3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, 2-Hydroxy-1(2)- methylethyl carbamate blocked

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

2-(carbamoyloxy)propyl N-[(3-{[3,5-bis({5-[({[2-(carbamoyloxy)propoxy]carbonyl}amino)methyl]-1,3,3-trimethylcyclohexyl}methyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]methyl}-3,5,5-trimethylcyclohexyl)methyl]carbamate

EC Number:

921-910-2

IUPAC Name:

2-(carbamoyloxy)propyl N-[(3-{[3,5-bis({5-[({[2-(carbamoyloxy)propoxy]carbonyl}amino)methyl]-1,3,3-trimethylcyclohexyl}methyl)-2,4,6-trioxo-1,3,5-triazinan-1-yl]methyl}-3,5,5-trimethylcyclohexyl)methyl]carbamate

Test material form:

solid

Details on test material:

- Analytical purity: 83.9 g/100g
- Composition of test material, percentage of components: 0.2g/100g water, 13.4g/100g n-butyl acetate, 2.5g/100g 1,2-propanediol, monocarbamate
- Lot/batch No.: 389-48
- Expiration date of the lot/batch: 2019-05-23

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: 16/0437-1, Batch 389-48

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final preparation of a solid: The solid test substance was ground in a mortar with pestle before application.

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V., Inc. Postbus 6174, 5960 AD Horst, The Netherlands
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: 8 weeks
- Weight at study initiation: 17.9 - 19.9 g (pretest), 17.8 - 20.7 g (main test)
- Housing: Polycarbonate cages type MII with mesh wire tops (Becker Co.)
- Diet (e.g. ad libitum): Kliba mouse/rat maintenance diet "GLP" supplied by Provimi Kliba SA; ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days before the first test substance application

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12
- IN-LIFE DATES: From: Apr 2017 To: May 2017

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

10, 25, 50%

No. of animals per dose:

5

Details on study design:

MAIN STUDY
- Irritation/systemic toxicity: Obvious signs of systemic toxicity and/or local inflammation at the application sites were noted for each animal in the raw data.
- Mortality: A check for moribund and dead animals was made twice each workday (beginning and end) and once on Saturdays, Sundays and on public holidays.
- Form of application: Epicutaneous application is simulating dermal contact with the compound which is possible to occur under practical use conditions.
- Application volume: 25µl per ear
- Site of application: dorsal part of both ears
- Frequency of application: 3 consecutive applications (day 0-2) to the same application site
- Body weight determination: Individual body weights on day 0 prior to the first application and on day 5 prior to the sacrifice of the animals.
- 3G Thymidine injection: On study day 5, 20µCi 3H-thymidine in 250µl sterile saline were injected into the tail vein of the mice.
- Section: The animals were sacrificed on study day 5 about 5h after 3H-thymidine injection by cervical dislocation under Isoflurane anesthesia.
- Ear weight: Immediately after the death of each animal, a circular piece of tissue was punched out of the apical part of each ear of all animals. The weight of the pooled punches was determined for each animal. These measurements serve for detecting a potential inflammatory ear swelling.
- Lymph nodes: Immeadiately after removal of the ear punches, the left and right auricular lymph nodes from both sides was determined for each animal.
- Cell count: After weight determination, the pooled lymph nodes of each animal were stored in PBS in an ice water bath until further preparation. A single cell suspension was prepared as soon as possible after dissection by carefully passing the lymph nodes through an iron mesh into 6ml PBS. For determination of cell counts, an aliquot of each suspension was further diluted and cell count was determined using a cell counter.
- Measuremtn of 3H-thymidine: The remaining cell suspensions were washed with PBS and precipitated with 5% TCA. Each precipitate was transferred to scintillation fluid and incorporation of 3H-thymidine into the cells was measured in a ß-scintillation counter.

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: A test item is regarded as a sensitizer in the LLNA if exposure to at least one concentration of the test item results in an incorporation of 3H-thymidine at least 3-fold or greater than that recorded in control mice as indicated by the stimulation index (SI ≥ 3.0). However, the biological relevance of the obtained stimulation indices is considered in conjunction with the other assessed end points (i.e. lymph node cell counts, lymph node weights, ear weights). Hereby, the thresholds used for assessment of cell counts and ear weights are represented by stimulation indices (SI) of 1.5 and 1.25, respectively. If applicable, the EC (estimated concentration) leading to the respective SI values were
calculated by linear or semi-logarithmical regression between the data points directly below and above the SI if possible or by using the two nearest points below or above the SI.

Positive control substance(s):

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

Statistics:

Mean values and standard deviations of the measured parameters were calculated for the test and control groups from the individual values. The stimulation indices of 3H-thymidine incorporation, cell count, lymph node weight and ear weight measurements were calculated as the ratio of the test group mean values for these parameters divided by that of the vehicle control group.

Results and discussion

Positive control results:

Positive controls are valid.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.01

Test group / Remarks:

Mean (10% in MEK)

Remarks on result:

other: based on 3H-thymidine incorporation

Results 2

Parameter:

SI

Value:

1.13

Test group / Remarks:

Mean (25% in MEK)

Remarks on result:

other: based on 3H-thymidine incorporation

Results 3

Parameter:

SI

Value:

1.55

Test group / Remarks:

Mean (50% in MEK)

Remarks on result:

other: based on 3H-thymidine incorporation

Any other information on results incl. tables

Pre-Test / Irritation Screening:

No signs of systemic toxicity were observed in the pretest. After application of the 50% and 5% test substance concentration no relevant increases in ear weights (compared to current historical vehicle values) were noticed. However, severe increase (< 25%) in ear thickness was noticed at the 50% concentration in one animal on day 5 only. However, as the severe increase in ear thickness was only observed for one ear while the other ear showed no mentionable increase in thickness, the severe increase was considered to be incidental. Additionally, slight to moderate residues of the test substance and slight loss of hair were observed on the application sites of the 50% concentration during the observation period. At the tested 5% concentration, no signs of local irritation were observed.

Main test:

When applied as 10%, 25% and 50% preparation in MEK, the test substance did not induce a biologically relevant (no increase above the cut-off Stimulation Index of 3) or statistically significant increase of 3H-thymidine incorporation into the cells from the auricular lymph nodes.

Concomitantly, all concentrations did not induce a biologically relevant (no increase to 1.5 -fold or above of control value = stimulation index (SI) >= 1.5) or statistically significant response in the auricular lymph node cell counts.

Statistically significant increases in lymph node weights were noted at the 25% and 50% concentration but were considered not to be biologically relevant, as the corresponding S.I.s for 3H-thymidine incorporation and cell counts did not reach or exceed the cut-off values.

The 50% test substance concentration caused a statistically significant but not biologically relevant increase (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation.

The expected body weight gain was generally observed during the study.

No signs of systemic toxicity were noticed in all animals during general observation.

Slight test compound residues were noted in all animals at the 50% concentration during the observation period.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the test conditions chosen, the test item does not exhibit a skin sensitizing potential.

Executive summary:

The skin sensitizing potential of the test substance was assessed by using the radioactive Murine Local Lymph Node Assay. The assay simulates the induction phase for skin sensitization in mice. It determines the response of cells in the auricular lymph nodes to repeated application of the test substance to the dorsal skin of the ears. 

Groups of 5 female CBA/CaOlaHsd mice each were treated with 10%, 25% and 50% (w/w) preparations of the test substance in methyl ethyl ketone (MEK) or with the vehicle alone.
The study consisted of 3 test groups and 1 control group. Each test animal was treated with 25 µL per ear of the appropriate test-substance preparation applied to the dorsal surfaces of both ears on three consecutive days. The control group was treated with 25 µL per ear of the vehicle alone. 

Three days after the last application, 20 µCi³H-thymidine in 250 µL sterile saline were injected into the tail vein of the mice. About 5 hours after the³H-thymidine injection, the mice were sacrificed and the auricular lymph nodes were removed. Lymph node response was evaluated by measuring³H-thymidine incorporation (indicator of cell proliferation). Cell counts and weights of each animal’s pooled lymph nodes were also determined. In addition, a 0.8cm diameter sample was punched out of the apical part of each ear and for each animal the weight of the pooled punches was determined to obtain an indication of possible skin irritation. 

No signs of systemic toxicity were noticed in all animals during general observation.

When applied as 10%, 25% and 50% preparation in MEK, the test substance did not induce a biologically relevant (no increase above the cut-off Stimulation Index of 3) or statistically significant increase of³H-thymidine incorporation into the cells from the auricular lymph nodes. 

Concomitantly, all concentrations did not induce a biologically relevant (no increase to 1.5-fold or above of control value = stimulation index (SI) ≥ 1.5) or statistically significant response in the auricular lymph node cell counts.

Statistically significant increases in lymph node weights were noted at the 25% and 50% concentration but were considered not to be biologically relevant, as the corresponding S.I.s for³H-thymidine incorporation and cell counts did not reach or exceed the cut-off values. 

The 50% test-substance concentration caused a statistically significant but not biologically relevant increase (SI > 1.25) in ear weights demonstrating the absence of relevant ear skin irritation. Slight test compound residues were noted in all animals at the 50% concentration during the observation period. 

Thus, it is concluded that the test substance does not exhibit a skin sensitizing potential in the Murine Local Lymph Node Assay under the test conditions chosen.