPS) revealed no indication of gene mutagenic activity.

Genetic toxicity - chromosome aberration:

The test item 1-(3-sulphonatopropyl)pyridinium, dissolved in deionised water, was assessed for its potential to induce micronuclei in human lymphocytes in vitro (Bohnenberger, 2012). Cultured human lymphocytes were used and the following study design was performed: experiment 1without S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 2 without S9 -mix (exposure period 20 hrs, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours), experiment 1 and b with S9 -mix (exposure period 4 hrs, recovery 16 hours, Cytochalason B exposure 20 hours, preparation interval 40 hours, total culture period 88 hours). In each experimental group two parallel cultures were analysed. 1000 binucleate cells per culture were scored for cytogenic damage on coded slides. The highest applied concentration in the pre-test on toxicity (2013.0 µg/mL of the test item, approx. 10 mM) was chosen with regard to the molecular weight of the test item and with respect to the current OECD Guideline 487. Dose selection of the cytogenetic experiment was performed considering the toxicity data in accordance with OECD Guideline 487.In the absence and presence of S9 mix, no cytotoxicity was observed up to the highest applied concentration.In the absence and the presence of S9 mix, no increase in the number of micronucleated cells was observed after treatment with the test item. However, statistically significant increases were observed in Experiment I and II in the presence of S9 mix after treatment with 2013.0 µg/mL (0.70, 0.75 % micronucleated cells, respectively). The values are clearly within the range of the historical control data (0.20 - 1.70 % micronucleated cells) and therefore regarded as biologically irrelevant. Appropriate mutagens were used as positive controls. The positive controls mitomycin C, demecolcin and cyclophosphamide induced statistically significant increases in cells with micronuclei and demonstrated the sensitivity of the test system and the activity of the used S9 mix. .

In conclusion, it can be stated that under the experimental conditions reported, the test item did not induce micronuclei as determined by the in vitro micronucleus test in human lymphocytes. Therefore, 1-(3-sulphonatopropyl)-pyridinium is considered to be non-mutagenic in this in vitro micronucleus test, when tested up to the highest required concentration.

Prediction using TOXTREE

Genetic toxicity potential of 3-(1-Pyridinio)-1-propanesulfonate was assessed using the Toxtree (version 2.5.0.) modelling tool. Toxtree was developed by IDEA Consult Ltd (Sofia, Bulgaria) and is approved and recommended by the EU Joint Research Center in Ispra (Italy) (LINK:http://ecb.jrc.ec.europa.eu/qsar/qsar-tools/index.php?c=TOXTREE). According to the modelling results of Toxtree, no structural alerts were identified for 3-(1-Pyridinio)-1-propanesulfonate, which might induce positive response in in-vivo micronucleus assay.


Justification for selection of genetic toxicity endpoint
well documented GLP-Guideline study according to OECD 487

Justification for classification or non-classification

According to the European regulation (EC) No. 1272/2008, the test material does not meet the criteria for classification and will not require labelling as a mutagen.