PDI oligomers, isocyanurate (3x)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

8 March - 3 april 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The in vivo LLNA study was performed to fulfill global data requirements.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

22 July 2010

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

1,3,5-tris(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione

Cas Number:

119934-71-3

Molecular formula:

C21 H30 N6 O6

IUPAC Name:

1,3,5-tris(5-isocyanatopentyl)-1,3,5-triazinane-2,4,6-trione

Constituent 2

Reference substance name:

2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate

Molecular formula:

C18 H30 N4 O5

IUPAC Name:

2-methylpropyl N-(5-isocyanatopentyl)-N-(5-isocyanatopentylcarbamoyl)carbamate

Test material form:

liquid: viscous

Details on test material:

Appearance: colourless or light yellow viscous liquid
Storage conditions: at room temperature, container tightly closed, flushed with nitrogen.

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/N

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Central Lab. Animal Inc., Republic of Korea
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 9 weeks (dose range finding studies), 11 weeks (main study)
- Weight at study initiation:15.8–20.9 g (dose range finding studies), 17.5–23.1 g (main study)
- Housing: Polysulfone cage, with 2-3 animals per cage during study
- Diet: Pelleted rodent chow (Teklad Certified Irradiated Global 18% Protein Rodent Diet 2918C), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.6–24.5
- Humidity (%): 43.8 - 58.7
- Air changes (per hr): 10–15
- Photoperiod (hrs dark / hrs light): 12/12
- IN-LIFE DATES: From: 8 March To: 3 april 2017

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

Dose range finding study 1: 5, 10, 25, 50 and 100% (w/v);
Dose range finding study 2: 0.25, 0.5, and 1% (w/v);
Main study 1: 0.25, 0.5, 1 and 25% (w/v)

No. of animals per dose:

Dose range finding studies: 2
Main study: 4

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test substance dissolved in acetone: olive oil (4:1 v/v) in a preliminary solubility test. Therefore, this was utilized as vehicle for this study. Dose selection was based on the consecutive doses and dose levels were selected from a series of appropriate concentrations such as 100, 50, 25, 10 and 5%. Excessive local irritation was observed at all doses in the first dose range finding study. Therefore, a second dose range finding study was carried out with three dose levels set at 0.25, 0.5 and 1%. The dose range finding studies were conducted using the same method as in the main study, but lymph node proliferation measurement not performed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay: BrdU-ELISA
- Criteria used to consider a positive response: A substance that elicits an SI ≥ 1.6 is considered positive.

TREATMENT PREPARATION AND ADMINISTRATION:
The required amount of the test substance was weighed and placed in a bottle. A small amount of vehicle was added and mixed using a vortex mixer until dissolved. The vehicle was gradually added to yield the desired concentration. The required amount of the positive control was placed in a bottle. A small amount of vehicle was added and mixed using a vortex mixer until dissolved. The vehicle was gradually added to yield the desired concentration (25%). All preparations were conducted just prior to use.

A volume of 25 μL was applied to the dorsum of both ears of all animals daily for three consecutive days. The dose level of the positive substance was selected at 25%, which is the dose recommended in the guideline. Negative control animals were dosed with the vehicle.

MAIN STUDY
All animals were observed for mortality, general condition and clinical signs for 6 days.
Body weights were recorded prior to dosing (Day 1) and on the day of necropsy (Day 6).
Both ears of each mouse were observed for erythema and scored for 6 days.
Ear thickness measurement was taken using a thickness gauge on Day 1 (pre-dose), Day 3 (approximately 48 hours after the first dose) and Day 6 (the day of necropsy).
A volume of 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL PBS) solution was injected inter-peritoneally on Day 5.
Approximately 24 hours (24 h) after BrdU injection, the animals were euthanized under CO2 gas. Using a punch for skin biopsy of 6 mm in diameter, centering around both ears and avoiding the flexed part of the inner ear, the inner tissue of the ear were removed about 1 mm from the outside of the ear and tissues of both sides were weighed together. The draining auricular lymph nodes from each mouse ear were excised and processed separately in PBS. A single-cell suspension of lymph node cells was prepared and cellular proliferation was determined by measuring BrdU incorporation.

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Statistical analysis was conducted using a statistical program (version 9.3, SAS Institute Inc., U.S.A.) for the data including body weight, erythema score, ear thickness, ear weight and stimulation index. Bartlett’s test was employed on homogeneity of variance (significance level: 0.05) for body weights, ear thickness, ear weight and stimulation index data. One-way analysis of variance (ANOVA) was employed on homogeneous data. Dunnett’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, onetailed) between the negative control group and each of the test substance groups or positive substance group. Kruskal-wallis test was employed on heterogeneous data and Steel’s test was applied for multiple comparisons (significance levels: 0.05 and 0.01, one-tailed) between the negative control group and each of the test substance groups or positive substance group.

Results and discussion

Positive control results:

In the positive control group at 25%, the mean stimulation index was 3.41 (significant increase compared to the negative control group (p<0.05)).

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

2.95

Test group / Remarks:

0.25%

Results 2

Key result

Parameter:

SI

Value:

3.16

Test group / Remarks:

0.5%

Results 3

Key result

Parameter:

SI

Value:

7.31

Test group / Remarks:

1%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The mean BrdU labelling index was found to be 0.14 (SD 0.01), 0.41 (SD 0.11), 0.44 (SD 0.05), 1.02 (SD 0.26) and 0.48 (SD 0.05) for groups exposed to respectively 0, 0.25, 0.5, 1% test substance and the positive control group.

DETAILS ON STIMULATION INDEX CALCULATION
In the negative control group, the mean stimulation index was 1.00. In the test substance groups at 0.25, 0.5 and 1%, the mean stimulation index was 2.95, 3.16 and 7.31, respectively. There were significant increases when compared to the negative control group (p<0.05: 0.25, 0.5 and 1%). In the positive control group at 25%, the mean stimulation index was 3.41. There was a significant increase when compared to the negative control group (p<0.05).

CLINICAL OBSERVATIONS:
There were no abnormal clinical signs or deaths in any dosing group during the observation periods. In the negative control group, the mean erythema score was 0 from Day 1 to Day 6 after dosing. In the test substance groups at 0.25, 0.5 and 1%, the mean erythema scores were 0–0, 0–1 and 0–1, respectively. There were significant increases when compared to the negative control group (p<0.05: Day 3 (1%), Day 4 (1%), Day 5 (0.5 and 1%) and Day 6 (0.5 and 1%)). In the positive control group at 25%, the mean erythema score was 0–2. There were significant increases when compared to the negative control group (p<0.05: Days 3, 4, 5 and 6). In the test substance groups at 0.25, 0.5 and 1%, the mean ear thickness was significantly increased when compared to the negative control group (p<0.05: Day 6 (0.25, 0.5 and 1%), p<0.01: Day 3 (1%)). In the positive control group at 25%, the ear thickness was significantly increased when compared to the negative control group (p<0.05: Day 6, p<0.01: Day 3). In the test substance groups at 0.25, 0.5 and 1%, the mean ear weights were 14.1, 14.4 and 15.7 mg, respectively. There was a significant increase when compared to the negative control group (p<0.01: 1%). In the positive control group at 25%, the mean ear weight was 14.8 mg (13.4 mg for the control group). There was a significant increase when compared to the negative control group (p<0.05).

BODY WEIGHTS
In the negative control group, the mean body weight was 20.0–19.1 g from Day 1 to Day 6 after dosing. In the test substance groups at 0.25, 0.5 and 1%, the mean body weights were 19.5– 19.1, 19.6–19.0 and 19.9–19.5 g, respectively. There were no significant differences when compared to the negative control group.
In the positive control group at 25%, the mean body weight was 19.5–19.2 g. There were no significant differences when compared to the negative control group.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on the results of an in vivo skin sensitisation assay performed according to OECD guideline 442B (Local lymph node assay: BrdU-ELISA) and GLP principles it is concluded that STABiO D-376N is considered to be a sensitizer.

Executive summary:

An in vivo sin sensitisation assay was performed with STABiO D-376N according to OECD guideline 442B (Local lymph node assay: BrdU-ELISA) and GLP principles. Based on the results of the dose range finding study, the high dose level of the main study was selected at 1%, and two additional lower dose levels (0.5 and 0.25%) were established. In the main study, no clinical signs and abnormalities were observed in any animal. In the test substance groups, the body weights were not significantly different when compared to the negative control group. The erythema score, ear thickness, ear weights and stimulation index were significantly different when compared to the negative control group. In the positive control group, the body weights were not significantly different when compared to the negative control group. In the test substance groups at 0.25, 0.5 and 1%, the mean stimulation index was 2.95, 3.16 and 7.31, respectively.

The erythema score, ear thickness, ear weights and stimulation index were significantly different when compared to the negative control group. The stimulation index was above the threshold for sensitizers of 1.6 in the positive control group demonstrating the validity of the study. Based on the result of this study (Local lymph node assay: BrdU- ELISA), STABiO D-376N produced a stimulation index of ≥1.6 in the all dose groups, therefore, it is considered to be a sensitizer.