Registration Dossier

Administrative data

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)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 September - 23 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
: The deviations did not, in any case, influence the development and the results of the study. (for details see "any other information on materials and methods incl. tables")
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
: The deviations did not, in any case, influence the development and the results of the study. (for details see "any other information on materials and methods incl. tables")
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source : Centre de Production animale (F-45160 Olivet)
- Age at study initiation : not stated
- Weight at study initiation : 238g - 318g
- Housing : not stated
- Diet (e.g. ad libitum) : guinea pig breeding diet , feeding frequency not stated
- Water (e.g. ad libitum) : not stated
- Acclimation period : 5 days

ENVIRONMENTAL CONDITIONS
- Temperature : 18°C - 22°C
- Humidity : 39% - 79%
- Air changes (per hr) : not stated
- Photoperiod (hrs dark / hrs light) : not stated
Route:
other: intradermal injection and topical application
Vehicle:
other: Preliminary studies : olive oil and paraffin oil ; Main study induction phase : olive oil and physiological saline solution ; Main study challenge phase : paraffin oil
Concentration / amount:
Preliminary studies
Maximum Non Necrotizing Concentration (M.N.N.C.) determination :
- The test item was injected by intradermal route at the following concentrations: 100%, 50%, 25%, 12.5%, 6.25% and 3.125% diluted in olive oil.
Pre -Maximum Non Irritant Concentration (M.N.I.C.) determination :
- The item was applied under an occlusive dressing during 24 hours, at the following concentrations pure (100%) and diluted 50%, 25% and 12.5% in paraffin oil.
Maximum Non Irritant Concentration (M.N.I.C.) determination :
- After induction by intradermal injection with olive oil and by topical application with paraffin oil and a 17-days rest phase, the challenge phase under occlusive dressing for 24 hours consists in a single topical application of the test item at the following concentrations diluted 50%, 25%, 12.5% and 6.25% in paraffin oil.

Main study
1st induction:
- 2 intradermal injections of the item diluted at 12.5% in olive oil.
- 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in a physiological saline solution.
- 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the item diluted at 25% in olive oil.
2nd induction:
- topical application, on the same zone, with the item at 100% , 24 hours after brushing with 0.5 ml of a solution of Sodium lauryl sulfate at 10%.
Challenge phase :
- topical application under occlusive dressing at the following concentrations : 25% & 12.5%.
Route:
other: topical application under occlusive dressing
Vehicle:
other: Preliminary studies : olive oil and paraffin oil ; Main study induction phase : olive oil and physiological saline solution ; Main study challenge phase : paraffin oil
Concentration / amount:
Preliminary studies
Maximum Non Necrotizing Concentration (M.N.N.C.) determination :
- The test item was injected by intradermal route at the following concentrations: 100%, 50%, 25%, 12.5%, 6.25% and 3.125% diluted in olive oil.
Pre -Maximum Non Irritant Concentration (M.N.I.C.) determination :
- The item was applied under an occlusive dressing during 24 hours, at the following concentrations pure (100%) and diluted 50%, 25% and 12.5% in paraffin oil.
Maximum Non Irritant Concentration (M.N.I.C.) determination :
- After induction by intradermal injection with olive oil and by topical application with paraffin oil and a 17-days rest phase, the challenge phase under occlusive dressing for 24 hours consists in a single topical application of the test item at the following concentrations diluted 50%, 25%, 12.5% and 6.25% in paraffin oil.

Main study
1st induction:
- 2 intradermal injections of the item diluted at 12.5% in olive oil.
- 2 intradermal injections of Freund’s Complete Adjuvant diluted at 50% in a physiological saline solution.
- 2 intradermal injections of a mixture with equal volumes - Freund’s Complete Adjuvant at 50% and the item diluted at 25% in olive oil.
2nd induction:
- topical application, on the same zone, with the item at 100% , 24 hours after brushing with 0.5 ml of a solution of Sodium lauryl sulfate at 10%.
Challenge phase :
- topical application under occlusive dressing at the following concentrations : 25% & 12.5%.
No. of animals per dose:
Negative control group : 5
Treated group : 10
Details on study design:
PRELIMINARY STUDIES
Maximum Non Necrotizing Concentration (M.N.N.C.) determination : 2 female guinea pigs identified C8584 & C8585 were used.
-The test item was injected by intradermal route at the following concentrations: 100%, 50%, 25%, 12.5%, 6.25% and 3.125% diluted in olive oil.
Pre -Maximum Non Irritant Concentration (M.N.I.C.) determination : 2 female guinea pigs identified C8584 & C8585 were used.
-The item was applied under an occlusive dressing during 24 hours, at the following concentrations pure (100%) and diluted 50%, 25% and 12.5% in paraffin oil.
Maximum Non Irritant Concentration (M.N.I.C.) determination : 3 female guinea pigs identified C8605 to C8507 were used.
-After induction by intradermal injection with olive oil and by topical application with paraffin oil and a 17-days rest phase, the challenge phase under occlusive dressing for 24 hours consists in a single topical application of the test item at the following concentrations diluted 50%, 25%, 12.5% and 6.25% in paraffin oil.


MAIN STUDY
GROUP 1 (negative control) : 5 female guinea pigs identified n° C8635 to C8639;
GROUP 2 (treated) : 10 female guinea pigs identified n° C8640 to C8649;
Note : The results of the 3 latest positive group (Reference substance : neomycin sulfate Test 6 and benzocaine Test 4 and 5) carried out as method sensibility, were presented in "any other information on results incl. tables"

CHALLENGE PHASE
-topical application under occlusive dressing at the following concentrations : 25% & 12.5%.
- Evaluation (hr after challenge) : 24h and 48h
Positive control substance(s):
yes
Remarks:
The results of the 3 latest positive group (Reference substance : neomycin sulfate Test 6 and benzocaine Test 4 and 5) carried out as method sensibility, were presented in "any other information on results incl. tables"
Positive control results:
See "any other information on results incl. tables"
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Concentration : 25%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Concentration : 25%. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Concentration : 25%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Concentration : 25%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Concentration : 25%
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Concentration : 25%. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Concentration : 25%
No. with + reactions:
1
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Concentration : 25%. No with. + reactions: 1.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
Concentration : 12.5%
No. with + reactions:
4
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Concentration : 12.5%. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
Concentration : 12.5%
No. with + reactions:
1
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Concentration : 12.5%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Concentration : 12.5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Concentration : 12.5%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Concentration : 12.5%
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Concentration : 12.5%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Positive control results :

Test substances :

-Benzocaine (5thtest)

-Neomycin sulfate (6th& 7thtest)

Application date (test 5) : 02/20/01 ; Animals : 10

Application date (test 6) : 01/15/02 ; Animals : 10

Application date (test 7) : 03/25/03 ; Animals : 9

Overall results
Macroscopic evaluations (reading at 24 and 48 hours) of cutaneous reactions

Test  Reading Time  Concentrations  Quotations           % of Positive Responses 
   
Treated Group Test 5  24 h 12,5%  40% 
  48 h  12,5%  50% 
  24 h 6,25%  40% 
  48 h  6,25%  50% 
Treated Group Test 6  24 h  75%  40% 
  48 h  75%  40% 
  24 h  38% 30% 
  48 h  38%  40% 
Treated Group Test 7  24 h  75%  50% 
  48 h  75%  38% 
  24 h  38%  25% 
  48 h  38%  38% 

In conclusion, in view of these results, under these experimental conditions, the substances NEOMYCIN SULFATE and BENZOCAINE :

must be classified R 43 "may sause sensitization by skin contact" in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C.Directives 67/548, 2001/59 and 99/45.These products must be characterised by the symbol "Xi" and the warning label "Irritant".

PRELIMINARY STUDIES :

MNNC determination :

No necrosis has been observed since the concentrations of 12.5%, the first induction has been carried out by intradermal injection at the same concentration. Macroscopic evaluation of cutaneous reaction

Injection  No. of animals  Concentrations                
    100%  50%  25%  12.5%  6.25%  3.125% 
Intradermic injection  C8584  Ne  Ne  Ne 
Intradermic injection  C8585  Ne  Ne  Ne 

Ne : necrosis

Maximal Non Necrotizing Concentration (MNNC) = 12.5%

Pre-MNIC determination :

24 hours after the removal of the occlusive dressings, it was recorded a slight erythema on the treated area at 100% in one animal. In view of these results, the concentrations selected were pure (100%) for the 2nd induction of the main study and the MNIC began at the concentration of 50%. Macroscopic evaluation of cutaneous reactions
Application  No. of animals      Concentrations                  
    100%  50%  25%  12,5% 
Topical application under occlusive dressing  C8584  1(E) 
Topical application under occlusive dressing  C8585 

E = Erythema

MNIC deterrnination :

24 hours after removal of the occlusive dressings, it was recorded a slight erythema on the treated area at 50% in two animals. In view of this result, the concentrations selected were 25% (MNIC) and 12.5% (1/2 MNIC) for the challenge phase.
Application  No. of animals      Concentrations                  
    50%  25%  12.5%  6.25% 
Topical application under occlusive dressing  C8605 
Topical application under occlusive dressing  C8606  1(E) 
Topical application under occlusive dressing  C8607  1(E) 

E = Erythema

Maximal Non Irritant Concentration (MNIC) = 25%

MAIN STUDY :

Induction phase :

The induction phase was performed by intradermal injection at D0 with the test item diluted at 12.5% and by topical application at D7 with the test item at 100% .

Challenge phase :

The test item has been used diluted at 25% and diluted at 12.5% in paraffin oil (1/2 MNIC).

Assessment of the sensitising potential :

Overall results (Erythema & Oedema)

Macroscopic evaluation (readings at 24 and 48 hours) of cutaneous reactions

Groups  Reading Time  Concentrations  Quotations           % of Positive Responses 
  3 or >   
Negative control group  24 h 25%  0% 
  48 h  25%  1 0% 
  24 h 12.5%  0% 
  48 h  12.5%  0% 
Treated group  24 h  25%  0% 
  48 h  25%  0% 
  24 h  12.5% 0% 
  48 h  12.5%  0% 

Note : The animals which died during the test are not counted

Grading scales :

Erythema

0 = No visible modification

1 = Slight or patches of erythema

2 = Moderate confluent erythema

3 = Internal erythema and swelling

Oedema

0 = No visible modification

1 = Slight oedema

2 = Moderate oedema

3 = Important oedema

Individual results

Macroscopic evaluation (readings at 24 and 48 hours) of cutaneous reactions

N° of animals  MNIC (25%)           1/2 MNIC (12.5%)          
  24 hours     48 hours     24 hours     48 hours    
  Er  Oe  Er  Oe  Er  Oe  Er  Oe 
Negative control group (Distilled water)                 
N° C8635 F 
N° C8636 F 
N° C8637 F             
N° C8638 F 
N° C8639 F
Treated group (Test item)  Er  Oe  Er  Oe  Er  Oe  Er  Oe 
N° C8640 F 
N° C8641 F 
N° C8642 F 
N° C8643 F 
N° C8644 F                 
N° C8645 F 
N° C8646 F 
N° C8647 F 
N° C8648 F 
N° C8649 F 

Er = Erythema ; Oe = Oedema ; F = Female

C8637 & C8644 : died at D7 (this mortality is not attributable to the test item administration)

No macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase) from the animals of the treated group with the test item .

No cutaneous intolerance reactions was recorded in animals from the negative control group .

Weight evolution

No abnormality was recorded in the weight growth of the negative control group and the treated group .

Interpretation of results:
other: not classified
Remarks:
Criteria used for interpretation of results: EU
Conclusions:
The study was performed according to the Magnusson and Kligman method (J. Invest. Dermatol. 1969. 52, 268-276) and in accordance with O.E.C.D. Guideline N° 406 of July 17th, 1992, and the test method B.6 of the 96/54 E.E.C. Directive and therefore considered to be of the highest quality (reliability Klimisch 1).
In conclusion, in view of the results, under these experimental conditions, the item Nicotinsäurepentylester HF, in accordance with the criteria for classification, packaging and labelling of dangerous substances of the E.E.C. Directives 67/548, 2001/59 and 99/45, must not be classified.
Executive summary:

After induction (intradermic injection and topical application) of 10 Guinea Pigs (female) of treated group with the test item Nicotinsäurepentylester HF and a 18 -days rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 25% and 12.5% in paraffin oil, according to the experimental protocol established from the O.E.C.D. guideline n°406 dated July 17th, 1992 and the method B.6 of the E.E.C. n°96/54 dated July 30th,1996.

No macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase) from the animals of the treated group with the test item. No cutaneous intolerance reaction was recorded in animals from the negative control group.

In conclusion, in view of these results, under these experimental conditions, the item Nicotinsäurepentylester HF, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45, must not be classified.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

After induction (intradermic injection and topical application) of 10 Guinea Pigs (female) of treated group with the test material and a 18-days rest phase, the challenge phase, under occlusive dressing for 24 hours, consisted to a single topical application of the test item diluted at 25% and 12.5% in paraffin oil, according to the experimental protocol established from the O.E.C.D. guideline n°406 dated July 17th, 1992 and the method B.6 of the E.E.C. n°96/54 dated July 30th,1996. No macroscopic cutaneous reactions attributable to allergy was recorded during the examination following the removal of the occlusive dressing (challenge phase) from the animals of the treated group with the test item. No cutaneous intolerance reaction was recorded in animals from the negative control group. In conclusion, in view of these results, under these experimental conditions the test material, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the E.E.C. Directives 67/548, 2001/59 and 99/45, must not be classified.


Migrated from Short description of key information:
Phycher, 2003, skin sensitisation according to the guinea pig maximisation method of Magnusson and Kligman, Dunking-heartley, solvent distilled water

Justification for selection of skin sensitisation endpoint:
GLP guideline study

Justification for classification or non-classification