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Toxicological information

Eye irritation

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Administrative data

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
Cross-reference
Reason / purpose:
reference to other study
Remarks:
OECD 405
Reference
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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
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)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
- Name of test material (as cited in study report) : 1-(2-Hydroxy-3-sulfopropy1)-pyridinium-betain
- Physical state : white powder
- Storage condition of test material : room temperature 20 ± 5 °C

Test animals / tissue source

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.

Test system

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.

Results and discussion

In vitro

Results
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

Applicant's summary and conclusion

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.