N,N'-bis{3-[di-(branched and cyclic) alkylamino]alkyl}alkanediamide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-01 to 2012-12-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Reference

Name:

Unnamed

Type:

Constituent

Details on test material:

- Physical state: dark orange coloured liquid
- Expiration date of the lot/batch: 18 October 2013
- Storage condition of test material: Room temperature in the dark

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/Ca (CBA/CaOlaHsd)

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories UK Ltd., Oxon, UK.
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 15-23 g
- Housing: Animals were individually housed in suspended solid floor polypropylene cages furnished with softwood woodflakes.
- Diet: Food (2014C Teklad Global Rodent diet supplied by Harlan Laboratories UK Ltd., Oxon, UK), ad libitum
- Water: Mains tap water, ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: 15 changes per hour
- Photoperiod: 12 h dark / 12 h light

IN-LIFE DATES: From: 2012-11-01 to 2012-12-05

Study design: in vivo (LLNA)

Vehicle:

other: butanone

Concentration:

Preliminary test: 100%
Main test: 25 and 50 % v/v in butone and 100 %

No. of animals per dose:

Preliminary screening test: 1 female/dose
Main test: 5 females/dose

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: Test item was used undiluted and freshly prepared as a solution at the concentrations of 25 and 50 % in butanone
- Irritation: No signs of sysemic toxicity or irritation indicated by an equal to or greater than 25% increase inmean ear thickness were noted. Very slight erythema was noted on both ears on Days 2 to 4. Based on this information the undiluted test item and the test item at concentrations of 50% or 25% v/v in butanonewere selected for the main test.

MAIN STUDY
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: The test item will be regarded as a sensitizer if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared to control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a "non-sensitizer."

TREATMENT PREPARATION AND ADMINISTRATION: The mice were treated by daily application of 25 µL of test material at concentrations of 0 (vehicle control), 25, 50 and 100 % to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). On Day 6, all mice were injected via the tail vein with 250 μL of phosphate buffered saline (PBS) containing 3H-methyl thymidine (3HTdR: 80 μCi/mL, specific activity 2.0 Ci/mmoL, ARC UK Ltd) giving a total of 20 μCi to each mouse. Five hours following the administration of 3HTdR all mice were killed by carbon dioxide asphyxiation. The draining auricular lymph nodes from the five mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes. A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. Lymph node cells were washed with PBS and precipitated with 5 % (w/v) trichloroacetic acid (TCA) for radioactive material at 4 °C. Pellets were re-suspended in 1 mL TCA and transferred to 10 mL of scintillation fluid (Optiphase ‘Trisafe’). 3HTdR incorporation was measured by β -scintillation counting. The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per animal and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index).

Positive control substance(s):

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

Statistics:

No data

Results and discussion

Positive control results:

Stimulation index for α-hexylcinnamaldehyde at 15 % v/v in butanone was 11.92 and classified as a sensitiser.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

7.47

Test group / Remarks:

25%

Results 2

Key result

Parameter:

SI

Value:

13.6

Test group / Remarks:

50%

Results 3

Key result

Parameter:

SI

Value:

13.05

Test group / Remarks:

100%

Results 4

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: DPM per group for vehicle, 25, 50 and 100 % were 1610.55, 12035.69, 21194.12, and 21015.39, respectively.

Any other information on results incl. tables

See attached document for Tables.

There were no death. No signs of systemic toxicity or irritation indicated by an equal to or greater than 25% increase in mean ear thickness were noted in the test or control animals during the test.

Very slight erythema on the ears was noted on Day 1 in three animals treated with the undiluted test item and persisted in two animals on Day 2. No signs of local irritation were noted in the remaining test animals or vehicle control animals during the test.

Bodyweight: On animal treated with the test item at a concentration of 50% v/v in butanone showed a greater than expected bodyweight loss. Bodyweight changes of the remaining test animals beween Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions,the substance is classified as sensitising to the skin according to CLP Regulation (EC) No. 1272/2008

Executive summary:

Test Guidance

OECD Guideline 429 and EC Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Method and materials

Groups of CBA/Ca (CBA/CaOlaHsd) mice (5 females/dose) were topically applied with 50 µL (25 µL per ear) of the undiluted test item or the test item as a solution in butanone at concentrations of 50 % or 25 % v/v. A further group of five animals was treated with vehicle alone. On Day 6, the animals received a single intravenous injection of tritiated methyl thymidine (3HTdR). Five hours later, the animals were sacrificed and the auricular lymph nodes were excised. The proliferation of lymphocytes in the lymph node draining the application site was measured by incorporation of 3HTdR. The results were expressed as disintegrations per minute (dpm) per group and the obtained values were used to calculate Stimulation Indices (SI). Animals were observed for mortality, clinical signs and body weight during the study. The test concentrations for the main study were determined from a preliminary study using one female mouse at the concentration of 100 %.

Results

No mortality and no clinical signs were observed during the observation period. On animal treated with the test item at a concentration of 50% v/v in butanone showed a greater than expected bodyweight loss. Bodyweight changes of the remaining test animals beween Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

DPM per group for vehicle, 25, 50 and 100 % were 1610.55, 12035.69, 21194.12, and 21015.39, respectively. Stimulation index for 25, 50 and 100 % were 7.47, 13.6 and 13.05, respectively. Positive control (α-hexylcinnamaldehyde, 15%) exhibited evidence of sensitisation indicating the validity of the study. In this study, the substance is a skin sensitiser in mice.

Conclusion

Under these test conditions, the substance is classified as sensitizing to the skin according to the CLP Regulation (EC)No. 1272/2008).