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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
GPMT
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From November, 1986 to December 1986
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Conducted before the requirement of LLNA method was implemented.
Specific details on test material used for the study:
Batch number: NBW 7993/9
Physical state: Red purple powder, having moisture content of 9.0% (w/w)
Storage: Substance stored in sealed container at room temperature
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
- Test animal: Albino male guinea pigs (Dunkin harley)
- Supplier: Animal breedign unit, Imperial chemicla industries, UK
- Weight: At the beginning of the study the guinea pigs weighed 300-382 g
- Housing: The guinea pigs were housed individually in suspended cages (37cm length x 32cm width x 20cm height). The floor and the back of each cage were made of approximately 1cm square stainless steel mesh, the sides were made of solid stainless steel and the front was made of polycarbonate.
- Food and water: The animals were allowed free access to food and were given access to unlimited water via an automatic system.
- Temperature: 19 ± 2°C
- Relative humidity: 50±10%
- Air-exchange system: 20-30 air-changes per h
- Lighting: 12 h of artificial light and 12 h of darkness.
- Acclimatisation: The guinea pigs were acclimatised to the animal laboratory for a minimum of 6 d prior to the study.
Route:
intradermal
Vehicle:
other: water bi-distilled
Concentration / amount:
Induction: 75% test substance in water (0.05 to 0.1 mL)
Adequacy of induction:
not specified
Route:
epicutaneous, occlusive
Vehicle:
other: Water bi-distilled
Concentration / amount:
Induction: 75% test substance in water (0.2 to 0.3 mL)
Day(s)/duration:
48
Adequacy of induction:
not specified
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: Water bidistilled
Concentration / amount:
Induction: 75% test substance in water
Challenge: 75, 30, 0.3% in water
Day(s)/duration:
24 h
Adequacy of challenge:
not specified
No. of animals per dose:
Test group: 20 animals
Control group: 10 animals
Details on study design:
The sensitising properties of the test substance were assessed using the maximisation test of Magnusson and Kligman. Each animal was selected at random and given a number, unique within
the study, which was written both on a small area of clipped flank, using a black waterproof marker-pen, and on the cage card. The bodyweight of each animal at the start and at the end of the study was recorded.

The dose levels for each of the three stages of the main study were determined by a 'sighting' study in which groups of two or more guinea pigs were used and up to two dose levels were tested on each group of animals.

The procedure was as follows:
i) Intradermal injection (induction): preparations of the test substance in deionised water were tested to determine the highest concentration, up to 5% (w/v), that could be well tolerated locally and systemically.

ii) topical application (induction): preparations of the test substance in deionised water of up to a concentration of 75% (w/v) were tested to determine the highest concentration which did not produce excessive inflammation.

iii) topical application (challenge): preparations of the test substance in deionised water were tested to determine the highest concentration which did not produce irritation in animals that had been injected with Freund's Complete Adjuvant at least fourteen days previously.

A group of 30 male guinea pigs were used for the main study, 20 test and 10 control.

Two main procedures were involved;
(a) an induction of a response and
(b) a challenge of that response

(a) Induction: Induction of the test animals: the hair was removed from an area approximately 5cm x 5cm on the scapular region of each animal with a pair of veterinary clippers and a row of three injections (0.05-O.lml each) was made on each side of the mid-line.

The injections were:
(i) Top: Freund's Complete Adjuvant plus deionised water in the ratio 1:1.
(ii) Middle: a 1% (w/v) preparation of the test substance in deionised Water.
(iii) Bottom a 1% (w/v) preparation of the test substance in a 1:1 preparation of Freund's Complete Adjuvant plus deionised water.

One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 75% (w/v) preparation in deionised water. This preparation (0.2-0.3mL) was applied on filter paper (approximate size 4cm x 2cm) which was held in place by a piece of surgical tape. The tape was covered by a strip of adhesive bandage (approximate size 20-30cm x 5cm) and secured by a piece of self-adhesive PVC tape. This occlusive dressing was kept in place for 48 h.

Induction of the control animals: intradermal injections were administered using an identical procedure to that used for the test animals, except that the injections were:

(i) Top: Freund's Complete Adjuvant plus deionised water in the ratio 1:1.
(ii) Middle: deionised water.
(iii) Bottom: Freund's Complete Adjuvant plus deionised water in the ratio 1:1

The topical applications followed the same procedure as for the test animals except that deionised water only was applied to the filter paper.

(b) Challenge: Two weeks after the topical inductions, an area, approximately 15cm x 5cm, on both flanks of each animal, both test and control, was clipped free of hair with a pair of veterinary clippers. An occlusive dressing was prepared which consisted of 3 pieces of filter paper (approximate size 1cm x 1.5-2.0cm) stitched to a piece of rubber sheeting (approximate size 12cm x 5cm)

A 75% (w/v) preparation of the test sample (0.05-0.1ml) was applied to one of the pieces of filter paper, a 30%(wlv) preparation was applied to the second and a 0.3% (w/v) preparation was applied to the third piece of filter paper. 0.05-0.1 mL of the preparation in deionised water was applied in each case. The dressing was placed onto the guinea pig so that the 15% and 30% (wlv) preparations were on the left shorn flank and the 0.3% (wlv) preparation was on the right shorn flank. It was then covered with a strip of adhesive bandage (approximate size 25-40cm x 7.5cm) which was secured by self-adhesive PVC tape.

After 24 h, the dressing was carefully cut, using blunt-tipped scissors, removed and discarded. The position of the pieces of filter paper on the skin were identified using a black waterproof marker-pen. The skin, at the site of application, was cleansed free of any residual test substance using clean swabs of absorbent cotton wool soaked in clean warm water and was then dried' gently with clean tissue paper. After a further 24 and 48 h, any erythematous reactions were quantified, when possible, using the four-point scale shown below and the number of positive responses was recorded.

Scale:-
0 - no reaction
1 - scattered mild redness
2 - moderate diffuse redness
3 - intense redness and swelling

To classify the sensitisation response, the percentage of the control animals that responded was subtracted from the percentage of test animals that responded and the net response was compared with the following scheme:

% net response description
0 Not a sensitiser
>0- 8 Weak sensitiser
>8-28 Mild sensitiser
>28-64 Moderate sensitiser
>64-80 Strong sensitiser
>80-100 Extreme sensitiser

At the end of the study, the skin at the challenge sites of both test and control animals was examined histopathologically. Skin samples were pinned out on wax and fixed in Bouin's solution then embedded in wax and sectioned at Sum. Sections were stained with haematoxylin and eosin and examined under the light microscope.
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
0.3%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
30%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Erythema could not able to evalauate due to red coloration, inflamation aboserved
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
75%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Erythema could not able to evalauate due to red coloration, inflamation aboserved
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
0.3%
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
30%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Erythema could not able to evalauate due to red coloration, inflamation aboserved
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
75%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Erythema could not able to evalauate due to red coloration, inflamation aboserved
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0
No. with + reactions:
0
Total no. in group:
0
Remarks on result:
not measured/tested

After challenge with a 0.3%(wlv)preparation of the test sample in deionised water, none of the animals, test or control, showed an erythematous response.

 

After challenge with 75% and 30%(wlv)preparations of the test sample in deionised water, erythema could not be assessed in any animal, either test or control, due to red staining of the challenge sites by the test sample. Histopathological examination of the 75% and 30% (w/v) challenge sites, from both test and control animals, showed that challenge with both concentrations of the test sample elicited a sensitisation response. Slight acanthosis was seen in animals from both the test and controlgroups at each site of application, indicating minimal irritation. This was confirmed by the finding in two control animals of minimal focal inflammation at the 75%(wlv)application site. However, the incidence and severity of acanthosis and, more importantly, the incidence of minimal to slight focal or multifocal inflammation, was higher in the test animals. Inflammation was seen in 50% of the test animals at the site of application of both the 75%(wlv)and 30% (w/v) concentrations. In a small number of cases the inflammation was spreading from its subepithelial position into the epithelium, usually in association with epithelial swelling and early bullous formation. This observation was

also restricted to the test group.

 

In conclusion, therefore, challenge application of 75% and 30% (w/v) preparations of test substance indeionised water elicited a sensitisation response in a group of previously-induced guinea pigs. Challenge application of a 0.3%(wlv)preparation did not elicit a sensitisation response.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the study conditions, the substance was sensitizing to Guinea pig skin.
Executive summary:

A study was conducted to determine the skin sensitisation potential of the substance according to a method similar to OECD Guideline 406, in compliance with GLP. The experiment was performed on male Guinea pigs. Induction of the test animals: the hair was removed from an area approximately 5 cm x 5 cm on the scapular region of each animal with a pair of veterinary clippers and a row of three injections (0.05-0.l ml each) was made on each side of the mid-line. The injections were: (i) Top: Freund’s Complete Adjuvant plus deionised water in the ratio 1:1. (ii) Middle: a 1% (w/v) preparation of the test substance in deionised Water. (iii) Bottom a 1% (w/v) preparation of the test substance in a 1:1 preparation of Freund’s Complete Adjuvant plus deionised water. One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 75% (w/v) preparation in deionised water. This preparation (0.2-0.3 ml) was applied on filter paper (approximate size 4 cm x 2 cm) which was held in place by a piece of surgical tape. This occlusive dressing was kept in place for 48 h. Induction of the control done as like test substance induction instead of test substance water was used. Challenge: Two weeks after the topical inductions, an area, approximately 15 cm x 5 cm, on both flanks of each animal, both test and control, was clipped free of hair with a pair of veterinary clippers. An occlusive dressing was prepared which consisted of 3 pieces of filter paper (approximate size 1 cm x 1.5-2.0 cm) stitched to a piece of rubber sheeting (approximate size 12 cm x 5 cm). A 75% (w/v) preparation of the test sample (0.05-0.1 ml) was applied to one of the pieces of filter paper, a 30% (w/v) to the second and a 0.3% (w/v) to the third piece of filter paper. 0.05-0.1 mL of the preparation in deionised water was applied in each case. The dressing was placed onto the guinea pig so that the15% and 30% (w/v) preparations were on the left shorn flank and the 0.3% (w/v) preparation was on the right shorn flank. It was then covered with a strip of adhesive bandage (approximate size 25-40 cm x 7.5 cm) which was secured by self-adhesive PVC tape. After 24 h, the adhesive was cut and removed. After a further 24 and 48 h, any erythematous reactions were quantified, when possible, using the four-point scale shown below and the number of positive responses was recorded.  After challenge with a 0.3% (w/v) preparation of the test sample in deionised water, none of the animals, test or control, showed an erythematous response. After challenge with 75% and 30% (w/v) preparations of the test sample in deionised water, erythema could not be assessed in any animal, either test or control, due to red staining of the challenge sites by the test sample. Histopathological examination of the 75% and 30% (w/v) challenge sites, from both test and control animals, showed that challenge with both concentrations of the test sample elicited a sensitisation response. Slight acanthosis was seen in animals from both the test and control groups at each site of application, indicating minimal irritation. This was confirmed by the finding in two control animals of minimal focal inflammation at the 75% (w/v) application site. However, the incidence and severity of acanthosis and, more importantly, the incidence of minimal to slight focal or multifocal inflammation, was higher in the test animals. Inflammation was seen in 50% of the test animals at the site of application of both the 75% (w/v) and 30% (w/v) concentrations. In a small number of cases the inflammation was spreading from its subepithelial position into the epithelium, usually in association with epithelial swelling and early bullous formation. This observation was also restricted to the test group. Under the study conditions, the substance was sensitizing to Guinea pig skin (Botham, 1987).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was conducted to determine the skin sensitisation potential of the substance according to a method similar to OECD Guideline 406, in compliance with GLP. The experiment was performed on male Guinea pigs. Induction of the test animals: the hair was removed from an area approximately 5 cm x 5 cm on the scapular region of each animal with a pair of veterinary clippers and a row of three injections (0.05-0.l ml each) was made on each side of the mid-line. The injections were: (i) Top: Freund’s Complete Adjuvant plus deionised water in the ratio 1:1. (ii) Middle: a 1% (w/v) preparation of the test substance in deionised Water. (iii) Bottom a 1% (w/v) preparation of the test substance in a 1:1 preparation of Freund’s Complete Adjuvant plus deionised water. One week later, the scapular area was clipped again and treated with a topical application of the test sample as a 75% (w/v) preparation in deionised water. This preparation (0.2-0.3 ml) was applied on filter paper (approximate size 4 cm x 2 cm) which was held in place by a piece of surgical tape. This occlusive dressing was kept in place for 48 h. Induction of the control done as like test substance induction instead of test substance water was used. Challenge: Two weeks after the topical inductions, an area, approximately 15 cm x 5 cm, on both flanks of each animal, both test and control, was clipped free of hair with a pair of veterinary clippers. An occlusive dressing was prepared which consisted of 3 pieces of filter paper (approximate size 1 cm x 1.5-2.0 cm) stitched to a piece of rubber sheeting (approximate size 12 cm x 5 cm). A 75% (w/v) preparation of the test sample (0.05-0.1 ml) was applied to one of the pieces of filter paper, a 30% (w/v) to the second and a 0.3% (w/v) to the third piece of filter paper. 0.05-0.1 mL of the preparation in deionised water was applied in each case. The dressing was placed onto the guinea pig so that the15% and 30% (w/v) preparations were on the left shorn flank and the 0.3% (w/v) preparation was on the right shorn flank. It was then covered with a strip of adhesive bandage (approximate size 25-40 cm x 7.5 cm) which was secured by self-adhesive PVC tape. After 24 h, the adhesive was cut and removed. After a further 24 and 48 h, any erythematous reactions were quantified, when possible, using the four-point scale shown below and the number of positive responses was recorded.  After challenge with a 0.3% (w/v) preparation of the test sample in deionised water, none of the animals, test or control, showed an erythematous response. After challenge with 75% and 30% (w/v) preparations of the test sample in deionised water, erythema could not be assessed in any animal, either test or control, due to red staining of the challenge sites by the test sample. Histopathological examination of the 75% and 30% (w/v) challenge sites, from both test and control animals, showed that challenge with both concentrations of the test sample elicited a sensitisation response. Slight acanthosis was seen in animals from both the test and control groups at each site of application, indicating minimal irritation. This was confirmed by the finding in two control animals of minimal focal inflammation at the 75% (w/v) application site. However, the incidence and severity of acanthosis and, more importantly, the incidence of minimal to slight focal or multifocal inflammation, was higher in the test animals. Inflammation was seen in 50% of the test animals at the site of application of both the 75% (w/v) and 30% (w/v) concentrations. In a small number of cases the inflammation was spreading from its subepithelial position into the epithelium, usually in association with epithelial swelling and early bullous formation. This observation was also restricted to the test group. Under the study conditions, the substance was sensitizing to Guinea pig skin (Botham, 1987).

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on an vivo skin sensitisation study, classification as Skin Sens. 1B - H317 (May cause an allergic skin reaction) is warranted according to CLP (EC 1272/2008) criteria.