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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:
2019
Reliability:
1 (reliable without restriction)
Qualifier:
according to guideline
Guideline:
OECD Guideline 442B (Skin sensitisation: Local Lymph Node Assay: BrdU-ELISA or –FCM)
Version / remarks:
Adopted : 25 June 2018
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA): BrdU-ELISA
Species:
mouse
Strain:
other: CBA/JCrHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: KOATECH Co., Ltd. (181-21, Jinwi-ro, Jinwi-myeon, Pyeongtaek-si, Gyeonggi-do, Korea)
- Females (if applicable) nulliparous and non-pregnant: not specified
- Microbiological status of animals, when known: not specified
- Age at study initiation: 9 weeks
- Weight at study initiation: 21.73 g – 24.16 g (Pre-screen test); 21.83 g – 25.25 g (Main test)
- Housing:
Cage style : Polysulfonate cage
Cage size : (180W × 300D × 140H) mm
Animal per cage : Less than 5
Laboratory animal bedding aspen [ABEDD, Austria] was used after high-pressure steam
sterilization (121 ℃, 20 minutes) in the laboratory.
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature : (21.7 – 22.7) ℃
- Relative humidity : (49.0 – 63.6) % R.H.
- Air exchange : (10 – 20) times/h
- Light cycle : Light 12 h (08:00 - 20:00)
- Dark 12 h (20:00 - 08:00)
- Illumination : (150 - 300) Lux

- IN-LIFE DATES:
From: 2019-03-07
To: 2019-03-25
Vehicle:
dimethyl sulphoxide
Concentration:
0.25 %, 0.5 % and 1 % (v/v)

The positive control was dissolved in acetone / olive oil (4:1, v/v).
No. of animals per dose:
6
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: Solution in dimethyl sulfoxide
- Systemic toxicity: Animals exposed to concentrations of 25 %, 50 % and 100 % did not survive.
- Ear thickness measurements: The animals showed increased ear thickness at concentrations of 5 % and 10 % which increased further at 25 %.
- Erythema scores: Severe erythema (score 3) was observed in animals exposed to concentrations of 5% and 10%.
Animals exposed to concentrations of 2.5 % and 1 % observed well-defined erythema on both ears (score 2), and ones exposed to 0.5 % concentration observed very slight erythema on both ears (barely perceptible) (score 1).

A pre-screen test was conducted in ten groups of CBA/J mice to determine the concentrations of test substance to be used in the main study. Animals exposed to concentrations of 25 %, 50 % and 100 % did not survive whereas those exposed to 10 % and 5 % concentration observed severe erythema (score 3) on both ears.
Further, animals exposed to concentrations of 2.5 % and 1 % observed well-defined erythema on both ears (score 2), and ones exposed to 0.5 % concentration observed very slight erythema on both ears (barely perceptible) (score 1). The animals showed increased ear thickness at concentrations of 5 % and 10 % which increased further at 25 %. No changes in body weight were observed in all animals. The test concentrations used for the main study were determined based on systemic toxicity and local dermal irritation studies. Concentrations (v/v) of 0.25 %, 0.5 % and 1 % were finally chosen for the main study.

MAIN STUDY
The study was performed according to OECD 442B.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response:
Skin responses was evaluated according to Erythema scores.
Skin sensitization was evaluated by calculating the mean stimulation index (SI) for each test group.
A positive response is indicated when an SI ≥ 1.6.

TREATMENT PREPARATION AND ADMINISTRATION:
Administration of substance: Day 1, Day 2, Day 3
The test article (Test substance, Vehicle control, Positive control) was spread over the entire dorsal surface of the ear using a micropipette at 25 μL/ear.

No treatment: Day 4
BrdU solution administration: Day 5
The mice were injected with BrdU solution at a dose of 0.5 mL per mouse inter peritoneally.

Auricular lymph node extraction: Day 6
Approximately 24 hours after BrdU solution injection, each mouse was sacrificed using CO2 asphyxiation, and the lymph nodes were collected.

Preparation of cell suspension: Day 6
From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through 70 μm nylon mesh (Falcon, USA). Volume of the LNC suspension was adjusted to a 18 mL using PBS.

Cellular proliferation measurement: Day 6
Cell suspension was measured by cell proliferation assay using a commercial BrdU kit. Absorbance of each well was measured using Microplate reader (Synergy HT, BioTek) at 370 nm with a reference wavelength of 492 nm.

Clinical signs were carefully observed once each day.

Body weight
Individual mouse body weight was measured at animal receipt day, Day 1, Day 6.

Ear thickness
Individual mouse left and right ear thickness was measured at Day 1, Day 3, Day 6.

Observation of test substance application site
Skin responses to site of administration were observed once each day according to Erythema scores.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The body weights and ear thickness of main test were presented as mean ±S.D. and analysis was conducted by using SPSS (Ver 19.0) program. The Levene's test was performed for homogeneity of variances. As a result of ANOVA test, the significance of the test groups has been identified and then Dunnett's T3 Post-test was conducted. The confidence interval was set to 95 %.
Positive control results:
The positive control group (25 % solution v/v of alpha-hexyl cinnamic aldehyde in acetone / olive oil (4:1, v/v)) caused the expected increased stimulation index (SI) of 3.3.
Key result
Parameter:
SI
Value:
3.6
Test group / Remarks:
0.25 % test item in DMSO
Parameter:
SI
Value:
5.5
Test group / Remarks:
0.5 % test item in DMSO
Parameter:
SI
Value:
5.2
Test group / Remarks:
1 % test item in DMSO
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA
The stimulation index (SI) was calculated to be 3.6, 5.5, and 5.2 for the test substance concentrations of 0.25 %, 0.5 %, and 1 % compared to the vehicle control (DMSO).
The stimulation index (SI) of the positive control was calculated to be 3.3 compared to the vehicle control (acetone: olive oil (4:1, v/v)).
Ear thickness increased by 11.11% and 16.67% in both ears of each mouse in the 0.5% and 1% test groups, respectively, although these changes were not statistically significant.

DETAILS ON STIMULATION INDEX CALCULATION
Skin sensitization was evaluated by calculating the mean stimulation index (SI) for each test group.

BrdU L.I. = Blank370 - Blank492

S.I. = (Mean of BrdU L.I. in test substance group) / (Mean of BrdU L.I. in negative control group)

L.I. = Labelling Index,
Blank370: ABSem-ABSblankem,
Blank492: ABSref-ABSblankref,
ABS: Absorbance,
em: emission wavelength (370 nm),
ref: reference wavelength (492 nm)

A positive response is indicated when an SI ≥ 1.6.

CLINICAL OBSERVATIONS:
No mortality was observed relating to test substance treatment.
No treatment-related clinical signs were observed in any treated animals.

BODY WEIGHTS
Animals of all treatment groups showed a normal change in body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness).
In the lowest dose group (0.25%), no skin reactions were observed on the ears.
In the middle dose group (0.5%), skin reactions were observed; they were very mild (barely noticeable) erythema (score 1) on both ears.
In the highest dose group (1 %), well-defined erythema (score 2) was observed on both ears.

Ear thickness increased by 11.11% and 16.67% in both ears of each mouse in the 0.5% and 1% test groups, respectively, although these changes were not statistically significant.

Summary of the LLNA results  (means of 5 animals per group)


 



























































 

Parameter investigated



Vehicle


Control
DMSO



Vehicle


Control
AOO



Dose
0.25 %



Dose


0.5 %



Dose


1 %



Positive


control



BrdU labelling index (mean)
(S.D.)



0.141
(0.020)



0.143
(0.037)



0.512
(0.037)



0.777
(0.058)



0.728
(0.032)



0.472
(0.105)



Stimulation index
 



1.0



1.0



3.6



5.5



5.2



3.3



Ear thickness in mm on
day 1



0.18


 



0.18


 



0.18


 



0.18


 



0.18


 



0.18


 



Ear thickness in mm on
day 6



0.18


 



0.18


 



0.18


 



0.20


 



0.21


 



0.18


 



Ear thickness,
Change in % (day 6 - day 1)



0.00


 



0.00


 



0.00


 



11.11


 



16.67


 



0.00


 



 


Ear thickness increased by 11.11% and 16.67%  in the 0.5% and 1% test groups, respectively, although these changes were not statistically significant.


The body weights of the animals were not affected by any treatment.


 

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Executive summary:

The test item was investigated in the local lymph node assay (LLNA:BrdU-ELISA) on female mice according to OECD TG 442B. Concentrations of 0 (vehicle control), 0.25 %, 0.5 % and 1 % formulated in dimethyl sulfoxide (DMSO) were tested.


The acute inflammatory skin reaction was observed once each day according to Erythema scores and ear thickness measurements on test day 1, day 3 and day 6 to identify skin irritation properties of the test item. It is important to determine if a positive test result is due to the skin irritation potential of the test item or due to its sensitising properties.


Stimulation indices were calculated for the lymph node cell proliferation by dividing the average values of the BrdU labelling index per group of the test item treated animals by the vehicle treated ones.


Values >= 1.6 (stimulation index to identify sensitisation) are considered positive.


 


In the main study treatment with PDI at the concentration of 0.25 %, 0.5 % and 1 % led to an increase of the stimulation index above the threshold level of 1.6. Stimulation indices of 3.6, 5.5 and 5.2 were determined for the concentrations of 0.25 %, 0.5 % and 1 %, respectively. Concentrations of 0.5% lead to skin reactions; they were very mild (barely noticeable) erythema (score 1) on both ears.


Concentrations of 1 % lead to well-defined erythema (score 2) on both ears.


Ear thickness increased by 11.11% and 16.67% in both ears of each mouse in the 0.5% and 1% test groups, respectively, although these changes were not statistically significant.


The positive control group caused the expected increases in stimulation index (3.3). Therefore, the study can be regarded as valid.


 


No signs of systemic intolerance were recorded.


In conclusion, under the present test conditions, PDI at the concentrations of 0.25 %, 0.5 % and 1 % revealed signs of skin sensitisation potential. The stimulation indices for the concentration of 0.25 %, 0.5 % and 1 % were 3.6, 5.5 and 5.2 and hence, the test item PDI is classified to be skin sensitising in this test system.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

The test item was investigated in the local lymph node assay (LLNA:BrdU-ELISA) on female mice according to OECD TG 442B. In the main study treatment with the test item at the concentration of 0.25 %, 0.5 % and 1 % led to an increase of the stimulation index above the threshold level of 1.6. Stimulation indices of 3.6, 5.5 and 5.2 were determined for the concentrations of 0.25 %, 0.5 % and 1 %, respectively.
Concentrations of 0.5% lead to skin reactions; they were very mild (barely noticeable) erythema (score 1) on both ears. Concentrations of 1 % lead to well-defined erythema (score 2) on both ears.


Ear thickness increased by 11.11% and 16.67% in both ears of each mouse in the 0.5% and 1% test groups, respectively, although these changes were not statistically significant.


The positive control group caused the expected increases in stimulation index (3.3). Therefore, the study can be regarded as valid.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Information on respiratory sensitization is not available for the substance. However, the database for the structurally similar substance 1,6 -diisocyanatohexane (HDI; CAS 822 -06 -0) indicates a potential for respiratory sensitization (Classification as resp. sens. Cat. 1). Based on read across this is also valid for 1,5-diisocyanatopentane.

Justification for classification or non-classification

Skin sensitisation


According to Regulation (EC) No 1272/2008, Annex I, classification is warranted for skin sensitisation as Cat. 1 (H317: May cause an allergic skin reaction). Based on the available data an allocation into sub-categories for skin sensitisation cannot be justified.


 


 


Respiratory sensitisation


According to Regulation (EC) No 1272/2008, Annex I, classification is warranted for respiratory sensitisation as Cat. 1 (H334: May cause allergy or asthma symptoms or breathing difficulties if inhaled). Based on the available data (read across) an allocation into sub-categories for respiratory sensitisation cannot be justified.


 


 


For classification of 1,5-diisocyanatopentane the following considerations were taken into account:


Skin sensitisation


There is some inconsistency regarding the irritation properties that could influence the outcome of skin sensitisation studies. 1,5-Diisocyanatopentane is classified as corrosive and it is questionable, whether the LLNA is over-predictive for irritants.


The LLNA result indicates high potency, but as this test system reveals positive results for skin as well as respiratory sensitisers (and the substance is in fact classified for respiratory sensitisation) it can not be conclusively evaluated whether the indicated potency directly relates to skin sensitisation. Additionally, ICCVAM concluded in its report of 2011 (NIH Publication No. 10-7512 http://iccvam.niehs.nih.gov/methods/immunotox/LLNA-app/TMER.htm) that the “LLNA cannot be considered a stand-alone assay to determine skin sensitization potency categories. … Among the 21 substances that produced a LLNA EC3 ≤ 2 %, 67 % (14/21) were correctly identified as strong sensitizers, but 33 % (7/21) were incorrectly overclassified as strong skin sensitizers based on available human test data.”


The result of the LLNA (according to OECD 442B) of 1,5-diisocyanatopentane , classified as respiratory sensitiser with corrosive properties, point to a high potency (correlate to sub-category 1A).


Overall as the data on potency of 1,5-diisocyanatopentane are limited it is concluded that the available data currently do not allow a solid assessment of the potency.


According to Annex I to Regulation (EC) No 1272/2008 (CLP) paragraph 3.4.2.2.1.1“skin sensitisers shall be classified in Category 1 where data are not sufficient for sub-categorisation.”.


Therefore, a classification of 1,5-diisocyanatopentane in Category 1 of hazard class “skin sensitisation” is appropriate here.


 


Respiratory sensitisation


Information on respiratory sensitisation is not available for the substance. However, the database for the structurally similar substance 1,6-diisocyanatohexane (HDI; CAS 822-06-0) indicates a potential for respiratory sensitisation (Classification as resp. sens. Cat. 1). Based on read across this is also valid for 1,5-diisocyanatopentane.

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