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Skin sensitisation

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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:
1 Sep 2000 - 25 Sep 2000
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was conducted according to OECD Guideline 406 and in compliance with GLP

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2001
Report Date:
2001

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Justification for performing the GPMT instead of the LLNA:

In recently published articles in peer reviewed journals, see reference list, it is clearly demonstrated that irritants and surfactants are more likely to give rise to false positives in the LLNA.
Consequently, in the evaluation of such substances for sensitizing properties the LLNA test is not an appropriate assay and would not represent an optimum use of test animals. It is therefore recommended that the GPMT is used instead. This is also supported by the TG OECD 406 "In addition, test substance classes or substances containing functional groups shown to act as potential con founders (Basketter et al., 2009) may necessitate the use of guinea pig tests".

References:
1. Kreiling et al., 2008
Comparison of the skin sensitizing potential of unsaturated compounds as assessed by the murine local lymph node assay (LLNA) and the guinea pig maximization test (GPMT).
Food Chem. Toxicol. 46. 1896-1904.
2. Basketter et al., 2009
Application of a weight of evidence approach to assessing discordant sensitisation datasets: Implications for REACH
Regul Toxicol Pharmacol. 55: 90–96
3. Ball et al., 2010
Comparative testing for the identification of skin-sensitizing potentials of nonionic sugar lipid surfactants.
Regul Toxicol Pharmacol. 58(2):301-307.
4. Ball et al., 2011
Evaluating the sensitization potential of surfactants: Integrating data from the local lymph node assay, guinea pig maximization test, and in vitro methods in a weight-of-evidence approach
Regul Toxicol Pharmacol. 60: 389–400

Additional:
Kreiling et al., 2017, In chemicio, in vitro and in vivo comparison of the skin sensitizing potential of eight unsaturated and one saturated lipid compounds.
Regulatory Toxicology and Pharmacology 90 (2017) 262-276

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Certificate of analysis included in report.
Name: Base 136
description: Sel thioclycolate dímidazoline
CASno:: 68991-84-4
Batch: Lot AB771
mw = 581 (pure salt)
Aspect: brown solid (@25ºC)
pH: 9,25 (5% inwater)
acid number: 122 mg KOH/g
Specific details on test material used for the study:
name: BASE 136
batch number: AB771
description: thick brown liquid
pH: about 9 (5 % in water)
container: one smoked glass flask
storage conditions: at room temperature and protected from light
composition: see analytical certificate
expiry date: July 2002.

In vivo test system

Test animals

Species:
guinea pig
Strain:
Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
Hartley Cd: (HA) BR, Caesarian obtained, Barrier sustained - Virus Antibody Free (COBS - VAF®)
- Source: Charles River France, 76410 Saint-Aubin-lès-Elbeuf, France.
- Age at study initiation: 1-3 months
- Weight at study initiation: 379 ± 13 g for the males and 374 ± 13 g for the females.
- Housing: 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.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 30 to 70%
- Air changes (per hr): 12 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: From: 1 Sep 2000 To: 25 Sep 2000

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
physiological saline
Concentration / amount:
The dosage form preparation which could pass through a needle and into the dermis had a maximum concentration of 10% (w/w).
Intradermal induction: 0.1%
epicutaneous induction: 5%
epicutaneous challenge: 1%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
physiological saline
Concentration / amount:
The dosage form preparation which could pass through a needle and into the dermis had a maximum concentration of 10% (w/w).
Intradermal induction: 0.1%
epicutaneous induction: 5%
epicutaneous challenge: 1%
No. of animals per dose:
Control: 5 animals/sex
Treated group: 10 animals /sex
Details on study design:
RANGE FINDING TESTS:
Series of test substance concentrations were tested by intra dermal (0.1, 1, 5, 10% with and without FCA) and epicutaneous route (0.1, 1, 5, 10, 50 100%)
Intradermal: Concentrations of 1% and higher were corrosive. 0.1% resulted to irritation visible during all reported observation day from 24h to 6 days. This concentration was selected for the main study.
Epicutaneous: 5% was the lowest concentration leading to irritation (grade 2: moderate and confluent erythema). This concentration was used for epidermal induction. 1% was without signs of irritation, and was select for challenge concentration.

MAIN STUDY
A. INDUCTION EXPOSURE
-Day 1
The scapular region was clipped and three pairs of intradermal injections (0.1 mL/site) were made in this area as follows:
A) A 1:1 w/w mixture of FCA (Sigma, France) with 0.9% NaCl.
B) The test substance at a 50% concentration in 0.9% NaCl.
C) A 1:1 w/w mixture of the undiluted test substance and FCA.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
Controls were treated similarly, but vehicle instead of testsubstance was used.

Day 7
The scapular area between the injection sites was clipped .

Day 8
a pad of filter paper (approximately 8 cm2) was fully-loaded with the test substance at the concentration of 5% (w/w) and was then applied to the interscapular region of the animals of the treated group.
The animals of the control group received an application of the vehicle alone under the same experimental conditions.
The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster.

B. CHALLENGE EXPOSURE
Day 21: Clipped and shaved each flank.
Day 22: the animals of treated and control groups received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chamber®) was fully-loaded with the test substance at the concentration of 1% (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank.
Twenty-four and 48 hours after removal of the dressing of the challenge application, both flanks of the treated and control animals were observed in order to evaluate cutaneous reactions, according to the following scale:
• 0 - no visible change
• 1 - discrete or patchy erythema
• 2 - moderate and confluent erythema
• 3 - intense erythema
Any observed oedema and other lesions were noted..

In life examinations:
At least one a day for clinical signs and mortality
Body weights: Prior to start and at termination of the study.

Positive control substance(s):
yes

Results and discussion

Positive control results:
The sensitivity of the experimental technique is regularly assessed using a known moderate sensitizer, MERCAPTOBENZOTHIAZOLE. In a recent study performed under CIT experimental conditions, the strain of guinea pigs used showed a satisfactory sensitization response in 70% animals.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
1% w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Two animals skin injured by dressing, making scoring difficult/impossible; one of these animals also showed crusts.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 1% w/w. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Two animals skin injured by dressing, making scoring difficult/impossible; one of these animals also showed crusts..
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
5% w/w
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Two animals skin injured by dressing, making scoring difficult/impossible.
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 5% w/w. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Two animals skin injured by dressing, making scoring difficult/impossible..
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
5%
No. with + reactions:
19
Total no. in group:
19
Clinical observations:
One animal died study day 11 (before challenge). Dryness of skin and/or crusts observed in most animals.
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5%. No with. + reactions: 19.0. Total no. in groups: 19.0. Clinical observations: One animal died study day 11 (before challenge). Dryness of skin and/or crusts observed in most animals..
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
5% w/w
No. with + reactions:
19
Total no. in group:
19
Clinical observations:
Dryness of skin and/or crusts observed in all animals.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5% w/w. No with. + reactions: 19.0. Total no. in groups: 19.0. Clinical observations: Dryness of skin and/or crusts observed in all animals..
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20% (w/w)
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
dryness ofthe skin & oedema
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information
Conclusions:
Reactions were observed in all animals of treatment group and none in the control. Cutaneous effects were attributed to delayed contact hypersenisitivity.
Executive summary:

The potential of the test substance BASE 136 (batch No. AB77l) to induce delayed contact hypersensitivity was evaluated in guinea pigs according to the maximization method of Magnusson and Kligman and to OECD (No. 406, 17th July 1992) and EC (96/S4/EEC, B.6, 30 July 1996) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

 

Thirty 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% 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 (treated group) or vehicle at the concentration of SO% in a mixture FCAl/0.9% NaCl 50/50 (control group).

 

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 hours.

 

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 hours.

Skin reactions were evaluated approximately 24 and 48 hours after removal of the dressing.

 

Test substance concentrations were as follows:

 

Induction (treated group)

-       intradermal injections (day 1): BASE 136 at the concentration of 0.1% (w/w) in sterile isotonic saline solution (0.9% NaCl),

-       topical application (day 8): BASE 136 at the concentration of 5% (w/w) in sterile isotonic saline solution (0.9% NaCl).

 

Challenge (all groups)

-       topical application (day 22): BASE 136 at the concentration of 1% (w/w) in sterile isotonic saline solution (0.9% NaCl).

 

At the end of the study, animals were killed without examination of internal organs.

Skin samples were taken from the challenge application sites of all the animals.

No histological examination was performed.

 

Results

No clinical signs and no deaths related to treatment were noted during the study.

After the challenge application, no relevant cutaneous reactions were observed in the animals of the control group.

In the treated group, a discrete or moderate erythema was noted in all animals. An oedema was recorded in 11/19 animals. Dryness of the skin and/or crusts were observed all animals. The observed cutaneous were attributed to delayed contact hypersensitivity.

 

Conclusion

Under our experimental conditions and according to the maximization method of Magnusson and Kligman, the test substance BASE 136 (batch No. AB771) induces delayed contact hypersensitivity in 19/19 (100%) guinea pigs.

According to the classification criteria laid down in Directive 93/21/EEC (27th April 1993) adapting to technical progress for the eighteenth time Council Directive 67/548/EEC, the test substance should be considered as a skin sensitizer.

These results are valid also for tall oil + TEPA, as Fatty acids C16-18, C18 unsat and Tall oil are only marginally differing in chain lengths distributions, and it is considered that both their reaction products with TEPA are principally the same when evaluating for sensitisation.

These results lead to classification according to CLP (ATP 2): Skin sensitiser Cat.1A (GPMT ≥ 30% positive at ≤ 0.1% i.d. induction)