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Skin sensitisation

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Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
2001-09-07 - 2001-10-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-guideline study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
see target record
Cross-reference
Reason / purpose for cross-reference:
read-across source
Remarks:
link to target
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
2001-09-07 - 2001-10-12
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Klimisch 1 source record, but 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 for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission Directive 96/54/EC Method B6
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
No new study has been conducted; data from a existing GLP-guideline study (2001) has been used as this study is totally sufficient to cover this endpoint. This study was conducted prior to the acceptance of alternative methods or REACH.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 300 to 450g,
- Housing: housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Free access to and food (Certified Guinea Pig Diet (Code 5026) supplied by PMI Nutrition International, Nottingham, UK) was allowed throughout the study. The diet was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study. The drinking water was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

OTHER:
- each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen
- the animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study
Route:
intradermal
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 5% w/w in distilled water
Topical Induction: 75% w/w in distilled water
Topical Challenge: 75% and 50% w/w in distilled water
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 5% w/w in distilled water
Topical Induction: 75% w/w in distilled water
Topical Challenge: 75% and 50% w/w in distilled water
No. of animals per dose:
Ten test and five control animals
Details on study design:
RANGE FINDING TESTS:
Selection of Concentrations for Main Study (Sighting Tests)
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
Selection of Concentration for Intradermal Induction: Intradermal injections (0.1 mL/injection site) were made on the clipped shoulder of two guinea pigs, using concentrations of 1% and 5% w/w in distilled water. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.
Selection of Concentration for Topical Induction: Two guinea pigs (intradermally injected with Freund's Complete Adjuvant eight days earlier) were treated with four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.
Selection of Concentration for Topical Challenge: Four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
intradermal injection
Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line into a 20 mm x 40 mm area. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% w/w formulation of the test material in distilled water
c) a 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated
- No. of exposures: 1
- Exposure period: 24 / 48
- Test groups: 10 males
- Control group: 5 males
- Site: an area approximately 40 mm x 60 mm on the shoulder region of each animal
- Frequency of applications: once
- Duration: 24 / 48
- Concentrations: 5 % w/w formulation of the test material in distilled water and 5 % w/w formulation of the test material in a 1:1 preparation of Freud's Complete Adjuant plus distilled water
topical induction
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No. 4: approximate size 40 mm x 20 mm), saturated with the test material formulation (75% w/w in distilled water) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.
- No. of exposures: one
- Day(s) of induction: day 7 after induction
- Exposure period: 48 hours
- Site: the same area on the shoulder region used previously for intradermal injections
- Concentrations: 75 % w/w formulation in distilled water
- Evaluation (hr after topical induction): The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers. A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm), saturated with the test material formulation at the maximum non-irritant concentration (75% w/w in distilled water) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% w/w in distilled water was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
- No. of exposures: one
- Day(s) of challenge: day 21 after induction
- Exposure period: 24 hours
- Site: an area of approximately 50 mm x 70 mm on both flanks of each animal
- Concentrations: 75 % w/w formulation in distilled water and on the right flank: 50 % w/w formulation in distilled water
- Evaluation (hr after challenge): Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified

OTHER: Determination by analysis of the concentration, homogeneity and stability of the test material preparations was not conducted.
Positive control substance(s):
yes
Remarks:
parallel studies, either 2-Mercaptobenzothiazole or alpha-Hexylcinnamaldehyde
Positive control results:
2-Mercaptobenzothiazole or alpha-Hexylcinnamaldehyde were shown to be sensitizer.
Key result
Reading:
other: Reading 24 hours after intradermal induction
Group:
test chemical
Dose level:
5 % w/w formulation in distilled water
Clinical observations:
Discrete or patchy erythema was noted at the intradermal induction sites of test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 24 hours after intradermal induction. Group: test group. Dose level: 5 % w/w formulation in distilled water. Clinical observations: Discrete or patchy erythema was noted at the intradermal induction sites of test group animals. .
Reading:
other: Reading24 hours after intradermal induction
Group:
negative control
Dose level:
distilled water
Clinical observations:
Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.
Remarks on result:
other: Reading: other: Reading24 hours after intradermal induction. Group: negative control. Dose level: distilled water. Clinical observations: Discrete or patchy erythema was noted at the intradermal induction sites of control group animals. .
Reading:
other: Reading 1 hour after topical induction
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
Clinical observations:
Discrete or patchy erythema was noted at the topical induction sites of test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 1 hour after topical induction. Group: test group. Dose level: 75 % w/w and 50 % w/w formulation in distilled water. Clinical observations: Discrete or patchy erythema was noted at the topical induction sites of test group animals. .
Reading:
other: Reading 1 hours after topical induction
Group:
negative control
Clinical observations:
No skin reactions were noted at the topical induction sites of control group animals.
Remarks on result:
other: Reading: other: Reading 1 hours after topical induction. Group: negative control. Clinical observations: No skin reactions were noted at the topical induction sites of control group animals. .
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: reading & time point not stated
Group:
positive control
Dose level:
2-Mercaptobenzothiazole, 5% resp. 50% intradermal or topical induction, 50% and 25% challenge
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
not stated
Remarks on result:
positive indication of skin sensitisation
Reading:
other: reading & time point not stated
Group:
positive control
Dose level:
alpha-Hexylcinnamaldehyde 5% resp. 100% intradermal or topical induction, 100% and 75% challenge
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
not stated
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
not sensitising
Remarks:
Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitizer to guinea pig skin. Criteria used for interpretation of results: EU-GHS
Conclusions:
The GLP-study was performed according to the OECD Guideline 406, guinea pig maximisation method according to Magnusson and Kligman without deviations and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did not induce any response. The test material was considered to be not sensitising under the conditions of the test since no evidence of sensitizing or allergenic properties was found.
Executive summary:

The test item was tested for its sensitising potential according to OECD Guideline 406 in guinea pigs (Driscoll, 2001). The procedure used was performed according to the guinea pig maximisation method - Magnusson and Kligman. The test included three phases: an intradermal induction phase, when test material (0.1mL of a 5 % w/w formulation of the test material in distilled water and 5 % w/w formulation of the test material in a 1:1 preparation of Freud's Complete Adjuvant plus distilled water) is applied intradermally to the test animals; after an one-week rest period, a topical induction with 5 % w/w formulation in water for 24 hours was conducted. Finally after a two-week rest period, a challenge was performed (on day 21 after intradermal induction) with 75 % w/w or 50 % w/w formulation in distilled water for 24 hours. 10 test animals and 5 control animals were used in the study. Treated skin sites were scored according scale as described in the Guinea Pig Maximisation Test of Magnusson B & Kligman. 24- and 48-hour readings were performed after the challenge applications. Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate. Therefore the substance is not considered to be as skin sensitizer and was classified as a non-sensitizer to guinea pig skin.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report date:
2001

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
Commission Directive 96/54/EC Method B6
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
No new study has been conducted; data from a existing GLP-guideline study (2001) has been used as this study is totally sufficient to cover this endpoint. This study was conducted prior to the acceptance of alternative methods or REACH.

Test material

Constituent 1
Reference substance name:
3-(1-Pyridinio)-1-propanesulfonate
IUPAC Name:
3-(1-Pyridinio)-1-propanesulfonate
Constituent 2
Chemical structure
Reference substance name:
1-(3-sulphonatopropyl)pyridinium
EC Number:
239-491-3
EC Name:
1-(3-sulphonatopropyl)pyridinium
Cas Number:
15471-17-7
Molecular formula:
C8H11NO3S
IUPAC Name:
3-pyridinium-1-ylpropane-1-sulfonate
Test material form:
solid
Details on test material:
- Name of test material (as cited in study report): PPS solid
- Substance type: organic salt
- Physical state: white solid
- Storage condition of test material: room temperature in the dark

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited, Burton-on-Trent, Staffordshire, UK
- Age at study initiation: approximately eight to twelve weeks
- Weight at study initiation: 300 to 450g,
- Housing: housed singly or in pairs in solid-floor polypropylene cages furnished with woodflakes
- Diet (e.g. ad libitum): Free access to and food (Certified Guinea Pig Diet (Code 5026) supplied by PMI Nutrition International, Nottingham, UK) was allowed throughout the study. The diet was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Water (e.g. ad libitum): Free access to mains tap water was allowed throughout the study. The drinking water was routinely analysed and was considered not to contain any contaminant that could reasonably be expected to affect the purpose or integrity of the study
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17 to 23°C
- Humidity (%): 30 to 70%
- Air changes (per hr): at least fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light (06:00 to 18:00) and twelve hours darkness

OTHER:
- each animal was selected at random and given a number unique within the study which was written on a small area of clipped rump using a black indelible marker-pen
- the animals were provided with environmental enrichment items which were considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 5% w/w in distilled water
Topical Induction: 75% w/w in distilled water
Topical Challenge: 75% and 50% w/w in distilled water
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: distilled water
Concentration / amount:
Based on the results of sighting tests, the concentrations of test material for the induction and challenge phases were selected as:
Intradermal Induction: 5% w/w in distilled water
Topical Induction: 75% w/w in distilled water
Topical Challenge: 75% and 50% w/w in distilled water
No. of animals per dose:
Ten test and five control animals
Details on study design:
RANGE FINDING TESTS:
Selection of Concentrations for Main Study (Sighting Tests)
The concentrations of test material to be used at each stage of the main study were determined by 'sighting tests' in which groups of guinea pigs were treated with various concentrations of test material. The procedures were as follows:
Selection of Concentration for Intradermal Induction: Intradermal injections (0.1 mL/injection site) were made on the clipped shoulder of two guinea pigs, using concentrations of 1% and 5% w/w in distilled water. The degree of erythema at the injection sites was assessed approximately 24, 48, 72 hours and 7 days after injection. The degree of oedema was not evaluated. Any evidence of systemic toxicity was also recorded. The highest concentration that caused only mild to moderate skin irritation, and which was well tolerated systemically, was selected for the intradermal induction stage of the main study.
Selection of Concentration for Topical Induction: Two guinea pigs (intradermally injected with Freund's Complete Adjuvant eight days earlier) were treated with four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water). Applications were made to the clipped flanks under occlusive dressings for an exposure period of 48 hours. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest concentration producing only mild to moderate dermal irritation was selected for the topical induction stage of the main study.
Selection of Concentration for Topical Challenge: Four preparations of the test material (75%, 50%, 25% and 10% w/w in distilled water) were applied to the clipped flanks of two guinea pigs under occlusive dressings for an exposure period of 24 hours. These guinea pigs did not form part of the main study but had been treated identically to the control animals of the main study, up to Day 14. The degree of erythema and oedema was evaluated approximately 1, 24 and 48 hours after dressing removal. The highest non-irritant concentration of the test material and one lower concentration were selected for the topical challenge stage of the main study.

MAIN STUDY
A. INDUCTION EXPOSURE
intradermal injection
Shortly before treatment on Day 0 the hair was removed from an area approximately 40 mm x 60 mm on the shoulder region of each animal with veterinary clippers. A row of three injections (0.1 mL each) was made on each side of the mid-line into a 20 mm x 40 mm area. The injections were:
a) Freund's Complete Adjuvant plus distilled water in the ratio 1:1
b) a 5% w/w formulation of the test material in distilled water
c) a 5% w/w formulation of the test material in a 1:1 preparation of Freund's Complete Adjuvant plus distilled water.
Approximately 24 and 48 hours after intradermal injection the degree of erythema at the test material injection sites (ie. injection site b) was evaluated
- No. of exposures: 1
- Exposure period: 24 / 48
- Test groups: 10 males
- Control group: 5 males
- Site: an area approximately 40 mm x 60 mm on the shoulder region of each animal
- Frequency of applications: once
- Duration: 24 / 48
- Concentrations: 5 % w/w formulation of the test material in distilled water and 5 % w/w formulation of the test material in a 1:1 preparation of Freud's Complete Adjuant plus distilled water
topical induction
On Day 7 the same area on the shoulder region used previously for intradermal injections was clipped again and treated with a topical application of the test material formulation. A filter paper patch (WHATMAN No. 4: approximate size 40 mm x 20 mm), saturated with the test material formulation (75% w/w in distilled water) was applied to the prepared skin and held in place with a strip of surgical adhesive tape covered with an overlapping length of aluminium foil. The patch and foil were further secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal. This occlusive dressing was kept in place for 48 hours.
- No. of exposures: one
- Day(s) of induction: day 7 after induction
- Exposure period: 48 hours
- Site: the same area on the shoulder region used previously for intradermal injections
- Concentrations: 75 % w/w formulation in distilled water
- Evaluation (hr after topical induction): The degree of erythema and oedema was quantified one and twenty-four hours following removal of the patches

B. CHALLENGE EXPOSURE
Shortly before treatment on Day 21, an area of approximately 50 mm x 70 mm on both flanks of each animal, was clipped free of hair with veterinary clippers. A square filter paper patch (WHATMAN No. 4: approximate size 20 mm x 20 mm), saturated with the test material formulation at the maximum non-irritant concentration (75% w/w in distilled water) was applied to the shorn right flank of each animal and was held in place with a strip of surgical adhesive tape. To ensure that the maximum non-irritant concentration was used at challenge, the test material at a concentration of 50% w/w in distilled water was similarly applied to a skin site on the left shorn flank. The patches were occluded with an overlapping length of aluminium foil and secured with a strip of elastic adhesive bandage wound in a double layer around the torso of each animal.
After 24 hours, the dressing was carefully removed and discarded. The challenge sites were swabbed with cotton wool soaked in distilled water to remove residual material. The position of the treatment sites was identified by using a black indelible marker-pen.
Prior to the 24-hour observation the flanks were clipped using veterinary clippers to remove regrown hair.
- No. of exposures: one
- Day(s) of challenge: day 21 after induction
- Exposure period: 24 hours
- Site: an area of approximately 50 mm x 70 mm on both flanks of each animal
- Concentrations: 75 % w/w formulation in distilled water and on the right flank: 50 % w/w formulation in distilled water
- Evaluation (hr after challenge): Approximately 24 and 48 hours after challenge dressing removal, the degree of erythema and oedema was quantified

OTHER: Determination by analysis of the concentration, homogeneity and stability of the test material preparations was not conducted.
Positive control substance(s):
yes
Remarks:
parallel studies, either 2-Mercaptobenzothiazole or alpha-Hexylcinnamaldehyde

Results and discussion

Positive control results:
2-Mercaptobenzothiazole or alpha-Hexylcinnamaldehyde were shown to be sensitizer.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
other: Reading 24 hours after intradermal induction
Group:
test chemical
Dose level:
5 % w/w formulation in distilled water
Clinical observations:
Discrete or patchy erythema was noted at the intradermal induction sites of test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 24 hours after intradermal induction. Group: test group. Dose level: 5 % w/w formulation in distilled water. Clinical observations: Discrete or patchy erythema was noted at the intradermal induction sites of test group animals. .
Reading:
other: Reading24 hours after intradermal induction
Group:
negative control
Dose level:
distilled water
Clinical observations:
Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.
Remarks on result:
other: Reading: other: Reading24 hours after intradermal induction. Group: negative control. Dose level: distilled water. Clinical observations: Discrete or patchy erythema was noted at the intradermal induction sites of control group animals. .
Reading:
other: Reading 1 hour after topical induction
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
Clinical observations:
Discrete or patchy erythema was noted at the topical induction sites of test group animals.
Remarks on result:
other: see Remark
Remarks:
Reading: other: Reading 1 hour after topical induction. Group: test group. Dose level: 75 % w/w and 50 % w/w formulation in distilled water. Clinical observations: Discrete or patchy erythema was noted at the topical induction sites of test group animals. .
Reading:
other: Reading 1 hours after topical induction
Group:
negative control
Clinical observations:
No skin reactions were noted at the topical induction sites of control group animals.
Remarks on result:
other: Reading: other: Reading 1 hours after topical induction. Group: negative control. Clinical observations: No skin reactions were noted at the topical induction sites of control group animals. .
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
75 % w/w and 50 % w/w formulation in distilled water
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
other: reading & time point not stated
Group:
positive control
Dose level:
2-Mercaptobenzothiazole, 5% resp. 50% intradermal or topical induction, 50% and 25% challenge
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
not stated
Remarks on result:
positive indication of skin sensitisation
Reading:
other: reading & time point not stated
Group:
positive control
Dose level:
alpha-Hexylcinnamaldehyde 5% resp. 100% intradermal or topical induction, 100% and 75% challenge
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
not stated
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate and was classified as a non-sensitizer to guinea pig skin. Criteria used for interpretation of results: EU-GHS
Conclusions:
The GLP-study was performed according to the OECD Guideline 406, guinea pig maximisation method according to Magnusson and Kligman without deviations and is considered to be reliable without restrictions (reliability Klimisch 1). The test material did not induce any response. The test material was considered to be not sensitising under the conditions of the test since no evidence of sensitizing or allergenic properties was found.
Executive summary:

The test item was tested for its sensitising potential according to OECD Guideline 406 in guinea pigs (Driscoll, 2001). The procedure used was performed according to the guinea pig maximisation method - Magnusson and Kligman. The test included three phases: an intradermal induction phase, when test material (0.1mL of a 5 % w/w formulation of the test material in distilled water and 5 % w/w formulation of the test material in a 1:1 preparation of Freud's Complete Adjuvant plus distilled water) is applied intradermally to the test animals; after an one-week rest period, a topical induction with 5 % w/w formulation in water for 24 hours was conducted. Finally after a two-week rest period, a challenge was performed (on day 21 after intradermal induction) with 75 % w/w or 50 % w/w formulation in distilled water for 24 hours. 10 test animals and 5 control animals were used in the study. Treated skin sites were scored according scale as described in the Guinea Pig Maximisation Test of Magnusson B & Kligman. 24- and 48-hour readings were performed after the challenge applications. Under the conditions of the test, the test material produced a 0% (0/10) sensitisation rate. Therefore the substance is not considered to be as skin sensitizer and was classified as a non-sensitizer to guinea pig skin.