Reaction mass of 2-ethylhexyl (6-isocyanatohexyl)-carbamate and bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Oct. 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

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:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

EC Number:

247-735-5

EC Name:

2-ethylhexyl (6-isocyanatohexyl)-carbamate

Cas Number:

26488-60-8

Molecular formula:

C16H30N2O3

IUPAC Name:

6-Isocyanatohexylamino 3-ethylheptanoate

Constituent 2

Reference substance name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

EC Number:

278-583-8

EC Name:

Bis(2-ethylhexyl) 1,6-hexan-1,6-diylbiscarbamate

Cas Number:

76977-79-2

Molecular formula:

C24H48N2O4

IUPAC Name:

6-(2-Ethylhexyloxycarbonylamino)hexylamino 3-ethylheptanoate

impurity 1

Reference substance name:

Hexamethylene diisocyanate

EC Number:

212-485-8

EC Name:

Hexamethylene diisocyanate

Cas Number:

822-06-0

Molecular formula:

C8H12N2O2

IUPAC Name:

1,6-diisocyanatohexane

Test material form:

liquid

Specific details on test material used for the study:

- Stability under test conditions: A stability test in the formulation at 1 and 50 % (w/w) revealed no significant degradation of the test item up to at least 2 hours (A 01/0207/05 LEV).

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
- Source: Harlan Winkelmann GmbH, 33176 Borchen, Germany
- Age at study initiation: no data
- Weight at study initiation: 25-32 g
- Housing: during the study period animals were kept singly in Makrolon 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

Remarks:

(dried with molecular sieve)

Concentration:

0 (vehicle control), 3, 10, and 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 concentrations used were based on the experiences with the test system and the properties of the test substance. 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 (absolute and 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 so-called SI (or LLN-) index - is calculated by dividing the absolute number of weight or cell counts of the substance treated lymph nodes by the vehicle treated ones. Thus, in case of no stimulating effect the index is always about 1.00 (+/- standard deviation). The “positive level” was defined in that study as 1.3.

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 the Mann Whitney or the Wilcoxon significance test (Rank Sum Test or One Way ANOV A or Kruskal-Wallis ANOV A) at significance levels of 5 % (one-tailed for LLNAIIMDS or PNLA (larger)). Outlying values in the LN/ear weights or LN cell counts were eliminated at a probability level of 99 % by Nalimovs method. In addition, for the LLNA/IMDS the smallest significant differences in the means were calculated by Scheffe's method, which according to Sachs can be used for both equal and unequal sample sizes.

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Remarks:

relative Cell Count (compared to vehicle control)

Value:

3.51

Test group / Remarks:

3 %

Remarks on result:

other: The positive level, which is 1.3, has been exceeded.

Results 2

Parameter:

SI

Remarks:

relative Cell Count (compared to vehicle control)

Value:

4.76

Test group / Remarks:

10 %

Remarks on result:

other: The positive level, which is 1.3, has been exceeded.

Results 3

Parameter:

SI

Remarks:

relative Cell Count (compared to vehicle control)

Value:

5.33

Test group / Remarks:

30 %

Remarks on result:

other: The positive level, which is 1.3, has been exceeded.

Any other information on results incl. tables

All dosed groups (3 %, 10 % and 30 %) of the NMRI mice showed a clear increase in the weights of the draining lymph nodes and clear increases in the stimulation indices for cell counts compared to control animals after application of the test item.
The "positive level", which is 1.3 for cell count indices, has been exceeded in all dose groups. This increase is of statistical significance. The increase of cell counts and lymph nodes weights was dose dependent.
The "positive level" of ear swelling which is 2x10-2 mm increase has been exceeded or reached in all dose groups. In all dose groups there were a statistically significant increase of the ear weights and ear swelling compared to control animals.

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

Applicant's summary and conclusion

Executive summary:

A modified LLNA (IMDS = Integrated Model for the Differentiation of Skin Reactions) was performed according to OECD TG 429 with 6 female NMRI mice per dose group using substance formulations of 0 % (vehicle control), 3 %, 10 % and 30 %. The IMDS method 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 / LN weights) with the immediate nonspecific acute skin reaction (ear swelling / ear weight).

After treatment with the test substance there was a clear dose-dependent increase compared to controls regarding the weights of the draining lymph nodes and a clear increase in the stimulation indices for cell counts of all dose groups. The "positive level" which was defined in that test as 1.3 for cell counts has been exceeded in all dose groups. These increases were of statistical significance. Thus, a sensitizing potential can be assumed from the increases in cell proliferation in the draining lymph nodes.

The "positive level" of ear swelling, which is 2xl0exp-2 mm has also been exceeded or reached in all dose groups. An increase in this parameter would point to an acute irritating (inflammatory) response. However, such an irritating property can also be combined with a strong skin sensitizing potential of a test compound.

In conclusion, the study result revealed a skin sensitizing potential of the substance. The EC1.3 is expected to be in any case below 1 %.