Registration Dossier

Administrative data

Description of key information

LLNA, not a skin sensitizer (read-across substance Resinoid of Boswellia Carterii (Burseraceae) obtained from exudate by hexane extraction; 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
27 July to 06 September 2017
Justification for type of information:
The source substance and the target substance have the same botanical origin
Reason / purpose:
read-across source
Positive control results:
The positive control α Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (13.45) 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:
1.67
Test group / Remarks:
5% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
1.59
Test group / Remarks:
10% w/w in acetone/olive oil 4:1
Key result
Parameter:
SI
Value:
2.79
Test group / Remarks:
25% w/w in acetone/olive oil 4:1
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA:
See Table 7.4.1/1 below.

DETAILS ON STIMULATION INDEX CALCULATION
Stimulation index for 5, 10 and 25% v/v in acetone/olive oil 4:1 were 1.67, 1.59 and 2.79, respectively.

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

1081.68

1206.96
(±303.86)

na

na

1-2

1038.55

1-3

1645.81

1-4

887.26

1-5

1381.52

Test Item
5w/win
acetone/olive oil 4:1

2-1

1353.33

2020.02*
(±584.14)

1.67

Negative

2-2

2581.54

2-3

1722.25

2-4

2688.00

2-5

1754.99

Test Item
10w/win
acetone/olive oil 4:1

3-1

1203.98

1922.29
(±665.24)

1.59

Negative

3-2

1795.78

3-3

1422.01

3-4

2390.86

3-5

2798.84

Test Item
25w/win
acetone/olive oil 4:1

4-1

2140.11

3371.42**
(±1073.79)

2.79

Negative

4-2

3989.73

4-3

4888.40

4-4

2875.62

4-5

2963.22

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

5-1

12499.31

16238.89**
(±4556.94)

13.45

Positive

5-2

21524.30

5-3

20373.82

5-4

15387.17

5-5

11409.83


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
5w/win
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
10w/win
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
25w/win
acetone/olive oil 4:1

4-1

0

0

0

0Rt

0

0Rt

0

0

0

4-2

0

0

0

0Rt

0

0Rt

0

0

0

4-3

0

0

0

0Rt

0

0Rt

0

0

0

4-4

0

0

0

0Rt

0

0Rt

0

0

0

4-5

0

0

0

0Rt

0

0Rt

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

Rt = Sticky residual test item on the ears

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
5w/win
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
10w/win
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
25w/win
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:
GHS criteria not met
Conclusions:
Under the test conditions, test item is considered to be a non-sensitizer under the conditions of the test. Therefore the registered substance is also considered to be a non-sensitizer.
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 at a concentration of 50% w/w in which a greater than 25% increase in ear thickness was noted, a further screening test at a concentration of 25% w/w was performed. No clinical signs of toxicity or excessive local irritation were noted at a concentration of 25% w/w and therefore 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 4:1 at concentrations of 25%,10% or 5% w/w. A further group of five animals was treated withacetone/olive oil 4:1 alone. 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 25%, 10% or 5% w/w in acetone/olive oil 4:1 were 2.79, 1.59 and 1.67, respectively. Calculation of an EC3 value was not possible due to the negative result obtained at each concentration tested. No signs of systemic toxicity or excessive local skin irritation were noted at the concentrations of 25%,10% or 5% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (13.45) 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 item was considered to be a non-sensitizer under the conditions of the test.

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:

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 at a concentration of 50% w/w in which a greater than 25% increase in ear thickness was noted, a further screening test at a concentration of 25% w/w was performed. No clinical signs of toxicity or excessive local irritation were noted at a concentration of 25% w/w and therefore 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 4:1 at concentrations of 25%,10% or 5% w/w. A further group of five animals was treated withacetone/olive oil 4:1 alone. 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 25%, 10% or 5% w/w in acetone/olive oil 4:1 were 2.79, 1.59 and 1.67, respectively. Calculation of an EC3 value was not possible due to the negative result obtained at each concentration tested. No signs of systemic toxicity or excessive local skin irritation were noted at the concentrations of 25%,10% or 5% w/w.

The positive control α-Hexylcinnamaldehyde, tech., 85% gave a Stimulation Index of greater than 3 (13.45) 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 item was considered to be a non-sensitizer under the conditions of the test.

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 not classified as a skin sensitizer according to the Regulation (EC) No. 1272/2008 (CLP) and to the Globally Harmonised System of classification and labelling of chemicals (GHS).

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