Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2005-09-01 to 2005-09-27
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
adopted 2002-04-24
Deviations:
yes
Remarks:
deviations from the study plan, see below
Principles of method if other than guideline:
Deviations from the Study Plan:
Test Item Preparation: The dilutions were prepared serially.
Animal Species: the age of the mice was 6-7 weeks.
Husbandry: The relative humidity in the animal room was between approximately 30 - 84%.

According to the authors, this deviation to the study plan did not affect the validity of the study.
GLP compliance:
yes (incl. certificate)
Remarks:
RCC Cytotest Cell Research GmbH
Type of study:
mouse local lymph node assay (LLNA)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Caffeine, anhydrous
- Physical state: solid, crystalline white powder
- Analytical purity: >= 99.5 % (99.5 -100.5 by titration)
- Impurities (identity and concentrations): chloride, sulfate, heavy metals; sum of all impurities max. 0.14%
- Purity test date: 2005-08-05; certificate No. 2434 (BASF AG)
- Lot/batch No.: 402841AX1E; date of production: 2004-06-11
- Expiration date of the lot/batch: 2008-06-11
- Storage condition of test material: room temperature
- Other: supplier: BASF AG

In vivo test system

Test animals

Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
female CBA/CaOlaHsd mice
- Source: Harlan Netherlands
- Age at study initiation: 6 - 7 weeks (beginning of acclimatization)
- Weight at study initiation: mean body weight: 19.8 +/- 1.3 g (range: 17.7 - 22.5 g)
- Housing: single caging in Makrolon Type I, with wire mesh top
- Diet: pelleted standard diet, ad libitum (Harlan Winkelmann GmbH, D-33178 Borchen)
- Water: tap water, ad libitum, (Gemeindewerke, D-64380 Rossdorf)
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25 °C
- Humidity (%): 30 - 84 %
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: no data

Study design: in vivo (LLNA)

Vehicle:
other: ethanol:water, 70:30 % (v/v)
Concentration:
0 (vehicle control), 0.5, 2, 5%
No. of animals per dose:
4 female mice per dose and control
Details on study design:
RANGE FINDING TESTS:
To determine the highest non-irritant test concentration or the highest technically applicable concentration, a non-GLP pretest was performed in two mice with concentrations of 0.625, 1.25, 2.5 and 5% (w/v) (pretest excluded from Statement of Compliance).
- Compound solubility:
Solubility in water: Approximately 20 g/L at room temperature; good solubility in vehicle

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
The animals were assigned to 4 groups of 4 animals.

- Criteria used to consider a positive response:
A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
- First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the stimulation index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

TREATMENT PREPARATION AND ADMINISTRATION: see freetext
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables.
A statistical analysis was conducted for assessment of the dose-response relationship, and the EC3 value was calculated according to the equation
EC3 = (a-c) [(3-d)/(b-d)] + c
where EC3 is the estimated concentration of the test item required to produce a 3-fold increase in draining lymph node cell proliferative activity; (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.

Results and discussion

Positive control results:
SI values:
5%: SI = 1.06
10%: SI = 1.81
25%: SI = 4.00
EC3 = 18.2% (w/v)

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: 0.5% (w/v): 0.91 2% (w/v): 0.92 5% (w/v): 0.87 The EC3 Value could not be calculated, since all SI's are below 3.
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: DPM minus background per lymph node (mean of n=8 lymph nodes) vehicle control: 408.4 0.5% (w/v): 372.0 2% (w/v): 373.8 5% (w/v): 354.0

Any other information on results incl. tables

Calculation and Results of Individual Data

Vehicle: Ethanol:water (7:3) (v/v)

Test item

Concentration

[% (w/v)]

Group

Measurement

DPM

Calculation

Result

DPB-BGa

number of

lymph nodesb

DPM per

lymph node

S.I.

---

BG I

28.22

---

---

---

---

---

BG II

25.88

---

---

---

---

---

CG 1

3294.49

3267.4

8

408.4

 

0.5

TG 2

3002.67

2975.6

8

372.0

0.91

2

TG 3

3017.35

2990.3

8

373.8

0.92

5

TG 4

2859.08

2832.0

8

354.0

0.87

The EC3 Value could not be calculated, since all SI´s are below 3.

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

CG = Control Group

TG = Test Group

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

 

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.

Results of the Positive Control GLP Study (RCC-CCR No. 884900)

Positive control substance: alpha-Hexylcinnamaldehyde

Vehicle: acetone:olive oil, 4:1 (v/v)

Test item

Concentration

[% (w/v)]

Group

Measurement

DPM

Calculation

Result

DPB-BGa

number of

lymph nodesb

DPM per

lymph node

S.I.

---

BG I

11.1

---

---

---

---

---

BG II

4.8

---

---

---

---

---

CG 1

5836.1

5828.2

8

728.5

 

5

TG 2

6162.5

6154.6

8

769.3

1.06

10

TG 3

10534.7

10526.8

8

1315.8

1.81

25

TG 4

23323.7

23315.8

8

2914.5

4.00

EC3 = 18.0 % (w/v)

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

CG = Control Group

TG = Test Group

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.

Applicant's summary and conclusion

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The test item, Caffeine Anhydrous, was not a skin sensitiser in this assay.
Executive summary:

Anhydrous caffeine (purity >=99.5%) was tested for skin sensitization in the Mouse Local Lymph Node Assay. The test substance was dissolved in ethanol/water (70/30) to give final concentrations of 0.5, 2, and 5%. Groups of 4 female CBA mice were applied the respective test solutions or the vehicle onto the dorsal surface of each auricle for 3 consecutive days. On the 5th day after the first application, all mice were i.v. injected with ³H-TdR and sacrificed. The auricular draining lypnh nodes were excised and pooled per group. Incorporation of ³H-TdR was measured by beta-scintillation counting. The Stimulation Index was calculated.

Treatment with the test substance did not produce an SI exceeding 3 at any concentration tested. Nor was there a dose-related increase in SI.

The test substance was not a skin sensitizer in this test.