1-(3-sulphonatopropyl)pyridinium

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2001-09-07 - 2001-10-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2001

Report date:

2001

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

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.

Test material

Test material information

Constituent 1

Reference substance name:

3-(1-Pyridinio)-1-propanesulfonate

IUPAC Name:

3-(1-Pyridinio)-1-propanesulfonate

Constituent 2

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:

1-(2-hydroxy-3-sulphonatopropyl)pyridinium

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
- Other: date receied: 19. July 2001

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)

Induction

Induction 1

Route:

intradermal

Vehicle:

other: distilled water

Induction 2

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

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: distilled water

Challenge 2

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 prepartion 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.

Results and discussion

In vivo (non-LLNA)

Results

Results 1

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. .

Results 2

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. .

Results 3

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. .

Results 4

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. .

Results 5

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:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 75 % w/w and 50 % w/w formulation in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Results 6

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:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 75 % w/w and 50 % w/w formulation in distilled water. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: none.

Any other information on results incl. tables

Skin Reactions Observed After Intradermal Induction:

- Discrete or patchy erythema was noted at the intradermal induction sites of test group animals.

- Discrete or patchy erythema was noted at the intradermal induction sites of control group animals.

Table 1	Intradermal Sighting Test- Summary of Results
VEHICLE:     Distilled Water
Animal Identification	Time of Observation	Concentration of Test Material(%w/w)	Grade of Erythema at Injection Sites	Evidence of Systemic Toxicity
A	24 Hours	1	2	None
	48 Hours		1	None
	72 Hours		0	None
	7 Days		0	None
B	24 Hours	5	2	None
	48 Hours		1-2	None
	72 Hours		0	None
	7 Days		0	None
The concentration of the test material selected for the intradermal induction stage of the main study was 5% w/w in distilled water

Table 2 - Intradermal Induction-Individual Skin Reactions
Group	Animal Number	Grade of Erythema at Observation Site
		24Hours		48Hours
		Left Side	Right Side	Left Side	Right Side
TEST	1	1	1	0	0
	2	1	1	1	0
	3	1	1	0	0
	4	1	1	0	0
	5	1	1	0	0
	6	1	1	1	1
	7	1	1	0	1
	8	1	1	1	1
	9	1	1	0	0
	10	1	1	0	0
CONTROL	11	0	0	0	0
	12	0	0	0	0
	13	1	1	0	0
	14	1	1	0	0
	15	1	1	0	0

Skin Reactions Observed After Topical Induction:

- Discrete or patchy erythema was noted at the topical induction sites of test group animals.

- No skin reactions were noted at the topical induction sites of control group animals.

- Bleeding from the intradermal injection sites was noted in six test group animals with residual test material noted at the intradermal injection sites of four test group animals at the 1-hour observation.

Table 3	Topical Sighting Test for Induction Application (48-Hour Exposure) - Individual Skin Reactions
VEHICLE:	Distilled Water
Animal Identification	Concentration of	Skin Reactions (Hours After Removal of Patches)
	Test Material(% w/w)	1			24			48
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
C	75	1	0	-	0	0	-	0	0	-
	50	1	0	-	0	0	-	0	0	-
	25	0	0	-	0	0	-	0	0	-
	10	0	0	-	0	0	-	0	0	-
D	75	1	0	-	0	0	-	0	0	-
	50	1	0	-	0	0	-	0	0	-
	25	0	0	-	0	0	-	0	0	-
	10	0	0	-	0	0	-	0	0	-
The concentration of the test material selected for the main study topical induction was 75% w/w in distilled water
Er = Erythema
Oe = Oedema
- = No other reactions noted

Table 4	Topical Induction - Individual Skin Reactions
Group	Animal Number	Skin Reactions (Hours After Removal of Dressing)
		1 Hour			24 Hours
		Er	Oe	Other	Er	Oe	Other
TEST	1	1	0	Bs	1	0	-
	2	1	0	BsRt	1	0	-
	3	1	0	-	0	0	-
	4	1	0	Bs	1	0	-
	5	1	0	BsRt	0	0	-
	6	1	0	-	0	0	-
	7	1	0	BsRt	0	0	-
	8	1	0	BsRt	1	0	-
	9	1	0	-	1	0	-
	10	1	0	-	1	0	-
CONTROL	11	0	0	-	0	0	-
	12	0	0	-	0	0	-
	13	0	0	-	0	0	-
	14	0	0	-	0	0	-
	15	0	0	-	0	0	-
Er = Erythema
Oe = Oedema
Bs = Bleeding from intradermal injection sites
- = No other reactions noted
Rt = Residual test material

Skin Reactions Observed After Topical Challenge:

- 75% w/w in Distilled Water: No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

- 50% w/w in Distilled Water: No skin reactions were noted at the challenge sites of the test or control group animals at the 24 or 48-hour observations.

Table 5	Topical Sighting Test for Challenge Application (24-Hour Exposure) - Individual Skin Reactions
VEHICLE:	Distilled Water
Animal Identification	Concentration of Test Material(% w/w)	Skin Reactions (Hours After Removal of Patches)
		1			24			48
		Er	Oe	Other	Ers	Oe	Other	Er	Oe	Other
E	75	0	0	-	0	0	-	0	0	-
	50	0	0	-	0	0	-	0	0	-
	25	0	0	-	0	0	-	0	0	-
	10	0	0	-	0	0	-	0	0	-
F	75	0	0	-	0	0	-	0	0	-
	50	0	0	-	0	0	-	0	0	-
	25	0	0	-	0	0		0	0	-
	10	0	0	-	0	0	-	0	0	-
The concentrations of the test material selected for the main study topical challenge were 75% and 50% w/w in distilled water
Er = Erythema
Oe = Oedema
- = No other reactions noted

Table 6	Individual Skin Reactions at Challenge
CHALLENGE CONCENTRATIONS:   75%and50%v/v
VEHICLE:		Distilled Water
Group	Animal Number	Skin Reactions (Hours after Removal of Dressings)
		24Hours						48Hours
		50%			75%			50%			75%
		Er	Oe	Other	Er	Oe	Other	Er	Oe	Other	Er	Oe	Other
TEST	1	0	0	-	0	0	-	0	0	-	0	0	-
	2	0	0	-	0	0	-	0	0	-	0	0	-
	3	0	0	-	0	0	-	0	0	-	0	0	-
	4	0	0	-	0	0	-	0	0	-	0	0	-
	5	0	0		0	0		0	0	_	0	0
	6	0	0	-	0	0	-	0	0	-	0	0	-
	7	0	0	-	0	0	-	0	0	-	0	0	-
	8	0	0	-	0	0	-	0	0	-	0	0	-
	9	0	0	-	0	0	-	0	0	-	0	0	-
	10	0	0	-	0	0	-	0	0	-	0	0	-
CONTROL	11	0	0	-	0	0	-	0	0	-	0	0	-
	12	0	0	-	0	0	-	0	0	-	0	0	-
	13	0	0	-	0	0	-	0	0	-	0	0	-
	14	0	0	-	0	0	-	0	0	-	0	0	-
	15	0	0	-	0	0	-	0	0	-	0	0	-
Er =Erythema
Oe=Oedema
- =No other reactions noted

Table 7	Summary of Positive Control Data for the Magnusson and Kligman Maximisation Study
Project Number	Date Start	Date End	Number of Animals and Sex*		Positive Control Material	Concentration			Incidence of Sensitisation
						Induction		Challenge
			Test	Control		Intradermal	Topical
039/333	22/12/98	05/02/99	10 Female	5 Female	2-Mercaptobenzothiazole	10% in arachis oil BP	50%in acetone: PEG400 (70:30)	50% and 25% in acetone: PEG400 (70:30)	90% (9/10)
039/370	14/06/99	17/07/99	10 Male	5 Male	2-Mercaptobenzothiazole	5% in arachis oil BP	50%in acetone: PEG400 (70:30)	50% and 25% in acetone: PEG400 (70:30)	100% (10/10)
039/422	12/01/00	05/02/00	10 Female	5 Female	2-Mercaptobenzothiazole	5% in arachis oil BP	50%in acetone: PEG400 (70:30)	50% and 25% in acetone: PEG400 (70:30)	100% (10/10)
039/444	29/06/00	22/07/00	10 Male	5 Male	2-Mercaptobenzothiazole	5% in arachis oil BP	50%in acetone: PEG400 (70:30)	50% and 25% in acetone: PEG400 (70:30)	100% (9/9)
039/446	28/06/00	06/08/00	10 Male	5 Male	cc-Hexylcinnamaldehyde	5% in arachis oil BP	100%	100% and 75% in arachis oil BP	50% (5/10)
039/458	25/01/01	25/02/01	10 Male	5 Male	oc-Hexylcinnamaldehyde	5% in arachis oil BP	100%	100% and 75% in arachis oil BP	40% (4/10)
*All animals supplied bv David Hall Ltd. Burton-on-Trent. Staffordshire. UK

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information 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: other: 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.