Registration Dossier

Toxicological information

Skin sensitisation

Currently viewing:

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
other: Data sharing dispute
Adequacy of study:
key study
Study period:
2010-06-18 to 2010-08-09
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study was conducted as per OECD 429 and EU B.42 test method guidelines. The test was done under GLP compliance.
Cross-referenceopen allclose all
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(also corresponds to EU B.42 test method on skin sensitization)
Deviations:
no
Principles of method if other than guideline:
not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
Identity: Charcoal (Probe 1: C-Fix = 73.3%)
CAS No.: 16291-96-6
Batch No.: 19062009 1PP
Purity: C-Fix: 73.3%; dose calculation not adjusted to purity
Storage: At room temperature, moisture protected
Expiration Date: December 2030

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
Animals: Female mice of 8-12 weeks of age and with 19-23 g of body weight were included in the study; Housing: Animals were individually housed; Lighting: 12 h light/12 h dark; Temperature: 22 ± 2°C; Relative humidity: 35 to 85%; Food: Animals had access to pelleted standard diet, ad libitum; Water: Tap water, ad libitum; Bedding: Granulated soft wood bedding; Acclimatisation: Study animals were acclimated to their housing for 5 days prior to their first day of dosing.

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Remarks:
(purity: 99%)
Concentration:
3 groups of 4 female mice were treated on the dorsal surface of both ears once per day for 3 days with the test article, charcoal (Probe 1: C-Fix = 73.3%) at 2.5, 5, and 10% (w/w) in the vehicle propylene glycol.
No. of animals per dose:
4 female mice
Details on study design:
ANIMAL ASSIGNMENT AND TREATMENT:
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response.

TREATMENT PREPARATION AND ADMINISTRATION:
On each day of dosing, the test article was prepared at the appropriate concentrations (w/w) by suspending the appropriate amount of test article in propylene glycol. The application volume of 25 µL was used.

4 groups of 4 female mice were treated on the dorsal surface of both ears once per day for 3 consecutive days as follows:
Group 1: vehicle, propylene glycol; Group 2: Charcoal, 2.5% (w/w); Group 3: Charcoal, 5% (w/w); and Group 4: Charcoal 10% (w/w)

On day 6, the mice were injected, i.v., with 20.4 µCi of 3H-Methyl thymidine (3HTdR) per mouse. Five hours later, the mice were euthanized and the draining auricular lymph nodes were removed. The lymph node cells were treated with 5% trichloroacetic acid (TCA) to precipitate the DNA. The resulting pellets were counted in a β-scintillation counter to determine incorporation of 3HTdR.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated for the body weight parameter.

Results and discussion

Positive control results:
The positive control, HCA at 10% and 25% resulted in a stimulation index (SI) of 3.41 and 6.14, respectively. A 3-fold or greater increase in proliferative activity relative to the concurrent vehicle control is considered a positive response.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: SI: Vehicle control = 1.00; Charcoal (2.5% w/v) = 0.65; Charcoal (5% w/v) = 0.72; and Charcoal (10% w/v) = 1.11
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM per lymph node: Vehicle control = 295.6; Charcoal (2.5% w/v) = 192.7; Charcoal (5% w/v) = 211.4; and Charcoal (10% w/v) = 329.3

Any other information on results incl. tables

DPM and SI:

 

Test item concentration % (w/w)

Group

Measurement DPM

Calculation

Result

DPM-BGa)

number of lymph nodes

DPM per lymph nodeb)

S.I.

---

BG I

20

---

---

---

---

---

BG II

17

---

---

---

---

0

1

2383

2365

8

295.6

1.00

2.5

2

1560

1542

8

192.7

0.65

5

3

1710

1692

8

211.4

0.72

10

4

2653

2635

8

329.3

1.11

BG=  Background (1 ml 5% trichloroacetic acid) in duplicate

1    =  Control Group

2-4=  Test Groups

S.I.=  Stimulation Index

a)   =  The mean value was taken from the figures BG I and BG II

b)    =  Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled

The estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity (termed the EC3 value) can be calculated according to the equation [EC3=(a-c) [(3-d)/(b-d)] + c]. Here, (a, b) and (c, d) are respectively the co-ordinates of the two pair of data lying immediately above and below the S.I. value of 3 on the local lymph node assay dose response plot.

The EC3 value could not be calculated, since all S.I.´s are below 3.

Viability / Mortality:

No deaths occurred during the study period.

Clinical Signs:

No symptoms of local toxicity at the ears of the animals and no systemic findings were observed during the study period.

Body Weights:

The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information up to 10% (w/w) of charcoal (Probe 1: C-Fix 73.3%) tested
Conclusions:
The test item, charcoal (Probe 1: C-Fix = 73.3%) was not a skin sensitiser under the test conditions of this study.
Executive summary:

In order to study a possible contact allergenic potential of Charcoal (Probe 1: C-Fix = 73.3%), three groups each of four female mice were treated daily with the test item at concentrations of 2.5, 5, and 10% (w/w) in propylene glycol by topical application to the dorsum of each ear (left and right) for three consecutive days. A control group of four mice was treated with the vehicle (propylene glycol) only. Five days after the first topical application the mice were injected intravenously into a tail vein with 3HTdR. Approximately 5 h 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 3HTdR measured in a beta-scintillation counter.

All treated animals survived the scheduled study period and no signs of toxicity were observed.

A test item is regarded as a sensitiser in the LLNA if the exposure to one or more test concentration resulted in 3-fold or greater increase in incorporation of 3HTdR compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated concentration of test item required to produce a S.I. of 3 is referred to as the EC3 value. In this study Stimulation Indices of 0.65, 0.72, and 1.11 were determined with the test item at concentrations of 2.5, 5, and 10%, respectively, in propylene glycol. The EC3 value could not be calculated, since none of the tested concentrations induced an S.I. greater than 3.

The test item, charcoal (Probe 1: C-Fix = 73.3%) was not a skin sensitiser under the test conditions of this study.