Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1999
Report Date:
1999

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed before the implementation of the REACH regulation

Test material

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

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
The choice of the vehicle was based on tests to check the homogeneity (visual check) of the preparation (for cutaneous application and intradermal injections) and its free passage through a needle (for intradermal injections). The highest concentrations which satisfied these criteria were called the maximal practicable concentrations.The vehicle used was corn oil.

Strain and sanitary status: Hartley Crl: (HA) BR, Caesarian obtained, Barrier sustained - Virus; Antibody Free (COBS- VAF@)
Reason for this choice: species generally accepted by regulatory authorities for this type of study. The strain used has been shown to produce a satisfactory sensitization response using known positive sensitizers.
Breeder: Charles River France, 76410 Saint-Aubin-lès-Elbeuf, France.
Main test: 30 animals (15 males and 15 females) for the main test. Females were nulliparous and non-pregnant.
Weight: on day 1, the animals of the main test were approximately 3 months old and had a mean body weight ±standard deviation of 358 ± 10 g for the males and 362 ± 14 g for the females. Acclimatization: at least 5 days before the beginning of the study.
Identification of the animals: ear-tattoo.

Environmental conditions:
. temperature: 21 ± 2°C
. relative humidity: 30 to 70%
. light/dark cycle: 12 h/12 h
. ventilation: approximately 12 cycles/hour of filtered, non-recycled air.

Food and water ad libitum. No contaminants were known to have been present in the diet, drinking water or bedding material at levels which may be expected to have interfered with or prejudiced the outcome of the study.

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
corn oil
Concentration / amount:
10%
Day(s)/duration:
D1
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 ml
Day(s)/duration:
D8
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.5 mL
Day(s)/duration:
D22
Adequacy of challenge:
highest non-irritant concentration
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
50%
Day(s)/duration:
D33
No. of animals per dose:
5 males and 5 females
Control group : 10 males and 10 females
Details on study design:
RANGE FINDING TESTS:
Preliminary test: one male and one female.
By intradermal route:
24 hours before treatment, the dorsal region of the animals was clipped,
intradermal administrations of the test substance formulation (0.1 ml) at different concentrations were performed in the interscapular region, cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections
By cutaneous route:
24 hours before treatment, both flank regions of the animals were clipped, a volume of 0.5 ml of the undiluted test substance or test substance formulation at the chosen concentration(s) was placed on a dry gauze pad (approximately 4 cm2 which was then applied to the skin and held in place by an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings


MAIN STUDY


Induction by intradermal route:
Three injections of 0.1 ml were made into each side of this interscapular region (i.e. three pairs of sites), as follows:

Injection sites Treated group Control group

Anterior 1: FCA diluted at 50% (v/v) with 0.9% NaCl 1: FCA diluted at 50% (v/v) with 0.9% NaCl

Middle 2: test substance at 10% (w/w) in corn oil 2: vehicle

Posterior* 3: test substance at 10% (w/w) in a mixture FCA /0.9% NaCI 50/50 (v/v) 3: vehicle at 50% (w/w) in a mixture FCA /0.9% NaCl
50150 (v/v)

Induction by topical administration:
As the test substance was shown to be non-irritant during the preliminary test, the animals were treated at day 7 with 0.5 ml of sodium lauryl sulfate at the concentration of 10% (w/w) in vaseline, in order to induce local irritation.
Duration exposure for topical application: 48 hours, occlusive dressing

Challenge:
Topical application, duration exposure: 24 hours, occusive dressing
Positive control substance(s):
yes
Remarks:
DNCB (CIT/Study No. 17335 TSG) - September 1998

Results and discussion

Positive control results:
According to the Magnusson and Kligman method, the test substance DNCB at the concentration of 1 % (w/w) induced positive skin sensitization
reactions in 90% guinea-pigs.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
5
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
4
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100 %
No. with + reactions:
12
Total no. in group:
20
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100 %
No. with + reactions:
6
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
4
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
50 %
No. with + reactions:
13
Total no. in group:
20
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
50 %
No. with + reactions:
5
Total no. in group:
20
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
1%
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
1%
No. with + reactions:
7
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
TBEC was considered as non-sensitizing in the Guinea Pigs Maximalisation Test.
Executive summary:

The delayed contact hypersensivity of TBEC was evaluated in Guinea pigs according to OECD N°406 guideline (July 17th1992 - Magnusson and Kligman test). The induction phase has been realized both by intradermal route on day 1, at the concentration of 10 % in corn oil

and by cutaneous route on day 8 (undiluted) in 2 groups of guinea pigs: 5 males and 5 females for control group and 10 males and 10 females for treated group. The challenge phase was realized on day 22 by cutaneous application of TBEC (50% in corn oil); the cutaneous reactions were scored 24 and 48 hours after the challenge phase. As equivocal cutaneous reactions were noted, a second challenge application was perforrned under the same experimental conditions after a rest period of 10 days, except that the test substance and the vehicle were applied to the left and right flanks, respectively. At the end of the study, animals were killed and skin samples were taken from the challenge application sites of all the animals showing skin reactions. An histological examination was performed on the preserved tissue samples. After the first challenge application, at the 24-hour reading, a very slight erythema was observed in 4/10 animals of the control group and in 8/20 animals of the treated group. A well-defined erythema was noted in 1/10 animals of the control group and in 4/20 animals of the treated group. Most of these cutaneous reactions, associated with dryness of the skin, persisted at the 48-hour reading. A well-defined erythema was also recorded on the left control flank of 1/20 animals of the treated group.After the second challenge application, at the 24-hour reading, a very slight erythema was still observed in 3/10 animals of the control group and in 8/20 animals of the treated group. A well-defined erythema was noted in 1/10 animals of the control group (but was already noted before the second challenge application) and in 5/20 animals of the treated group. A few cutaneous reactions, associated with dryness of the skin, persisted at the 48-hour reading. As the cutaneous reactions observed in the animals of the treated group after both challenge applications were of similar incidence and severity when compared to those recorded in the animals of the control group, they were attributed to irritant properties of the test substance but not to delayed contact hypersensitivity. This was supported by the fact that most of the observed cutaneous reactions decreased between the 24-hour reading and the 48-hour reading and by the absence of any observed oedema. Histhological examinations confirmed that TBEC was not sensitizing to guinea pig skin. In conclusion, under these experimental conditions, TBEC was considered as non-sensitizing in the Guinea Pigs Maximalisation Test.