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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:
29 June 2018 to 10 August 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: JMAFF Guidelines
Version / remarks:
2000
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Based on the results of the in vitro test battery, it was considered that there was need to perform an in vivo skin sensitisation study. The guinea pig Maximisation test was selected since the test material is a fatty acid and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for fatty acids.

Test material

Constituent 1
Chemical structure
Reference substance name:
Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)
EC Number:
814-602-3
Cas Number:
190454-06-9
Molecular formula:
Variable
IUPAC Name:
Fatty Acids, C16-18 and C18-unsatd., diesters with 2-(3-hydroxypropyl)-6-[(3-hydroxypropyl)amino]-1H-benz[de]isoquinoline-1,3(2H)-dione)
Test material form:
liquid
Details on test material:
- Appearance: Amber liquid

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 5 weeks old
- Weight at study initiation: 296 to 353 g
- Housing: On arrival and following assignment to the study, animals were group housed (up to 5 animals of the same sex and same dosing group together) in labelled cages (74 cm x 54 cm x 25 cm height) containing sterilised sawdust as bedding material equipped with water bottles.
- For psychological/environmental enrichment, animals were provided with shelters except when interrupted by study procedures/activities.
- Diet: Complete maintenance diet for guinea pigs was provided ad libitum, except during designated procedures. In addition, hay was provided at least twice a week.
- Water: ad libitum
- Acclimation period: at least 5 days
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24 °C
- Humidity: 39-67 %
- Air changes: Ten or more air changes per hour with 100 % fresh air (no air recirculation)
- Photoperiod: 12-hour light/12-hour dark cycle was maintained

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
Intradermal: 0.1 mL of the test material at 50 %
Epicutaneous: 0.5 mL of 100 % test material
Day(s)/duration:
Intradermal on Day 1 and epicutaneous on Day 8 for 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
0.1 mL of 100 % test material
Day(s)/duration:
Day 21 for 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 females in the experimental group and 5 females in the control group.
Details on study design:
RANGE FINDING TESTS:
- A preliminary irritation study was conducted in order to select test material concentrations to be used in the main study. The selection of concentrations was based on the following criteria: the concentrations are well-tolerated systemically by the animals, for the induction exposures: the highest possible concentration that produced mild to moderate irritation (grades 2 - 3) and for challenge exposure: the maximum non-irritant concentration.
- A series of test material concentrations were tested. Practical feasibility of administration determined the highest starting-concentration for each route. The starting- and subsequent concentrations were taken from the series: 100 (undiluted), 50, 20, 10, 5, 2 and 1 %.
- The test system and procedures were identical to those used during the main study, unless otherwise specified. The four animals selected were 5 weeks old. No body weights were determined.
- Intradermal injections: A series of three test material concentrations was tested (the undiluted test material could not be dosed intradermally). Two animals received one or two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The injection sites were assessed for irritation 24 and 48 hours after treatment.
- Epidermal application: A series of four test material concentrations was tested, the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each or an equivalent amount when dosed with a spatula) per animal to the clipped flank, using Metalline patches (2x3 cm) mounted on Medical tape which were held in place with Micropore tape and subsequently Coban elastic bandage. The animals receiving intradermal injections were treated with the lowest concentrations and two other animals with the highest concentrations. After 24 hours, the dressing was removed and the skin cleaned of residual test material using vehiculum. The treated skin areas were assessed for irritation 24 and 48 hours after removal of the dressings.

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 Freunds' Complete Adjuvant with water for injection.
B) The test material at a 50 % concentration.
C) A 1:1 w/w mixture of the test material, at twice the concentration used in (B) and Freunds' Complete Adjuvant.
Note: One of each pair was on each side of the midline and from cranial A) to caudal C).
- Day 3: The dermal reactions caused by the intradermal injections were assessed for irritation.
- Day 7: The scapular area between the injection sites was clipped and subsequently rubbed with 10 % sodium-dodecyl-sulphate in vaseline using a spatula. This concentration of SDS provokes a mild inflammatory reaction.
- Day 8: The 10 % SDS treated area between the injection sites was treated with 0.5 mL or an equivalent amount when dosed with a spatula of a 100 % test material concentration using a Metalline patch (2x3 cm) mounted on Medical tape, which was held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 48 hours exposure, the skin cleaned of residual test material using vehicle and the dermal reactions caused by the epidermal exposure were assessed for irritation.
- Control group: The control animals were treated as described for the experimental animals except that, instead of the test material, the vehicle was administered.

B. CHALLENGE EXPOSURE
- Day 21: One flank of all animals was clipped and treated by epidermal application of a 100 % test material concentration and the vehicle (0.1 mL each or an equivalent amount when dosed with a spatula, using Patch Test Plasters. The patches were held in place with Micropore tape and subsequently Coban elastic bandage. The dressing was removed after 24 hours exposure and the skin cleaned of residual test material and vehicle using vehicle. The treated sites were assessed for challenge reactions 24 and 48 hours after removal of the dressing. After termination, animals were sacrificed using isoflurane and an intracardial injection of Euthasol® 20 %.

OBSERVATIONS AND MEASUREMENTS
- Mortality/Moribundity Checks: Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
- Toxicity: Observations for toxicity were performed once daily throughout the study.
- Body Weights: Animals were weighed individually on Day 1 (pre-dose) and at termination of the study.
- Irritation: Irritation observations were performed as described in according to the following numerical scoring system:
Grading Irritation Reactions:
Erythema and eschar formation:
No erythema = 0
Slight erythema (barely perceptible) = 1
Well-defined erythema = 2
Moderate erythema = 3
Severe erythema (beet redness) to slight eschar formation (injuries in depth) = 4
Oedema formation:
No oedema = 0
Slight oedema (barely perceptible) = 1
Well-defined oedema (edges of area well-defined by definite raising) = 2
Moderate oedema (raised approximately 1 millimetre) = 3
Severe oedema (raised more than 1 millimetre and extending beyond the area of exposure) = 4
* Intradermal reactions will be scored for erythema only or, if necrosis is present, the diameter of necrosis.

Grading Challenge Reactions:
No visible change = 0
Discrete or patchy erythema = 1
Moderate and confluent erythema = 2
Moderate erythema and swelling = 3
Intense erythema and swelling = 4

- Terminal Procedures: No necropsy for gross macroscopic examination was performed according to study plan.

OTHER:
TEST MATERIAL FORMULTIONS
- Test material dosing formulations (w/w) were homogenised to visually acceptable levels at appropriate concentrations to meet dose level requirements.
- The dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were stirred until and during dosing.
- No adjustment was made for specific gravity of the vehicle. No correction was made for the purity/composition of the test material.
- Any residual volumes were discarded.
Challenge controls:
Challenge animals were treated with the vehicle alone in the induction phase but the test material in the challenge phase.
Positive control substance(s):
yes
Remarks:
Alpha- Hexylcinnamaldehyde

Results and discussion

Positive control results:
The skin reactions observed in all experimental animals in response to the 50 % positive control substance concentration in the challenge phase were considered indicative of sensitisation, based on the absence of any response in the control animals. These results lead to a sensitisation rate of 100 percent to the 50 % concentration. From these results, it was concluded that the female guinea pig of the Dunkin Hartley strain is an appropriate animal model for the performance of studies designed to evaluate the sensitising potential of a test material in a Maximisation type of test.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
10
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 %
No. with + reactions:
0
Total no. in group:
5
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50 %
No. with + reactions:
10
Total no. in group:
10
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50%
No. with + reactions:
10
Total no. in group:
10

Any other information on results incl. tables

Preliminary Irritation Study

- Staining by the test material prevented scoring for erythema at the intradermal injection sites. As no signs of necrosis were present, a 50 % concentration was selected for the intradermal induction in the main study. A 100 % concentration was selected for the epidermal induction exposure.

- No signs of irritation were observed to the highest test material concentration epidermally tested. Therefore, the test site of all animals of the main study were treated with 10 % SDS approximately 24 hours before the epidermal induction, to provoke a mild inflammatory reaction.

- A 100% test material concentration was selected for the challenge phase.

 

Main study

- Induction Phase: The reactions noted in the experimental and control animals after the epidermal induction exposure were considered to be enhanced by the SDS treatment.

- Challenge Phase: No skin reactions were evident after the challenge exposure in the experimental and control animals. Yellow staining was observed at the test material treated skin sites, 24 and 48 hours after challenge. This staining did not hamper the scoring of the skin reactions.

- Toxicity / Mortality: No mortality occurred and no symptoms of systemic 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.

Applicant's summary and conclusion

Interpretation of results:
other: Not classified in accordance with EU criteria
Conclusions:
Under the conditions of this study, the test material did not cause skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100 % test material concentration in the challenge phase. This result indicates a sensitisation rate of 0 per cent.
Executive summary:

The skin sensitisation potential of the test material was investigated in accordance with the standardised guidelines OECD 406, EU Method B.6, OPPTS 870.2600 and JMAFF Guidelines, under GLP conditions.

The objective of the study was to evaluate whether the test material induces contact hypersensitivity in guinea pigs after intradermal and epidermal exposure.

Test material concentrations selected for the main study were based on the results of a preliminary study.

In the main study, ten experimental animals were intradermally injected with a 50 % concentration and epidermally exposed to a 100 % concentration. Five control animals were similarly treated, but with vehicle alone (corn oil). Approximately 24 hours before the epidermal induction exposure all animals were treated with 10 % SDS.

Two weeks after the epidermal application all animals were epidermally challenged with a 100 % test material concentration and the vehicle.

No skin reactions were evident after the challenge exposure in the experimental and control animals.

Under the conditions of this study, the test material did not cause skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 100 % test material concentration in the challenge phase. This result indicates a sensitisation rate of 0 per cent.