Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
01 Mar 2016 to 08 Apr 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study without restrictions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2016
Report Date:
2016

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
(adopted 1992)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
(Official Journal of the European Union No. L142, May 2008, including most recent amendments)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(March 2003)
Deviations:
no
Qualifier:
according to
Guideline:
other: JMAFF Guidelines (12 Nousan, Notification No 8147, Nov 2000, including the most recent revisions)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): MIPA-Cocoyl-Sarkosinat
- Substance type: organic (UVCB)
- Physical state: clear yellow-brown viscous liquid
- Analytical purity: 100%, according to definition of UVCB
- Impurities (identity and concentrations): no impurities, UVCB
- Lot/batch No.: 070715
- Expiration date of the lot/batch: 31 Dec 2016
- Stability under test conditions: stable in vehicle water
- Storage condition of test material: at room temperature
- Other: pH 5.7 - 6.0 (1% in water)

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L'Arbresle, France
- Age at study initiation: approx. 4 weeks old
- Weight at study initiation: 248 - 288 g
- Housing: Group housing of maximally 5 animals per labeled Noryl cage (Tecniplast; 74 cm x 54 cm x 25 cm height) containing sterilised sawdust as bedding material (Lignocel S 8-15. JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and shelters (CS3B02A Play tunnels (90 mm x 5 mm x 125 mm), Datesand, Manchester, UK) as cage enrichment.
- Diet: Complete maintenance diet for guinea pigs (SSNIFF Spezialitäten GmbH, Soest, Germany). In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Tap water, ad libitum
- Acclimation period: at least 2 days prior to start of treatment under laboratory conditions

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24 °C
- Humidity (%): 40 - 70%
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
Induction:
Intradermal: 0.1%
Epicutaneous: 2%

Challenge:
Epicutaneous: 2%
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
Induction:
Intradermal: 0.1%
Epicutaneous: 2%

Challenge:
Epicutaneous: 2%
No. of animals per dose:
10 test group
5 control
Details on study design:
RANGE FINDING TESTS:
Series of test item concentrations were tested. The starting and subsequent concentrations were taken from the series: 100% (undiluted), 50%, 20%, 10%, 5%, 2%, 1%, and further lower concentrations using the same steps.
A series of eight test item concentrations was tested for intradermal injection, the highest concentration being the maximum technically injectable concentration (20%). Each of four animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 h after treatment.
A series of four test item concentrations was tested for epidermal application, the highest concentration being the maximum concentration that could technically be applied (100%). Two different concentrations were applied (0.5 mL each) per animal to the clipped flank, using Metalline patches (2 x 3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. After 24 h occlusive, the dressing was removed and the skin cleaned of residual test item using water. The treated skin areas were assessed for irritation 24 and 48 h after removal of the dressings. Based on the results in the initially treated animals, two additional animals were epidermally treated in a similar manner with two lower concentrations at a later stage.
The concentrations and induction method in the main study were selected based on the results of the preliminary irritation study.


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal and epidermal)
- Exposure period: Day 1 to Day 10 (Intradermal induction on Day 1, epidermal exposure from Day 8 to Day 10)
- Test group: Intradermal: 3 pairs of injections in the clipped scapular region (0.1 mL/site): A) 1:1 w/w mixture of Freunds' Complete Adjuvant with water for injection B) Test item at 0.1% C) 1:1 w/w mixture of test item at 0.2% and Freunds' Complete Adjuvant
Epidermal: Application of 0.5 mL of 2% test item concentration under occlusive dressing using a Metalline patch (2 x 3cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage.
- Control group: Treated as described for test group except that, instead of test item, the vehicle was administered in analogous fashion.
- Site: Clipped scapular region
- Frequency of applications: Intradermal injection once, epidermal application once
- Duration: Epidermal application for 48 h under occlusive dressing
- Concentrations: Intradermal 0.1%, epidermal 2%

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 21
- Exposure period: 24 hours
- Test groups: Epidermal application of 2% test item concentration and vehicle (0.1 mL each) under occlusive conditions, using two Patch Test Plasters (Curatest, Lohmann, Almere, The Netherlands) approximately 2 cm apart, mounted on Medical tape. The patches were held in place with Micropore tape and subsequently Coban elastic bandage.
- Control group: Treated in analogous fashion to the test group.
- Site: Clipped flank (one side only)
- Concentrations: 2%
- Evaluation (hr after challenge): 24 and 48 hours after removal of the dressing (i.e. 48 and 72 hours after challenge application)
Challenge controls:
The control group actually satisfied the requirements of a challenge control as these animals had not been in contact with the test item prior to challenge, and the treatment with water and FCA in the induction phase is not supposed to induce any sensitisation reactions due to lack of antigens. However, FCA is known to have the potential to lower the irritation threshold which could be reflected by observation of irritation reactions at the challenge concentration determined as non-irritating in the range-finding study. That is a plausible explanation for the observation of scaliness in 1/5 control animals 48 hours after removal of the challenge patch.
Positive control substance(s):
yes
Remarks:
alpha-hexylcinnamaldehyde, assessed in regular intervals to check the reliability of the experimental techniques (last check April 2016: Intradermal induction 1% concentration, epidermal induction 50% concentration, challenge 20% concentration)

Results and discussion

Positive control results:
In the positive control experiment a sensitisation rate of 33% to a challenge concentration of 20% alpha-hexylcinnamaldehyde was observed.

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
2% challenge
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 2% challenge. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
2% induction + 2% challenge
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Scaliness in animal with positive reaction (grade 1)
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 2% induction + 2% challenge. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: Scaliness in animal with positive reaction (grade 1).
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% induction + 20% challenge
No. with + reactions:
3
Total no. in group:
9
Clinical observations:
no data
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 50% induction + 20% challenge. No with. + reactions: 3.0. Total no. in groups: 9.0. Clinical observations: no data.
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
2% challenge
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Scaliness in 1/5 animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 2% challenge. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: Scaliness in 1/5 animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
2% induction + 2% challenge
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Scaliness in 6/10 animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 2% induction + 2% challenge. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: Scaliness in 6/10 animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% induction + 20% challenge
No. with + reactions:
1
Total no. in group:
9
Clinical observations:
no data
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 50% induction + 20% challenge. No with. + reactions: 1.0. Total no. in groups: 9.0. Clinical observations: no data.

Any other information on results incl. tables

Observations:

Toxicity/Mortality: No mortality occurred and no symptoms of toxicity were observed in the animals of the main study.

Body weights: Body weights and body weight gain of experimental animals remained in the same range as controls over the study period (see Table 1).

Table 1: Body weights (gram)

Group

Animal no.

Day 1

Day 24

Control

 

26

266

371

 

27

265

383

 

28

264

385

 

29

268

450

 

30

252

381

 

Mean ± SD

263 ± 6

394 ± 32

Test

 

31

257

414

 

32

248

379

 

33

280

408

 

34

275

389

 

35

283

417

 

36

288

419

 

37

248

357

 

38

273

393

 

39

273

409

 

40

266

401

 

Mean ± SD

269 ± 14

399 ± 20

 

Conclusion:

The skin reactions other than scaliness observed in response to a 2% test item concentration in one (out of the ten) test animal in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results indicate a sensitisation rate of 10 percent.

Based on these results the test item does not have to be considered as sensitiser according to the criteria outlined In OECD guideline 406.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU