Registration Dossier

Administrative data

Description of key information

LLNA (OECD 429): sensitising

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:
24 Jan 2014 - 24 Feb 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(adopted 22 Jul 2010)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Version / remarks:
(adopted 30 May 2008)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
(adopted Mar 2003)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Bayerisches Landesamt fuer Gesundheit und Lebensmittelsicherheit, Erlangen, Germany
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 Laboratories GmbH, Venray, The Netherlands
- Age at study initiation: 9-10 weeks
- Housing: in groups of 5 in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding (lot no. 131113)
- Diet: Altromin 1324 maintenance diet for rats and mice (lot no. 0801), ad libitum
- Water: tap water, sulphur acidified to a pH value of approximately 2.8 (drinking water, municipal residue control, microbial control at regular intervals), ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3
- Humidity (%): 55±10
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
dimethyl sulphoxide
Concentration:
17, 33 and 67% (w/v)
No. of animals per dose:
3 in prescreen test (2 in test group and 1 treated with vehicle alone as negative control)
5/group in main study
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: DMSO was selected as vehicle due to the solubility properties of the test item. The maximum technically applicable concentration of the test item in DMSO was determined to be 67%.
- Irritation: No irritation was observed at the maximum technically feasible concentration of 67% test item in DMSO and after treatment with DMSO alone.
- Lymph node proliferation response: Not conducted in pre-screen test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-Methyl Thymidine (3HTdR) incorporation method
- Criteria used to consider a positive response: A substance is regarded as a "sensitiser" in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3HTdR incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (i.e. Stimulation index equal or greater than 3.0).

TREATMENT PREPARATION AND ADMINISTRATION:
Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. Topical applications were performed once daily over three consecutive days.
Five days after the first topical application all mice were dosed with 20 µCi 3HTdR by intravenous injection (Tail vein) of 250 µL of 3HTdR, diluted to a working concentration of 80 µCi/mL.
Approximately 5 h after the injection of 3HTdR all mice were sacrificed. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph node per animal) and collected in phosphated buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS: The washing procedure was repeated. After the final wash each pellet was resuspended in approximately 1 mL 5% trichloroacetic acid (TCA) at approximately 4°C for approximately 18 h for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.
3HTdR incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3HTdR levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.
Positive control substance(s):
other: p-phenylenediamine (CAS 106-50-3, Sigma, purity >98%, lot no. SLBC7171V), 1% in DMSO
Positive control results:
The recent reliability check was performed in Nov 2013. The DPM/node were 1891.9±387.6 for the negative control animals and 18051.7±1056.2 for the positive control animals. SI of the positive control animals was 9.5±0.6. The sensitivity of the test system was thus confirmed.
Key result
Parameter:
SI
Value:
5
Variability:
± 1.4
Test group / Remarks:
17% solution
Key result
Parameter:
SI
Value:
6
Variability:
± 0.6
Test group / Remarks:
33% solution
Key result
Parameter:
SI
Value:
8.9
Variability:
± 3.2
Test group / Remarks:
67% solution
Key result
Parameter:
EC3
Value:
4.51
Parameter:
other: disintegrations per minute (DPM)/node
Value:
1 539.4
Variability:
± 353.1
Test group / Remarks:
negative control
Parameter:
other: disintegrations per minute (DPM)/node
Value:
7 706.9
Variability:
± 2165.6
Test group / Remarks:
17% solution
Parameter:
other: disintegrations per minute (DPM)/node
Value:
9 204.6
Variability:
± 898.8
Test group / Remarks:
33% solution
Parameter:
other: disintegrations per minute (DPM)/node
Value:
13 683.1
Variability:
± 9886.7
Test group / Remarks:
67% solution

Pre-screen test

Neither signs of systemic toxicity nor signs of irritation at any application site could be detected in any animal. One animal showed weight loss of 4 g. All other animals showed the expected weight development, which includes a weight loss of up to 2 g throughout the duration of the pre-screen test.

Main study

All animals survived throughot the study period. On Day 6, one animal of the 33% group showed bodyweight loss, hypothermia, dehydration, piloerection and lymph nodes with a small size. All other test group animals showed enlarged lymph nodes, and their body weights developed as expected. Eschar was observed in all animals of the test groups on Day 6. As only one animal of the mid dose group (33%) showed clinical signs, a clear test item relation cannot be concluded.

Interpretation of results:
Category 1B (indication of skin sensitising potential) based on GHS criteria
Conclusions:
CLP: Cat 1B, H317
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

Skin sensitisation of Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate was investigated by means of Local Lymph Node Assay (LLNA). In the available key study (CBMM Europe BV, Key, 2014, LLNA) the ears of 5 mice per group were topically treated with concentrations of 17, 33 and 67% of the test item in DMSO for 3 consecutive days. Five days after the first treatment the mice received an intravenous injection of 3H-Methyl Thymidine (3HTdR). Five hours after treatment the mice were euthanised, the cells of the draining auricular lymph nodes were dissected, and the incorporated radioactivity was determined as number of disintegrations per minute (DPM) by means of a β-scintillation counter. Stimulation Indices were calculated as ratio of respective DPM of the individual animals from the treated groups versus the mean DPM of the control group. Subsequently, group mean Stimulation Indices of 5.0±1.4, 6.0±0.6 and 8.9±3.2 were determined for the 17, 33 and 67% group, respectively. According to the criteria of OECD Guideline 429 the test item has to be regarded as a skin sensitiser.

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available
Additional information:

A study assessing the potential risk of respiratory sensitisation is not required according to Annex VII-X of Regulation (EC) No 1907/2006.

Justification for classification or non-classification

The available data are reliable and suitable for classification. Based on these data, Reaction mass of ammonium diaqua[bis(oxalate)]oxoniobate(1-) hydrate and ammonium hydrogen oxalate oxalic acid (1:1:1) dihydrate meets the criteria to be classified for skin sensitisation (Cat 1B, H317) according to EC/1272/2008.