Registration Dossier

Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
12 August - 17 September 2008
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP - guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2008
Report Date:
2008

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
Husbandry: relative humidity in the animal room was between appr. 30 - 100% for few hours; historical positive control
GLP compliance:
yes (incl. certificate)
Remarks:
Hessisches Ministerium für Umwelt, ländlichen Raum und Verbraucherschutz, Wiesbaden
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): Chromium (III) acetate
- Analytical purity: > 95%
- Lot/batch No.: 1000141379
- Expiration date of the lot/batch: 03 July 2009
- Storage condition of test material: room temperature, moisture protected

In vivo test system

Test animals

Species:
mouse
Strain:
other: CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Netherlands, B:V. Postbus 6174, NL - 5960 AD Horst / The Netherlands
- Age at study initiation: 8 - 12 week, nulliparous and non-pregnant
- Weight at study initiation: 18.5 - 23.5 g
- Housing: single caging; cage type: Makrolon Type I, with wire mesh top (EHRET GmbH, D-79302 Emmendingen); bedding: granulated soft wood bedding (Harlan Winkelmann GmbH, D-33178 Borchen)
- Diet: pelleted standard diet (Harlan Winkelmann GmbH)
- Water: local tap water
- Acclimation period: at least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 100%
- Photoperiod (hrs dark / hrs light): 12 / 12

Study design: in vivo (LLNA)

Vehicle:
dimethylformamide
Concentration:
10, 25, and 50%
No. of animals per dose:
4
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: solubility test according to OECD 429 performed; highest test item concentration, which can be technically used was a 50% suspension in dimethylformamide
- Irritation: no severe irritant effects were tolerated choosing the test concentrations

To determine the highest non-irritant test concentration, a pre-test was performed in two animals with concentrations of 5, 10, 25 and 50% on one ear each on three consecutive days. No signs of irritation or systemic toxicity could be observed.


MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: The test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled: (a) 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, (b) 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:
Each test group was treated by application to the dorsal surface of each ear lobe (left and right). 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. The control group was treated with an equivalent volume of the relevant vehicle alone.
Five days after the first topical application, all mice were administered with 250 µL 3H-methyl thymidine (3HTdR; corresponds to 20.1 µCi 3HTdR per mouse) by intravenous injection via a tail vein. Approximately five hours after treatment with 3HTdR all mice were euthanized by intraperitoneal injection of Pentobarbital-Natrium.
The draining lymph nodes were rapidly excised and pooled per group (8 nodes per group). Single cell suspensions of pooled lymph node cells were prepared and the level of 3HTdR incorporation was measured on a ß-scintillation counter. The ß-scintillation counter expresses 3HTdR as the number of radioactive disintegrations per minute (DPM).
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)

Results and discussion

Positive control results:
The EC3 value was estimated to be < 5%. A precise calculation of the EC3 value was not possible since the S.I. was above the threshold of 3 even at the lowest tested concentration. The obtained values are within the expected range and the positive control experiment is considered fully valid.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Remarks on result:
other: see tables below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see tables below

Any other information on results incl. tables

Table: Results of individual data

Test item concentration [%]

DPM (a)

Stimulation Index

0

179.9

10

1335.4

7.42

25

1304.9

7.25

50

2447.8

13.60

DPM: desintegrations per minute per lymph node

(a): 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

OBSERVATIONS

In addition to the sensitising reactions the following observation and data were recorded during the test and observation period:

Mortality/viability: No deaths occurred 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.

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

In summary, all treated animals survived the scheduled study period and no signs of toxicity were observed. Under the described test conditions, the test item was found to be a skin sensitiser.

Applicant's summary and conclusion

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: Skin Sens. 1B
DSD: Xi, R43