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Toxicological information

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:
From August 28 to September 29, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The guinea pig maximisation test (OECD 406) was performed instead of LLNA, because test substance is an organic-metal salt (it contains Zn), which could be the cause of false negative results in the LLNA (OECD 429).

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: BFA albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Luboš Sobota, U nádraží 901, 289 03 Městec Králové, Czech Republic, monitored breeding farm, RČH CZ 21760039
- Females nulliparous and non-pregnant: yes
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30-70 %
- Photoperiod: 12 hrs dark / light cycle

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
1 %
Day(s)/duration:
day 0
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50 %
Day(s)/duration:
day 6 for 48 h
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
50 %
Day(s)/duration:
day 20 for 24 h
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
3 males for pilot experiment
20 animals (10 males and 10 females) in treatment group
10 animals (6 males and 4 females) in control group
Details on study design:
RANGE FINDING TEST
5 % suspension of test substance in physiological saline was the highest dose of test substance which was possible to apply by injection. Prepared suspensions of test substance in physiological saline were mixed by magnetic stirrer during application.
50 % test substance in vaseline was the highest dose of test substance which could be prepared and locally applied.

Induction - intradermal injections
5 % suspension of test substance in physiological saline: euthanasia 3 hours after application (jerking of limbs, opistotonus, position on the side, tachypnoe)
1 % solution of test substance in physiological saline: moderate erythema of the skin
Note: 1 % solution of test substance in physiological saline was chosen for the main study for induction – intradermal injections.

Induction (topical application) and challenge (topical application)
50 % test substance in vaseline: non-irritant dose
25 % test substance in vaseline: non-irritant dose

Note: 50 % test substance in vaseline was chosen for the main study for induction (treated 10 % sodium lauryl sulphate in vaseline 24 hours before topical induction application) and for challenge (topical application).


MAIN STUDY
A. INDUCTION EXPOSURE - INTRADERMAL
Day 0 - treated group
Three pairs of intradermal injections of 0.1 ml volume were given in the shoulder region, which was cleared of hair so that one of each pair lies on each side of the midline.
Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/physiological saline
Injection 2: 1 % suspension of test substance in physiological saline
Injection 3: 1 % suspension of test substance in a 1:1 mixture (v/v) FCA/physiological saline

In injection 3, test substance was dissolved in the aqueous phase prior to mixing with FCA.

Day 0 - control group
Three pairs of intradermal injections of 0.1 ml volume were given in the same sites as in the treated animals.
Injection 1: a 1:1 mixture (v/v) Freunds Complete Adjuvant (FCA)/physiological saline
Injection 2: physiological saline
Injection 3: physiological saline in a 1:1 mixture (v/v) FCA/physiological saline

A. INDUCTION EXPOSURE - TOPICAL APPLICATION
Day 5 - treated group and control group
Test area was cleared of hair and treated with 0.5 g of 10 % sodium lauryl sulphate in vaseline, in order to create a local irritation.

Day 6 - treated group
A filter paper (2 × 4 cm) with 50 % test substance in vaseline was applied to test area and held in contact by an occlusive dressing for 48 hours.

Day 6 – control group
A filter paper (2 × 4 cm) with vaseline only was applied in a similar manner to test area and held in contact by an occlusive dressing for 48 hours.


B. CHALLENGE EXPOSURE
Day 20 - treated and control groups
The flanks of treated and control animals were cleared of hair before application. In treated and control animals a filter paper (2 × 2 cm) with the 50 % test substance in vaseline was applied to the left flank of the animals and a filter paper (2 × 2 cm) with vaseline only was applied to the right flank. The patches were hold in contact by an occlusive dressing for 24 hours.

Observation of skin reactions
Day 22-23 - treated and control groups
Approximately 21 hours after removing the patch, challenge areas were cleaned and shaved.
Approximately 3 hours later (approximately 48 hours from the start of application of the challenge application), skin reactions were observed and recorded according to the “Magnusson and Kligman grading scale”.
Approximately 24 hours after this observation a second observation (72 hours) were made and skin reaction was again recorded.

Magnusson and Kligman grading scale for the evaluation of challenge patch test reactions
0 no visible change
1 discrete or patchy erythema
2 moderate and confluent erythema
3 intense erythema and swelling

Observation of skin reaction after induction: all skin reactions and any unusual findings, including systemic reactions, resulting from induction procedures were also observed and recorded.

Time schedule of observations
Clinical signs of intoxication and health condition: daily
Mortality/viability: daily
Body weight: 0 and 24th day
Skin reaction:
24, 48, 72 and 96 hours after intradermal injection
48, 72, 96 hours after induction – topical application
48 and 72 hours after challenge

Body weight: one day before the first application (day 0) and after observation of skin reaction (day 24). The average of group was counted from individual body weight values of animals.

Clinical observation: during experimental part of study, clinical observation of animals was practised every day. Clinical symptoms of intoxication and health condition of animals were observed.
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

Positive control results:
Reliability of the experimental technique is checked periodically in about six-month interval by experiment with the known sensitiser 2-mercaptobenzothiazole.
Result of last experiment (March 2017): positive skin reaction in 7/10 animals, i.e. 70 %. According to the guideline the minimal number of animals with positive skin reaction should be 30 %. The result was satisfactory.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no changes of skin
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
50 %
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no changes of skin
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
1 % intradermal induction, 50 % epicutaneous application
No. with + reactions:
7
Total no. in group:
20
Clinical observations:
discrete erythema in the left flank
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test group
Dose level:
1 % intradermal induction, 50 % epicutaneous application
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
discrete erythema in the left flank (persisted in 5 animals and appeared in 3 new animals)
Remarks on result:
positive indication of skin sensitisation
Reading:
other: all
Group:
positive control
Remarks on result:
other: not available

Any other information on results incl. tables

Body weight of animals increased through the study. The exposition to test substance had no influence on body weight of animals.

Clinical observation

In the course of the main experiment the animals did not show clinical symptoms of intoxication. At the end of experiment the animals were sacrificed (ether narcosis).

 

Evaluation of skin reactions after induction

Intradermal injections

The skin reaction evaluation of test area of all animals from the exposed group showed moderate erythema around all injection sites. These changes around the second and third injection sites gradually faded away 48 hours after the first evaluation of the injection sites and around the first injection sites faded away 96 hours after the first evaluation of the injection sites.

The skin reaction evaluation of test area of all animals from the control group showed moderate erythema around the first injection sites. There were no visible changes around the second and third injection sites.These changes faded away after 72 hours after the first evaluation of the injection sites.

 

Topical application

The evaluation of skin reactions of exposed group showed in all animals moderate erythema in test area which faded away 48 hours after the first evaluation of topical application. All animals were painted with 0.5 g 10 % sodium lauryl sulphate in vaseline 24 hours before the topical induction application, because test substance is not a skin irritant.

The evaluation of skin reactions in the control group with vaseline showed moderate erythema (caused by the sodium lauryl sulphate in vaseline) in test area which faded away 48 hours after the first evaluation of topical application.

Applicant's summary and conclusion

Interpretation of results:
other: category 1B according to the CLP Regulation (EC 1272/2008)
Conclusions:
Skin sensitising potential.
Executive summary:

Method

Test was performed according to the EU method B.6, analogous to OECD guideline 406.

The pilot experiment was executed on 3 animals. The main test was performed on 20 treated and 10 control animals.

The experiment proceeded in three phases: two induction phases (intradermal injections on day 0 and topical application on day 6 for 48 h) and the challenge phase (topical application on day 20 for 24 h). Potential skin reactions were evaluated 48 and 72 h after start of challenge

 

Results

Evaluation of skin reactions in the exposed group 48 hours after the start of the challenge phase showed discrete erythema in 7 animals in left flank where test substance was applied. Evaluation at 72 hours after start of the challenge phase showed discrete erythema in next 3 animals and in 5 animals the discrete erythema persisted from the previous day.

Body weight of animals increased through the study and it was not affected by the treatment. Exposed animals showed no other negative clinical symptoms throughout the main experiment.

Test substance caused a positive skin reaction in 10 of 20 animals, i.e. in 50 % of animals,which were exposed to test substance. Therefore, test substance proved to be a contact allergen in guinea pigs.