2,2,4,4-tetramethyl-7-oxa-3,20-diazadispiro[5.1.11.2]-henicosan-21-one

Endpoint summary

Description of key information

The sensitization potential of the test substance was investigated according to OECD Guideline 429. The test item concentrations chosen for the LLNA test were 10, 25 and 50 %.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

21. Jan - 7. June 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline-conform study under GLP without deviations.

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

One animal was accidentally killed. This event did not affect the validity of the study.

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V.; AD Horst, The Netherlands
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 20.6-16.6 g
- Housing: single in Macrolon Type I cages with wire mesh top with granulated soft wood bedding
- Diet (e.g. ad libitum): pelleted standard diet, (ALTROMIN, Lage/Lippe, Germany), ad libitum
- Water (e.g. ad libitum): tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22+/-3°C
- Humidity (%): 20-70%
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 19.01.To: 03.03.2004

No. of animals per dose:

5

Positive control substance(s):

yes

Vehicle:

propylene glycol

Concentration:

Two independent experiments were performed.
First experiment: 50, 25 and 10 %
Second experiment: 10, 5 and 2.5 %

No. of animals per dose:

First experiment: 4 animals per dose level
Second experiment: 5 animals per dose level

Details on study design:

RANGE FINDING TEST:
To determine the highest non-irritant and technically applicable test item concentration, a non-GLP pretest was performed in two mice with concentrations of 50, 25, 10 and 5%. The concentration of 50% was the highest technically achievable concentration whilst avoiding systemic toxicity and excessive local irritation.
Measurement of DPM: In the first experiment the draining lymph nodes were pooled for each dose level, whereas in the second experiment the draining lymph nodes were processed individually for each animals.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay (LLNA)
- Criteria used to consider a positive response: The sensitivity and reliability of the experimental technique employed was assessed by use of a
substance which is known to have skin sensitisation properties in CBA/CaOlaHsd mice (α-Hexylcinnamaldehyde in acetone:olive oil, 4:1 )

TREATMENT PREPARATION AND ADMINISTRATION:
The study encompassed two seperately performed main experiments.
In the first experiment the test item was used at 50, 25 and 10% (w/v) formulated in Propylene glycol. Each test group consisted of 4 females. The determination of Thymidine incorporation was carried out with lymph nodes of the mice, which were pooled per test group.
In the second experiment the test item was used st 10, 5 and 2.5% (w/v) formulated in Propylene glycol. Each test group consisted of 5 females. The determination of Thymidine incorporation was carried out with lymph nodes of the mice measured individually.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µl was spread to the entire dorsal surface of each ear of each mouse once daily for three consecutive days. On day 6 an injection of 250 µl phosphate buffered saline (PBS) containing 19.9 µCi of 3H-methyl thymidine (3H-TdR) was made into the tail vein of each experimental mouse. Five hours
later, the draining Auricular lymph node of each ear was excised and in the first experiment pooled per group (8 nodes per group) into PBS. In the second experiment the lymph nodes of the animals were pooled individually. A single cell suspension of lymph node cells was prepared from each group. After washing two times with phosphate buffered saline the lymph node cells were resuspended in 5 % trichloroacetic acid and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid and transferred to plastic scintillation vials. The level of 3HTdR incorporation was then measured on a ß-scintillation counter.

Positive control substance(s):

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

Statistics:

The mean values and atandard deviations were calculated.

Positive control results:

At concentration of 5, 10 and 25 % of positive control (hexyl cinnamic aldehyde in aceton: olive oil) the SI values amounted to 0.87, 2.26 and 6.01 respectively. Estimated concentration for EC3 was 12.96%.

Key result

Parameter:

SI

Value:

2.5

Remarks on result:

other: First experiment: SI of 4.3, 2.5 and 2.3 were obtained with the test item concentrations of 10, 25 and 50% respectively. Second experiment: SI of 1.3, 1.1 and 1.3 were obtained with the test item concentrations of 2.5, 5 and 10% respectively.

Key result

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: see Remark

Remarks:

First experiment: No dose responce relation was set up wihin the values of treated animals. At concentration of 10%, the DPM value was increased by more than 3-fold (790.1) of the control values, whereas, concentartion of 25 and 50% lead to DPM values of 470.6 and 432.6, respectivley. Second experiment: DPM values of treated animals were comparable to those of control animals.

In the first experiment SI of 4.3, 2.5 and 2.3 were obtained with the test item concentrations of 10, 25 and 50%. The data showed that a treatment with 10% test item led to a relevant increase of SI value whereas no increase was observed at higher concentrations. In order to address the relevance of this observation a second experiment was performed repeating the treatment with 10% and also using lower concentrations of the test item.

In the second experiment a treatment with 2.5, 5 and 10 % test item gave SI of 1.3, 1.1 and 1.1 respectively. The result of the treatment with 10% test item in the first experiment could not be reproduced.

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

The test item is not sensitizing in LLNA.

Executive summary:

The sensitizing potential of the test item was investigated in LLNA in two separately performed experiments.

In the first experiment SI of 4.3, 2.5 and 2.3 were obtained with the test item concentrations of 10, 25 and 50%. The data showed that a treatment with 10% test item led to a relevant increase if SI.. In order to address the relevance of this observation a second experiment was performed repeating the treatment with 10% and also using lower concentrations of the test item.

In the second experiment a treatment with 2.5, 5 and 10 % test item gave SI of 1.3, 1.1 and 1.1 respectively. The results of the treatment with 10% test item in the first experiment could not be reproduced and a dose dependent effect could not be observed.

Overall, it could be shown that the test item did not induce a lymph node cell proliferation.

The test item is not sensitizing in LLNA.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

The sensitizing potential of the substance was investigated in LLNA. The substance is not a skin sensitizer in LLNA. Furthermore, The animals did not show any clinical signs during the course of the study and no cases of mortality were observed

.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

The test item did not show skin sensitizing properties in the Local Lymph Node Assay according to the current OECD guidelines. Allergic skin reactions or case reports of acute contact dermatitis to the substance have not been described in the literature. Therefore, the substance does not have to be classified as a skin sensitizer.