Registration Dossier

Administrative data

Description of key information

Skin irritation: Skin corrosion in vitro, RHE-model: not corrosive (OECD 431, EpiDerm, GLP)

Skin irritation: Skin irritation in vivo, New Zealand White rabbit, m/f, clipped, occlusive, 1g, 50% solution in PEG400:Saline 70:30, 4h exposure: not irritating (read-across from PPS, OECD 404, GLP)

Eye irritation: Eye irritation/corrosion in vitro, HET-CAM, 6 eggs, 301 s incubation, 275 µg/egg (undiluted): mean irritation score = 6.53, moderately irritant (HET-CAM, GLP)

Eye irritation: Eye irritation in vivo, New Zealand White rabbit, m/f, 0.1g single instillation, no wash-out: not irritating (read-across from PPS, OECD 405, GLP)

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2008-12-01 - 2009-02-11
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline study
Reason / purpose:
reference to other study
Remarks:
OECD 404
Qualifier:
according to
Guideline:
OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)
Version / remarks:
OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Original Guideline adopted April 13, 2004)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))
Version / remarks:
Commission Directive 2000/33/EC, OJL136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40.
Deviations:
no
GLP compliance:
yes (incl. certificate)
Test system:
human skin model
Source species:
human
Cell type:
non-transformed keratinocytes
Cell source:
other: not stated
Source strain:
other: not applicable
Details on animal used as source of test system:
not applicable
Details on test system:
RECONSTRUCTED HUMAN EPIDERMIS (RHE) TISSUE
Epi-200 kits and MTT-100 assays diluent were purchased from MatTek Corporation (Ashland, MA 01721, USA). The EpiDerm™ tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm diameter).
EpiDerm™ tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24-well plate and reached the lab on February 28, 2008. Tissues were stored at 4 °C until February 29, 2008 prior to use. On day of receipt EpiDerm™ tissues were kept in the refrigerator until use. 1 – 1.5 hours before starting the assay, tissues were transferred to 6-well plates with assay medium, which was immediately replaced before the test was started.

TEMPERATURE USED FOR TEST SYSTEM
- Temperature used during treatment / exposure:
Prewarming of Epiderm™ tissues
1-1.5 hours before dosing, EpiDerm™ tissues were removed from the refrigerator. Under sterile conditions using sterile forceps, the inserts were transferred into 6-well plates containing the pre-warmed assay medium. Two 24-well plates were prepared as holding plates containing 300 μL assay medium. The holding plates were pre-warmed in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.
Treatment
Duplicate EpiDerm™ tissues were treated with the test item, positive, and negative controls for 2 different treatment intervals: 3 minutes and 1 hour.
After pre-incubation of EpiDerm™ tissues was completed (each 1 hour for both treatments) medium was replaced by 0.9 mL fresh assay medium in all four 6-well plates. 50 μL deionised water (negative control) were added into the first insert atop the EpiDerm tissue. The procedure was repeated with the second tissue. It was proceeded with test item and the positive control in the same manner until all tissues of the same treatment interval were dosed. The 6-well plates were placed into the incubator (37 ± 1.5 °C, 5 ± 0.5% CO2).

REMOVAL OF TEST MATERIAL AND CONTROLS
-Volume and number of washing steps: After the end of the treatment interval the first insert was removed immediately from the 6-well plate. Using a wash bottle the tissue was gently rinsed with PBS to remove any residual test material. Excess PBS was removed by gently shaking the insert and blotting the bottom with blotting paper. The insert was placed in the prepared holding plate. It was proceeded with test item and the positive control in the same manner until all EpiDerm™ tissues were dosed.
- Observable damage in the tissue due to washing: none stated

MTT DYE USED TO MEASURE TISSUE VIABILITY AFTER TREATMENT / EXPOSURE
The MTT concentrate was prepared on the day of testing and diluted with the MTT diluent. The remaining MTT solution was stored in the dark at 4 °C for later use on the same day (not until next day). Two 24-well plates were prepared before end of the tissue prewarming period. 300 μL of the MTT solution were added to each well and the plates were kept in an incubator (37 ± 1.5 °C, 5 ± 0.5% CO2) until use.
After the treatment procedure was completed for all tissues of each time point cell culture inserts were transferred from the holding plates to the MTT-plates. After a 3 hour incubation period (37 ± 1.5 °C, 5 ± 0.5% CO2) MTT solution was aspirated from the wells and wells were rinsed three times with PBS. Inserts were transferred into new 24 well plates. The inserts were immersed into extractant solution by gently pipetting 2 mL extractant solution (isopropanol) into each insert. The level rised above the upper edge of the insert, thus completely covering the tissue from both sides. The 24 well plate was sealed to inhibit isopropanol evaporation. The formazan salt was extracted for about 68 hours.
After the extraction period was completed for both treatment intervals the inserts were pierced with an injection needle to allow the extract to run into the well from which the insert was taken. Afterwards the insert was discarded. 24 well plates were placed on a shaker for approx. 15 minutes until solution was homogeneous in colour.
Per each tissue 3 × 200 μL aliquots of the blue formazan solution were transferred into a 96-well flat bottom microtiter plate, both from the 3 minutes exposure and from the 1 hour exposure. OD was read in a microplate reader (Versamax® Molecular Devices, D-85737 Ismaning) at 570 nm without reference filter. Mean values were calculated from the 3 wells per tissue.
Some test chemicals may reduce MTT, which will result in a blue colour without any involvement of cellular mitochondrial dehydrogenase. Although in the present assay the test chemicals were rinsed off and the DMEM medium beneath the tissues was changed before contact with MTT medium, some amount of a test chemical may be released by the tissues into the MTT medium and directly reduce the MTT, which would be interpreted as "tissue viability". To check MTT reducing capability a solution of MTT in DMEM (1.0 mg/mL) was prepared and about 50mg of the test item were added to 1 mL MTT medium. If the mixture turned blue/purple after about 1 hour at room temperature, the test material would have been presumed to have reduced the MTT. No colour change could be observed in the present study.

NUMBER OF REPLICATE TISSUES: duplicate tissues (therein: 3 replicates) for each treatment and incubation time

CONTROL TISSUES USED IN CASE OF MTT DIRECT INTERFERENCE
not applicable

NUMBER OF INDEPENDENT TEST SEQUENCES / EXPERIMENTS TO DERIVE FINAL PREDICTION: 2 incubation times

PREDICTION MODEL / DECISION CRITERIA
Interpretation of Results
The mean OD value obtained for the duplicate tissues per test item were used to calculate a percentage viability relative to the negative control, which was arbitrarily set at 100%.
The test item is considered to be corrosive to skin:
(1) if the viability after 3 minutes exposure is less than 50%, or
(2) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is less than 15%.
The test item is considered to be non-corrosive to skin:
(1) if the viability after 3 minutes exposure is greater or equal than 50% and the viability after 1 hour exposure is greater than or equal to 15%.
Control samples:
yes, concurrent negative control
yes, concurrent positive control
Amount/concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): Each about 25 mg of test material were wetted with 25 μL deionised water and spread to match size of tissue.

NEGATIVE CONTROL
- Amount(s) applied (volume or weight): Deionised water was used as the negative control. 50 μL were applied to each of duplicate tissues for 3 minutes and 1 hour, respectively.

POSITIVE CONTROL
- Amount(s) applied (volume or weight): Potassium Hydroxide as 8.0 N ready made solution (Cat.No. 17-8, Sigma 82024 Taufkirchen, Lot No. 096K6091) was used as positive reference. 50 μL were applied to each of duplicate tissues for 3 minutes and 1 hour, respectively.
Duration of treatment / exposure:
3 min and 1h
Duration of post-treatment incubation (if applicable):
3h MTT incubation
Number of replicates:
duplicate tissues (therein: 3 replicates) for each treatment and incubation time
Species:
other: a three-dimensional human epidermis model was used
Strain:
other: Epi-200
Details on test animals and environmental conditions:
Epi-200 kits and MTT-100 assays diluent were purchased from MatTek Corporation (Ashland, MA 01721, USA). The EpiDermTM tissue consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDermTM tissues (surface 0.6 cm2) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm diameter).
EpiDermTM tissues were shipped at 4 °C on medium-supplemented agarose gels in a 24- well plate and reached RCC-CCR GmbH on February 28, 2008. Tissues were stored at 4 °C until February 29, 2008 prior to use. On day of receipt EpiDermTM tissues were kept in the refrigerator until use. 1 – 1.5 hours before starting the assay, tissues were transferred to 6-well plates with assay medium, which was immediately replaced before the test was started.
Type of coverage:
open
Preparation of test site:
other: not applicable
Vehicle:
other: wetted with deionised water
Controls:
other: negative control : deionised water ; positive control : 8.0 N KOH
Amount / concentration applied:
Each about 25 mg of test material were wetted with 25 μL deionised water and spread to match size of tissue.
For the positive and negative controls 50 μL were dosed per tissue.
Duration of treatment / exposure:
3 min and 60 min
Number of animals:
not applicable
Details on study design:
The MTT (Methylthiazoletetrazolium) method was used to determine the values of viability after 3 min or after 60 min of incubation.
Irritation / corrosion parameter:
% tissue viability
Remarks:
Rel. Absorbance [% of Negative Control]
Run / experiment:
mean of Tissue 1 + 2. Time point: 3 min.
Value:
78.8
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not corrosive
Remarks:
viability after 3 minutes exposure is greater or equal than 50%
Irritation / corrosion parameter:
% tissue viability
Remarks:
Rel. Absorbance [% of Negative Control]
Run / experiment:
mean of Tissue 1 + 2. Time point: 60 min.
Value:
90.4
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: not corrosive
Remarks:
viability after 60 minutes exposure is greater or equal than 15%
Other effects / acceptance of results:
After treatment with the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain the relative absorbance values did not decrease relevantly each after 3 minutes (78.8%) and after 1 hour treatment (90.4%). Both values are well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment.
Interpretation of results:
other: not corrosive
Remarks:
Criteria used for interpretation of results: EU-GHS
Conclusions:
The study was performed according to the OECD Guideline 431 with no deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. After treatment with the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain the relative absorbance values did not decrease relevantly each after 3 minutes (78.8%) and after 1 hour treatment (90.4%). Both values are well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment. Hence, no classification as corrosive to the skin, i.e. Skin Corr. Cat. 1 as indicated by Regulation 1272/2008, is triggered. However, the model is not suitable to distinguish between classification as Skin Irr. Cat. 2 and non-classification, so to make this final assessment, in vivo data on a suitable read-across substance will be taken into account.
Executive summary:

This study was conducted according to the procedures indicated by the following internationally accepted guidelines and recommendations:

-Commission Directive 2000/33/EC, OJL136200, dated June 08, 2000, adopting the 27th time to technical progress the Dangerous Substances Directive 67/548/EEC, Annex V, part B40.

-OECD Guideline for Testing of Chemicals 431: In vitro Skin Corrosion: Human Skin Model Test (Original Guideline adopted April 13, 2004)

This in vitro study was performed to assess the corrosive potential of 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain by means of the Human Skin Model Test. Two tissues of the human skin model EpiDermTM were treated with either the test item, the negative or the positive control for 3 minutes and 1 hour, respectively. About 25 mg of the test item were applied to each tissue, wetted with 25μL deionised water, and spread to match the tissue size. 50μL of either the negative control (deionised water) or the positive control (8.0 N KOH) were applied to each tissue. After treatment with the negative control the absorbance values were well above the required acceptability criterion of mean OD ≥ 0.8 for both treatment intervals thus showing the quality of the tissues. Treatment with the positive control induced a decrease in the relative absorbance as compared to the negative control to 21.9% for the 3 minutes treatment interval and 8.2% for the 1 hour treatment interval thus ensuring the validity of the test system. After treatment with the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain the relative absorbance values did not decrease relevantly each after 3 minutes (78.8%) and after 1 hour treatment (90.4%). Both values are well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain is non corrosive to skin.

Endpoint:
skin irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1982-11-25 - 1982-12-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
non-GLP, read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

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

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

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
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 404 (Acute Dermal Irritation / Corrosion)
Deviations:
yes
Remarks:
only 4 days of acclimatisation
Qualifier:
according to
Guideline:
other: ETAD (ecological and toxicological association of the dye stuffs manufacturing industries) Methods 001-003 (1979)
Deviations:
no
GLP compliance:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, 4414 Fuellingsdorf, Switzerland
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 2.3 - 3.0 kg
- Housing: cages individually in stainless steel cages with automatic drinking water supply and cleaning system (Dipl. Ing. W. Ehret GmbH/Versuchstiertechnik, 7830 Mendingen, Germany).
- Diet (e.g. ad libitum): pelleted standard KLIBA 23/341/1 rabbit maintenance dies (Klingentalmuehle AG, 4303 Kaiseraugust, Switzerland)
defined for acceptable contaminant level, ad libitum.
- Water (e.g. ad libitum): tap water ad libitum (water quality according to the requirements of the "schweiz. Lebensmittelbuch".
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 55 +/- 10 %
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

IDENTIFICATION: individually by numbered ear tags (Eisenhut Vet. AG, 4123 Allschwil, Switzerland) and cage number.
Type of coverage:
occlusive
Preparation of test site:
other: back and flanks of each rabbit were closely clipped with electric clippers
Vehicle:
other: polyethylene glycol (PEG400) + Saline (70 : 30 parts)
Controls:
other: the not-treated skin served as control site
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 1 gram
- Concentration (if solution): 50% solution of the test substance in polyethylene glycol (PEG400) + Saline (70 : 30 parts)

VEHICLE
- Concentration (if solution): polyethylene glycol (PEG400) + Saline (70 : 30 parts)
Duration of treatment / exposure:
The bandages and gauze patches remained in place for 4 hours. The animals were not restrained during this period.
Observation period:
72 hours
Number of animals:
3 (2 males, 1 female)
Details on study design:
TEST SITE
- Area of exposure: Twenty four hours before treatment the back and flanks of each rabbit were closely clipped with electric clippers, exposing an area of skin of approximately 10 X 10 cm.

SCORING SYSTEM:
1. Erythema and Eschar formation
no erythema = 0
very light erythema (barely detectable) = 1
well-defined erythema = 2
moderate to severe erythema = 3
severe erythema (beet redness) to slight eschar formation (injuries in depth) = 4
total possible erythema score: 4
2. Oedema
no oedema = 0
very light oedema (barely perceptible) = 1
light oedema (edges of area well defined by definitive raising) = 2
moderate oedema (raised approximately 1 mm) = 3
severe oedema (raised more than 3 mm and extending beyond area of exposure) = 4
total possible oedema score: 4

The skin reaction of the treated site was observed 1 hour/ 48 / 72 hours following removal of bandages and gauze patch.
In addition the corrosion effect to the skin of the rabbits was recorded and the number of affected animals was registered.
Mortality and clinical symptoms were monitored once daily.

SACRIFICE AND NECROPSY
Termination and post-mortem examination: the study was terminated 72 hours after substance application. All rabbits were killed by an intravenous injection of T61 (Hoechst) in the ear vein. Due to the results obtained, no macroscopical organ examination was indicated.
Irritation parameter:
other: Index of colouration
Basis:
mean
Time point:
other: 1, 24, 48, 72 hours
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
erythema score
Basis:
mean
Time point:
other: 1 h
Score:
1
Max. score:
1
Reversibility:
fully reversible
Irritation parameter:
erythema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
edema score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
primary dermal irritation index (PDII)
Basis:
mean
Time point:
other: 1, 24, 48, 72 h
Score:
0.2
Max. score:
1
Reversibility:
fully reversible within: 1 day
Remarks on result:
no indication of irritation
Irritant / corrosive response data:
1-(3-Sulfopropyl)-Pyridinium-Betain (PPS) showed no irritation, when applied to intact rabbit skin.
No destructions or irreversible alterations of the treated skin were observed. Thus it was concluded that no corrosion effect had occurred on the skin. In the area of application no discolouration f the skin was observed which could be related to compound effects. No acute toxicological signs were observed in the animals during the test period.
Other effects:
No other effects were reported.
Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU-GHS
Conclusions:
The study was performed according to the OECD Guideline 404 with only negligible deviations and even though no information was available whether it was performed according to the good laboratory practice principles, it is considered to be of high quality (reliability Klimisch 2). The criteria of validity of the test system are fulfilled. The test material did not induce any irritation or corrosion on the intact skin of rabbits. The test material was considered to be not irritating under the conditions of the test. Both edema and erythema scores were 0.0 (mean of 24, 48, and 72h), so no classification as Skin Irr. according to Regulation 1272/2008 is triggered.
Executive summary:

The skin irritation potential of the test substance was investigated in New Zealand White rabbits according to OECD TG404 (Claus and Ullmann, 1982). The test substance (1 gram of a 50% dilution of the test substance in polyethylene glycol/water) was applied occlusively to the intact skin for 4 hours; thereafter the skin reactions were monitored for 72 hours. Under the conditions of this experiment the test material was found to cause no irritation. In the area of application no discoloration of the skin was observed in the rabbits which could be related to compound effects. No corrosion effect had occurred on the skin at each measuring interval. The calculated primary irritation index was found to be 0.2 (Mean irritation index for intact skin).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Referenceopen allclose all

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 April 2008
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline-study
Reason / purpose:
reference to other study
Remarks:
OECD 405
Qualifier:
according to
Guideline:
other: ICCVAm Test Method Evaluation Report: Appendix G ICCVAM recommended HET-CAM Method Protocol (Nov. 2006)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Species:
other: Chicken eggs
Strain:
other: Lohmann selected Leghorn chicken's eggs
Details on test animals or tissues and environmental conditions:
Freshly laid Lohmann Leghorn chicken eggs were obtained from the LSL Rhein Main Geflügelvermehrungsbetrieb (Dieburg, Germany) on 08. April 2008 (nine days before the start of the test). The eggs were incubated at 37.5 ± 0.5 °C for eight days. During incubation, the eggs were rotated to prevent an attachment of the embryo to one side of the egg. 24 hours before the start of the experiment (eight days after obtaining the eggs), the chicken eggs were candled and non-viable eggs were discarded. The rest of the eggs were placed upward in the incubator until the next day.
Vehicle:
unchanged (no vehicle)
Controls:
other: 3 eggs with an 1% solution of sodium dodecyl sulfate and 0.1 n NaOH respectively were used as positive controls. 3 eggs with Physiological sodium chloride solution were used as negative control.
Amount / concentration applied:
275 µg (The test item was tested directly, without dilution or preparation of a solution.)
Duration of treatment / exposure:
300s
Observation period (in vivo):
5 min.
Number of animals or in vitro replicates:
6 eggs
Details on study design:
Test Vessels
All vessels used are made of glass or sterilizable plastic. They were sterilised before use by heating to 180 °C (two hours) or autoclavation.
The following vessels were used:
♦Schott-bottles, glass vials, and culture flasks for solutions and media

Instruments and Devices
The following instruments were used in the performance of the study.
♦Autoclave MLS 3020
♦Candling light
♦Mortar and pestle
♦Stop clock
♦Analytical scales MT AB 184 SA
♦Incubation chamber Binder
♦Orbital shaker GFL 3005
♦Adjustable pipette with sterile tips, LAUS No. 8 (0.2-2 mL)
♦Glass measuring flasks, 25, 500 and 1000 mL

Preparation of Test System
24 hours before the start of the experiment (eight days after obtaining the eggs), the chicken eggs were candled and non-viable eggs were discarded. The rest of the eggs were placed upward in the incubator until the next day.

Experimental Parameters
-Date of treatment : 17. April 2008
-Incubation time : 300 seconds
-Positive controls : Na-dodecylsulfate, 1% ; NaOH, 0.1 mol/L

Method Description
On the day of the test, the eggs were removed from the incubator for use in the assay.
At first, the eggs were candled and the air bubble was marked. The egg was opened on the air bubble with a forceps. The inner membrane was moistened with 0.9 % NaCI, then the egg was left to stand into the incubator for 30 minutes. After this period, the solution was decanted and the inner membrane was carefully removed with a forceps. Then the test item was given directly onto the CAM surface. 300 µL (equalling to 275 µg) were measured using a centrifuge vial. The test item was given on the membrane in such a manner that 50% of the surface of the membrane were covered with test item. The reactions on the CAM were observed over a period of 301 seconds. The following endpoints are described in the guideline:
♦haemorrhage (bleeding from the vessels)
♦vascular lysis (blood vessel disintegration)
♦coagulation (intra- and extra-vascular protein denaturation)
The time for the appearance of each of the noted endpoints was monitored and recorded in seconds.
Irritation parameter:
other: Mean irritation score
Run / experiment:
mean of 6 eggs / after 301 seconds
Value:
6.53
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: moderately irritant
Interpretation of results:
moderately irritating
Conclusions:
The study was performed according to the following guideline : ICCVAm Test Method Evaluation Report: Appendix G ICCVAM recommended HET-CAM Method Protocol (Nov. 2006) with no deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain was brought onto the surface of the CAM of a hen's egg which had been incubated at 37 °C for nine days. The test item showed moderate effects an the blood vessels of the CAM. A mean irritation score of 6.53 was calculated, corresponding to a classification as "moderately irritant". However, the study is not suitable to determine absolutely the classification according to Regulation 1272/2008. Hence, the results of an OECD 405 study on a suitable read-across substance will be regarded. Further, the present test item is most likely to contain a certain amount of NaCl, which may have contributed to the result, and which is not relevant for the registered substance as such.
Executive summary:

This "in vitro" study was performed to assess the irritating potential of 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain by detection of damages in blood vessels under the chorioallantoic membrane of nine day incubated chicken eggs. Observation time was five minutes at room temperature.. The study was performed according to the following guideline : ICCVAm Test Method Evaluation Report: Appendix G ICCVAM recommended HET-CAM Method Protocol (Nov. 2006) with no deviations and according to the good laboratory practice principles. Physiological sodium chloride solution was used as negative control, sodium dodecyl sulfate (1% solution ) and sodium hydroxide (0.1-n solution) were used as positive controls. The positive controls induced a severe irritation on the blood vessels. The negative control showed no irritation, the irritation score for the positive controls lay within the demanded range. The test item was tested pure. A mean irritation score of 6.53 was calculated, corresponding to a classification as "moderately irritant". No observations were made which might cause doubts concerning the validity of the study outcome. The test is considered valid.

In conclusion, it can be stated that in this study and under the experimental conditions reported, the test item 1-(2-Hydroxy-3-sulfopropyl)-pyridinium-betain possesses a moderate irritation potential.

Endpoint:
eye irritation: in vivo
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1982-11-25 - 1982-12-02
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
GLP-Status not stated, performed on read-across substance
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH

1. HYPOTHESIS FOR THE ANALOGUE APPROACH

According to ECHA’s guidance document on information requirements and chemical safety assessment Chapter R.6 „QSARs and grouping of chemicals”, there are two techniques for grouping chemicals known when reading across to cover data gaps, i.e., category approach and analogue approach [ECHA, 2008].
A chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity (or other similarity characteristic). The term analogue approach is used when the grouping is based on a very limited number of chemicals, where trends in properties are not apparent. Categories of chemicals are selected based on the hypothesis that the properties of a series of chemicals with common structural features will show coherent trends in their physico-chemical properties, and more importantly, in their toxicological (human health / ecotoxicity) effects or environmental fate properties [ECHA, 2008].
As set out in the guidance document, a chemical category is a group of chemicals whose physico-chemical and human health and/or environmental toxicological properties and/or environmental fate properties are likely to be similar or follow a regular pattern as a result of structural similarity. The similarities may be based on the following:
- common functional group(s) (e.g. aldehyde, epoxide, ester, specific metal ion);
- common constituents or chemical classes, e.g., similar carbon range numbers;
- an incremental and constant change across the category (e.g. a chain-length category), often observed in physico-chemical properties, e.g. boiling point range;
- the likelihood of common precursors and/or breakdown products, via physical or biological processes, which result in structurally similar chemicals (e.g. the metabolic pathway approach of examining related chemicals such as acid/ester/salt) [ECHA, 2008].

It is aimed to combine similarity patterns in order to cover data gaps for PPSOH. One rational for the analogue approach is the high structural similarity between the source and the target substance. 3-pyridinium-1-ylpropane-1-sulfonate (PPS) (source) and 1-(2-hydroxy-3-sulphonatopropyl)pyridinium, inner salt (PPSOH) (target) are structurally identical except an additional hydroxyl group on position 2 of the propyl moiety of the target substance. Despite the fact that a hydroxyl group may alter the toxicological or toxicokinetic behaviour of a substance, this effect is considered minor as there are three common groups in the molecules which are considered more relevant for their toxicological behaviour, i.e. the sulfo-group, the propyl moiety and the pyridine. Due to the similarities in structure, similar physico-chemical properties of the substances are to be expected, which would result in a similar toxicokinetic behaviour and most likely also in very similar toxicodynamic and toxicological behaviour. Second, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization (ToxTree estimation, Ideaconsult Ltd (2004-2013). Estimation of Toxic Hazard – A decision Tree approach, version 2.6.6, http://toxtree.sourceforge.net/), but they also share common metabolites, as shown from additional modelling of the source chemical metabolites (see respective table in the attachment).
Further, both substances show similar (eco-)toxicological properties in the endpoints for which data for both substances is available, which is considered proof of the suitability of the analogue approach, i.e. cross-reading from PPS to PPSOH.

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

Source Chemical: 3-pyridinium-1-ylpropane-1-sulfonate / Pyridinium, 1-(3-sulfopropyl)-, hydroxide, inner salt / CAS 15471-17-7 / EC 239-491-3 (PPS), SMILES [O-]S(=O)(=O)CCC[n+]1ccccc1, MW 201.2428, C8H11NO3S

Target Chemical: Pyridinium, 1-(2-hydroxy-3sulfopropyl)-, hydroxide, inner salt / 2-hydroxy-3-pyridinium-1-ylpropane-1-sulfonate / CAS 3918-73-8 / EC 223-485-2 (PPSOH) SMILES OC(C[n+]1ccccc1)CS(=O)(=O)[O-], MW 217.2422, C8H11NO4S

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
Comparing the actually available information on the substances with regard to their physico-chemical properties, the minor influence of the additional hydroxyl group of the target chemical becomes obvious. All relevant information on similar metabolites can be retrieved from the respective table, in brief, the target substance is not only a metabolite of the source chemical, resulting from CYP450 metabolization, but they also share common metabolites, as shown from additional modelling of the source chemical metabolites. Considering the non-metabolized source and target chemicals only, the molecular weight only differs in the weight of a hydroxyl group and is hence in the same range, i.e. 201.24 g/mol and 217.24 g/mol, indicating per se the potential for absorption.
Both substances are solids which melt under decomposition at rather high temperatures, i.e. ≥ 245°C and have hence a negligible vapour pressure. Both compounds are very soluble in water, and their logPow is in a negative range.

In general, absorption of a chemical is possible, if the substance crosses biological membranes. In case where no transport mechanisms are involved, this process requires a substance to be soluble, both in lipid and in water, and is also dependent on its molecular weight (substances with molecular weights below 500 are favourable for absorption). Relevant for the endpoint acute toxicity dermal and skin sensitisation is the absorption resp. retention in the skin. In order to cross the skin, a compound must first penetrate into the stratum corneum and may subsequently reach the epidermis, the dermis and the vascular network. The stratum corneum provides its greatest barrier function against hydrophilic compounds, whereas the epidermis is most resistant to penetration by highly lipophilic compounds. Substances with a molecular weight below 100 are favourable for penetration through the skin and substances above 500 are normally not able to penetrate. The substance must be sufficiently soluble in water to partition from the stratum corneum into the epidermis. Therefore if the water solubility is below 1 mg/L, dermal uptake is likely to be low. Additionally logPow values between 1 and 4 favour dermal absorption. In the case of both the target and source chemical, due to their high water solubility and very low logPow, their absorption is very likely to be hindered in the stratum corneum. Nevertheless, once reaching the epidermis, i.a. due to their common small size, their absorption is favoured.
Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar toxicological behaviour. Both substances are relatively non-toxic, with oral LD50 values >5000 mg/kg bw, and are non-irritating the skin and eyes.
Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, toxicokinetic and toxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its acute dermal toxicity and skin-sensitizing properties compared to the source chemical can be expected.
As indicated by studies on gene mutations in bacteria (both substances), chromosome aberrations in mammalian cells (PPS) and gene mutations in mammalian cells (PPSOH), both substances are not genotoxic. It can hence be reasonable concluded that a positive result in a chromosome mutation test on PPSOH can be excluded and read-across is justified, an underestimation of the actual hazard for genotoxic insults is unlikely. Further, as both substances are not acutely toxic, i.e. oral LD50 values are >5000 mg/kg, due to their physico-chemical properties a relevant accumulation in the body can be neglected, and no systemic or reprotoxic effects at all were noted in the OECD 422 study on PPS at the limit dose of 1000 mg/kg, the target chemical PPSOH does not need to be regarded as harmful upon repeated exposure or reproductive toxicant, too.

Besides the common physico-chemical and toxicokinetic properties, they exhibit a similar ecotoxicological behaviour. Both substances are relatively non-toxic towards aquatic invertebrates, both 48h EC50 values and even NOECs were above the limit value for classification, the EC50(48h) was even shown to be > 1000 mg/l for PPSOH. PPS showed results of LC50 (96h) > 1000 mg/L and NOEC (96h) > 1000 mg/L in the trout in an acute fish toxicity study acc. OECD 203. The EC50(72h) in algae in a study acc. OECD 201 is also above 100 mg/l, allowing in summary the conclusion that acute toxicity testing in fish would also not indicate any hazardous properties of PPSOH, so the assumption of a similar ecotoxicity profile and so read-across from PPS is also justified here.
In consequence, a similar behaviour can be expected in microorganisms. PPS is non-toxic to microorganisms, in a OECD 209 no toxicity was observed at a concentration of 1000 mg/l, so the following values were obtained for activated sludge: EC50(3h) > 1000 mg/L, NOEC(3h) = 1000 mg/L. This allows the conclusion that the substance is relatively non-toxic towards microorganisms.

Hence, due to the above-mentioned similarities of the source and target chemical, with regard to their structure, functional groups, common metabolites, toxicokinetic and ecotoxicological behaviour, it can be reasonably concluded that a similar behaviour of the target chemical regarding its ecotoxicological and toxicological properties compared to the source chemical can be expected. In summary, the target chemical PPSOH needs to be regarded as relatively non-toxic.


4. DATA MATRIX
The following table shows the available data relevant to justify the read-across from the source to the target chemical for several endpoints in order to omit testing for animal welfare:

Endpoint Source: PPS Target: PPSOH
Molecular weight 201.24 g/mol 217.24 g/mol
Physical state solid solid
Partition coefficient logPow < -2.78 at 21.5°C logPow < -2
Water solubility 240.5 g/L at 25°C (EpiSuite estimation) 1280 g/l at 23°C
Biodegradation 86 % degradation after 28 days Not readily biodegradable: no degradation observed (DOC) (OECD 301E)
readily biodegradable
Hydrolysis Not expected to undergo hydrolysis Hydrolysis can be excluded
Short-term toxicity to fish LC50 (96h) > 1000 mg/L, n/a
NOEC (96h) > 1000 mg/L (trout, OECD 203)
Short-term toxicity to aquatic invertebrates 24&48h NOEC ≥ 100 mg/L EC50(48h) > 1000 mg/l
24&48h EC50 > 100 mg/L (OECD 202) NOEC(48h) = 1000 mg/l (OECD 202)
Short-term toxicity to aquatic algae n/a EC50(72h) > 100 mg/l (OECD 201)
Toxicity to microorganisms EC50(3h) > 1000 mg/L, n/a
NOEC(3h) = 1000 mg/L (activated sludge, OECD 209)
MIC = 0.12 g/mL (Pseudomonas putida)
Acute toxicity oral LD50 > 5000 mg/kg (rat, OECD 401) LD50 > 5000 mg/kg (rat, OECD 423))
Acute toxicity dermal LD50 > 2000 mg/kg (rat, OECD 402) n/a
Skin irritation Not irritating (in vivo, rabbit) not corrosive (OECD 431, EpiDerm)
Eye irritation Not irritating (in vivo, rabbit) moderately irritant (HET-CAM, GLP)
Skin sensitization Not sensitizing (GPMT, OECD 406) n/a
Gene mutation in bacteria Negative ± S9 (OECD 471) negative ± S9 (OECD 471)
Chromosome aberration in mammalian cells Negative ± S9 (OECD 487) n/a
Gene mutation in mammalian cells n/a negative ± S9 (OECD 490)
Repeated dose toxicity NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Toxicity to reproduction NOAEL ≥ 1000 mg/kg (rat, OECD 422) n/a
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Version / remarks:
May 12, 1981
Deviations:
no
Qualifier:
according to
Guideline:
other: ETAD (Ecological and toxicological association of the dye stuffs manufacturing industry, Methods 001-003 (1979)
Qualifier:
according to
Guideline:
other: VCI (Verband der chemischen Industrie, BRD) Sicherheitsdatenblatt
GLP compliance:
not specified
Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
TEST ANIMALS
- Source: Kleintierfarm Madoerin AG, 4414 Fuellinsdorf, Switzerland
- Age at study initiation: approximately 14 weeks
- Weight at study initiation: 2.3 - 3.0 kg
- Housing: caged individually in stainless steel cages, with automatic drinking water supply and cleaning system (Dipl. Ing. W. Ehret GmbH, Versuchstier Technik, 7830 Emmendingen, Germany).
- Diet (e.g. ad libitum): pelleted standard KLIBA 23/341/1, rabbit maintenance diet (Klingentalmuehle AG, 4303 Kaiseraugust /- Switzerland). Defined for acceptable contaminant level, ad libitum
- Water (e.g. ad libitum): tap water ad libitum (water quality according to the requirements of the "SCHWEIZ. LEBENSMITTELBUCH")
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2°C
- Humidity (%): 55 +/- 10 %
- Photoperiod (hrs dark / hrs light): 12 hours light, 12 hours dark

Other:
- Identification: individually by numbered ear tags (Eisenhut Vet AG, 4123 Allschwil, Switzerland) and cage numbers.
Vehicle:
unchanged (no vehicle)
Controls:
other: the right eye served as untreated control
Amount / concentration applied:
0.1g
Duration of treatment / exposure:
single application into the conjunctival sac of the left rabbit eye, without rinsing out
Observation period (in vivo):
1/ 24/ 48/ 72 hours after treatment
Number of animals or in vitro replicates:
3 in total (2 males, 1 female) - the same animals were used for the skin and eye irritation experiment.
Details on study design:
SCORING SYSTEM:

Cornea

A) opacity and the degree of opacity (most dense area scored).
No opacity = 0
Scattered or diffuse area (details of iris clearly visible) = 1
Easily discernible translucent areas, details of iris slightly obscured =2
Opalescent areas, no details of iris visible, size of pupil barely discernible = 3
Opal opacity, iris invisible = 4

B) area of cornea involved
No area involved = 0
One quarter (or less, but not 0) = 1
more than one quarter, less than half = 2
greater than half, but less than three quarters = 3
greater than three quarters up to the whole area = 4
A x B x 5 Total maximum = 80

Iris

A. Values
Normal = 0
Fold above normal, congestion, swelling, circumcornea injection (any or all of these or combination of any thereof) Iris still reacting to light (sluggish reaction is positive) = 1
No reaction to light, haemorrhage, gross destruction (any or all of these) = 2
A x 5 = Total Highest number = 10

Conjunctiva

A) Redness (refers to palpebral and bulbar conjunctiva excluding cornea and iris)
Vessels normal = 0
Vessels definitively injected more than normal = 1
More diffuse, deeper crimson red, individual vessels not easily discernible = 2
Diffuse beefy red = 3

B) Chemosis
No swelling = 0
Any swelling above normal (includes nictitating membrane) = 1
Obvious swelling with partial eversion of the lids = 2
Swelling with lids about half closed = 3
Swelling with lids about half closed to completely closed = 4

C) Discharge
No discharge = 0
Any amount different from normal (does not include small amounts observed in the inner canthus of normal animals) = 1
Discharge with moistening of lids and hairs just adjacent to lids = 2
Discharge with moistening of the lids and hairs, and considerable area around the eye = 3
(A + B + C) x 2 Total maximum = 20

In addition the coloration of the sclera/cornea was recorded in connection with the colour of the test material.
The total score for the eye is the sum of all points for cornea, iris and conjunctiva.
Irritation parameter:
overall irritation score
Basis:
mean
Time point:
other: 24, 48 and 72 h
Score:
1.4
Max. score:
110
Reversibility:
other: not applicable
Remarks on result:
other: no irritation or corrosivity
Irritation parameter:
other: mean reaction score
Basis:
mean
Time point:
other: 1 hour
Score:
5.3
Reversibility:
fully reversible
Irritation parameter:
other: mean reaction score
Basis:
mean
Time point:
other: 24 hours
Score:
0.6
Reversibility:
fully reversible
Irritation parameter:
other: mean reaction score
Basis:
mean
Time point:
other: 48 hours and 72 hours
Score:
0
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
other: animal 1, 2 and 3
Time point:
other: 1 hour, 24 hours, 48 hours and 72 hours
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
iris score
Basis:
other: animal 1, 2 and 3
Time point:
other: 1 hour, 24 hours, 48 hours and 72 hours
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal #1
Time point:
24/48/72 h
Score:
0.33
Max. score:
1
Reversibility:
fully reversible within: 48h
Irritation parameter:
conjunctivae score
Remarks:
redness
Basis:
animal: 2 as well as 3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
chemosis score
Basis:
mean
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Remarks on result:
no indication of irritation
Irritation parameter:
other: conjunctiva redness
Basis:
other: animal 1, 2 and 3
Time point:
other: 1 hour,
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: conjunctiva redness
Basis:
animal #1
Time point:
other: 24 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 48 hours
Irritation parameter:
other: conjunctiva redness
Basis:
other: animal 2 and 3
Time point:
other: 24 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
other: conjunctiva chemosis
Basis:
animal #1
Time point:
other: 1 hour
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
other: conjunctiva chemosis
Basis:
other: animal 2 and 3
Time point:
other: 1 hour
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
other: conjunctiva chemosis
Basis:
other: animal 1, 2 and 3
Time point:
other: 24 hours, 48 hours and 72 hours
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
other: conjunctiva discharge
Basis:
other: animal 1 and 3
Time point:
other: 1 hour
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
other: conjunctiva discharge
Basis:
animal #2
Time point:
other: 1 hour
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 24 hours
Irritation parameter:
other: conjunctiva discharge
Basis:
other: animal 1, 2 and 3
Time point:
other: 24 hours, 48 hours and 72 hours
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritant / corrosive response data:
1 -(3 -sulfopropyl)-pyridinium-betain (PPS) showed no irritation when applied to the rabbit eye mucosa.
No discoloration of the cornea and conjunctivae was observed in the rabbits during the entire test period which could be related to effects of the test material.
No corrosion effect was observed at each of the measuring intervals.
No acute toxicological signs were observed in the test animals during the test period.
Other effects:
no other effects reported
Interpretation of results:
not irritating
Remarks:
Criteria used for interpretation of results: EU-GHS
Conclusions:
The study was performed according to the OECD TG405 without deviations and even though no information was available whether it was performed according to the good laboratory practice principles, it is considered to be of high quality (reliability Klimisch 2). The criteria of validity of the test system are fulfilled. The test material did not induce any irritation or corrosion on rabbit eyes. The test material was considered to be not irritating under the conditions of the test. Mean conjunctivae score (redness, mean over 24, 48, and 72h) was 0.33 in animal 1, all other scores over 24, 48, and 72h relevant for classification acc. Regulation 1272/2008, i.e. cornea opacity score, iris score, chemosis score and conjunctivae score (redness, in animals 2 and 3) were consistently 0.0. Hence, the criteria for classification as eye irritant acc. Regulation 1272/2008 are not met.
Executive summary:

The test substance 1 -(3 -sulfopropyl)-pyridinium-betain (PPS) was investigated for its potential to cause irritation in the rabbit eye according to OECD TG405. 0.1 g of pure substance were instilled into the conjunctival sac, the untreated eye served as control. Under the conditions of this experiment 1 -(3 -sulfopropyl)-pyridinium-betain (PPS) was found to cause no irritation when applied to the rabbit eye mucosa. No corrosion effect was observed at each of the measuring intervals. No discoloration of the cornea and conjunctivae which could be related to effects of the test material was observed. Additionally no corrosive or acute toxicological signs were observed in the animals during the test period. The primary irritation index was found to be for the unrinsed eye: 1.4. Mean conjunctivae score (redness, mean over 24, 48, and 72h) was 0.33 in animal 1, all other scores over 24, 48, and 72h relevant for classification acc. Regulation 1272/2008, i.e. cornea opacity score, iris score, chemosis score and conjunctivae score (redness, in animals 2 and 3) were consistently 0.0. Hence, the criteria for classification as eye irritant acc. Regulation 1272/2008 are not met.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Justification for classification or non-classification

With regard to skin irritation, the available OECD 431 study revealed that the relative absorbance values did not decrease relevantly each after 3 minutes (78.8%) and after 1 hour treatment (90.4%)., being hence well above the threshold of 50% for 3 minutes treatment and above 15% for 1 hour treatment for classification as corrosive. Hence, no classification as corrosive to the skin, i.e. Skin Corr. Cat. 1 as indicated by Regulation 1272/2008, is triggered. However, the model is not suitable to distinguish between classification as Skin Irr. Cat. 2 and non-classification, so in vivo data on a suitable read-across substance will be taken into account. In the available OECD 404 study on PPS, the test material did not induce any irritation or corrosion on the intact skin of rabbits. The test material was considered to be not irritating under the conditions of the test. Both edema and erythema scores were 0.0 (mean of 24, 48, and 72h), so no classification as Skin Irr. Cat. 2according to Regulation 1272/2008 is triggered.

Regarding eye irritation, the available HET-Cam test on the registered substance itself, the test item showed moderate effects an the blood vessels. A mean irritation score of 6.53 was calculated, corresponding to a classification as "moderately irritant". However, the study is not suitable to determine absolutely the classification according to Regulation 1272/2008. Hence, the results of an OECD 405 study on a suitable read-across substance will be regarded. Further, the present test item is most likely to contain a certain amount of NaCl, which may have contributed to the result, and which is not relevant for the registered substance as such. In the available OECD 405 study on PPS, the test material did not induce any irritation or corrosion on rabbit eyes. Mean conjunctivae score (redness, mean over 24, 48, and 72h) was 0.33 in animal 1, all other scores over 24, 48, and 72h relevant for classification acc. Regulation 1272/2008, i.e. cornea opacity score, iris score, chemosis score and conjunctivae score (redness, in animals 2 and 3) were consistently 0.0. Hence, the criteria for classification as eye irritant acc. Regulation 1272/2008 are not met.