Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 24, 2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2013
Report Date:
2013

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Remarks:
no major deviations only minor deviations that have no impact on results
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
- Diet: FD1 (P) SQC SDS/DIETEX feed was available ad libitum. The criteria for acceptable levels of contaminants in the feed supply were within the limits of the analytical specifications established by the diet manufacturer.
- Water:Drinking water was available ad libitum in polycarbonate bottles with a stainless steel nipple. A specimen of water is obtained approximately every 6 months and sent to Laboratoire de la Touraine - ZA n°1 du Papillon - Rue de l0Aviation - 37210 Parçay-Meslay - France, for analysis. The criteria for acceptable levels of contaminants in the water supplied were within the limits of the analytical specifications.
-Housing: Observations were performed at the time of delivery of the animals and daily during the period of acclimatisation. Animals were housed in cages of standard dimensions (5 animals per cage at maximum) with sawdust bedding. The animals were placed in an air-conditioned (20-24°C) animal house kept at relative humidity between 45% and 65% (except during the cleaning slot) with nonrecycled filtered air changed 10-15 times per hour. These parameters are continuously recorded using a computerised acquisition system. The artificial day/night cycle involved 12 hours light and 12 hours darkness with light on at 7.30 a.m.
- IN-LIFE DATES: Start of the study (signature of the study plan by the Study Director): 07 May 2013, Start of the experimental period: 14 May 2013, End of the experimental period: 14 Jun 2013.
- Body weight: Between 361.5 g and 452.5 g at the start of the experiment
- Preparation of animals: The day prior to each treatment, an area of approximately 24 cm2 (4 cm x 6 cm) on the retro-scapular region on either side of the vertebral column (induction area) or on both flanks (challenge area) was clipped free of hair using an electric clipper. Only healthy animals with an intact skin were used for the experiment.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Concentration / amount:
Preliminary results
Intradermal injection (primary induction): The concentrations of 0.25%, 0.5%, 1%, 2.5% 5% and 10% were tested. No skin reaction was seen whatever the concentration tested. In conclusion, the maximum concentration administered which led to slight to moderate irritation when tested by intradermal injection was "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10%. It was therefore the concentration used during the primary induction on D1.
Cutaneous application (second induction): No skin reaction was seen with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10% and
100%.
In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the second induction phase on D9.
Cutaneous application (challenge): No skin reaction was seen in animals treated with "hydrate de carbone et sucres, hexitol, anhydro"
LAB 4623 at 10% and 100%, 24 hours and 48 hours after removal of the dressing. In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the challenge on D22.

Study day Phase Administration route Concentration of test item Vehicle
D1 primary induction intradermal injection 10% 0.9% NaCl

D9 sensitisation phase topical application 100% /
D22 challenge topical application 100% /

Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Remarks:
0.9% NaCl
Concentration / amount:
Preliminary results
Intradermal injection (primary induction): The concentrations of 0.25%, 0.5%, 1%, 2.5% 5% and 10% were tested. No skin reaction was seen whatever the concentration tested. In conclusion, the maximum concentration administered which led to slight to moderate irritation when tested by intradermal injection was "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10%. It was therefore the concentration used during the primary induction on D1.
Cutaneous application (second induction): No skin reaction was seen with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10% and
100%.
In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the second induction phase on D9.
Cutaneous application (challenge): No skin reaction was seen in animals treated with "hydrate de carbone et sucres, hexitol, anhydro"
LAB 4623 at 10% and 100%, 24 hours and 48 hours after removal of the dressing. In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the challenge on D22.

Study day Phase Administration route Concentration of test item Vehicle
D1 primary induction intradermal injection 10% 0.9% NaCl

D9 sensitisation phase topical application 100% /
D22 challenge topical application 100% /

No. of animals per dose:
3 males for the preliminary study and 15 males (including a negative control group of 5 males) for the main study. The Guinea pig was chosen because of its recognition by regulatory authorities as a predictor of skin sensitising effects in man.
Details on study design:
A. INDUCTION EXPOSURE
- No. of exposures: Intradermal induction = 1st contact between the body and "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 (D1)
• Topical induction = 2nd contact with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623; in case of sensitising item recognition of allergen with multiplication of cellular and/or humoral transformation processes (D9)
• Topical challenge = 3rd contact with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 (on an area of skin having never previously been in contact with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623) leading to the occurrence of a cutaneous clinical manifestation if "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 has a sensitising potential (D22)
- choice of the dose: The concentration of test item used for each induction exposure was well-tolerated systemically and was the highest to cause mild-to-moderate skin irritation. The concentration used for the challenge exposure was the highest non-irritant dose. The appropriate concentrations were determined from the preliminary study using three animals. Consideration was given to the use of FCA-treated animals for this purpose. The dose was expressed in mL of the test item diluted or not. The standard amounts applied were not adjusted to body weight.
1) Preliminary study
For intradermal injections (primary induction of the sensitisation phase): The chosen concentration was the maximum concentration injectable under acceptable conditions which caused slight to moderate irritation with neither necrosis nor ulcerative lesion formation and without having any systemic toxic effect on the animal.
For topical applications (second induction of the sensitisation phase and challenge): For the second induction phase of the sensitisation phase, the chosen concentration was the maximum concentration which caused slight to moderate irritation with neither necrosis nor ulcerative lesion formation and without having any systemic toxic effect on the animal. For challenge, the chosen concentration was the maximum concentration causing no irritation in any of the animals (MNIC = Maximum Non-Irritant Concentration) and was also free of any systemic toxic effect.
Determination of the maximum injectable concentration by intradermal injection One male received intradermal injections of 0.1 mL in the retro-scapular region at 6 sites (one concentration per site, i.e. 6 concentrations per animal, i.e. 0.25%, 0.5%, 1%, 2.5%, 5% and 10%).
The skin reaction at each concentration was graded approximately 24 hours after injection using the following grading system in accordance with the OECD Guideline No. 406.
- Exposure: One male received a cutaneous application over an area of 8 cm2 (4 cm x 2 cm), using one concentration on each flank, i.e. two concentrations per animal (i.e. 10% and 100%). A filter paper (Whatman International Ltd, UK - grade 1 - 8 cm2) was soaked until saturation in the test item preparation. The
filter paper was deposited on the skin, protected by a piece of gauze and was held in place for 48 hours using an occlusive dressing made up of surgical tape BLENDERM 3M - 5 cm width covered with adhesive tape ELASTOPLASTE.
- The skin reaction at each concentration was graded approximately one hour after the removal of dressings using the grading system as shown above.
- For each concentration, the mean index was calculated: since the mean index at time 1 hour was lower than 1, the test item was considered as non irritant and a topical application of sodium lauryl sulfate (SLS) was performed on D8.
The maximum slight to moderate irritant concentration determined by topical application was used during the second induction phase performed by the topical route on D9 (i.e. test item at 100%).

2) Main study
The study design included 15 animals allocated to one group of 10 males treated with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 and to one negative control group of 5 males.
- Primary induction by intradermal route (D1): Each Guinea pig received 6 injections in the retroscapular region on either side of the vertebral column,
using an area previously clipped free of hair.
- Treatment of negative control:
• Site 1: 2 injections of 0.1 mL of a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
• Site 2: 2 injections of 0.1 mL of the vehicle
• Site 3: 2 injections of 0.1 mL of the vehicle diluted at 50% (v/v) in a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
- Treatment of positive control:
• Site 1: 2 injections of 0.1 mL of a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
• Site 2: 2 injections of 0.1 mL of HCA solution (5% in corn oil)
• Site 3: 2 injections of 0.1 mL of an emulsion of equal volume of HCA solution (10% in corn oil) and of a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
- Treatment of test item animals:
• Site 1: 2 injections of 0.1 mL of a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
• Site 2: 2 injections of 0.1 mL of "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at the concentration determined during the preliminary study by intradermal injection
• Site 3: 2 injections of 0.1 mL of "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at the concentration determined during the preliminary study by intradermal injection in a mixture 1:1 (v/v) of Freund's complete adjuvant (FCA) and sterile and pyrogen-free isotonic sodium chloride solution (0.9% NaCl)
- Second induction phase by topical route Preparatory phase (D8) - creation of local irritation: Since the maximum concentration determined during the preliminary study did not produce irritation, the area was managed with 0.5 mL of a suspension of 10% sodium lauryl sulphate (dodecyl-hydrogenosulphate sodium salt) in paraffin oil. On D8, this suspension was applied topically to the skin at the 6 injection sites utilised on D1, over an area of approximately 8 cm² previously clipped free of hair in order to create a local irritation.
Attempted sensitisation (D9): The 2nd induction on D9 involved topical application at the 6 injection sites of D1. Treatment group animals received "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 preparation at the maximum slight to moderate irritant concentration determined during the preliminary study by topical application. A filter paper (Whatman International Ltd, UK - grade 1 - 8 cm²) was soaked until saturation in the test item preparation. The filter paper was deposited on the skin, protected by a piece of gauze and was held in place for 48 hours using an occlusive dressing made up of surgical tape BLENDERM 3 - 5 cm width covered with adhesive tape ELASTOPLASTE. Under the same experimental conditions, negative control animals received the vehicle (and positive control animals received HCA solution, 90% in corn oil).
Expression phase (D10 to D21): Animals remained untreated from D10 to D21 (12 days).
B. CHALLENGE EXPOSURE
1) Preliminary study
One male received a cutaneous application, using one concentration on each flank, i.e. two concentrations per animal. A Finn Chamber cupule containing a filter paper of 1.1 cm diameter was used. The filter paper was soaked until saturation in the test item preparation. The filter paper was deposited in the cupule, which was then deposited on the skin. The Finn Chamber was held in place for 24 hours using an occlusive dressing made up of surgical tape BLENDERM 3M - 5 cm width covered with adhesive tape ELASTOPLASTE.
The skin reaction at each concentration was graded approximately 24 and 48 hours after the removal of dressings using the grading system as shown above.
The Maximum Non-Irritant Concentration (MNIC) determined by topical administration was used in the challenge phase on D22 (i.e. test item at 100%).
2) Main study
-Topical challenge (D22): On D22, the test item was applied topically to the right lateral abdominal region of the treated animals, on an area never previously in contact with the test item at the MNIC determined during the preliminary study. A Finn Chamber cupule containing a filter paper of 1.1 cm diameter was used. The filter paper was soaked until saturation in the test item preparation. The filter paper was deposited in the cupule, which was then deposited on the skin. The Finn Chamber was held in place for 24 hours using an occlusive dressing made up of surgical tape BLENDERM 3M - 5 cm width covered with adhesive tape ELASTOPLASTE. Under the same conditions as above, negative control animals received "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at the MNIC determined during the preliminary study (and positive control animals received HCA solution, 90% in corn oil).

- Mortality - Morbidity: Animals were examined in the home cage daily throughout the study period.
- General observations: Animals were examined in the home cage daily throughout the study period.
- Reading of skin reaction (D24 and D25): Approximately 24 hours after removal of the dressing, the skin reaction was evaluated and graded using the scale given below. Approximately 24 hours later, the site was scored again (i.e. 48 hours after removal of the dressing). Results were graded for each animal and by group using the scoring syste.
- Weighing: Animals were weighed on the following days: on the day of randomisation, and on the last day of the study.
At the end of the measurements, animals were euthanased by inhalation in a CO2 chamber.
No necropsy was performed at the end of the study.






Challenge controls:
The study involved 3 males for a preliminary test and 15 males (including a negative control group of 5 males) for the main study.
The control group was used to confirm that under the experimental conditions adopted, the appearance of skin reactions in the treated group in the absence of any reaction in the controls were due to an allergic response and not to skin irritation.
Reliability check : A positive control group of 5 males was tested in parallel to validate the ongoing sensitivity of the method used (CERB 20130022RT). This confirmed that hexyl cinnamic aldehyde (HCA), a test item recognised as being allergenic in the Guinea pig, continued to fully exert its sensitising properties under the experimental conditions employed.
Positive control substance(s):
yes
Remarks:
HCA (hexyl cinnamic aldehyde)

Results and discussion

Positive control results:
In the positive control group treated with HCA, there was no change in body weight gain and there were no clinical signs. At time 24 hours, 3/5 animals showed moderate erythema (score 2) and 2/5 animals showed intense erythema (score 3). At time 48 hours, 2/5 animals showed discrete erythema (score 1)
and 3/5 animals showed moderate erythema (score 2).

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no skin changes
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: no skin changes.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no skin changes
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 100%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no skin changes.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
10%
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
no change in body weight no clinical signs
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 10%. No with. + reactions: 5.0. Total no. in groups: 5.0. Clinical observations: no change in body weight no clinical signs.

Any other information on results incl. tables

Preliminary result:

Intradermal injection (primary induction): The concentrations of 0.25%, 0.5%, 1%, 2.5% 5% and 10% were tested. No skin reaction was seen whatever the concentration tested.

In conclusion, the maximum concentration administered which led to slight to moderate irritation when tested by intradermal injection was "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10%. It was therefore the concentration used during the primary induction on D1.

Cutaneous application (second induction): No skin reaction was seen with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 10% and 100%.

In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the second induction phase on D9.

Cutaneous application (challenge): No skin reaction was seen in animals treated with "hydrate de carbone et sucres, hexitol, anhydro"

LAB 4623 at 10% and 100%, 24 hours and 48 hours after removal of the dressing.

In conclusion, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 at 100% was used during the challenge on D22.

Main study results

Mortality: There was no mortality during the study.

General observations: There was no clinical finding during the study in animals treated with "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 or in animals of the positive control group.

Body weight: There was no change in body weight gain between predose and D25.

Main study: Summary of results of cutaneous reactions: negative control, positive control and treated groups

Treatment

Time

Number of animals score 0

Number of animals score 1

Number of animals score 2

Number of animals score 3

% of sensitised animals

Negative control

24h

5

0

0

0

0

48h

5

0

0

0

0

Test Item

24h

10

0

0

0

0

48h

10

0

0

0

0

Positive control

24h

0

0

3

2

100%

48h

0

2

3

0

100%

There was no skin change at times 24 and 48 hours in the negative control group or in the group treated with the test item. In the positive control group treated with HCA, there was no change in body weight gain and there were no clinical signs. At time 24 hours, 3/5 animals showed moderate erythema (score 2) and 2/5 animals showed intense erythema (score 3). At time 48 hours, 2/5 animals showed discrete erythema (score 1) and 3/5 animals showed moderate erythema (score 2).

Skin lesions - Individual scores - Negative control - Local observations

 

 

 

Animal serial number

 

Clinical signs (range of scores; normal score)

 

Time

1

2

3

4

5

Skin lesions

(0-3; 0)

D24

0

0

0

0

0

D25

0

0

0

0

0

 

Individual data are presented as a score attributed to each sign.

D: Day


 Skin lesions - Individual scores - Test item - Local observations

 

 

Animal serial number

 

Clinical signs (range of scores; normal score)

 

Time

1

2

3

4

5

6

7

8

9

10

Skin lesions

(0-3; 0)

D24

0

0

0

0

0

0

0

0

0

0

D25

0

0

0

0

0

0

0

0

0

0

Skin lesions - Individual scores - Positive Control Males - Local observations

 

 

 

Animal serial number

 

Clinical signs (range of scores; normal score)

 

Time

1

2

3

4

5

Skin lesions

(0-3; 0)

D24

2

3

3

2

2

D25

1

2

2

1

2

 

Individual data are presented as a score attributed to each sign.

D: Day

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under the experimental conditions adopted, "hydrate de carbone et sucres, hexitol, anhydro" LAB 4623 was found to be a non sensitiser in the Guinea pig.
Classification according to Council Directive No. 67/548/EEC and subsequents amendments and CLP Regulation No. 1272/2008:
Symbol: none is required.
Indication of danger: none is appropriate.
Risk phrase: none is applicable.
Executive summary:

The aim of the study was to determine the possible skin sensitising potential of "hydrate de carbone et sucres, hexitol, anhydro" LAB4623 in the Guinea pig.

The experimental technique applied is based on that of Magnusson and Kligman, in which sensitisation is potentiated by the injection of Freund's Complete Adjuvant (FCA) (Guinea Pig Maximisation Test (GPMT)), in accordance with the General Requirements of OECD Guideline No. 406 (July 17, 1992) and method B6 of Commission Directive No. 96/54/EC (July 30, 1996) adapting to technical progress for the 22nd time Council Directive No. 67/548/EEC and subsequent amendments and of Council Regulation No. 440/2008 (30th May 2008) and Regulation No. 1907/2006 (REACH).

Experimental procedure: The study involved 3 males for the preliminary study and 15 males (including a negative control group

of 5 males) for the main study. Based on the results obtained in the preliminary study, the doses chosen for the main study were as

follows:

Study day

Phase

Administration route

Concentration of

test item

 

Vehicle

D1

primary induction

intradermal injection

10%

0.9% NaCl

 

D9

sensitisation phase

topical application

100%

 

/

D22

challenge

topical application

100%

 

/

The sensitivity and the reliability of the experimental method are verified at least every six months, by use of a positive control group in which animals are treated with HCA. Determination of the degree of allergenicity at times 24 and 48 hours was based upon the percentage of animals in the group showing a reaction, rather than on the severity of the latter.

Results:

Preliminary study: Since there was no local change after indradermal injection or cutaneous application, whatever the concentration tested, the doses chosen for the main study were test item at 10% in 0.9% NaCl for the first induction and test item at 100% for the second induction and the challenge.

Main study:

Positive control: There was no change in body weight gain and no clinical signs. At time 24 hours, 3/5 animals showed moderate erythema (score 2) and 2/5 animals showed intense erythema (score 3). At time 48 hours, 2/5 animals showed discrete erythema (score 1) and 3/5 animals showed moderate erythema (score 2). Consequently, all results obtained with the positive control showed that the study could be considered as valid.

Clinical findings: There was no clinical sign from D1 up to D25.

Local examination: The application of the test item induced a brown colouration of the application site. The application site was rinsed using a waterlogged gauze. Grading of any skin change was therefore possible. There was no local change at times 24 and 48 hours in the negative control group or in the treated group.

Body weight: There was no change in body weight gain.

Conclusion: Under the experimental conditions adopted, "hydrate de carbone et sucres, hexitol, anhydro" LAB4623was found to be a non sensitiser in the Guinea pig.