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Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
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
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)
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
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)
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.
Parameter:
SI
Remarks on result:
other: see tables below
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see tables below

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.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
CLP: Skin Sens. 1B
DSD: Xi, R43
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

To investigate the skin sensitising properties of Acetic acid, chromium salt, basic, a Local Lymph Node Assay (LLNA) according to OECD 429 and GLP was performed (Meurer and Honarvar, 2008). In a preliminary range-finding test, the maximum technically applicable concentration of the test substance was found to be 50% in dimethylformamide. Therefore, test substance concentrations of 10, 25 and 50% were applied once daily to the dorsal surface of both ears of 4 mice per group for 3 consecutive days. On Day 5, all mice were dosed with ³H-Methylthymidine. Five hours thereafter, the mice were sacrificed and their draining auricular lymph nodes were excised, pooled per group and a single cell suspension was prepared. The ³H-Methylthymidine incorporation was determined in a β-counter.

All animals survived the study period and no signs of toxicity or local skin irritation were observed. Stimulation indices of 7.42, 7.25 and 13.60 were recorded for test substance concentrations of 10, 25 and 50%, respectively. The EC3 for the test substance is < 10%.

Under the conditions tested, the substance was considered to be a skin sensitiser.

Migrated from Short description of key information:
Skin sensitisation (OECD 429, LLNA): sensitising (EC3 < 10%)

Justification for selection of skin sensitisation endpoint:
There is only one study available.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The available data on skin sensitisation of the test substance are conclusive and meet the criteria for classification. The substance has to be classified as skin sensitiser Cat. 1B according to Regulation (EC) 1272/2008 and according to Directive 67/548/EEC as Xi, R43.