[No public or meaningful name is available]

Endpoint summary

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:

Between 09 November 2015 and 10 December 2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study performed according to OECD test guideline No. 406 and in compliance with GLP.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

not applicable

GLP compliance:

yes (incl. QA statement)

Remarks:

Inspected on: 23-24 April 2015 / Signed on 23 October 2016

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The substance is considered to have non‐ionic surface-active potential and hence is potentially confounding to the LLNA result. Irritation has been reported to induce Langerhans cells (LC) to migrate to the regional lymph nodes which may induce nonspecific lymph node proliferation and subsequently result in a false positive result (Ball et al., 2011; Kreiling et al., 2008; Strauss et al., 2014). Therefore, skin irritation may be a confounding factor in LLNA. The registered substance is classified as a skin irritant Category 2, which suggests that the LLNA may produce an irritant-driven false positive result. Thus, the LLNA according to OECD TG 429 is considered to be incompatible with the substance. Therefore, a Guinea‐pig maximisation test (GPMT) according to OECD TG 406 (OECD, 1992) is considered exceptionally justified for the registered substance.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS:
- Source: HARLAN/ENVIGO (Kreuzelweg 53, 5961 NM HORST - The Netherlands)
- Age at study initiation: 3 or 4 weeks old
- Weight at study initiation: Control: 277.2 ± 12.9 Test: 274.8 ± 5.4 g
- Housing: individually or in groups of 2 in polycarbonate containers, the flooring of which was covered with dust-free cuttings and the top fitted with a stainless steel lid with a feeding device and drinking device of 500 mL.
- Diet (e.g. ad libitum): free access to "SAFE, 106"
- Water (e.g. ad libitum): drinking water filtered by a FG Millipore membrane (0.22 µm) ad libitum. Contaminants-free. Ad libitum. Microbiological and chemical analyses of the water were carried out once every six months by Eurofins IPL Atlantique (Bordeaux).
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19°C to 25°C
- Humidity (%): 30 to 70 %. A relative humidity lower than 30% was registered on 22, 23, 24 November 2015 and 02 December 2015. The minimum value measured was 24%. This deviation is considered as without impact on the conclusion of the study.
- Air changes (per hr): at least ten changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

Route:

intradermal and epicutaneous

Vehicle:

other: olive oil for intradermal induction, liquid paraffin for epicutaneous challenge

Concentration / amount:

20% w/w for intradermal injections; undiluted for topical induction and 50% for challenge

Route:

epicutaneous, occlusive

Vehicle:

other: olive oil for intradermal induction, liquid paraffin for epicutaneous challenge

Concentration / amount:

20% w/w for intradermal injections; undiluted for topical induction and 50% for challenge

No. of animals per dose:

10 treated/sex, 5 control/sex

Details on study design:

RANGE FINDING TESTS:
- Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): Two animals received a volume of 0.1 mL of the test item, on both sides of the spine, at 4 concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in olive oil in view to determine the MNNC.
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): The test item was applied on the dorso-lumbar zone of two guinea pigs shorn beforehand, with occlusive dressing for 24 hours, at 4 different concentrations: undiluted (100%) and diluted at 50%, 20% and 10% in liquid paraffin.
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC): Three guinea pigs were treated according to the same treatment as animals from GROUP 1 (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: diluted at 50%, 20%, 10% and 5% in liquid paraffin.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2, intradermal injections + topical application
- Exposure period: 48 hours for topical application
- Test groups:
INTRADERMAL: 3 pairs of intradermal injection (0.1 mL) on Day 0 as follows:
- 1/ Freund’s Complete Adjuvant diluted at 50 % in olive oil
- 2/ test item at 20% in olive oil
- 3/ a test mixture in equal volumes v/v : Freund’s Complete Adjuvant at 50% and the test item at 40% in olive oil
On Day 6, 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: A topical application under occlusive dressing (25mm x 25mm 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 hours was performed on the injection sites of each animal.
GROUP 1 (control): 0.5 mL of liquid paraffin.
GROUP 2 (treated): 0.5 mL of the test item at 100%
- Control group: similarly treated with the exception that olive oil was topically applied for the 2nd intradermal injection instead of the test substance.
- Site: scapular zone
- Occlusive dressing removal on Day 9

B. CHALLENGE EXPOSURE
- No. of exposures: 1
- Day(s) of challenge: Day 20
- Exposure period: 24 hours
- Test and control groups: The experimental procedure of this phase was identical for both groups GROUP 1 (Control) and GROUP 2 (Treated) submitted to this experimentation: on the previously shorn dorso-lumbar zone, an application, under occlusive dressing, was performed during 24 hours: - 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing the test item diluted at 20% in liquid paraffin (MNIC) and 1 sample cup (allergEAZE® clear patch test chamber - SmartPractice®) containing liquid paraffin.
- Concentrations: 50 %
- Evaluation (hr after challenge): approximately 48 and 72 hours (24 and 48 hours after removal of the dressing)

Challenge controls:

None

Positive control substance(s):

yes

Remarks:

alpha-Hexylcinnamaldehyde (recent reliability check)

Positive control results:

In recent studies, alpha-Hexylcinnamaldehyde was classified in category 1 “Skin sensitisation” sub-category 1B.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

12.5%

No. with + reactions:

9

Total no. in group:

10

Clinical observations:

1 with discrete or patchy erythema; 2 with moderate and confluent erythema; 5 with intense erythema and swelling

Remarks on result:

other: other: reference substance : alpha-hexylcinnamaldehyde : 90% of animals sensitized.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

12.5%

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

4 with discrete or patchy erythema; 5 with intense erythema and swelling

Remarks on result:

other: reference substance : alpha-hexylcinnamaldehyde : 80% of animals sensitized.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50%

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

discrete or patchy erythema (score 1) in 1/10 animals

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50%. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: discrete or patchy erythema (score 1) in 1/10 animals.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

0

Total no. in group:

20

Clinical observations:

None

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 0.0. Total no. in groups: 20.0.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

None

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0%

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

1 male with discrete erythema and dryness of the skin

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: 1 male with discrete erythema and dryness of the skin.

Preliminary studies

- MNNC determination:

24 hours after the injections, a moderate erythema and a discrete erythema have been observed in the animals at the tested concentration of 20%. Discrete erythema has been observed in all animals at the tested concentration of 10%. In view of these results, the first induction of the Group 2 has been carried out by intradermal injection at the maximal non necrosing concentration of 20%

- Pre MNIC determination:

24 hours after the removal of the occlusive dressings, a moderate erythema and a discrete erythema have been observed in the animals at the tested concentration of 100%. No cutaneous reaction was noted at the other tested concentrations 50%, 20% and 10%. In view of these results, the concentration selected was 100% for the 2nd induction of the Group 2 and the MNIC determination began at the concentration of 50%. As a discrete erythema was noted in one animal at the test concentration of 100%, SLS application was necessary, 24 hours before the topical induction.

- MNIC determination:

24 hours after the removal of the occlusive dressings, no cutaneous reaction was noted at all concentrations.

In view of this result, the concentrations selected were 50% (MNIC) and 0% (liquid paraffin) for the challenge phase.

Main study

- Induction phase Group 2 (treated):

Moderate erythema was noted in five animals (5/10) and discrete erythema was noted in five animals (5/5) 24 hours after the first induction. Dryness was noted in three animals (3/10) and scab was noted in seven animals (7/10) 24 hours after the second induction.

- Induction phase Group 1 (control):

No cutaneous reaction was recorded after the first induction. Dryness was noted in all animals (5/5) 24 hours after the second induction.

- Challenge phase Groups 1 & 2:

In the treated group (treatment dose of 50%), 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 50%), no cutaneous intolerance reaction was recorded after the challenge phase. No cutaneous reaction was recorded in animals from the treated group after the challenge phase, on the treated area with liquid paraffin (control item).

No cutaneous reaction was recorded in animals from the control group after the challenge phase, on the treated area with liquid paraffin (control item).

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:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Based on the overall sensitisation rate, the test material is not classified as skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

Executive summary:

In a dermal sensitisation study performed according to the OECD test method No. 406 and in compliance with GLP, the test material was tested in male and female Hartley guinea-pigs using the Guinea-Pig Maximisation Test method (10 treated animals + 5 controls).

The preliminary study determined the concentration to be used for the induction and challenge phases of the main study.

The test material diluted in olive oil at 20 %( w/w) was administered by injection for intradermal induction. On Day 6, the animals received a topical application of sodium lauryl sulfate in vaseline (10% w/w), in order to induce local irritation. On Day 7, topical induction was performed with the test material as supplied. For the challenge, on Day 20, the test material was tested diluted in liquid paraffin at 50% (w/w).

In the treated group (treatment dose of 50%), 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 50%), no cutaneous intolerance reaction was recorded after the challenge phase.

No cutaneous reaction was recorded in animals from the treated or control group after the challenge phase, on the treated area with liquid paraffin (control items).

The results of the 3 last positive control groups carried out in order to assess the sensitivity of the strain of guinea pig used at these laboratories to a known sensitizer are satisfactory. The experimental technique used was therefore considered to be valid.

Based on the overall sensitisation rate, the test material is not classified as skin sensitizer according to the annex VI of the Regulation EC No. 1272/2008 (CLP).

This study is considered as acceptable and satisfies the requirement for the skin sensitisation endpoint.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Existing human data (HRIPT, Biosearch, 1981)) concluded that the registered substance has no skin sensitization potential at 4%. However, the reliability of this data was not considered adequate to meet the REACH requirements and to make a conclusion on classification and labelling.

For new in vivo testing of skin sensitisation potential, the murine local lymph node assay (LLNA) is the REACH Annex VII-endorsed method. Despite the advantages of the LLNA over TG 406, it has been recognised that there are certain limitations that may necessitate the use of TG 406, e.g. false positive findings with certain skin irritants [such as some surfactant type chemicals]. This is applicable to the registered substance which is a skin irritant and is surface-active. The GPMT was therefore considered more appropriate for this test item.

This GPMT (Phycher, 2016, Rel.1) was performed according to OECD Guideline 406 and in compliance with GLP (10 treated animals + 5 controls). The preliminary study determined the concentration to be used for the induction and challenge phases of the main study.

The test material diluted in olive oil at 20 %( w/w) was administered by injection for intradermal induction (0.1 mL 20% w/w for intradermal injections followed by topical induction with 0.5 mL 100%). On Day 6, the animals received a topical application of sodium lauryl sulfate in vaseline (10% w/w), in order to induce local irritation. On Day 7, topical induction was performed with the test material as supplied. For the challenge, on Day 20, the test material was tested diluted in liquid paraffin at 50% (w/w).

In the treated group (treatment dose of 50%), 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 50%), no cutaneous intolerance reaction was recorded after the challenge phase.

No cutaneous reaction was recorded in animals from the treated or control group after the challenge phase, on the treated area with liquid paraffin (control items).

The results of the 3 last positive control groups carried out in order to assess the sensitivity of the strain of guinea pig used at these laboratories to a known sensitizer are satisfactory. The experimental technique used was therefore considered to be valid.

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

Migrated from Short description of key information:
- GPMT, not sensitising (OECD 406, GLP, K, Rel. 1)
- Not sensitising in humans at 4 % (HRIPT).

Justification for selection of skin sensitisation endpoint:
Only one study available. The key-study is GLP-compliant and of high quality (Klimisch score = 1).

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).

No data was available regarding respiratory sensitisation.