Copper thiocyanate

Skin sensitisation

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

Test material information

Constituent 1

Reference substance name:

Copper thiocyanate

EC Number:

214-183-1

EC Name:

Copper thiocyanate

Cas Number:

1111-67-7

Molecular formula:

CHNS.Cu

IUPAC Name:

copper thiocyanate

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)

Induction

Induction 1

Route:

intradermal

Vehicle:

paraffin oil

Concentration / amount:

10% w/v

Day(s)/duration:

Day 0

Induction 2

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)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 2

Reading:

2nd reading

Hours after challenge:

24

Group:

test chemical

Dose level:

25% w/v in paraffin oil

No. with + reactions:

0

Total no. in group:

20

Results 3

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

20

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

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%	1	1	2	2	2	2
5%	1	1	2	2	2	2
2%	1	1	2	2	2	2
1%	1	1	2	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	0
10%	0	0	0	0	0	0
5%	0	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	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.