Cyclohexane, 5-isocyanato-1-(isocyanatomethyl)-1,3,3-trimethyl-, homopolymer, 3,5-dimethyl-1H-pyrazole-blocked

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002). Modified LLNA (IMDS; Integrated Model for the Differentiation of Skin Reactions). Modifications are authorised in the OECD TG 429 and in the Note for Guidance SWP/2145/00 of the CPMP (2001). Information on validation of IMDS and scientific justification is given in: Vohr HW et al., Arch. Toxicol., 73, 501-509 (2000); Ehling G et al., Toxicology 212, 60-68 and 69-79 (2005).

Deviations:

yes

Remarks:

- Measurement of cell counts instead of radioactive labeling. In addition, ear swelling and ear weights are determined to discriminate the irritating potential from the sensitizing potential of the test substance.

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:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 3,5-dimethyl-1H-pyrazole

EC Number:

606-423-2

Cas Number:

200295-52-9

Molecular formula:

>= C51H78N12O6 (IPDI-Isocyanurate n=3, blocked with 3,5-dimethylpyrazole)

IUPAC Name:

3-Isocyanatomethyl-3,5,5-trimethylcyclohexyl isocyanate, oligomers, reaction products with 3,5-dimethyl-1H-pyrazole

Test material form:

solid: crystalline

Specific details on test material used for the study:

- Stability under test conditions: The stability of the substance in the formulation was analytically verified for at least 4 days.

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Strain: Hsd Win:NMRI (SPF-bred)
- Source: Harlan Winkelmann GmbH, 33176 Borchen, Germany
- Age at study initiation: 10 weeks
- Weight at study initiation: 25-32 g
- Housing: During the study period the animals were single-housed in type II cages.
- Diet and water: ad libitum
- Acclimation period: at least 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 2
- Humidity (%): 40 -70
- Air changes (per hr): about 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:

methyl ethyl ketone

Concentration:

0 (vehicle control), 3, 10, 30 % (due to the solubility of the test item the highest feasible concentration was 30%)

No. of animals per dose:

6

Details on study design:

TREATMENT PREPARATION AND ADMINISTRATION:
The test item in the formulation was applied epicutaneously onto the dorsal part of both ears of the animals. This treatment was repeated on three consecutive days (d1, d2 and d3). The volume administered was 25 µL/ear. The following concentrations were used: 0 (vehicle control), 3, 10 and 30 %. Due to the solubility of the test item the highest concentration had to be limited to 30%. For negative control a dose group treated only with the vehicle in the above described manner was used.
The animals were anaesthetized by inhalation of carbon dioxide and sacrificed one day after the last application (d4). The appropriate organs were then removed. Lymphatic organs (the auricular lymph nodes) were transferred into physiological saline (PBS).
The following endpoints were determined:
- weight of draining lymph nodes (relative to vehicle control)
- cell counts in draining lymph nodes (relative to vehicle control)
- ear swelling (Day 1 absolute, Day 4 absolute and relative to vehicle control)
- ear weight (based on a 8 mm diameter piece punched from the ears; Day 4 absolute and relative to vehicle control)
- body weights (Day 1 and 4, absolute)

The relative indices are calculated by dividing the values of the test groups by the values of the vehicle control group, e.g. cell counts of substance-treated lymph nodes divided by cell counts of vehicle-treated lymph nodes. Thus, in case of no stimulating effect the index is about 1.00 (± relative standard deviation). By comparing the specific immune reaction induced by the test item in the draining lymph nodes (LN cell counts / LN weights) with the immediate non-specific acute skin reaction (ear swelling / ear weight) this IMDS method allows for discrimination of the irritant potential from the sensitizing potential of the compound tested.
The “positive level” of 1.4 for relative cell counts is exclusively defined for the NMRI outbreed mice used in this study. Such positive limits have to be calculated for each strain of mice individually.

The methodological reliability is checked in regular intervals using alpha cinnamic aldehyde as positive control. The closest reliability test using alpha hexyl cinnamic aldehyde formulated in acetone/olive oil (4:1) at concentrations of 3 %, 10 % and 30 % clearly demonstrated the sensitizing potential of the test item.

Positive control substance(s):

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

Statistics:

When it was statistically reasonable, the values from treated groups were compared with those from the control group by a one-way analysis of variance (ANOVA) when the variances are considered homogeneous according to a homogeneity testing like Cochran's test. Alternatively, if the variances are considered to be heterogenous (p<=0.05), a non-parametric Kruskal-Wallis test has been used (Kruskal-Wallis ANOVA) at significance levels of 5 %. Two sided multiple test procedures were done according to Dunnett or Bonferroni-Holm, respectively. Outlying values in the LN weights were eliminated at a probability level of 99 % by Nalimov's method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffels method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

Positive control results:

After treatment with Alpha Hexyl Cinnamic Aldehyde in Acetone/Olive Oil (4: 1) at concentrations of 3 %, 10 % and 30 % the sensitising potential of the substance was clearly confirmed.

In vivo (LLNA)

Results

Parameter:

SI

Remarks:

relative Cell Count (compared to vehicle control)

Value:

1.24

Test group / Remarks:

30 %

Remarks on result:

other: The cut-off for cell count of 1.4 was never reached or exceeded in any dose group: 1.00 (0 %) / 1.12 (3 %) / 0.81 (10 %) / 1.24 (30 %)

Cellular proliferation data / Observations:

Compared to vehicle treated animals none of the parameter measured, i.e. cell counts and weights of the draining lymph nodes, ear weights and ear swelling reached or exceeded the "positive levels" defined for this assay. These results show that there is no indication for a skin sensitizing effect after administration of a concentration of up to 30 % test item in this test system. A statistical significant increase compared to vehicle treated animals regarding the weights of the draining lymph nodes was detected in the highest dose group (30 %). An increase in this parameter could point to an irritant property of the test item.

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

Any other information on results incl. tables

The mice did not show increases for cell counts of the draining lymph nodes after application of the test item. (Cell count indices: 1.0 (0 %), 1.12 (3 %), 0.81 (10 %), 1.24 (30 %))

A slight statistical significant increase compared to vehicle treated animals regarding the weights of the draining lymph nodes was detected in the highest dose group (30%). An increase in this parameter could point to an irritant property of the test item. (Weight indices: 1.00 (0 %) / 1.10 (3 %) / 0.81 (10 %) / 1.29 (30 %))

The positive level of ear swelling which is 2 x 10exp-2 mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group (mm x 10 exp-2). (Day 1 = 18.00 (0 %) / 18.33 (3 %) / 17.92 (10 %) / 18.58 (30 %); Day 4 = 18.42 (0 %) / 18.75 (3 %) / 18.75 (10 %) / 19.33 (30 %); Index Day 4 = 1.00 (0 %) / 1.02 (3 %) / 1.02 (10 %) / 1.05 (30 %))

No substance specific effects were determined for ear weights either. (Day 4 = 10.80 (0 %) / 10.75 (3 %) / 10.33 (10 %) / 11.01 (30 %); Index Day 4 = 1.00 (0 %) / 1.00 (3 %) / 0.96 (10 %) / 1.02 (30 %))

Applicant's summary and conclusion

Interpretation of results:

other: no skin sensitization potential

Executive summary:

An LLNA according to OECD TG 429 was performed on 6 female NMRI mice per dose group using substance formulations in methyl ethyl ketone of 0 % (vehicle control), 3 %, 10 % and 30 % (due to the solubility of the test item the highest feasible concentration was 30 %). The assay was conducted as IMDS-modification (Integrated Model for the Differentiation of Skin Reactions), which allows for discrimination of the irritant potential by comparing the specific immune reaction induced by the test substance in the draining lymph nodes (LN cell counts) to the immediate non-specific acute skin reaction (ear swelling / ear weight).

With respect to a specific immune reaction, the "positive level" for cell counts, which is 1.4, has not been exceeded in any dose group. Compared to vehicle treated animals there was a slight statistical significant increase at the highest dose level (30 % concentration) for the weights of the draining lymph nodes. An increase in this parameter could point to an irritant property of the test item. The "positive level" of ear swelling which is 2 x 10 exp-2 mm increase, i.e. about 10 % of the control values, has not been reached or exceeded in any dose group. No substance specific effects were determined for ear weights either.

In conclusion, the study results did not reveal indications for a skin sensitizing potential up to and including a 30 % concentration of the substance, and also no conclusive indications for an irritant potential were found. 30 % turned out to be the NOEL in this study with respect to skin sensitization.