[No public or meaningful name is available]

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September 07, 2004 - September 27, 2004

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was performed before a non-animal testing strategy was available.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2002)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(2001)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

(2003)

Deviations:

no

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

453-140-3

EC Name:

-

Cas Number:

667465-46-5

Molecular formula:

C20H38N6O13 (not dissociated) C20H44N6O13 (dissociated in water)

IUPAC Name:

3,6,9-tris(2-{[(2-hydroxyethyl)amino]oxy}-2-oxoethyl)-3,6,9-triazaundecan-1,11-dioic acid

Constituent 2

Reference substance name:

Water

EC Number:

231-791-2

EC Name:

Water

Cas Number:

7732-18-5

Molecular formula:

H2O

IUPAC Name:

Water

Test material form:

liquid

Details on test material:

- Name of test material: DHX2
- CAS no.: 667465-46-5
- Storage condition of test material: at room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Sex:

female

Details on test animals and environmental conditions:

- Source: Charles River France, L'Arbresle Cedex, France
- Age at study initiation: Young adult animals (approx. 11 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean.
- Housing: Animals were individually housed in labelled Macrolon cages.
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.6 – 21.7
- Humidity (%): 38 - 76
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

Cleaning procedures in the room might have caused the temporary fluctuations above the optimal maximum level of 70% for relative humidity. Based on laboratory historical data, these fluctuations were considered not to have affected the study integrity.

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

0, 5, 25 and 100%

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
A preliminary irritation study was conducted in order to select the highest test substance concentration to be used in the main study. In principle, this concentration should be well tolerated systemically by the animals and may give moderate irritation (grade 2) at the highest.
Two test substance concentrations 50 and 100% were tested, the highest concentration being the maximum concentration that could technically be applied. Two young adult animals were selected (5-14 weeks old). Each animal was treated with one concentration on three consecutive days. Approximately 4 hours after the last exposure, the ear was cleaned of residual test substance with water and the irritation was assessed. Bodyweights were determined on day 3. No necropsy was performed after termination.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitiser, based on the test guideline and recommendations done by ICCVAM.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
Test substance preparation: The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels.
Rationale for vehicle: The vehicle was selected based on trial formulations performed at NOTOX.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Toxicity: At least once daily.
Body weights: On days 1 (pre-treatment) and 6.
Irritation: On day 3 (3-4 hours after treatment), the skin reactions were assessed. If possible, skin reactions were graded according to the guideline. Furthermore descriptions of all other (local) effects were recorded.

Necropsy: The animals were subjected to necropsy for gross macroscopic examination.

Positive control substance(s):

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

Statistics:

Not performed.

Results and discussion

Positive control results:

The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at NOTOX is an appropriate model for testing for contact hypersensitivity. See attached document 'Reliability check'.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

0.5

Test group / Remarks:

100%

Results 2

Parameter:

SI

Value:

1.8

Test group / Remarks:

25%

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

5%

Cellular proliferation data / Observations:

Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 100% were 669, 893 and 254, respectively. The mean DPM/animal value for the vehicle control group was 502.

Any other information on results incl. tables

Results Pre-screen test:

No irritation was observed in any of the animals examined.  

Based on the results, the test highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No irritation was observed in any of the animals examined.

 

All animals of the 25% dose group showed an increased size of the auricular lymph nodes. Nodes of the other animals were normal in size. In one animal of the control group the right lymph node was noted to be dark red.

 

Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.Slight weight loss noted among individual animals was considered to be of no toxicological relevance in the absence of a dose-related incidence.

    

No mortality occurred and no symptoms of systemic toxicity were observed in the animals of the main study.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an LLNA skin sensitisation study, performed according to OECD 429 test guideline, the substance was considered not be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 100%.

Executive summary:

DHX2 was tested in an LLNA skin sensitisation study (five female animals/group) at concentrations of 5, 25 and 100%, performed according to OECD 429 test guideline and GLP principles.

No irritation was observed in any of the animals examined. All animals of the 25% dose group showed an increased size of the auricular lymph nodes. Nodes of the other animals were normal in size. Mean DPM/animal values for the experimental groups treated with test substance concentrations 5, 25 and 100% were 669, 893 and 254 respectively. The mean DPM/animal value for the vehicle control group was 502. The SI values calculated for the substance concentrations 5, 25 and 100% were 1.3, 1.8 and 0.5 respectively.

It can be concluded that DHX2 was considered not be a skin sensitiser, as the SI appeared not to be ≥ 3 when tested up to 100%. DHX2 does not need to be classified according to Regulation (EC) No 1272/2008.