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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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation test was performed in accordance to the OECD guideline 406 and can be considered as a validated and reliable testing method for skin sensitization including a high statistical sample size and the challenge phase. Mid of 2001 the GPMT was the generally accepted in vivo model for the assessment of the skin sensitisation potential of substances in science and regulatory context. The adoption of the LLNA as a new Test Guideline is dated 24th April 2002.

Species:
guinea pig
Strain:
other: Hartley Crl: (HA) BR
Sex:
male/female
Details on test animals and environmental conditions:
Animals:
-Species and sex: Male and nulliparous and non-pregnant female guinea pigs.
-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 sensitizers.
-Breeder: CEGAV, Saint Mars d’Egrenne, France (preliminary test), Charles River France, 76410 Saint-Aubin-les-Elbeuf, France (main test).
-Number: Two males for the preliminary test, 30 animals (15 males and 15 females) for the main test.
-Allocation of the animals to the groups: On Day 1, the animals were weighed and randomly allocated to two groups: a control group of 10 animals (five males and five females) and a treated group of 20 animals (ten males and ten females).
-Age/weight: On Day 1, the animals of the main test were 1-3 months old and had a mean body weight + standard deviation of 360 ± 13 g for the males and 345 ± 19 g for the females.
-Acclimation: At least 5 d 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: Approx 12 cycles/h of filtered, non-recycled air. The temperature and relative humidity were under continuous control and recording. The records were checked daily and filed. In addition to these daily checks, the housing conditions and corresponding instrumentation and equipment are verified and calibrated at regular intervals. During the acclimation period and throughout the study, the animals were housed individually in polycarbonate cages with stainless steel lid (48 cm x 27 cm x 20 cm) equipped with a polypropylene bottle. Dust-free sawdust was provided as litter (SICSA, 94142 Alfortville, France). Sawdust is analysed by the supplier for composition and contaminant levels.

Food and water:
During the study, the animals had free access to “106 pelleted diet” (UAR, 91360 Villemoissonsur-Orge, France). Food is analysed regularly by the supplier for composition and contaminant levels. Drinking water filtered by a FG Millipore membrane (0.22 micron) was provided ad libitum. Bacteriological and chemical analyses of water are performed regularly by external laboratories.These analyses include the detection of possible contaminants (pesticides, heavy metals and nitrosamines). 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.
Route:
intradermal and epicutaneous
Vehicle:
other: Corn oil for intradermal injection and acetone for topical application
Concentration / amount:
Induction: 0.1%, 30%
Challenge application: 30%
Route:
epicutaneous, occlusive
Vehicle:
other: Corn oil for intradermal injection and acetone for topical application
Concentration / amount:
Induction: 0.1%, 30%
Challenge application: 30%
No. of animals per dose:
Test group: 20
Controls: 10
Details on study design:
30 guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On day 1, three pairs of intradermal injections were performed in the interscapular region of all animals:
-Freund’s complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups),
-Test substance at the chosen concentration in the chosen vehicle (treated group) or vehicle alone (control group),
-Test substance at the chosen concentration in a mixture FCA/0.9% NaCl 50/50 (v/v) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 (v/v) (control group).
On Day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On Day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 h. On Day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for
24 h. Skin reactions were evaluated approx 24 and 48 h after removal of the dressing.
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole
Positive control results:
Under the experimental conditions and according to the Magnusson and Kligman method, the test substance mercaptobenzothiazole at the concentration of 20% (w/w) induced positive skin sensitization reactions in 100% guinea-pigs (CIT/Study No.21085 TSG - January 2001).
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
30%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Discrete or moderate erythema (grade 1 or 2) was noted in 7/20 or 2/20 animals (according to the table with data in single animals)
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
30%
No. with + reactions:
10
Total no. in group:
20
Clinical observations:
Discrete or moderate erythema (grade 1 or 2) persisted in 4/20 or 2/20 animals got verse (grade 1 to grade 2). 3/20 In addition, dryness of the skin was observed at the 48 h reading in 10/20 animals of the treated group.
Remarks on result:
positive indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
10
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:
20%
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation

Choice of the vehicle: The test substance was not soluble in 0.9% NaCl: two phases were observed. The vehicle chosen for intradermal injections was corn oil: the dosage form preparation which could pass through a needle and into the dermis had a maximum concentration of 0.1% (w/w). For topical applications, the vehicle used was acetone: a homogeneous dosage form preparation was obtained at the maximum concentration of 30% (w/w).

Preliminary study:

-Administration by intradermal route:

Animal

number

Concentration of the

test substance % (w/w)

Scoring after treatment

24 h

48 h

6 d

Male 301

30

10

I

LI

I

LI

LI

LI

Male 302

30

10

I

LI

I

LI

LI

LI

FCA : mixture Freund’s Complete Adjuvant/0.9% NaCl 50/50 (v/v); I: irritation; LI: slight irritation

-Application by cutaneous route:

Animal

number

Concentration of the

test substance % (w/w)

 

Scoring after treatment

24 h

48 h

Male 301

30

10

RF

LF

0

0

0

0

Male 302

30

10

RF

LF

0

0

0

0

RF : right flank ; LF : left flank

On removal of the dressing, no residual test substance was observed. In order to respect the criteria for the selection of concentrations (the concentrations should be well-tolerated systemically and locally, cutaneous application for the induction should cause at most weak or moderate skin reactions or be the maximal practicable concentration, cutaneous application for the challenge phase should be the highest concentration which does not cause irritant effect), concentration chosen for the topical application of the induction phase (Day 8)and for the challenge application (Day 22) was 30% (w/w).

Main study:

Clinical examinations: No clinical signs and no deaths were observed during the study.

Body weight: The body weight gain of the treated animals was similar to that of the control animals.

Challenge phase: Scoring of cutaneous reactions: On removal of the dressing, no residual test substance was observed.

No cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24 h reading, a discrete or moderate erythema (grade 1 or 2) was noted in 8/20 and 2/20 animals, respectively. At the 48 h reading, discrete or moderate erythema (grade 1 or 2) persisted in 5/20 and 5/20 animals, respectively. In addition, dryness of the skin was observed at the 48 h reading in 10/20 animals of the treated group. The cutaneous reactions observed in the animals.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions and according to the maximization method of Magnusson and Kligman, MON 13900 induces delayed contact hypersensitivity in 10/20 (50%) guinea pigs.

Executive summary:

A study was conducted to assess the sensitizing potential of MON 13900 in a guinea-pig maximisation test according to OECD Guideline 406 and EU method B.6.

30 guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On Day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund’s complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups), test substance at the chosen concentration in the chosen vehicle (treated group) or vehicle alone (control group), test substance at the chosen concentration in a mixture FCA/0.9% NaCl 50/50 (v/v) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 (v/v) (control group). On Day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On Day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 h. On Day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 h. Skin reactions were evaluated approximately 24 and 48 h after removal of the dressing. No clinical signs and no deaths were noted during the study. After the challenge application, no cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24 h reading, a discrete or moderate erythema was noted in 8/20 and 2/20 animals, respectively. At the 48 h reading, discrete or moderate erythema persisted in 5/20 and 5/20 animals respectively. Dryness of the skin was observed at the 48 h reading in 10/20 animals of the treated group. The cutaneous reactions observed in the animals of the treated group at the 48 h reading were attributed to delayed contact hypersensitivity.

Under the study conditions, the test substance induced delayed contact hypersensitivity in 10/20 (50%) guinea pigs.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

A study was conducted to assess the sensitizing potential of MON 13900 in a guinea-pig maximisation test according to OECD Guideline 406 and EU method B.6.

30 guinea pigs were allocated to two groups: a control group of five males and five females and a treated group of ten males and ten females. On Day 1, three pairs of intradermal injections were performed in the interscapular region of all animals: Freund’s complete adjuvant (FCA) diluted at 50% (v/v) with 0.9% NaCl (both groups), test substance at the chosen concentration in the chosen vehicle (treated group) or vehicle alone (control group), test substance at the chosen concentration in a mixture FCA/0.9% NaCl 50/50 (v/v) (treated group) or vehicle at the concentration of 50% (w/v) in a mixture FCA/0.9% NaCl 50/50 (v/v) (control group). On Day 7, the same region received a topical application of sodium lauryl sulfate in vaseline (10%, w/w) in order to induce local irritation. On Day 8, the test substance (treated group) or the vehicle (control group) was applied topically to the same test site, which was then covered by an occlusive dressing for 48 h. On Day 22, all animals of the treated and control groups were challenged by a cutaneous application of the test substance to the right flank. The left flank served as control and received the vehicle only. Test substance and vehicle were maintained under an occlusive dressing for 24 h. Skin reactions were evaluated approximately 24 and 48 h after removal of the dressing. No clinical signs and no deaths were noted during the study. After the challenge application, no cutaneous reactions were observed in the animals of the control group. In the treated group, at the 24 h reading, a discrete or moderate erythema was noted in 8/20 and 2/20 animals, respectively. At the 48 h reading, discrete or moderate erythema persisted in 5/20 and 5/20 animals respectively. Dryness of the skin was observed at the 48 h reading in 10/20 animals of the treated group. The cutaneous reactions observed in the animals of the treated group at the 48 h reading were attributed to delayed contact hypersensitivity.

Under the study conditions, the test substance induced delayed contact hypersensitivity in 10/20 (50%) guinea pigs.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

MON 13900 induced delayed contact hypersensitivity in guinea pig maximisation study and is therefore considered to be a skin sensitizer. Hence, it qualifies for classification as Skin Sens 1B (H317-May cause an allergic skin reaction) according to Regulation (EC) 1272/2008.