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Administrative data

Description of key information

KeratinoSensTM assay: Negative

Skin sensitisation (OECD TG 406): not sensitising

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
Study period:
01 October 2019 - 29 November 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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)
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 (2000), including the most recent revisions.
Version / remarks:
2000
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The Dunkin Hartley guinea pig was chosen as the animal model for this study as recognized by international guidelines as a recommended test system (e.g. OECD, FDA, MHLW). The test method and number of animals are based on the test guidelines.
The guinea pig Maximization test was selected since the test item is a surfactant and the Local Lymph Node Assay as preferred alternative has shown to provide false positive results for surfactants.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, L’Arbresle, France
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: Young adult animals (approximately 5 weeks old)
- Weight at study initiation: 276 to 334 g
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in labelled Noryl cages containing sterilized sawdust as bedding material.
- Diet: Free access to complete maintenance diet for guinea pigs (MS-H, SSNIFF® Spezialdiäten GmbH, Soest, Germany) was provided ad libitum, except during designated procedures. In addition, hay (TecniLab-BMI BV, Someren, The Netherlands) was provided at least twice a week.
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24
- Humidity (%): 40 to 70
- Air changes (per hr): Ten or greater
- Photoperiod (hrs dark / hrs light): 12/12

- IN-LIFE DATES: From: 08 October 2019 To: 29 November 2019
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
1% for the intradermal induction and 5% for the epidermal induction.
Day(s)/duration:
Intradermal induction: 7 days. Epidermal induction: 48 hours.
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
5% for the challenge phase.
Day(s)/duration:
24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Test animals: 10
Control animals: 5
Details on study design:
RANGE FINDING TESTS:
Series of test item 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%, 1% and if needed, further lower concentrations using the same steps.
The test system and procedures were identical to those used during the main study, unless otherwise specified. The eight animals selected were 4 weeks old. No body weights were determined.
Intradermal injections:
Initially, a series of four test item concentrations was tested; the highest concentration was the maximum concentration that could technically be injected. Each of two animals received two different concentrations in duplicate (0.1 mL/site) in the clipped scapular region. The resulting dermal reactions were assessed 24 and 48 hours after treatment.
Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.
Epidermal application:
A series of four test item concentrations was tested; the highest concentration being the maximum concentration that could technically be applied. Two different concentrations were applied (0.5 mL each) 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 initially used 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 item using water.
The resulting dermal reactions were assessed for irritation 24 and 48 hours after removal of the dressings.
Based on the results in the initially treated animals, two additional animals were treated in a similar manner with four lower concentrations at a later stage.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 1
1) Intradermal injections on day 1:
- Site: scapular region. One of each pair was on each side of the midline and from cranial to caudal:
Three pairs of intradermal injections:
1) 0.1 mL: FCA (50% in water for injection)
2) 0.1 mL: test substance at a 1% concentration (control animals: 0.1 mL corn oil)
3) 0.1 mL: 1:1 mixture of the test substance at a 2% concentration + FCA (undiluted)
- Readings: on day 3 (48 hrs after the injections)

2) Topical application on day 8:
- Amount: 0.5 mL (control animals: 0.5 mL corn oil) 5% test substance
- Area: approximately 6 cm^2
- Exposure period: 48 hours (occlusive)
- Readings: scores were rated directly after patch removal

B. CHALLENGE EXPOSURE (all animals, with the 5% test substance and the vehicle)
- Day of challenge: day 21
- Exposure period: 24 hours (occlusive)
- Site: flank
- Amount: 0.1 mL
- Readings: scores were rated 24 and 48 hours after patch removal

OBSERVATIONS
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.
Toxicity: At least once daily.
Body weights: Animals were weighed individually on Day 1 (pre-dose) and after each challenge on Day 24.
Necropsy: No necropsy was performed.
Irritation: Skin reactions were graded according to OECD 406. The intradermal reactions were assessed for erythema only or, if necrosis is present, the diameter of necrosis. A description of all other local effects was recorded.
Challenge controls:
Not applicable.
Positive control substance(s):
yes
Remarks:
the results of the latest reliability check, performed in June 2019 with Alpha-Hexylcinnamaldehyde, are reported.
Positive control results:
The latest reliability check shows a sensitisation rate of 100%.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
5%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle
No. with + reactions:
0
Total no. in group:
5
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
50% Alpha- Hexylcinnamaldehyde, technical grade
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
50% Alpha- Hexylcinnamaldehyde, technical grade
No. with + reactions:
10
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
other: Not classified.
Remarks:
According to Regulation (EC) 1272/2008.
Conclusions:
In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and GLP principles, showing a sensitization rate of 0 per cent.
Executive summary:

In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and GLP principles.

Slight to moderate erythema was observed during the intradermal induction (conc. 1%) at the injection sites in control and test animals. Slight erythema was observed following the epidermal induction (conc. 5%) in 8 test animals. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

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

There was no evidence that the substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 5% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Based on these results the substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

A KeratinoSensTM assay was performed in accordance with Section 8.3 of Annex VII of Regulation (EC) No 1907/2006 as amended in Commission Regulation (EU) 2016/1688 of 20 September 2016 and the strategy presented in ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a.

A DEREK and DPRA could not be performed as the substance is a UVCB. The outcome of the KeratinoSensTM assay was negative. Performance of a U-SENSTM assay is considered to give no additional information as the cytotoxic properties of the substance are expected to preclude the outcome as positive. The KeratinoSensTM data alone is insufficient to conclude on the skin sensitization endpoint and as further in vitro testing will not lead to a conclusion, as a last resort, it is justified to continue with in vivo testing (LLNA or GPMT).

A KeratinoSensTM assay was performed with the substance according to OECD 442D and in accordance with GLP principles. Three independent experiments were performed. The stock and spike solutions were diluted 400-fold in the assay resulting in test concentrations of 0.20 – 400 μg/mL in the first experiment (2-fold dilution series), in test concentrations of 5.41 – 63 μg/mL in the second experiment (1.25-fold dilution series) and in test concentrations of 0.73 – 63 μg/mL in the third experiment (1.5-fold dilution series).

Positive controls and vehicle controls were included. All acceptability criteria were met and therefore the study was considered to be valid. No precipitate was observed at any dose level tested.

Toxicity was observed, no biologically relevant induction of the luciferase activity (no EC1.5 value) was measured, at any of the test concentrations in all three experiments.

The maximum luciferase activity induction (Imax) was 1.46 -fold, 0.37 -fold and 1.43-fold in experiment 1, 2 and 3 respectively. Based on these results, the substance is classified as negative in the KeratinoSensTM assay since negative results (<1.5-fold induction) were observed at test concentrations up to and including 400 μg/mL.

In a guinea pig maximisation test method the potential of the substance for skin sensitisation was tested according to OECD 406 guideline and GLP principles.

Slight to moderate erythema was observed during the intradermal induction (conc. 1%) at the injection sites in control and test animals. Slight erythema was observed following the epidermal induction (conc. 5%) in 8 test animals. No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

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

There was no evidence that the substance had caused skin hypersensitivity in the guinea pig, since no responses were observed in the experimental animals in response to a 5% test item concentration in the challenge phase. This result indicates a sensitization rate of 0 per cent.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the results the substance does not have to be classified and has no obligatory labelling requirement for sensitization by skin contact according to Regulation (EC) No 1272/2008 on classification, labelling and packaging of items and mixtures (including all amendments).