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Administrative data

Description of key information

- LLNA, Skin sensitizer (OECD 429, GLP, K, Rel. 1)

- Solubility assays: no suitable solvents for in vitro tests (Waiver)

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 11 to November 05, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
2010
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
EC No 640/2012 Part B. Skin Sensitization: Local Lymph Node Assay, July 2012.
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
2003
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA:J
Remarks:
Inbred, SPF-Quality
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, Le Genest-Saint-Isle, France
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 10 weeks
- Weight at study initiation: 21.7 to 26.1 g
- Housing: Animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages containing sterilized sawdust as bedding material.
- Diet: Pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum,
- Water: Municipal tap-water, ad libitum via water bottles.
- Acclimation period: at least 5 days
- Indication of any skin lesions: Before the initiation of dosing, a health inspection was performed and any assigned animal considered unsuitable for use in the study were replaced by alternate animals obtained from the same shipment and maintained under the same environmental conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-22°C
- Humidity (%): 44-71%
- Air changes (per hr): at least 10 with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): 12 / 12

- IN-LIFE DATES: From September 13 To October 29, 2017
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
Main test: 10, 25 and 50 % v/v in acetone/olive oil 4:1
No. of animals per dose:
5 females
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 100% and 50% concentration in AcOO 4:1.
- The test system, procedures and techniques were identical to those used in the main study except that the animals were approximately 12 weeks (at initiation of treatment) and that the assessment of lymph node proliferation and necropsy were not performed.
- Ear thickness measurements: Ear thickness measurements were conducted using a digital thickness gauge (Kroeplin C110T-K) prior to dosing on Days 1 and 3, and on Day 6.

MAIN STUDY
Groups of five mice were treated with the test material at concentrations of 10, 25 and 50 % v/v in acetone/olive oil 4:1. The mice were treated by daily application of 25 µL of the appropriate concentration of the test material to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). A further group of five mice received the vehicle alone in the same manner. Each animal was injected via the tail vein with 250 µL of sterile phosphate buffered saline (PBS) containing 20 μCi of 3H-methyl thymidine on Day 6. After five hours, all animals were killed by intraperitoneal injection (0.2 mL/animal) of Euthasol(R) 20%. The draining (auricular) lymph node of each ear was excised. The nodes from the five mice were excised and pooled for each animal in PBS. A single cell suspension of lymph node cells (LNC) excised bilaterally using the individual animal approach was prepared in PBS by gentle separation through stainless steel gauze. LNC were washed twice with an excess of PBS by centrifugation at 200 g for 10 minutes at 4°C. To precipitate the DNA, the LNC were exposed to 5% trichloroacetic acid (TCA) and then stored in the refrigerator until the next day.
At Day 7, precipitates were recovered by centrifugation, resuspended in 1 mL TCA and transferred to 10 mL of Ultima Gold cocktail as scintillation fluid. Radioactivity measurements were performed using a Packard scintillation counter (2800TR). Counting time was to a statistical precision of ± 0.2% or a maximum of 5 minutes whichever came first. The scintillation counter was programmed to automatically subtract background and convert Counts Per Minute (CPM) to Disintegrations Per Minute (DPM).

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The individual SI is the ratio of the DPM/animal compared to the DPM/vehicle control group mean. If the results indicate a SI ≥ 3, the test item may be regarded as a skin sensitizer.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No data
Positive control results:
The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
1.6
Test group / Remarks:
10 %
Key result
Parameter:
SI
Value:
2
Test group / Remarks:
25 %
Key result
Parameter:
SI
Value:
4.6
Test group / Remarks:
50 %
Key result
Parameter:
EC3
Value:
34.6
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
Mean DPM / animal for vehicle, 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 440, 687, 886 and 2037, respectively.

DETAILS ON STIMULATION INDEX CALCULATION
- Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.6, 2.0 and 4.6, respectively.
- A stimulation index of greater than 3 was recorded for the test material at concentrations of 50 % v/v in acetone/olive oil 4:1.
- A stimulation index of less than 3 was recorded for the test material at a concentration of 10 and 25 % v/v in acetone/olive oil 4:1.

EC3 CALCULATION
EC3 = 34.6 (> 2%)

CLINICAL OBSERVATIONS
No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study.
The very slight erythema of the ears as shown by one animal treated at 50% on Day 3 and the scaliness as shown by all animals treated at 50% between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes.

BODY WEIGHTS
Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Table 7.4.1/1: Disintegrations per Minute and Stimulation Index

Concentration (% v/v in acetone/olive oil 4:1)

dpm

Stimulation Index

Result

Vehicle

440

1.0

na

10

687

1.6

Negative

25

886

2.0

Negative

50

2037

4.6

Positive

 dpm = Disintegrations per minute

na = Not applicable

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
Under the test conditions, test material is classified as skin sensitiser (Category 1B) according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS.
Executive summary:

A study was performed to assess the skin sensitisation potential of test material in the CBA/J 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.

Test item concentrations selected for the main study were based on the results of a preliminary screening test.

Three groups, each of five females, were treated with 50 μL (25 μL per ear) of test material as a solution in acetone/olive oil 4:1 at concentrations of 10, 25 and 50 % v/v for 3 consecutive days. A further group of five animals was treated with acetone/olive oil 4:1 alone. The animals were allowed to rest without dosing on days 4 and 5. On day 6, the proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3H-Methyl Thymidine.

 

Mean DPM / animal for vehicle, 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 440, 687, 886 and 2037, respectively. Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.6, 2.0 and 4.6, respectively. A stimulation index of greater than 3 was recorded for the test material at concentrations of 50 % v/v in acetone/olive oil 4:1. A stimulation index of less than 3 was recorded for the test material at a concentration of 10 and 25 % v/v in acetone/olive oil 4:1. The EC3 value was calculated to be 34.6%

There were no deaths and no clinical signs. The very slight erythema of the ears as shown by one animal treated at 50% on Day 3 and the scaliness as shown by all animals treated at 50% between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

 

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity. The test system was therefore considered to be valid.

 

Under the test conditions, test material is classified as skin sensitizer (Category 1B) according to the annex I of the Regulation EC No. 1272/2008 (CLP) and to the GHS since EC3 is higer than 2%.

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

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Justification for type of information:
JUSTIFICATION FOR DATA WAIVING
In accordance with Column 2 of REACH Annex VIII, an in vitro or in chemico skin sensitisation study does not need to be conducted because the available in vitro/in chemico test methods are not applicable for the substance or are not adequate for classification and risk assessment according to point 8.3.
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

 Solubility assays were performed to determine the feasibility of in chemico and in vitro skin sensitisation studies. Moreover, the test substance is not into the applicability domain of h-CLAT based on its partition coefficient value which is higher than 3.5.

The results showed that the test substance could not be dissolved in any recommended solvent at the minimum concentration required in DPRA and KeratinoSensTM assays. Moreover, it is considered that no appropriate solvent for h-CLAT and U-SENS assays is available since the recommended solvents are the same as those used in KeratinoSensTMassay.

 

In this situation, only a positive result could be valid to conclude on sensitisation potential, an in vivo study would be necessary to assess the potency category of the test material.

Then, it was decided to perform directly an in vivo study (LLNA).

 

A key study was then identified (CRL, GLP, K, rel.1). This Local Lymph Node Assay was conducted according to the OECD test guideline No 429 and in compliance with GLP.

Following a preliminary screening test with test material at concentrations of 100 and 50% in acetone/olive oil 4:1, three groups, each of five females, were treated with the test material as a solution in acetone/olive oil 4:1 at concentrations of 10, 25 and 50 % for 3 consecutive days. A further group of five animals was treated with acetone/olive oil 4:1 alone.

The six-month reliability check with Alpha-hexylcinnamaldehyde indicates that the Local Lymph Node Assay as performed at Charles River Den Bosch is an appropriate model for testing for contact hypersensitivity.

Stimulation index for 10, 25 and 50 % v/v in acetone/olive oil 4:1 were 1.6, 2.0 and 4.6, respectively.

The EC3 value was calculated to be 34.6%

There were no deaths and no signs of systemic toxicity. The very slight erythema of the ears as shown by one animal treated at 50% on Day 3 and the scaliness as shown by all animals treated at 50% between Days 2 and 6 was considered not to have a toxicologically significant effect on the activity of the nodes. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

Under the test conditions, the test material is classified as skin sensitizer.

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.

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 GHS, since EC3 > 2%.

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