[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 2016-06-24 to 2016-12-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes (incl. QA statement)

Remarks:

2015-11-03

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

A GPMT was performed because these type of study was available in this chemiacal family.A comparison could be therefore done between difference substance of the family.

Test material

Test material information

Constituent 1

Reference substance name:

Tetrakis(hydroxymethyl)phosphonium sulphate (2:1) and its oligomerisation products with urea

Molecular formula:

Not applicable

IUPAC Name:

Tetrakis(hydroxymethyl)phosphonium sulphate (2:1) and its oligomerisation products with urea

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River France, L’arbresle Cedex, France.
- Females
- Age at study initiation: approx. 5-6 weeks old
- Weight: 288.0 to 353.0 g at initiation
- Housing: Noryl cage (Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and shelters (CS3B02A Play tunnels (90 mm x 5 mm x 125 mm), Datesand, Manchester, UK) as cage enrichment.
- Diet (e.g. ad libitum): Complete maintenance diet for guinea pigs (SSNIFF® Spezialdiäten GmbH, Soest, Germany). In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water (e.g. ad libitum): Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%
- Air changes (per hr): at least 10 air changes/hour
- Photoperiod (hrs dark / hrs light): 2-hour light/12-hour dark cycle. The light/dark cycle may be interrupted for study related activities
- IN-LIFE DATES: From: To: from 2016-07-01 to 2016-08-12

Study design: in vivo (non-LLNA)

Induction

Route:

intradermal

Vehicle:

water

Concentration / amount:

0.1%

Day(s)/duration:

22 days

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Challenge

Route:

epicutaneous, occlusive

Vehicle:

unchanged (no vehicle)

Concentration / amount:

100%

Day(s)/duration:

1 day

Adequacy of challenge:

not specified

No. of animals per dose:

15 (5 in control group and 10 for the treated group)

Details on study design:

Preliminary Irritation Study
A preliminary irritation study was conducted in order to select test item concentrations to be used in the main study. The selection of concentrations was based on the following criteria:
• The concentrations are well-tolerated systemically by the animals.
• For the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3).
• For challenge exposure: the maximum non-irritant concentration.
Series of test item concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1% and if needed, further lower concentrations using the same steps. The test system and procedures were identical to those used during the main study, unless otherwise specified. The eight animals selected were 5 weeks old. No body weights were determined.

Intradermal injections:
Initially, a series of four test item concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment. Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.

Epidermal application:
A series of four test item concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches# (2x3 cm) mounted on Medical tape#, which were held in place with Micropore tape# and subsequently Coban elastic bandage#. The initially used animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test item using water. The resulting dermal reactions were assessed for irritation 24 and 48 hours after removal of the dressings.

Main Study
The concentrations and induction method were selected based on the results of the preliminary irritation study.
Induction - Experimental animals
Day 1 The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of Freunds' Complete Adjuvant (Sigma-Aldrich, Steinheim, Germany) with water for injection (B.Braun Melsungen AG, Melsungen. Germany).
B) The test item at a 0.1% concentration.
C) A 1:1 w/w mixture of the test item, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Day 3 The dermal reactions caused by the intradermal injections were assessed for irritation.
Day 7 The scapular area between the injection sites was clipped and subsequently rubbed with 10% sodium-dodecyl-sulfate (SDS, Boom, Meppel, The Netherlands) in vaseline using a spatula. This concentration of SDS causes mild irritation of the skin.
Day 8 The 10% SDS treated area between the injection sites was treated with 0.5 mL of a 100% test item concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 48 hours exposure, the skin cleaned of residual test item using water and the dermal reactions caused by the epidermal exposure were assessed for irritation.

Induction - Control animals
The control animals were treated as described for the experimental animals except that, instead of the test item, the vehicle was administered.

Challenge - All animals
Day 22 One flank of all animals was clipped and treated by epidermal application of a 100% test item concentration and the vehicle (0.1 mL each), using Patch Test Plasters (Curatest F®, Lohmann, Almere, The Netherlands). The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
The dressing was removed after 24 hours exposure and the skin cleaned of residual test item and vehicle using water. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing.
After termination, animals were sacrificed using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and an intra-peritoneal injection of Euthasol® 20% (AST Farma BV, Oudewater, The Netherlands).

Challenge controls:

See detail on study design

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

1st reading

Hours after challenge:

72

Group:

negative control

Dose level:

100%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Results 2

Key result

Reading:

1st reading

Hours after challenge:

72

Group:

test chemical

Dose level:

100%

No. with + reactions:

10

Total no. in group:

10

Remarks on result:

positive indication of skin sensitisation

Results 3

Key result

Reading:

1st reading

Hours after challenge:

72

Group:

positive control

Remarks on result:

not measured/tested

Any other information on results incl. tables

PRELIMINARY IRRITATION STUDY

Skin reactions after intradermal injection

Animal number	Concentration %	24h after injection		48h after injection
		Erythema	Necrosis (Ø mm)	Erythema	Necrosis (Ø mm)
7	100		25		28
	50		20		20
8	20		15		17
	10		13		15
9	1		7		7
	0.5		3		3
10	0.2		1		2
	0.1	1		1

 

Skin reactions after intradermal injection

Animal number	Concentration %	24h after injection		48h after injection
		Erythema	Oedema	Erythema	Oedema
5	100	0	0	0	0
	50	0	0	0	0
6	100	0	0	0	0
	50	0	0	0	0
7	20	0	0	0	0
	10	0	0	0	0
8	20	0	0	0	0
	10	0	0	0	0

0 = No erythema

0 = No oedema

 

Induction reading

Control animals

	Intradermal injections (Readings Day 3)						Epidermal exposure (Reading day 10)
Animal number	1:1 Mixture of FCA and water for injection		Vehicle		1:1 Mixture of FCA and vehicle		Vehicle
	Erythema	signs of necrosis (Ø mm)	Erythema	signs of necrosis (Ø mm)	Erythema	signs of necrosis (Ø mm)	Erythema	Oedema
41		3	0		2		0	0
42	3		0		3		1	0
43	3		0		3		0	0
44	2		0		3		0	0
45	2		0		2		0	0

 

Treated animals

	Intradermal injections (Readings Day 3)						Epidermal exposure (Reading day 10)
Animal number	1:1 Mixture of FCA and water for injection		Vehicle		1:1 Mixture of FCA and vehicle		Vehicle
	Erythema	signs of necrosis (Ø mm)	Erythema	signs of necrosis (Ø mm)	Erythema	signs of necrosis (Ø mm)	Erythema	Oedema
46		3	1		2		1	0
47	2		0		3		1	0
48	2		0		3		1	0
49	3		0		3		1	0
50		4	0		2		0	0
51		3	1		3		1	0
52	3		1		3		1	0
53		3	1		3		1	0
54	3		1		3		1	0
55	3		1		3		0	0

FCA = Freunds' Complete Adjuvant

Grading erythema:

0 = No erythema

1 = Slight erythema (barely perceptible)

Grading Oedema:

0 = No oedema

 

Challenge reading

Animal number	Group	Day 24		Day 25
		Test concentration 100%	Vehicle	Test concentration 100%	Vehicle	Comments
41	Control	0	0	0	0
42		0	0	0	0
43		0	0	0	0
44		0s	0	0s	0
45		0	0	0	0
46	Experimental	1	0	1	0	sensitized
47		2	0	2	0	sensitized
48		2	0	1	0	sensitized
49		2	0	1	0	sensitized
50		2	0	2	0	sensitized
51		2	0	2	0	sensitized
52		1	0	1	0	sensitized
53		3k	0	2k	0	sensitized
54		4k	0	3k	0	sensitized
55		1	0	1	0	sensitized

s. Scaliness, k. Scabs

Grading challenge reactions:

0 = No visible change

1 = Discrete or patch erythema

2 = Moderate and confluent erythema

3 = Moderate erythema and swelling

4 = Intense erythema and swelling

 

Body weight (g)

Sex / group	Animal	Day 1	Day 25
Female Control	41	288.0	391.0
	42	290.0	422.0
	43	343.0	465.0
	44	341.0	495.0
	45	304.0	427.0
MEAN		313..2	440.0
ST DEV.		27.0	40.4
N		5	5
Female experimental	46	321.0	419.0
	47	347.0	484.0
	48	353.0	460.0
	49	330.0	459.0
	50	347.0	484.0
	51	318.0	436.0
	52	310.0	3980
	53	329.0	418.0
	54	309.0	427.0
	55	373.0	492.0
MEAN		333.7	447.7
ST DEV.		20.8	32.7
N		10	10

Applicant's summary and conclusion

Interpretation of results:

Category 1A (indication of significant skin sensitising potential) based on GHS criteria

Conclusions:

The skin reactions observed in response to a 100% test item concentration in ten (of the ten) experimental animals in the challenge phase were considered indicative of sensitization, based on the absence of any positive response in the control animals.

Executive summary:

In a dermal sensitization study with Phosphonium, tetrakis(hydroxymethyl)-, sulphate (2:1) (salt), polymer with urea. female albino guinea pigs (10 treated females + 5 controls) were tested in compliance with OECD 406, method B6 in Commission Directive 84/449/EEC and in compliance with GLP. 

Based on the results of the pilot tests, the concentrations of test material were 0.1 % (w/v) for the intradermal applications of the induction phase, respectively, and 100 % in the challenge phase.

10/10 animals were sensitized. Phosphonium, tetrakis(hydroxymethyl)-, sulphate (2:1) (salt), polymer with urea was therefore considered to be skin sensitizer cat 1A.