[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:

9 October 2006 to 20 February 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2007

Report date:

2007

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

1

Reference substance name:

-

EC Number:

482-480-5

EC Name:

-

Cas Number:

1187571-02-3

Molecular formula:

(R2C5H4NO2)(0-3)(R3C3H5O3)(0-3)H(0-2)B

IUPAC Name:

Coconut oil reaction products with boric acid and diethanolamine

In vivo test system

Test animals

Species:

mouse

Strain:

other: CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

Test Animals
Age: 8 weeks (beginning of acclimatization)
Body weight: 18.0 g – 21.8 g (at delivery)
Identification: Each cage by unique cage card.
Randomization: Randomly selected by computer algorithm at time of delivery.
Acclimatization: 7 days prior to the first topical application under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

Environmental conditions
Air-conditioned with ranges for room temperature 22 + 3 °C, relative humidity 30 - 70 % and 10 - 15 air changes per hour. Room temperature and humidity were monitored continuously. There was a 12 hour fluorescent light / 12 hour dark cycle with at least 8 hours music during the light period.

Accommodation: Individual in Makrolon type-2 cages with standard softwood bedding.

Diet: Pelleted standard Kliba 3433, batch no. 48/06 mouse maintenance diet available ad libitum. There was no contamination of the diet.

Water: Community tap water from Itingen, available ad libitum. There was no contamination of water.

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

The test item in the main study was assayed at three consecutive concentrations: 1 %, 2.5 %
and 5 % (w/v).

Concentrations were in terms of material as supplied.

No. of animals per dose:

4 females (nulliparous and non-pregnant)

Details on study design:

The test item was placed into a volumetric flask on a tared Mettler balance and the vehicle
DMF was quantitatively added. The weight/volume (w/v) dilutions were prepared individually
using a magnetic stirrer as homogenizer.

Test item formulations were made freshly before each dosing occasion and no more than
4 hours prior to application to the ears.

Homogeneity of the test item in the vehicle was maintained until start of treatment using an
appropriate homogenizer.

Each test group of mice was treated by topical (epidermal) application to the dorsal surface
of each ear lobe (left and right) with the test item at concentrations of 1 %, 2.5 % or 5 % (w/v)
in DMF. The application volume, 25 µl, was spread over the entire dorsal surface (diameter ∼
8 mm) of each ear lobe once daily for three consecutive days. A further group of mice was
treated with an equivalent volume of the relevant vehicle alone (control animals).

Five days after the first topical application, all mice were administered with 250 µl of
phosphate-buffered saline (PBS) containing 78.67 µCi/ml 3 HTdR (equal to 19.7 µCi 3 HTdR)
by intravenous injection via a tail vein.

Approximately five hours after treatment with 3 HTdR all mice were euthanized by inhalation
of CO2 (dry ice).

The draining lymph nodes were rapidly excised and pooled in PBS for each experimental
group (8 nodes per group). Single cell suspensions of pooled lymph node cells were
prepared by gentle mechanical disaggregation through stainless steel gauze (200 µm mesh
size). After washing twice with phosphate buffered saline (approx. 10 ml) the lymph node
cells were resuspended in 5 % trichloroacetic acid (approx. 3 ml) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules. The precipitates were then resuspended in 5 % trichloroacetic acid (1 ml) and transferred to glass scintillation vials with 10 ml of ‘Irga-Safe Plus’ scintillation liquid and thoroughly mixed.

The level of 3 HTdR incorporation was then measured on a β-scintillation counter. Similarly,
background 3 HTdR levels were also measured in two 1ml-aliquots of 5 % trichloroacetic acid.

The β-scintillation counter expresses 3 HTdR incorporation as the number of radioactive
disintegrations per minute (dpm).

Results and discussion

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.1

Test group / Remarks:

Group 4, 5% w/v

Results 2

Parameter:

SI

Value:

2.5

Test group / Remarks:

Group 3, 2.5% w/v

Results 3

Parameter:

SI

Value:

1.3

Test group / Remarks:

Group 2, 1% w/v

Any other information on results incl. tables

No deaths occurred during the study period.

 

Neither clinical / local signs nor other findings were observed in any animals of the control group, Group 2 (1 %) or Group 3 (2.5 %). One day after the first topical application, slight ear swelling and ear erythema were observed at both dosing sites in all mice of Group 4 (5 %), persisting for a total of four or three days.

 

The body weights of the animals, recorded prior to the first application and prior to sacrifice, were within the range commonly recorded for animals of the strain and age.

 

No abnormal findings were observed in the size or other physical features of the draining lymph nodes.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

In an OECD 429 study the test substance does not show an allergenic potential
when tested up to the concentration of 5 % (w/v) in DMF.

Executive summary:

In order to study the contact allergenic potential of the test substance, three groups each of four female mice were treated daily with the test item at concentrations of 1 %, 2.5 % and 5 % (w/v) in N,N-dimethylformamide (DMF) by topical application to the dorsum of each ear lobe (left and right) for three consecutive days. A control group of four female mice was treated with the vehicle DMF only.

Five days after the first topical application the mice were injected intravenously into the tail vein with radio-labelled thymidine (3 H-methyl thymidine, 3 HTdR).

Approximately five hours after intravenous injection, the mice were sacrificed, the draining auricular lymph nodes excised and pooled per group. Single cell suspensions of lymph node cells were prepared from pooled lymph nodes which were subsequently washed and incubated with trichloroacetic acid overnight. The proliferative capacity of pooled lymph node cells was determined by the
incorporation of 3 HTdR measured in a β-scintillation counter.

All treated animals survived the scheduled study period.

Neither clinical / local signs nor other findings were observed in any animals of the control group, Group 2 (1 %) or Group 3 (2.5 %). One day after the first topical application, slight ear swelling and ear erythema were observed at both dosing sites in all mice of Group 4 (5 %), persisting for a total of four or three days.

A test item is regarded as a sensitizer in the LLNA if the exposure to one or more test concentrations resulted in 3-fold or greater increase in incorporation of 3
HTdR compared with concurrent controls, as indicated by the STIMULATION INDEX (S.I.). The S.I. results obtained are reported in the following table. The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value.

	Test Item Concentration % (w/v)	S.I.
Group 2	1	1.3
Group 3	2.5	1.2
Group 4	5	1.1

 

No positive dose-response relationship was observed.

Calculation of the EC 3 value was not performed because no test concentrations produced a S.I. of 3 or higher.

 

Based on the test results, the test substance does not show an allergenic potential when tested up to the concentration of 5 % (w/v) in DMF.