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Skin sensitisation

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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 guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
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

Constituent 1
Reference substance name:
hydrogen sulfate;tetrakis(hydroxymethyl)phosphanium;urea
EC Number:
613-239-6
Cas Number:
63502-25-0
Molecular formula:
Not applicable
IUPAC Name:
hydrogen sulfate;tetrakis(hydroxymethyl)phosphanium;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)

Resultsopen allclose all
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
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
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.