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Toxicological information

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:
1995-10-31 - 1995-12-03
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1996
Report date:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitisation". Adopted 17 July 1992.
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig: Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A.
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Study was conducted prior to the implementation of REACH and adoption of the LLNA or suitable in vitro methods.

Test material

Constituent 1
Reference substance name:
bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide
EC Number:
605-708-9
Cas Number:
174125-93-0
Molecular formula:
n.a.
IUPAC Name:
bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide
Test material form:
liquid
Remarks:
Orange/brown
Specific details on test material used for the study:
STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: D. Hall, Newchurch, Staffordshire, England.
- Age at study initiation: four to five weeks
- Weight at study initiation: 294 to 327 g on arrival
- Housing: The guinea-pigs were housed in groups of five in suspended metal cages with wire mesh floors.
- Diet (e.g. ad libitum): A vitamin C enriched guinea-pig diet FD2 was provided ad libitum. Hay was given weekly.
- Water (e.g. ad libitum): ad libitum
- Acclimation period: All the guinea-pigs were acclimatised to the experimental environment for six days prior to allocation to the main study.
- Indication of any skin lesions: none stated

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Animal room temperature was maintained at approximately 21 °C.
- Humidity (%): Relative humidity was maintained at 30 - 70 %.
These environmental parameters were recorded daily.
- Air changes (per hr): Air exchange was maintained at approximately 15 air changes per hour.
- Photoperiod (hrs dark / hrs light): Lighting was controlled by means of a time switch to give 12 hours of artificial light (0700 - 1900 hours) in each 24 hours period.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Alembicol D
Concentration / amount:
0.1 ml of 0.5% solution
Day(s)/duration:
day 1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
No. 3 paper was saturated with approximately 0.4 ml of 100% test item
Day(s)/duration:
six days after intradermal challenge for 48 h
Adequacy of induction:
highest technically applicable concentration used
Challenge
No.:
#1
Route:
epicutaneous, semiocclusive
Vehicle:
other: Alembicol D
Concentration / amount:
Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item as supplied and 50% v/v solution
Day(s)/duration:
two weeks after the topical induction for 24h
No. of animals per dose:
Six animals for the preliminary investigations, ten test and five control animals were used.
Details on study design:
RANGE FINDING TESTS:
Preliminary study
The intradermal and topical irritancy of a range of dilutions of the test substance was investigated to identify where possible (a) concentrations of the test substance that would produce irritation suitable for the induction phase of the main study and (b) a maximum non-irritant concentration by the topical route of administration for the challenge phase.
Animals for these investigations were pre-treated with an intradermal injection of Freund's complete adjuvant, 50:50 with water for irrigation (Ph. Eur), approximately two weeks prior to the start of the preliminary investigations.
Selection of concentrations of test substance for the main study: Based on the results of the preliminary investigations, the following concentrations of the test item were selected:
Induction intradermal injection - 0.5% v/v in Alembicol D (A product of coconut oil, supplied by Alembic Products, Saltney, Chester, England): This was the highest practical concentration that caused irritation but did not adversely affect the animals.
Induction topical application - as supplied
Topical challenge - as supplied and 50% v/v in Alembicol D
This was the highest practical concentration that could be dosed topically and did not give rise to irritating effects.

MAIN STUDY
A. INDUCTION EXPOSURE
Induction intradermal injections - test animals
A 40 x 60 mm area of dorsal skin on the scapular region of the guinea-pig was clipped free of hair with electric clippers. Three pairs of intradermal injections were made into a 20 x 40 mm area within the clipped area as shown in Figure 1.
Injectables for the test animals were prepared as follows:
1. Freund's complete adjuvant** was diluted with an equal volume of water for irrigation (Ph.Eur.).
2. Test item, 0.5% v/v in Alembicol D.
3.Test item, 0.5% v/v in a 50 : 50 mixture of Freund's complete adjuvant and Alembicol D.
** Difco Laboratories, Detroit 1, Michigan, U.S.A.
Induction topical application - test animals
The preliminary investigations indicated that the maximum practical concentration of the test substance for topical application (as supplied) did not produce skin irritation. Therefore, six days after the injections, the same 40 x 60 mm interscapular area was clipped and shaved free of hair and the site was pre-treated by gentle rubbing with 0.5 mJ per site of 10% w/w sodium lauryl sulphate in petrolatum. Twenty-four hours later a 20 x 40 mm patch of Whatman No. 3 paper was saturated with approximately 0.4 ml of the test item, as supplied. The patch was placed on the skin of the test animals and covered by a length of impermeable plastic adhesive tape (50 mm width "Blenderm"). This in turn was firmly secured by elastic adhesive bandage (50 mm width "Elastoplast") wound round the torso of the animal and fixed with "Sleek" impervious plastic adhesive tape. The dressing was left in place for 48 hours.
Induction - control animals
During the induction phase, the control animals were treated similarly to the test animals with the exception that the test substance was omitted from the intradermal injections and topical application.

B. CHALLENGE EXPOSURE
Challenge - control and test animals
The control and test animals were challenged topically two weeks after the topical induction application using the test item, as supplied and 50% v/v in Alembicol D.
Hair was removed by clipping and then shaving from an area on the left flank of each guinea-pig. A 20 x 20 mm patch of Whatman No. 3 paper was saturated with approximately 0.2 ml of the test item, as supplied and applied to an anterior site on the flank. The test item, 50% v/v in Alembicol D was applied in a similar manner to a posterior site. The patches were sealed to the flank for 24 hours under strips of "Blenderm" covered by "Elastoplast" wound round the trunk and secured with "Sleek".
Observations were made 24, 48, and 72 h after challenge
Challenge controls:
yes, animals not induced with the test item
Positive control substance(s):
yes
Remarks:
The sensitivity of the guinea-pig strain used is checked periodically at the laboratory with hexyl cinnamic aldehyde, a known sensitiser.

Results and discussion

Positive control results:
In the available summary of 7 studies with the positive control, it induced the following positive responses (No. of animals with reactions / total number of animals): 10/10, 9/10, 10/10, 9/10, 10/10, 10/10, 8/10.
The positive control gave the appropriate results, and the used guinea pig strain showed the required sensitivity.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
undiluted & 50%
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
5
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
50%
No. with + reactions:
0
Total no. in group:
10
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
undiluted
No. with + reactions:
6
Total no. in group:
10
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
50%
No. with + reactions:
1
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
undiluted & 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
only a few incidences of dryness and sloughing of the epidermis
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
undiluted & 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
only a few incidences of dryness and sloughing of the epidermis
Remarks on result:
no indication of skin sensitisation
Reading:
other: 3rd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
undiluted & 50%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
only a few incidences of dryness and sloughing of the epidermis
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
other: reading, No. not specified
Group:
positive control
Dose level:
induction: 10% intradermal, topical as supplied; challenge: as supplied and 50%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Remarks:
Results in four studies; in 2 studies, 9/10 animals showed reacions, in one, 8/10 and 1 inconclusive result was revealed

Applicant's summary and conclusion

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
The study was conducted under GLP according to OECD TG 406 (guinea pig maximisation test) on the registered substance itself. The method is to be considered scientifically reasonable with no deficiencies. Positive and negative controls gave the appropriate response. Hence, the results can be considered as sufficiently reliable to assess the irritating properties of the test item.
In this study, the test item produced evidence of skin sensitisation (delayed contact hypersensitivity) in five of the ten test animals. One animal gave an inconclusive response and the remaining four animals gave negative responses.
According to Regulation 1272/2008 / 286/2011, Table 3.4.4 Animal test results for sub-category 1B, a substance must be classified as skin sensitizer if in the Guinea pig maximisation test ≥ 30 % to < 60 % of the animals are responding at > 0.1 % to ≤ 1 % intradermal induction dose or ≥ 30 % responding at > 1 % intradermal induction dose. Intradermal induction was 0.5%, and 50% responded. Hence, Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide must be regarded as skin sensitizer Cat. 1B.
Executive summary:

This study was performed to assess the skin sensitisation potential of Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide using the guinea-pig. The method followed was that described in: OECD Guideline for Testing of Chemicals No. 406 "Skin Sensitisation". Adopted 17 July 1992, and MAGNUSSON, B. and KLIGMAN, A.M. (1970) Allergic Contact Dermatitis in the Guinea-pig: Identification of contact allergens, Thomas, C.C., Springfield, Illinois, U.S.A.

Based on the results of a preliminary study and in compliance with the guideline, the following dose levels were selected:

Intradermal injection: 0.5% v/v in Alembicol D

Topical application: as supplied v/v in Alembicol D

Challenge application: as supplied and 50% v/v in Alembicol D

Ten test and five control guinea-pigs were used in this study.

In this study Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide produced evidence of skin sensitisation (delayed contact hypersensitivity) in five of the ten test animals. One animal gave an inconclusive response and the remaining four animals gave negative responses.

Bis(O,O-2-ethylhexyl-thiophosphoryl)polysulfide must be regarded as skin sensitizer Cat. 1B.