Registration Dossier

Administrative data

Description of key information

- GPMT, not sensitising (OECD 406, GLP, K, Rel. 1)
- Not sensitising in humans at 2 % (HRIPT).

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:
18 April to 19 May 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 406 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
23 October 2015
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Cf. attached justification
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo (Kreuzelweg 53, 5961 NM HORST - The Netherlands).
- Females - nulliparous and non-pregnant: Yes
- Age at study initiation: 3 or 4 weeks
- Housing: Animals were housed in groups of 3 at the maximum in polycarbonate containers.
- Diet: Food (ENVIGO, 2940), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days
- Indication of any skin lesions: None

ENVIRONMENTAL CONDITIONS
- Temperature: 19–25 °C
- Humidity: 30-70 %
- Air changes: 10 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: 18 April to 19 May 2016
Route:
intradermal
Vehicle:
olive oil
Concentration / amount:
20% in olive oil
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:
unchanged (no vehicle)
Concentration / amount:
100% (undiluted) for topical application
Day(s)/duration:
Day 8 / 48 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
paraffin oil
Concentration / amount:
100% (undiluted) and 10% in liquid paraffin
Day(s)/duration:
Day 21 : 24 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
10 and 5 animals for treatment and control groups, respectively
Details on study design:
PRELIMINARY STUDIES:
Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC) for main study - intradermal induction:
Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: 100% and diluted at 50, 20 and 10% in olive oil in order to determine the MNNC. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after the injections.
Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC) for main study - topical application:
Test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 h, at 4 different concentrations: 100% and diluted at 50, 20 and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 h after removal of the dressing.
Determination by topical application of the Maximal Non Irritant Concentration (MNIC) for main study - challenge:
Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (negative control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 hours at 4 different concentrations: 100% and diluted at 50, 20 and 10% in liquid paraffin. A macroscopic evaluation of the cutaneous reactions was conducted 24 and 48 h after removal of the occlusive dressing.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Two

INTRADERMAL INJECTION
- Test groups: After shearing the scapular zone, three (3) pairs of intradermal injections (ID) of 0.1 mL were performed on the scapular zone in such a way as an injection on each pair is placed to either side of the spine as follows:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: test item at 20% in olive oil
2 ID: a test mixture in equal volumes v/v: Freund's Complete Adjuvant at 50% and the test item at 40% in olive oil
- Control group:
2 ID: Freund's Complete Adjuvant diluted at 50 % in olive oil
2 ID: olive oil
2 ID: a mixture with equal volumes v/v: - Freund's Complete Adjuvant at 50% and olive oil
- Site: Each side of the mid-line on scapular zone
- Exposure period: Day 0-6
- Duration: 7 days
Day 7: The scapular zone of all the animals in each group, shorn beforehand, was brushed with a solution of sodium lauryl sulfate at 10% in thick Vaseline, in order to create a local irritation.

TOPICAL APPLICATION
- Exposure period: 48 h
Day 8: A topical application under occlusive dressing (25 mm x 25 mm non woven swab of 4-layer patch from MEDISTOCK held in contact with the skin by means of 50 mm wide hypoallergenic micropore™ adhesive tape from 3M and Blenderm™ from 3M) for 48 h was performed on the injection sites of each animal.
- Test groups: 0.5 mL of the test item at 100%
- Control group: 0.5 mL of liquid paraffin
- Site: Same intradermally injected area of scapular zone
- Frequency of applications: Single application
Day 10: Occlusive dressing removal

REST PHASE
- The animals of both groups were left for 10 days.

B. CHALLENGE EXPOSURE
- No. of exposures: One
- Day(s) of challenge: Day 21
- Exposure period: 24 h
- The experimental procedure of this phase was identical for both negative control and treated groups submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 h: - 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item at 100% (undiluted) (MNIC) and 1 sample cup (allergEAZE® clear patch test chamber SmartPractice®) containing the test item diluted at 10% in liquid paraffin (1/10 MNIC).
- Day 22: Occlusive dressing removal
- Evaluation (h after removal of challenge patch): 24 and 48 h (Day 23 and 24, respectively)
Challenge controls:
None
Positive control substance(s):
yes
Remarks:
α-hexylcinnamaldehyde
Positive control results:
α-Hexylcinnamaldehyde induced skin sensitisation indicating the validity of the study (Study No.: SMK-2015-001; SMK-2015-002; SMK-2016-001).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
100%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
100%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
10% in liquid paraffin
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
10% in liquid paraffin
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
10% in liquid paraffin
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None
Remarks on result:
no indication of skin sensitisation

PRELIMINARY STUDIES:

Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC) for main study - intradermal induction:

No cutaneous reaction has been observed at the concentration of 20 and 10%. Therefore, 20% was selected for first induction in the main study.

Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC) for main study - topical application:

No cutaneous reactions were noted up to 100%. In view of these results, the concentration selected was 100% for the 2nd induction and the MNIC determination began at the concentration of 100%.

Determination by topical application of the Maximal Non Irritant Concentration (MNIC) for main study - challenge:

24 hours after the removal of the occlusive dressings, moderate erythema was noted in one animal at the tested concentration of 50% and in two animals at the tested concentration of 20%, no cutaneous reaction was noted in the other animals and at the other tested concentration of 100%. 48 hours after the removal of the occlusive dressings, discrete erythema was noted in one animal at the tested concentration of 50% and in two animals at the tested concentration of 20%, no cutaneous reaction was noted in the other animals and at the other tested concentration of 100%. In view to confirm the reactions observed, a repeated test was performed under the same experimental conditions after a rest phase of 6 days. 24 and 48 hours after the removal of the occlusive dressings, no cutaneous reaction was noted whatever the tested concentrations.

In view of this result, the concentration selected was 100% (MNIC) for the challenge phase. As some reactions were noted during the MNIC determination on the treated area at 50% or 20%, it was decided following consultation to the sponsor to select the additional dose 10% in liquid paraffin for the challenge phase.

 

MAIN STUDY:

Induction phase Group 2: No cutaneous reaction was noted during the induction phase.

Induction phase Group1: No cutaneous reaction was noted during the induction phase.

Challenge phase Groups1 & 2:

In the treated group (treatment dose of 100%), no cutaneous allergic reaction was noted after the challenge phase.

In the control group (associated with the treatment dose of 100%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In the treated group (treatment dose of 10%), a discrete erythema was recorded in 10% (1/10) of the animals 24 hours after the challenge phase. No cutaneous allergic reaction was noted 48 hours after the challenge phase.

In the control group (associated with the treatment dose of 10%), a discrete erythema was recorded in 20% (1/5) of the animals 24 hours after the challenge phase. No macroscopic cutaneous intolerance reactions were recorded 48 hours after the challenge phase.

The reaction was observed at 10% but not at 100%. A similar trend was seen in the preliminary study for the determination of MNIC. It is therefore considered that the irritant reaction was not concentration dependant. It was often observed during skin absorption study that the percent of passage was higher with a diluted formulation rather than with an undiluted formulation. If the percent of dermal absorption is higher with the test item diluted at 10%; then it could explain the irritant reaction noted at this concentration.

 

Body weight evolution: No abnormality was recorded in the body weight gain of both groups.

Mortality: No mortality was registered during the main test.

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test substance is not classified as skin sensitiser according to the CLP Regulation (EC) N° (1272-2008) and to the GHS.
Executive summary:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, female Dunkin-Hartley guinea pigs were treated as follows:

According the results of the pre-tests, the induction phase (intradermic injection at 20% and topical application at 100%) was conducted with the test item to 10 Guinea pigs. After a 10-day rest phase, the challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 100% and diluted at 10% in liquid paraffin.

 

In the treated group (treatment dose of 100%), no cutaneous allergic reaction was noted after the challenge phase. In the control group (associated with the treatment dose of 100%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In the treated group (treatment dose of 10%), a discrete erythema was recorded in 10% (1/10) of the animals 24 hours after the challenge phase. No cutaneous allergic reaction was noted 48 hours after the challenge phase. In the control group (associated with the treatment dose of 10%), a discrete erythema was recorded in 20% (1/5) of the animals 24 hours after the challenge phase. No macroscopic cutaneous intolerance reactions were recorded 48 hours after the challenge phase.

 

No mortality was observed during the test. No abnormality was recorded in the body weight gain of both groups.

 

The sensitivity of the guinea-pig was checked with known sensitiser i.e., α-Hexylcinnamaldehyde indicating the validity of the study.

 

Under the test conditions, test substance is not classified as skin sensitiser according to the CLP Regulation (EC) N° (1272-2008) and to the GHS.

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

Since no key study was identified on the registered substance, the testing and assessment strategy, as described in ECHA R.7a Endpoint specific guidance (December 2016), was used to evaluate the skin sensitisation potential of the registered substance:

Element

Information

Conclusion

Comments

Existing data on physico-chemical properties

1

Is the substance a strong acid (pH≤ 2.0) or base (pH≥ 11.5), corrosive to the skin or (spontaneously) flammable in air or in contact with water or moisture at room temperature?

NO

Existing human data

2

Are there adequate existing human data, which provide evidence that the substance is a skin sensitiser?

NO

In the HRIPT (1973), there was no evidence of sensitisation to the substance at 2%

Existing animal data from sensitisation studies

3

Are there data from existing studies on skin sensitisation in laboratory animals (LLNA, GPMT, or Buehler test, EU B.42, B.50, B.51 and B.6/OECD TGs 429, 442A, 442B and 406), which provide sound conclusive evidence that the substance is a sensitiser, or non-sensitiser?

NO

Existing/new (Q)SAR data and read-across

4

Do “read-across” from structurally and mechanistically related substances and/or do suitable (Q)SAR predictions reliably indicate skin sensitisation potential or the absence thereof of the substance?

NO

Not applicable - UVCB substance

Existing in chemico and in vitro data

5a

Is there evidence/hypothesis of dermal bioavailability based on physico-chemical, in silico, in vitro or in vivo data?

NO

Not applicable - UVCB substance

5b

Has the substance demonstrated peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. OECD TG 442c)? (Key event 1 of the AOP), and/or
Has the substance demonstrated activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. OECD TG 442d)? (Key event 2 of the AOP), and/or
Has the substance demonstrated induction of the cell surface markers (CD54 and/or CD86) on monocytic cells in a validated in vitro test, e.g. h-CLAT? (Key event 3 of the AOP).
Data from in chemico/in vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

5c

Are there data from (a) non-validated in vitro test(s), which provide evidence that the substance may be a skin sensitiser?

NO

Weight-of- Evidence analysis

6

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-5) into account, is there sufficient information to meet the information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

NO

Generation of new non-animal data

7a

Does the substance demonstrate peptide/protein binding properties in an EU/OECD adopted in chemico test (e.g. B. 59/OECD TG 442c)? (Key event 1 of the AOP)
In chemico test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7b

Does the substance demonstrate activation of the Nrf2-Keap1-ARE toxicity pathway in an EU/OECD adopted in vitro test (e.g. B.60/OECD TG 442d)? (Key event 2 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7c

Does the substance demonstrate induction of the cell surface markers (CD54 and/or CD86) of monocytic cells in a validated in vitro test (e.g. h-CLAT)? (Key event 3 of the AOP)
In vitro test methods that have been validated and are considered scientifically valid but are not yet adopted by the EU and/or OECD may also be used if the provisions defined in Annex XI to the REACH Regulation are met.

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

7d

Is any additional testing/generation of data considered necessary in order to conclude on classification, or e.g. to explain the inconsistent data obtained in previous elements or to address the Key event 4 of the AOP (T-cell proliferation) with an in vitro test?

NO

(at the initiation of the dossier, no test was available and the in vitro testing was not the REACH standard requirement)

Weight-of-Evidence analysis

8

The “elements” described above may be arranged as appropriate. Taking all existing and relevant data (elements 1-7) into account, is there sufficient information to meet the respective information requirement of Section 8.3 of Annex VII and to make a decision on whether classification and labelling are warranted?

NO

Generation of new in vivo data for sensitisation as a last resort (Annex VII to the REACH Regulation)

8b

Does the substance demonstrate sensitising properties in an EU/OECD adopted in vivo test, the LLNA (EU B.42/OECD TG 429)?

YES

=> A GPMT assay was initiated: not a skin sensitizer [please refer to the justification attached to the corresponding ESR for test method selection]

 

Therefore, a new GPMT study was performed according to the OECD test guideline No. 429 and in compliance with GLP (Phycher, 2016, rel.1) [NB: at the time of test performance, i.e. before 11 October 2016, in vitro testing was not the REACH default requirement].

According the results of the pre-tests, the induction phase (intradermic injection at 20% and topical application at 100%) was conducted with the test item to 10 Guinea pigs. After a 10-day rest phase, the challenge phase conducted under occlusive dressing for 24 hours, consisted of a single topical application of the test item at 100% and diluted at 10% in liquid paraffin.

In the treated group (treatment dose of 100%), no cutaneous allergic reaction was noted after the challenge phase. In the control group (associated with the treatment dose of 100%), no macroscopic cutaneous intolerance reactions were recorded after the challenge phase.

In the treated group (treatment dose of 10%), a discrete erythema was recorded in 10% (1/10) of the animals 24 hours after the challenge phase. No cutaneous allergic reaction was noted 48 hours after the challenge phase. In the control group (associated with the treatment dose of 10%), a discrete erythema was recorded in 20% (1/5) of the animals 24 hours after the challenge phase. No macroscopic cutaneous intolerance reactions were recorded 48 hours after the challenge phase.

No mortality was observed during the test. No abnormality was recorded in the body weight gain of both groups.

The sensitivity of the guinea-pig was checked with known sensitiser i.e.,α-Hexylcinnamaldehyde indicating the validity of the study.

Under the test conditions, the test material is not classified as skin sensitizer.

A human repeated insult patch test in human has been performed with the substance at up to 2 % (Biotest, 1973). Under the conditions employed in this study, there was no evidence of sensitisation to the test material at 50 %.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No. 1272/2008 .

Self-classification:

Based on the available data, the substance is not classified as skin sensitizer according to the Annex VI of the Regulation (EC) No. 1272/2008 (CLP) and according to the GHS.

No data was available for respiratory sensitisation. However, this substance is not a skin sensitizer, therefore according to Figure R.7.3 -2 of the Chapter R.7 (V 4.1 - October 2015) the chemical is not considered as a respiratory sensitizer.