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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
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Study was conducted to internationally recognised guidelines but not to GLP.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report Date:
1988

Materials and methods

Test guideline
Guideline:
EPA OPP 81-6 (Skin Sensitisation)
Principles of method if other than guideline:
The study report claims compliance with OECD and EEC Guidelines, though the actual guidelines have not been stated.
GLP compliance:
no
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An LLNA study was not conducted because adequate data from a guinea pig maximisation test are already available.

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Specific details on test material used for the study:
Batch No. 88/3/1
Purity: Not provided.
Stability of active substance: Not provided
Storage: Dark ambient

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall Limited (Darly Oaks, Newchurch, Burton-on-Trent, Staffordshire, UK).
- Age at study initiation: Young adult, less than one year old.
- Weight at study initiation: 393-477g
- Housing: 20 test group and 20 control group guinea pigs were housed 5 to a cage. 6 dose ranging guinea pigs were housed in a ninth cage. Each aluminium cage had a grid floor, beneath which was a peat moss filled tray.
- Diet: FD1 guinea pig diet, supplied by Special Diet Services (1 Stepfield, Witham, Essex, UK, CM8 3AB), supplemented with hay and allowed ad libitum.
- Water: Tap water allowed ad libitum.
- Acclimation period: At least 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature: Maximum and minimum temperatures were 22°C and 19°C
- Humidity: Mean relative humidity was 49%.
- Air changes (per hr): Not reported.
- Photoperiod (hrs dark / hrs light): A 12 h light/dark cycle was in operation (light hours 0700-1900 h).

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
paraffin oil
Concentration / amount:
10% w/v
Day(s)/duration:
Day 0
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
25% w/v
Day(s)/duration:
Day 7
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
25% w/v
Day(s)/duration:
Day 21
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
6 range finding animals

20 test animals

20 control animals
Details on study design:
The Magnusson-Kligman Maximisation Test comprises 2 procedures; an induction procedure and a challenge procedure (Magnusson, B., Kligman, A.M. (1969), J. Invest. Dermat., 52, 268-276).

The induction procedure consists of an intradermal injection of the test material followed after one week by a topical application. The challenge procedure, which consists of a topical application, is carried out 3 weeks after commencement of the induction procedure.

The test was carried out using the following method:

RANGE FINDING TESTS:

A preliminary dose ranging test was carried out to assess suitable concentrations for injection and topical application on 4 previously untreated guinea pigs. Injections were in the shaved scapular region and topical application was on the shaved flanks. 2 guinea pigs were subjected to intradermal injections and a further 2 guinea pigs were exposed to topical application for 48 h of the test material at 4 different concentrations.
Injections - 10%, 5%, 2% and 1% w/v in paraffin oil.
Topical application - 25%, 10%, 5% and 2% w/v in paraffin oil.

The injection sites were assessed for irritancy 24, 48, and 72 h after injection. The topical application sites were assessed for irritancy 24, 48 and 72 h after patch removal.

MAIN STUDY:

MAIN TEST - Injection Phase
In the main test, hair was shaved from an area 4 cm x 6 cm across the scapular region of 42 guinea pigs. These animals were allocated as follows:
Test group: 20 guinea pigs
Control group: 20 guinea pigs
Challenge dose ranging group: 2 guinea pigs

The test group guinea pigs were each given 6 intradermal injections, 3 in a line each side of and parallel to the mid-line in the shaved region as follows:
0.10 ml Freund's Complete Adjuvant (anterior injection)
0.10 ml test material (middle injection)
0.05 ml test material emulsified with 0.05 ml Freund's Complete Adjuvant (posterior injection)

The test material was injected at a concentration of 10% w/v in paraffin oil. The 20 control group guinea pigs were similarly treated but with paraffin oil replacing the test material. Twenty four hours after injection, the treated sites of both test and control groups were assessed for irritancy using the scoring system described below.

The 2 guinea pigs intended for dose ranging for challenge were each given 0.01 ml intradermal injection of Freund's Complete Adjuvant on either side of the mid-line of the shaved scapular region.

MAIN TEST - Topical application phase
Six days after the injection phase, the injection site of each of the test and control group animals was shaved again and then wetted with 10% aqueous SLS to provoke a mild inflammatory response to enhance the possibility of sensitisation.

After 24h, a 2 cm x 4 cm patch of Whatman No. 3MM filter paper, charged with the test material at a concentration of 25% w/v in paraffin oil, was applied to the pretreated area of each of the test group animals and the patch covered by an overlapping piece of impermeable plastic adhesive tape (Blenderm). The whole area was then firmly bound with Sleek occlusive tape, and the dressing was left in place for 48 h before removal.

The control group guinea pigs were similarly treated but with paraffin oil replacing the test material.

Twenty four hours after patch removal, the treated sites of both test and control groups were assessed for irritancy using the scoring system described in the section below.

The 2 guinea pigs intended for dose range finding for challenge were untreated at this stage.

CHALLENGE EXPOSURE:

DOSE RANGING TEST FOR CHALLENGE
Before challenging the test animals, a maximum non-irritant concentration of the test material was determined in a preliminary experiment on 2 dose ranging guinea pigs. The animals were pretreated at the induction phase with Freund's Complete Adjuvant only.

The test material was applied to the shaved flanks of the guinea pigs for 24 h at concentrations of 25% and 10% w/v in paraffin oil using the same method as topical induction.

MAIN TEST - Challenge phase
Two weeks after the topical induction, both the test and control group guinea pigs were challenged with the test material at a concentration of 25% w/v in paraffin oil and the vehicle. A 5 cm x 5 cm area of the left flank was shaved and the test and control materials were applied to the prepared test site on 2 cm x 2 cm pieces of filter paper. The patches were held in place for 24 h using the same method as topical induction after which time they were removed.

ASSESSMENT OF RESPONSE
The degree of response was determined by trained assessors 24 h and 48 h after removal of the challenge patch when an allergic response would be at a peak. Any animal showing erythema at the site of challenge was considered to have shown a positive response.

Reactions are scored according to the following scale:
No visible change - 0
Slight or discrete erythema - 1
Moderate and confluent erythema - 2
Intense erythema and / or oedema - 3

The sensitisation potential of the test material is rated according to the percentage of animals sensitised, based on Magnusson-Kligman as follows:

SENSITISATION GRADE CLASSIFICATION
0-8% I Weak
9-28% II Mild
29-64% III Moderate
65-80% IV Strong
81-100% V Extreme
Positive control substance(s):
yes
Remarks:
2, 4-dinitro-chlorobenzene (DNCB)

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
24
Group:
test group
Dose level:
25% w/v in paraffin oil
No. with + reactions:
0
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
25% w/v in paraffin oil
No. with + reactions:
0
Total no. in group:
20

Any other information on results incl. tables

Dose Ranging Test for Induction

Details of reaction scores are given in Table 1.

Slight to moderate responses were noted at sites injected with Cuprous Thiocyanate at concentrations of 10%, 5%, 2% and 1% w/v in paraffin oil. A concentration of 10% w/v in paraffin oil was selected for the injection phase of induction in the test group.

Main Test - Induction

Details of reaction scores are given in Table 2.

Moderate irritation was noted in the test group with slight irritation noted in the control group.

Dose Ranging Test for Challenge

Details of reaction scores are given in Table 3.

No irritation was noted at any of the concentrations tested.

A concentration of 25% w/v in paraffin oil was selected for challenge.

Main Test - Challenge

Details of reaction scores are given in Table 4.

Following challenge with Cuprous Thiocyanate, at a concentration of 25% w/v in paraffin oil, none of the 20 test group animals showed positive reactions to this application. None of the 20 control group animals, which had previously only been exposed to the vehicle, paraffin oil showed a positive reaction to this application.

Body Weight

Details are given in Table 5.

Body weights were recorded at test commencement and on test completion. Body weight gains were within the acceptable range.

Clinical Signs

No clinical signs, other than skin reactions induced by treatment, were noted.

Table 1

Dose Ranging Test for Induction: Reaction Scores

Intradermal Injection

Time/Cage/Animal
24 h* 48 h* 72 h*
9 9 9
Concentration (w/v in Paraffin Oil) 1 2 1 2  1 2
10% 2 2 2
5% 2 2 2
2% 2 2 2
1% 1 1 2 2 2

 

Topical Application

Time/Cage/Animal

24 h*

48 h*

72 h*

9

9

9

Concentration (w/v in Paraffin Oil)

3

 4

3

4

3

4

25%

0

0

0

0

0

10%

0

0

0

0

0

5%

0

0

0

0

0

2%

0

 0

0

0

0

0

* = After injection/patch removal

Table 2

Main Test - Induction: Reaction Scores

 

 

 

 

 Injection site

 Topical site

 Group

 Material (w/v in Paraffin Oil)

 Cage

 Animal

 24 h*

 24 h*

 Test

 Cuprous Thiocyanate: Injection 10% Topical 25%

 1

 1

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 1

 2

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 1

 3

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 1

 4

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 1

 5

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 2

 1

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 2

 2

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 2

 3

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 2

 4

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 2

 5

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 3

 1

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 3

 2

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 3

 3

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 3

 4

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 3

 5

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 4

 1

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 4

 2

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 4

 3

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 4

 4

 2

 2

 Test

   Cuprous Thiocyanate: Injection 10% Topical 25%

 4  5  2  2
  Control  Paraffin Oil  5  1  1  1
Control   Paraffin Oil  5  2  1  1
  Control    Paraffin Oil  5  3  1  1
  Control    Paraffin Oil  5  4  1  1
  Control    Paraffin Oil  5  5  1  1
  Control    Paraffin Oil  6  1  1  1
  Control    Paraffin Oil  6  2  1  1
  Control    Paraffin Oil  6  3  1  1
  Control    Paraffin Oil  6  4  1  1
  Control    Paraffin Oil  6  5  1  1
  Control    Paraffin Oil  7  1  1  1
  Control    Paraffin Oil  7  2  1  1
  Control    Paraffin Oil  7  3  1  1
  Control    Paraffin Oil  7  4  1  1
  Control    Paraffin Oil  7  5  1  1
  Control    Paraffin Oil  8  1  1  1
  Control    Paraffin Oil  8  2  1  1
  Control    Paraffin Oil  8  3  1  1
  Control    Paraffin Oil  8  4  1  1
  Control    Paraffin Oil  8  5  1  1

* = After injection/patch removal

Table 3

Dose Ranging Test for Challenge: Reaction Scores

      Time/Cage/Animal
      24 h*
      9
 Concentration (w/v in Paraffin Oil)  5  6
 25%  0  0
 10%  0  0

* = After patch removal

Table 4

Main Test - Challenge: Reaction Scores

                Score    
          24 h*     48 h*    
 Group  Cage  Animal  25%  Vehicle  25%  Vehicle  Positive/ negative  % Positive
 Test  1  1  0  0  0  0  -  0
  Test  1  2  0  0  0  0  -  0
  Test  1  3  0  0  0  0  -  0
  Test  1  4  0  0  0  0  -  0
  Test  1  5  0  0  0  0  -  0
  Test  2  1  0  0  0  0  -  0
  Test  2  2  0  0  0  0  -  0
  Test  2  3  0  0  0  0  -  0
  Test  2  4  0  0  0  0  -  0
  Test  2  5  0  0  0  0  -  0
  Test  3  1  0  0  0  0  -  0
  Test  3  2  0  0  0  0  -  0
  Test  3  3  0  0  0  0  -  0
  Test  3  4  0  0  0  0  -  0
  Test  3  5  0  0  0  0  -  0
  Test  4  1  0  0  0  0  -  0
  Test  4  2  0  0  0  0  -  0
  Test  4  3  0  0  0  0  -  0
  Test  4  4  0  0  0  0  -  0
  Test  4  5  0  0  0  0  -  0
  Control  5  1  0  0  0  0  -  0
   Control  5  2  0  0  0  0  -  0
   Control  5  3  0  0  0  0  -  0
   Control  5  4  0  0  0  0  -  0
   Control  5  5  0  0  0  0 -  0
   Control  6  1  0  0  0  0  -  0
   Control  6  2  0  0  0  0  -  0
   Control  6  3  0  0  0  0  -  0
   Control  6  4  0  0  0  0  -  0
   Control  6  5  0  0  0  0  -  0
   Control  7  1  0  0  0  0  -  0
   Control  7  2  0  0  0  0  -  0
   Control  7  3  0  0  0  0  -  0
   Control  7  4  0  0  0  0  -  0
   Control  7  5  0  0  0  0  -  0
   Control  8  1  0  0  0  0  -  0
   Control  8  2  0  0  0  0  -  0
   Control  8  3  0  0  0  0  -  0
   Control  8  4  0  0  0  0  -  0
   Control  8  5  0  0  0  0  -  0

† = Test Material in vehicle

* = After patch removal

- = negative

Table 5

Body Weight

             Body Weight (g)
 Group  Cage  Animal  At Start of Study  At End of Study  Gain
  Test  1  1  427  588  161
   Test  1  2  426  519  93
   Test  1  3  438  592  154
   Test  1  4  426  504  78
   Test  1  5  454  659  205
   Test  2  1  435  557  122
   Test  2  2  426  580  154
   Test  2  3  464  592  128
   Test  2  4  426  545  119
   Test  2  5  393  568  175
   Test  3  1  455  568  131
   Test  3  2  452  607  155
   Test  3  3  465  584  119
   Test  3  4  409  535  126
   Test  3  5  457  588  131
   Test  4  1  435  600  165
   Test  4  2  430  601  171
   Test  4  3  477  586  109
   Test  4  4  455  656  201
   Test  4  5  464  621  157
   Control  5  1  426  571  145
   Control  5  2  472  619  147
   Control  5  3  452  575  123
   Control  5  4  402  544  142
   Control  5  5  410  565  155
   Control  6  1  398  485  87
   Control  6  2  435  562  127
   Control  6  3  442  578  136
   Control  6  4  411  545  134
   Control  6  5  443  554  111
   Control  7  1  459  642  183
   Control  7  2  453  615  162
   Control  7  3  439  583  144
   Control  7  4  447  598  151
   Control  7  5  405  530  125
   Control  8  1  420  593  173
   Control  8  2  425  609  184
   Control  8  3  412  526  114
   Control  8  4  455  604  149
   Control  8  5  410  600  190

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Conclusions:
There is no evidence from the test results that cuprous thiocyanate is a sensitiser in guinea pigs.
Executive summary:

The execution of this type of short-term study is not individually inspected by the Quality Assurance Department. The processes involved are inspected at intervals according to a pre-determined schedule.

The report has been audited by IRI Quality Assurance Personnel according to the appropriate Standard Operating Procedures and is considered to describe the methods and procedure used in the study. The reported results accurately reflect the original data of the study.

There is no evidence from the test results that cuprous thiocyanate is a sensitiser in guinea pigs.