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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:
2009
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The reliability is rated Klimish 1

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2009
Report date:
2009

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test

Test material

Constituent 1
Reference substance name:
Milled Clinker of Ciment Fondu®
IUPAC Name:
Milled Clinker of Ciment Fondu®
Details on test material:
- Name of test material (as cited in study report): Milled Clinker of Ciment Fondu®
- Substance type: inorganic
- Physical state: powder
- Analytical purity: 100%
- Lot/batch No.: 70398
- Expiration date of the lot/batch: 6 months after the preparation
- Storage condition of test material: at room temperature in accordance with the Sponsor's instructions

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories France - Domaine des Oncins - F-69592 L'Arbresle cédex
- Strain: Hartley Crl:HA
- Weight at study initiation: between 317.1g and 386.8g for the males and between 331g and 380.5g for the females on the day of randomisation
- Housing: Five male or female animals from each treatment group were housed in a cage of standard dimensions with sawdust bedding
(or equivalent)
- Diet (e.g. ad libitum): FD1 (P) SQC SDS/DIETEX ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: A minimum of five days in the laboratory animal house where the experiment took place. Daily observation was performed
at the delivery of animals and during the period of acclimatization.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24 (except during the cleaning slot)
- Humidity (%): 45-65 (except during the cleaning slot)
- Air changes (per hr): approximately 10-15 times per hour (non-recycled filtered)
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours darkness with light on at 7.30 a.m


IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
During the primary induction phase : 10% (w/w)
During the second induction phase : 50% (w/w)
During the challenge phase : 25% (w/w)
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
During the primary induction phase : 10% (w/w)
During the second induction phase : 50% (w/w)
During the challenge phase : 25% (w/w)
No. of animals per dose:
Preliminary study : 6 males and 6 females
Main study : treated group of 10 males and 10 females and a negative control group of 5 males and 5 females
Positive control group (at least every 6 months) : 5 males and 5 females
Details on study design:
RANGE FINDING TESTS:
Prerequisite concentrations for main study: For intradermal injections (primary induction of the sensitisation phase): The concentrations of 50% a
nd 25% were successively tested. However, due to injection trouble ( technical trouble during the injection; mainly setting in
mass of the suspension in the syringe and needle) for both concentrations, the chosen concentration at 10% was retained as 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 of 50% was the maximum concentration which caused a slight to moderate irritation with neither necrosis nor ulcerative
lesion formation and without any systemic toxic effect on the animal. For challenge, the chosen concentration of 25% 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.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2: intradermal and topical exposure
- Exposure period: 48 hours for the topical exposure
- Test groups: 30 animals allocated to a group of 10 males and 10 females treated with Milled Clinker of Ciment Fondu and a negative control group of 5 males and 5 females
- Control group: 5 males and 5 females
- Site: retro-scapular region on either side of the vertebral column for the primary and the second induction phases
- Frequency of applications:
- Duration: 10 days
- Concentrations: 10% (w/w) and 50% (w/w)


B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 22
- Exposure period: 24h
- Test groups: 30 animals allocated to a group of 10 males and 10 females treated with Milled Clinker of Ciment Fondu and a negative control group of 5 males and 5 females
- Control group: 5 males and 5 females
- Site: flank (right lateral abdominal region)
- Concentrations: 25% (w/w)
- Evaluation (hr after challenge): 24 h and 48h



OTHER:
Primary induction by intradermal route : D1
Preparatory phase : D8 - creation of local irritation:
Attempted sensitisation: D9
Expression phase: D10 to D21
Topical challenge : D22

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.

Second induction phase by topical route
Preparatory phase (D8)- creation of local irritation: As 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 mineral 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. After removal of any residual sodium lauryl sulfate, treatment group animals received
Milled Clinker of Ciment Fondu 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 or saturated with an
appropriate volume of 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 3r - 5 cm covered with adhesive tape ELASTOPLASTEr.
Under the same experimental conditions, negative control animals received the vehicle (and positive
control animals received HCA solution, 90% in corn oil).
Positive control substance(s):
yes
Remarks:
HCA solution

Study design: in vivo (LLNA)

Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
All data were registered as and when obtained using forms identified by the study number. Only clinical signs exhibited by at least one animal were
tabulated. Results of body weight change were analysed for each sex separately and were given as means ± SEM (Standard Error of the Mean). The
effects of the test item on the body weight change were compared with that of the control groups using an analysis of variance with a Dunnett's test
in case of significance (P60.05). Statistical tests were processed using RS/1 software (release 6.3, APPLIED MATERIALS).

Results and discussion

Positive control results:
In the positive control group in which animals were treated with HCA, all animals showed skin reaction (score 2 to 3) at 24 and 48 hours (non audited results). The positive test item HCA appeared as an extreme sensitiser.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No skin reactions noted. Brown colouration of the application site.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 25% (w/w). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No skin reactions noted. Brown colouration of the application site..
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
No skin reactions noted. Brown colouration of the application site.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 25% (w/w). No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: No skin reactions noted. Brown colouration of the application site..
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions noted. Brown colouration of the application site.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 25% (w/w). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions noted. Brown colouration of the application site..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
25% (w/w)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
No skin reactions noted. Brown colouration of the application site.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 25% (w/w). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No skin reactions noted. Brown colouration of the application site..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
90%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Score 2 and 3
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 90%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: Score 2 and 3.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
90%
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
score 2 an d3
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 90%. No with. + reactions: 10.0. Total no. in groups: 10.0. Clinical observations: score 2 an d3.

Any other information on results incl. tables

Reading of skin reactions:

The application of Milled Clinker of Ciment Fondu® induced a brown/black colouration of the application

site. In order to allow satisfactory conditions for grading, the application site was cleaned using

a waterlogged compress at the time of removal of the dressing. The absence of local reaction to the

rinsing procedure with sterile water was confirmed simultaneously.

Summary of results of cutaneous reactions: Positive control:

 Treatement  Time  Nb of animals score 0  Nb of animals score 1  Nb of animals score 2  Nb of animals score 3 % of sensitized animals
 HCA  24 h48h 00  00   99 11  100%100% 

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the experimental conditions, the test item Milled Clinker of Ciment Fondu® (Batch No. 70398) is not sensitizing in the
guinea pig
Executive summary:

The possible delayed sensitising capacity of Milled Clinker of Ciment Fondu® (Batch No. 70398) was evaluated in the guinea pig 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, of Council Regulation No.440/2008 (30th May 2008) and Regulation No.1907/2006 (REACH) and CLP Regulation No.1272/2008.

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. The application of the test item Milled Clinker of Ciment Fondu® induced brown colouration of the application site. In order to allow satisfactory conditions for grading, the application site was cleaned using a water logged compress at the time of removal of the dressing. The absence of local reaction to the rinsing procedure with sterile water was confirmed simultaneously.

 Treatments
 Study day Phase   Administration route  Concentration of the substance % (w/w)  vehicle
 D1  primary induction intradermal injection  10  sterile water 
 D9  sensitisation phase topical application  50  sterile water 
 D22  challenge topical application  25  sterile water 

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.

Under the experimental conditions adopted, results obtained were as follows:

No irritation reaction was noted at times 24 and 48 hours in animals of the negative control group or in animals treated during the challenge phase with the test item Milled Clinker of Ciment Fondu® at the Maximum Non-Irritant Concentration(MNIC).

Under the experimental conditions adopted, the test item Milled Clinker of Ciment Fondu® (Batch No.70398) is not sensitising in the guinea pig.