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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:
15 September - 23 October 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2003
Report date:
2003

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
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
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. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
No new study has been conducted; data from a existing GLP-guideline study (2003) has been used as this study is totally sufficient to cover this endpoint.

Test material

Constituent 1
Chemical structure
Reference substance name:
4-morpholinopropanesulphonic acid
EC Number:
214-478-5
EC Name:
4-morpholinopropanesulphonic acid
Cas Number:
1132-61-2
Molecular formula:
C7H15NO4S
IUPAC Name:
3-morpholin-4-ylpropane-1-sulfonic acid
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report) : MOPS HIGH PURITY
- Physical state : white powder
- Analytical purity : 100,3%
- Purity test date : not stated
- Lot/batch No. : 1190567/006
- Expiration date of the lot/batch : not stated
- Storage condition of test material : room temperature

In vivo test system

Test animals

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 - 366g
- 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

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: physiological saline solution and distilled water
Concentration / amount:
1st induction:
- 2 intradermal injections of the item diluted at 25% in a physiological saline solution.
- 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 50% in a physiological saline solution.
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 : 100% & 50%.
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: physiological saline solution and distilled water
Concentration / amount:
1st induction:
- 2 intradermal injections of the item diluted at 25% in a physiological saline solution.
- 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 50% in a physiological saline solution.
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 : 100% & 50%.
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 C8586 & C8587 were used.
-The test item was injected by intradermal route at the following concentrations: 50%, 25%, 12.5%, 6.25%, 3.125% and 1.562% diluted in a physiological saline solution.
Pre -Maximum Non Irritant Concentration (M.N.I.C.) determination :
-2 female guinea pigs identified C8586 & C8587 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 a physiological saline solution.
Maximum Non Irritant Concentration (M.N.I.C.) determination :
-3 female guinea pigs identified C8608 to C8610 were used.
-After induction by intradermal injection with a physiological saline solution and by topical application with distilled water 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: 100% diluted 50%, 25% and 12.5% in distilled water.


MAIN STUDY
GROUP 1 (negative control) : 5 female guinea pigs identified n° C8635 to C8639;
GROUP 2 (treated) : 10 female guinea pigs identified n° C8650 to C8659;
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 : 100% & 50%.
- 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"

Results and discussion

Positive control results:
See "any other information on results incl. tables"

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Concentration : 100%
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 : 100%. No with. + reactions: 4.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Concentration : 100%
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 : 100%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Concentration : 100%
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 : 100%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Concentration : 100%
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 : 100%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Concentration : 50%
No. with + reactions:
2
Total no. in group:
10
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Concentration : 50%. No with. + reactions: 2.0. Total no. in groups: 10.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Concentration : 50%
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 : 50%. No with. + reactions: 1.0. Total no. in groups: 10.0.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Concentration : 50%
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 : 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
Concentration : 50%
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 : 50%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Any other information on results incl. tables

Positive control results :

Test substances :

-Benzocaine (5th test)

-Neomycin sulfate (6th & 7th test)

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 25%, the first induction has been carried out by intradermal injection at the same concentration. Macroscopic evaluation of cutaneous reactions

Injection  No. of animals  Concentrations                
    50%  25%  12,5%  6,25%  3,125%  1,562% 
Intradermic injection  C8586  Ne 
Intradermic injection  C8587  Ne 

Ne : necrosis

Maximal Non Necrotizing Concentration (MNNC) = 25%

Pre MNIC deterrnination :

24 hours after the removal of the occlusive dressings, no cutaneous reaction was recorded. Macroscopic evaluation of cutaneous reactions

Application  No. of animals      Concentrations                  
    100%  50%  25%  12,5% 
Topical application under occlusive dressing  C8586 
Topical application under occlusive dressing  C8587 

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 100%.

MNIC deterrnination :

24 hours after removal of the occlusive dressings, no cutaneous reaction was recorded. Macroscopic evaluation of cutaneous reactions

Application  No. of animals      Concentrations                  
    100%  50%  25%  12,5% 
Topical application under occlusive dressing  C8608 
Topical application under occlusive dressing  C8609 
Topical application under occlusive dressing  C8610 

Maximal Non Irritant Concentration (MNIC) = 100%

In view of this result, the concentrations selected were 100% (MNIC) and 50% (1/2 MNIC) for the challenge phase.

MAIN STUDY :

Induction phase :

The induction phase was performed by intradermal injection at D0 with the test item diluted at 25% and by topical application at D7 with the test item at 100% .

Challenge phase :

The test item has been used at 100% and diluted at 50% in distilled water .

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 100%  0% 
  48 h  100%  0 0% 
  24 h 50%  0% 
  48 h  50%  0% 
Treated group  24 h  100%  0% 
  48 h  100%  0% 
  24 h  50% 0% 
  48 h  50%  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 (100%)           1/2 MNIC (50%)          
  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° C8650 F 
N° C8651 F 
N° C8652 F 
N° C8653 F 
N° C8654 F 
N° C8655 F 
N° C8656 F 
N° C8657 F 
N° C8658 F 
N° C8659 F 

Er = Erythema ; Oe = Oedema ; F = Female

C8637 : 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 .

Applicant's summary and conclusion

Interpretation of results:
other: not classified
Remarks:
Criteria used for interpretation of results: other: EU-GHS
Conclusions:
The study was performed according to the OECD TG406 without deviations and therefore considered to be of the highest quality (reliability Klimisch 1). The validity criteria of the test system are fulfilled. 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 MOPS High Purity, 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.
Executive summary:

After induction (intradermic injection and topical application) of 10 Guinea Pigs (female) of treated group with the test item MOPS High Purity 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 at 100% and diluted at 50% in distilled water, according to the experimental protocol established from the OECD guideline n°406 dated July 17th, 1992 and the method B.6 of the EEC 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 MOPS High Purity, in accordance with the criteria for classification, packaging and labelling of dangerous substances and preparations of the EEC Directives 67/548, 2001/59 and 99/45, must not be classified.