N-[3-({[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}methyl)-3,5,5-trimethylcyclohexyl]-2,2-dimethyl-3-(morpholin-4-yl)propan-1-imine

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2011-05-31 to 2011-07-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2011

Report date:

2011

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

31 May 2008)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

22 July 2010

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003.

Deviations:

no

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:

[2,2-dimethyl-3-(morpholin-4-yl)propylidene][(5-{[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}-1,3,3-trimethylcyclohexyl)methyl]amine

EC Number:

700-584-3

Cas Number:

1217271-02-7

Molecular formula:

C28H52N4O2

IUPAC Name:

[2,2-dimethyl-3-(morpholin-4-yl)propylidene][(5-{[2,2-dimethyl-3-(morpholin-4-yl)propylidene]amino}-1,3,3-trimethylcyclohexyl)methyl]amine

Test material form:

other: liquid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt. H-1103, Budapest, Cserkesz u. 90, Hungary
- Age at study initiation: Young adult mice
- Weight at study initiation: The weight variation in animals involved in the study will not exceed ± 20 % of the mean weight.
- Housing: grouped caging (4 animals/cage); type II. polypropylene/polycarbonate
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany, ad libitum
- Water: tap water, as for human consumption, from a bottle ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidity: 30 – 70 % Relative Humidity
- Air changes (per hr): not stated
- Photoperiod: 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Study design: in vivo (LLNA)

Vehicle:

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

Concentration:

75, 50, 25, 10 % (w/v).
The maximum concentration was not achievable because of high viscosity of test item, which did not allow the application of the undiluted test item on the ears of animals.

No. of animals per dose:

4 animals per dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The test item was a liquid. Because of the high viscosity, administration of undiluted substance on the ears of animals was not feasible. The maximal applicable concentration was 75 % (w/v).
- Irritation: None.
- Lymph node proliferation response: Not examined.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymoh Node Assay
- Criteria used to consider a positive response:
The stimulation index (SI = the DPN of a treated (positive control or test item) group divided by the DPN of the respective negative control group) for each treatment group was also calculated. A stimulation index of 3 or greater is an indication of a positive result.

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was topically treated with 25 μL of the appropriate formulation of 3 different concentrations of the test item, with the positive control substance (positive control groups) and with the vehicle(s) (negative control group) using a pipette, on the dorsal surface of each ear. Each animal was dosed once a day for three consecutive days (Days 1, 2 and 3). There was no treatment on Days 4, 5 and 6.

Positive control substance(s):

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

Statistics:

NA

Results and discussion

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

36

Test group / Remarks:

75 %

Results 2

Key result

Parameter:

SI

Value:

24.9

Test group / Remarks:

50 %

Results 3

Key result

Parameter:

SI

Value:

19.6

Test group / Remarks:

25 %

Results 4

Key result

Parameter:

SI

Value:

4.5

Test group / Remarks:

10 %

Results 5

Parameter:

other: disintegrations per minute (DPM)

Value:

>= 22 690.5 - <= 181 811.5

Remarks on result:

other: Please refer to the table under "Any other information on results incl. tables".

Cellular proliferation data / Observations:

Significant lymphoproliferative response (SI > 3) was noted for Sika Hardener MI at all concentrations tested. The stimulation index values of the test item were 36.1, 24.9, 19.6 and 4.5 at treatment concentrations of 75 %, 50 %, 25 % and 10 %, respectively. The stimulation index values were compatible with a linear biological dose-dependent response.

Any other information on results incl. tables

DPM, DPN and Stimulation Index Values for all Groups in the Main Test

Test Group	Measured DPM/Group	Group DPM	DPN (DPN/Node)	Stimulation Index Value
Negative control	5088	5038.5	629.8	1.0
Positive control 25% HCA in AOO	375158	37108.5	4638.6	7.4
SIKA Hardener MI 75 %	181861	181811.5	22726.4	36.1
SIKA Hardener MI 50 %	94329	94279.5	15713.3	24.9
SIKA Hardener MI 25 %	98709	98659.5	12332.4	19.6
SIKA Hardener MI 10 %	22740	22690.5	2836.3	4.5

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Based on results obtained from testing, Sika Hardener MI was considered as a skin sensitiser.

Executive summary:

The aim of this study was to determine the skin sensitization potential of Sika Hardener MI following dermal exposure in the Local Lymph Node Assay (OECD 429).

The test item was a liquid. Because of the high viscosity, administration of undiluted substance on the ears of animals was not feasible. The maximal applicable concentration was 75 % (w/v). A preliminary irritation/toxicity test was performed with test item concentrations of 75 % and 50 %. A slight loss of body weight was observed in both dose groups. The effect was considered to be not significant and 75 % was accepted as the maximum concentration to be tested. Ensuring validity of the test four consecutive concentrations were selected to be tested in the main test.

In the main assay 24 female CBA/Ca mice were allocated to six groups of four animals each:

-    three groups received Sika Hardener MI at concentrations of 75 %, 50 %, 25 % or 10 %,

-    the negative control group received the vehicle (AOO),

-    the positive control groups received α-Hexylcinnamaldehyde (HCA) at concentration of 25 %.

Each substance was applied on the external surface of each ear (25 mL/ear) of the animals for three consecutive days (Day 1, 2 and 3). There was no treatment on Days 4, 5 and 6. On Day 6, the cell proliferation in local lymph nodes was determined by measuring incorporation of tritiated methyl thymidine (3HTdR) and the obtained values were used to calculate stimulation indices (SI).

No cutaneous reactions were observed at the application site in any of the groups. Slight, but not significant erythema were observed in the 75 %, 50 % and 25 % test item treated groups at the base of ears. Loss of hair was observed (1/4 animals, top of the head of the animal) in the 75 % dose group on Day 6, but the affected area was small. The effects were considered to be not significant and had no effect on the proliferation results.

Animals showed clinical signs of systemic toxicity in the 75 % and 50 % dose groups. In the 75 % test item treated group the observed effects were piloerection (4/4 animals) and hunched back posture (4/4 animals) on Day 4; piloerection (4/4 animals), hunched back posture (1/4 animals) and decreased activity (3/4 animals) on Day 5; piloerection (4/4 animals), decreased activity (2/4 animals) and tremor (2/4 animals) on Day 6 (all symptoms were slight on Day 5 and 6). No mortality was observed in this dose group.

In the 50 % dose group slight piloerection (4/4 animals) was observed on Day 3 after the third treatment. One animal in this dose group showed more marked symptoms on Day 4: piloerection, increased respiration rate, decreased activity (laying, when not stimulated), abnormal posture and wobbling (when stimulated). Because of the severity of the symptoms (laying in an abnormal (contorted) position, decreased respiration rate, wobbling, paralysis) observed on the next day, this animal (animal No.: 1034) was considered to be moribund and humanely killed on Day 5. The remaining three animals showed slight symptoms on Day 4: piloerection (3/3 animals), decreased activity (3/3 animals) and hunched back posture (1/3 animals) was observed. The symptoms observed on these animals on Day 5 were decreased activity (3/3 animals), pilerection (1/3 animals) and tremor (intermittent, 1/3 animals). One of these animals was symptom-free on Day 6, while slight piloerection was observed on the same day on the other two animals. The observed bad health condition of the euthanized animal was considered to be caused by an individual sensitivity of the animal to the test item.

Despite of the observed symptoms of a systemic toxic effect no significant treatment related effects on animal body weights were observed in the 75 % and 50 % dose groups.

No mortality or clinical signs of systemic toxicity were observed in the 25 % and 10 % dose groups as well as in negative and positive control groups.

 

Stimulation index values of the test item were 36.1, 24.9, 19.6 and 4.5 at treatment concentrations of 75 %, 50 %, 25 % and 10 %, respectively. The stimulation index values were compatible with a linear biological dose-dependent response.

Although proliferation values were also obtained from concentrations causing systemic toxicity the proliferation effect of the test item was evaluated in all dose groups. There were no sign of systemic toxicity at concentrations of 25 % and 10 % or confounding effects of irritation. Therefore, the proliferation values obtained at these concentrations are considered to reflect the potential of the test item to cause lymphoproliferation. Further, a linear dose-response is given up to 75 %, thus indicating that the stimulation index is not impaired by the immunological response against test item concentrations with toxicological relevance. According to evaluation criteria of the OECD Guideline 429, the proliferation value above 3 at these test concentrations and the dose-related response observed are considered to be good evidence that Sika Hardener MI is a sensitizer.

Since no SI value below 3 was observed with the test item, no estimation of the EC3 value (dose calculated to induce a stimulation index of 3) was performed in this LLNA.

α-Hexylcinnamaldehyde, dissolved in AOO at concentration of 25 % (w/v) was used as positive control to demonstrate the appropriate performance of the assay. A significant lymphoproliferative response was noted for the positive control chemical with a stimulation index value of 7.4. The positive control results obtained confirmed the validity of the assay.