Registration Dossier

Administrative data

Description of key information

LLNA, not a skin sensitizer (OECD 429, GLP, K, Rel. 1)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 September to 15 December 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
GLP study conducted in compliance with OECD Guideline No. 429 without any deviation.
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 22 July 2010
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Remarks:
UK GLP Compliance Programme (inspected on July 05, 2016/ signed on October 28, 2016)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
- Storage conditions: At room temperature, protected from moisture (Dry area, unopened containers, optimum temp. 11-25 °C / 52-77 °F)
Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Envigo RMS B.V., Inc., Horst, The Netherlands.
- Females nulliparous and non-pregnant: Yes
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Envigo RMS (UK) Limited, Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: at least 15 changes per hour
- Photoperiod: 12 hours continuous light and 12 hours darkness
Vehicle:
other: Tetrahydrofuran
Concentration:
10%, 5% and 2.5 %
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: For the purpose of the study, solubility checks were performed in house. All of the vehicles available (acetone/olive oil, DMF, butanone, DMSO, acetone, 1% Pluronic L92 in distilled water, 7:3 ethanol/distilled water and propylene glycol) have been investigated and none have found to be suitable, down to a concentration of 5%, even after heating to 80°C. Therefore, Tetrahydrofuran (THF) was found to produce a suitable formulation at a maximum attainable concentration of 10%. THF was shown to be a suitable vehicle in a validation study for positive control (α Hexylcinnamaldehyde, tech., 85% in tetrahydrofuran; there was no toxicity and no irritation and it produced a very acceptable dose response).
- Preliminary Screening test: As no toxicological information was available regarding the systemic toxicity/irritancy potential of the test item, a preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item at a concentration of 10% in THF (maximum attainable concentration), to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily. Any clinical signs of toxicity, if present, were also recorded. The body weight of the mouse was recorded on Day 1 (prior to dosing) and on Day 6.
The thickness of each ear was measured using a Mitutoyo 547-300S gauge (Mitutoyo Corporation), pre-dose and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6. Any changes in the ear thickness were noted. Mean ear thickness changes were calculated between time periods Days 1 and 3 and Days 1 and 6. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitization.
- Irritation: no signs of local skin irritation were observed during the preliminary screening test at 10% in THF (maximum attainable concentration).
- Systemic toxicity: no signs of toxicity were observed during the preliminary screening test at 10% in THF (maximum attainable concentration).
- Ear thickness measurements: no marked increase in ear thickness was observed during the preliminary screening test at 10% in THF (maximum attainable concentration).

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay, individual method
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non sensitizer".

TREATMENT PREPARATION AND ADMINISTRATION:
The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette.
A further group of five mice received the vehicle alone in the same manner.
The positive control animals were similarly treated to the test animals except that 25 μL of the positive control item, α-Hexylcinnamaldehyde, tech., 85%, at a concentration of 25% v/v in Tetrahydrofuran (THF), was applied to the dorsal surface of each ear.
Local skin irritation was scored daily. The thickness of each ear was measured and recorded pre and post dose on Day 1, post dose on Days 2 and 3 and on Days 4, 5 and 6.
Five days following the first topical application of the test item, vehicle control item or positive control item (Day 6) all mice were injected via the tail vein with 0.25 mL (250 μL) of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse.
Termination: Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation followed by cervical separation. For each individual animal of each group the draining auricular lymph nodes were excised and processed. For each individual animal 1 mL of PBS was added to the lymph nodes.
Preparation of Single Cell Suspension: A single cell suspension of the lymph node cells for each individual animal was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL of PBS into a petri dish labeled with the study number and dose concentration. The lymph node cells suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).
Determination of 3HTdR Incorporation: After approximately 18 hours incubation at approximately 4 °C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by β-scintillation counting. The "Poly Q™" vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigorously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system (Beckman Instruments Inc, Fullerton, CA, USA).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Data was processed to give group mean values for disintegrations per minute and standard deviations where appropriate. Individual and group mean disintegrations per minute values were assessed for dose response relationships. Data was first assessed for suitability by analysis of normality and homogeneity of variance. If the assumptions that the data are both normally distributed and has homogeneity of variances, then parametric one way analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to determine statistical significance. If the assumptions were not met, non parametric Kruskal Wallis Rank Sum and Mann Whitney U test procedures were used.
Positive control results:
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.02) when tested at a concentration of 25% v/v in Tetrahydrofuran, thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
0.98
Test group / Remarks:
10% in Tetrahydrofuran
Key result
Parameter:
SI
Value:
0.98
Test group / Remarks:
5% in tetrathydrofuran
Key result
Parameter:
SI
Value:
1.03
Test group / Remarks:
2.5% in tetrathydrofuran
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
See Table 7.4.1/1 below.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 2.5, 5 and 10% in tetrahydrofuran were 1.03, 0.98 and 0.98, respectively.

EC3 CALCULATION
No EC3 calculation since the substance did not induced a stimulation index higher than 3 at tested cocnentrations (up to 10%).

CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity, visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the test or control animals during the test (See Table 7.4.2 & 7.4.3 below).

BODY WEIGHTS: Body weight change of the test animals between Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Table 7.4.1/1: Individual Disintegrations per Minute and Stimulation Index

Treatment Group

Animal Number

dpm/Animal (a)

Mean dpm/Animal
(Standard Deviation)

Stimulation Index(b)

Result

Vehicle
Tetrahydrofuran

1-1

1127.45

1570.55
(±317.83)

na

na

1-2

1740.78

1-3

1406.20

1-4

1956.74

1-5

1621.58

Test Item
2.5
in tetrahydrofuran

2-1

1766.36

1610.31
(±484.50)

1.03

Negative

2-2

1373.16

2-3

1889.09

2-4

2128.35

2-5

894.57

Test Item

5in tetrahydrofuran

3-1

1598.93

1535.81
(±634.38)

0.98

 

Negative

3-2

826.48

3-3

2454.61

3-4

1732.12

3-5

1066.91

Test Item

10% in tetrahydrofuran

4-1

1150.31

1541.74
(±511.17)

0.98

 

Negative

4-2

1971.18

4-3

2211.67

4-4

1251.51

4-5

1124.02

Positive Control Item
25% v/v in
acetone/olive oil 4:1

5-1

5147.55

6317.55
(±2172.17)

4.02

Positive

5-2

10006.96

5-3

6314.46

5-4

5662.22

5-5

4456.58


dpm=     Disintegrations per minute

a=        Total number of lymph nodes per animal is 2

b=        Stimulation Index of 3.0 or greater indicates a positive result

Table 7.4.1/2: Individual Clinical Observations and Mortality Data

Treatment Group

Animal Number

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Pre-Dose

Post Dose

Vehicle
acetone/olive oil 4:1

1-1

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

Test Item
10v/vin
acetone/olive oil 4:1

2-1

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

Test Item
25v/vin
acetone/olive oil 4:1

3-1

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

Test Item
50v/vin
acetone/olive oil 4:1

4-1

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

Positive Control Item
25% v/v in
acetone/olive oil 4:1

5-1

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0


0=   No signs of systemic toxicity

Table 7.4.1/3: Local Skin Irritation

Treatment Group

Animal Number

Local Skin Irritation

Day 1

Day 2

Day 3

Day 4

Day 5

Day 6

left

right

left

right

left

right

left

right

left

right

left

right

Vehicle
acetone/olive oil 4:1

1-1

0

0

0

0

0

0

0

0

0

0

0

0

1-2

0

0

0

0

0

0

0

0

0

0

0

0

1-3

0

0

0

0

0

0

0

0

0

0

0

0

1-4

0

0

0

0

0

0

0

0

0

0

0

0

1-5

0

0

0

0

0

0

0

0

0

0

0

0

Test Item
10v/vin
acetone/olive oil 4:1

2-1

0

0

0

0

0

0

0

0

0

0

0

0

2-2

0

0

0

0

0

0

0

0

0

0

0

0

2-3

0

0

0

0

0

0

0

0

0

0

0

0

2-4

0

0

0

0

0

0

0

0

0

0

0

0

2-5

0

0

0

0

0

0

0

0

0

0

0

0

Test Item
25v/vin
acetone/olive oil 4:1

3-1

0

0

0

0

0

0

0

0

0

0

0

0

3-2

0

0

0

0

0

0

0

0

0

0

0

0

3-3

0

0

0

0

0

0

0

0

0

0

0

0

3-4

0

0

0

0

0

0

0

0

0

0

0

0

3-5

0

0

0

0

0

0

0

0

0

0

0

0

Test Item
50v/vin
acetone/olive oil 4:1

4-1

0

0

0

0

0

0

0

0

0

0

0

0

4-2

0

0

0

0

0

0

0

0

0

0

0

0

4-3

0

0

0

0

0

0

0

0

0

0

0

0

4-4

0

0

0

0

0

0

0

0

0

0

0

0

4-5

0

0

0

0

0

0

0

0

0

0

0

0

Positive Control
15% v/v in
acetone/olive oil 4:1

5-1

0

0

0

0

0

0

0

0

0

0

0

0

5-2

0

0

0

0

0

0

0

0

0

0

0

0

5-3

0

0

0

0

0

0

0

0

0

0

0

0

5-4

0

0

0

0

0

0

0

0

0

0

0

0

5-5

0

0

0

0

0

0

0

0

0

0

0

0

Interpretation of results:
GHS criteria not met
Conclusions:
Under the test conditions, test material is classified as a non-sensitizer according to Regulation (EC) No. 1272/2008.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/Ca strain mouse following topical application to the dorsal surface of the ear. The method was conducted according to the OECD test guideline No 429 and in compliance with GLP. 

Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in Tetrahydrofuran (THF) at concentrations of 10%, 5% or 2.5% v/v. A further group of five animals was treated with THF alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in THF.

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

Stimulation index for 2.5%, 5% or 10% v/v in THF were 1.03, 0.98 and 0.98, respectively. There were no deaths. No signs of systemic toxicity or local skin irritation (visual and ear thickness measurements) were noted in the test or control animals during the test.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.02) when tested at a concentration of 25% v/v in THF, thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, test material is classified as a non-sensitizer according to Regulation (EC) No. 1272/2008.

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

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 (July, 2017), 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

(at the initiation of the dossier, no test was available)

Existing human data

2

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

NO

 

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

(at the initiation of the dossier, no test was available)

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, due to the mostly unknown composition (UVCB Type II 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

 

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)

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

According to OECD TG 442C, the DPRA “current prediction model cannot be used for complex mixtures of unknown composition or for substances of unknown or variable composition, complex reaction products or biological materials (i.e. UVCB substances) due to the defined molar ratio of test chemical and peptide”. Therefore, this method cannot be used for the registered substance.

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

According to the OECD TG 442D, “the Keratinosens test method is applicable to test chemicals soluble or that form a stable dispersion […] either in water or DMSO (including all of the test chemical components in the case of testing a multi-constituent substance or a mixture).” The substance is a complex mixture, which contains a large number of constituents with different solubility (often low to very low water solubility) and volatility. In addition, the composition of this complex substance is mostly unknown, and its physical state leads to further difficulties for its solubilisation, even in organic solvents: It is pasty and solubility issues are therefore anticipated with this test method. Thus, this test method is considered not to be applicable for the substance.
LuSens and SENS-IS methods are under validation assessment.

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

HCLAT, U-SENS and IL-8 Luc Assay: according to the OECD 442E, “limited information is currently available on the applicability of the test methods to multi-constituent substances/mixtures”. In addition, the methods are “applicable to test chemicals soluble or that form a stable dispersion (i.e. a colloid or suspension in which the test chemical does not settle or separate from the solvent/vehicle into different phases) in an appropriate solvent/vehicle”. As mentioned above, the substance is a complex mixture, which contains a large number of constituents with different solubility (often low to very low water solubility) and volatility. In addition, the composition of this complex substance is mostly unknown, and its physical state leads to further difficulties for its solubilisation, even in organic solvents: It is pasty and solubility issues are therefore anticipated with this test method. Thus, the suitability of this test method cannot be ensured.

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

 

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 LLNA assay was initiated: Not a skin sensitiser (tested up to 10% due to poor solubility)

Therefore, a new LLNA study was performed according to the OECD test guideline No. 429 and in compliance with GLP (Envigo, 2017, rel.1).

 

Following a preliminary screening test in which no clinical signs of toxicity or excessive local irritation were noted at a concentration of 10% w/w, this concentration was selected as the highest dose investigated in the main test of the Local Lymph Node Assay. Three groups, each of five animals, were treated with 50 µL (25 µL per ear) of the test item as a solution in Tetrahydrofuran (THF) at concentrations of 10%, 5% or 2.5% v/v. A further group of five animals was treated with THF alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde tech., 85%, at a concentration of 25% v/v in THF.

The proliferative response of the lymph node cells (LNC) from the draining auricular lymph nodes was assessed five days following the initial application, by measurement of the incorporation of 3H-methyl Thymidine (3HTdR) by β-scintillation counting of LNC suspensions. The response was expressed as radioactive disintegrations per minute per lymph node (dpm/node) and as the ratio of 3HTdR incorporation into LNC of test nodes relative to that recorded for control nodes (test/control ratio), termed as Stimulation Index (SI).

Stimulation index for 2.5%, 5% or 10% v/v in THF were 1.03, 0.98 and 0.98, respectively. There were no deaths. No signs of systemic toxicity or local skin irritation (visual and ear thickness measurements) were noted in the test or control animals during the test.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (4.02) when tested at a concentration of 25% v/v in THF, thus, demonstrating the sensitivity and reliability of the test system.

 Under the test conditions, test material is classified as a non-sensitizer according to the Regulation (EC) No. 1272/2008 and to the GHS.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Harmonized classification:

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

One of the constituents, cis-tricos-9-ene, has a CLH as Skin Sens.1B, which rings a Quality Check alert in the Technical Completeness Check. However, since an experimental study is available on the UVCB, it has priority over the calculation method. The study was evaluated as reliable without restriction (K1), despite conducted on a 10% solution and not as pure; this was due to technical limitation because of the low solubility of the substance. Therefore it is considered that actual exposure cannot exceed the tested concentration, so the result is relevant for the UVCB. Moreover, based on composition, cis-tricos-9-ene typically amounts to 0.06%, which is below the generic concentration limit of 1% for classification limit for a SS1B component, and even below the concentration limit of 0.1% for elicitation. Finally, cis-tricos-9-ene was identified in the volatile fraction only, so considering the % in the whole UVCB is a worst-case with regard to skin contact. As a conclusion, both experimental and calculation methods provide a consistent outcome for non-classification, and EUH208 labelling is not required either.

Self-classification:

Based on the available data, no additional classification is proposed regarding skin sensitisation according to the Regulation (EC) No. 1272/2008 (CLP) and 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.