Registration Dossier

Administrative data

Description of key information

LLNA, skin sensitizer Category 1B (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:
25 July to 22 August 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:
Identification: GALBANUM RDE SUPER INDE
Other name: Galbanum resinoid FIR; 944452
Batch: 1003396873
Purity: 100% UVCB
Physical state/Appearance: brown to brown red, very viscous liquid
Expiry Date: 18 May 2020
Storage Conditions: room temperature in the dark
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:
acetone/olive oil (4:1 v/v)
Concentration:
50%, 25% or 10% v/v
No. of animals per dose:
5
Details on study design:
PRE-SCREEN TESTS:
- Test Item preparation: For the purpose of the study, the test item was freshly prepared, as required, as a solution in acetone/olive oil 4:1. This vehicle was chosen as it produced the highest concentration that was suitable for dosing. The test item was formulated within 2 hours of being applied to the test system. It is assumed that the formulation was stable for this duration.
- 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 two mice, one mouse per test item concentration. The mice were treated by daily application of 25 µL of the undiluted test item or the test item at a concentration of 50% v/v in acetone/olive oil 4:1, to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mice were observed twice daily on Days 1, 2 and 3 and once daily on Days 4, 5 and 6. Local skin irritation was scored daily according to the scale included as Annex 4. Any clinical signs of toxicity, if present, were also recorded. The body weight of each 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.
- Results: Colorless, sticky residual test item on the ears was noted in both animals post-dose on Day 1 and up to Day 5. Fur loss was also noted in the animal treated with the undiluted test item pre-dose on Day 2 and for the remainder of the observation period.
No signs of systemic toxicity were noted.
Very slight erythema was noted on both ears and a greater than 25% increase in mean ear thickness were noted on Days 2 and 3 in the animal treated with the undiluted test item.
No visual local skin irritation or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the animal treated with the test item at a concentration of 50% v/v in acetone/olive oil 4:1.
Based on this information, and after consultation with the Sponsor, the dose levels selected for the main test were 50%, 25% and 10% v/v in acetone/olive oil 4:1.

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 acetone/olive oil 4:1, 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 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: AAfter 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 beta 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 (13.21) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.
Key result
Parameter:
SI
Value:
2.65
Test group / Remarks:
10% v/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
2.72
Test group / Remarks:
25% v/v in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
3.98
Test group / Remarks:
50% v/v in acetone/olive oil 4:1
Key result
Parameter:
EC3
Remarks:
%
Value:
31
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
See Table 7.4.1/1 below.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 10, 25 and 50% v/v in acetone/olive oil 4:1 were 2.65, 2.72 and 3.98, respectively.

EC3 CALCULATION
EC3 = c + [[(3-d)/(bd)] x (a-c)]
a=50
b=3.98
c=25
d=2.72

EC3 = 25 + [[(3-2.72)/(3.98-2.72)] x (50-25)] = 31
The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (EC3 value) was calculated to be 31%.


CLINICAL OBSERVATIONS: There were no deaths. No signs of systemic toxicity were noted in the test or control animals during the test (See Table 7.4.2 & 7.4.3 below).
EAR THICKNESS: No treatment group showed an equal to or greater than 25% increase in mean ear thickness over the observation period.
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 Indexb

Result

Vehicle
acetone/olive oil 4:1

1-1

952.08

1258.06
(±290.70)

na

na

1-2

1490.77

1-3

1455.73

1-4

1463.65

1-5

928.06

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

2-1

2459.57

3337.46**
(±895.14)

2.65

Negative

2-2

2437.85

2-3

3468.88

2-4

4516.19

2-5

3804.83

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

3-1

5945.86

3424.96**
(±1502.02)

2.72

Negative

3-2

2930.50

3-3

2612.58

3-4

2101.05

3-5

3534.83

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

4-1

4046.77

5009.06**
(±1289.01)

3.98

Positive

4-2

5035.10

4-3

5771.02

4-4

6696.78

4-5

3495.61

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

5-1

24688.01

16623.79**
(±7495.30)

13.21

Positive

5-2

8217.10

5-3

13632.79

5-4

12186.77

5-5

24394.30


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

**=       Significantly different from vehicle control group p<0.01

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 Item
25% 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:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as a contact sensitizer (Category 1B) 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 irritation were noted at a concentration of50v/v, 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 acetone/olive oil 4:1at concentrations of 50%,25% or10v/v. A further group of five animals was treated with acetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde, technical grade, 85%, at a concentration of 25% v/v inacetone/olive oil 4:1

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 50%, 25% or 10% v/vin acetone/olive oil 4:1 were 3.98, 2.72 and 2.65, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3value) was calculated to be 31%. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 50%, 25% or 10% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (13.21) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, the test material is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008.

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

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (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.

As the weight-of-evidence approach using a combination of alternative test methods cannot be applied and that there are no existing human, animal, (Q)SAR or read-across data to assess the skin sensitisation potential of the substance, an in vivo alternative study (LLNA) was therefore performed.  

A 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 irritation were noted at a concentration of50v/v, 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 acetone/olive oil 4:1at concentrations of50%,25% or10v/v. A further group of five animals was treated with acetone/olive oil 4:1alone. A concurrent positive control test, using a group of five animals, was also performed with the known sensitizer, α‑Hexylcinnamaldehyde, technical grade, 85%, at a concentration of 25% v/v in acetone/olive oil 4:1

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 50%, 25% or 10% v/vin acetone/olive oil 4:1 were 3.98, 2.72 and 2.65, respectively. The concentration of test item expected to cause a 3 fold increase in 3HTdR incorporation (extrapolated EC3 value) was calculated to be 31%. No sign of systemic toxicity or excessive local skin irritation were noted at the concentrations of 50%, 25% or 10% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (13.21) when tested at a concentration of 25% v/v in acetone/olive oil 4:1, thus, demonstrating the sensitivity and reliability of the test system.

 

Under the test conditions, the test material is classified as a contact sensitizer (Category 1B) according to Regulation (EC) No. 1272/2008.

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

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

Under the REACH regulation there is no legal standard information requirement in Annexes VII to X to perform any specific test for respiratory sensitisation. In addition, no validated or widely recognised in vitro or in vivo test methods specific to respiratory sensitisation are available yet. No human or animal data are available on the substance to address respiratory sensitisation. Due to the mostly unknown composition of the substance, it was not possible to use OECD QSAR Toolbox predictions.

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 classified as Skin Sens. 1B, H317 (May cause an allergic skin reaction) according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonised System of classification and labelling of chemicals (GHS), since EC3 is > 2% (31%).

No direct scientific data are available on the substance to address respiratory sensitisation.