2-Butenedioic acid (2Z)-, reaction products with ammonium di-.mu.3-hydroxyhexacosa-.mu.-oxododecaoxododecatungstate(6-) (6:1), ammonium octa-.mu.-oxodi-.mu.3-oxodi-.mu.4-oxododecaoxoheptamolybdate(6-) (6:1), nickel(2+) nitrate (1:2) and nickel(2+) sulfate (1:1)

Endpoint summary

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:

18 Jul - 7 Aug 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP-Guideline study (the selected test method is not appropriate for insoluble and metal-containing substances)

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Hess. Ministerium für Umwelt, Energie, Landwirtschaft und Verbraucherschutz, Wiesbaden, 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 B.V., AD Horst, The Netherlands
- Age at study initiation: 9 - 10 weeks
- Weight at study initiation: 19.4 - 23.1 g
- Housing: 4 animals per cage in Makrolon, type III L, cages with wire mesh top on granulated soft wood bedding
- Diet: 2018C Teklad Global 18% protein rodent diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 45 - 60
- Photoperiod (hrs dark / hrs light): 12 / 12

Vehicle:

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

Concentration:

2.5, 5 and 10% (w/v)

No. of animals per dose:

4 (main study), 1 (pre-screen test)

Details on study design:

RANGE FINDING TESTS:
- Compound solubility: The maximum technically applicable concentration was a 50% suspension in acetone:olive oil (4+1 v/v).
- Irritation:
1 mouse each was treated with 25 or 50% test item solution on 3 consecutive days. Animals were weighed prior to the first application of the test item and before sacrifice. Clinical signs of systemic toxicity or local irritation at the application site were recorded once daily. Ears of each mouse were observed for erythema/eschar formation and scored. Measurement of ear thickness was performed on Day 1 (pre-dose), Day 3 and before sacrifice using a micrometer (S0247). Additionally, the ears of both animals were punched after sacrifice at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm²) and pooled per animal before weighing.
Ear irritation was considered to be excessive if an erythema score ≥ 3 and/or an increase in ear thickness ≥ 25% was recorded.
The animal treated with 25% test item solution showed a decreased body weight of 10% during the study period. Moreover, ear thickening of 36% was determined on Day 6 after topical application of 25%. Although the animal treated with 50% test item solution did not show increased ear thickness above the threshold value of 25%, both animals showed visible swelling of the ears on Day 6. Ear weights of both animals were highly increased compared to historical vehicle values and well defined erythema of the ear skin (animal 1: score 2 on day 4, score 1 on days 5 and 6; animal 2: score 2 on days 4 and 5, score 1 on day 6) were observed. Therefore, a second pre-test was performed with test item concentrations of 5 and 10% in one animal each. Both animals did not show signs of systemic toxicity or excessive local skin irritation. According to this, concentrations of 2.5, 5 and 10% (w/v) were selected as test concentrations in the main test to avoid sytemic toxicity and excessive local skin irritation.
- Lymph node proliferation response: Lymph node proliferation was not assessed in the range-finding test.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: 3H-methyl thymidine incorporation determined by ß-scintillation
- Criteria used to consider a positive response: The test item was considered as a skin sensitiser, if exposure to at least one concentration of the test item resulted in an at least 3-fold or greater stimulation index in test animals compared to control mice (SI ≥ 3), and data are compatible with a dose-response, although allowance must be made especially at high concetrations for either local toxicity or immunological suppression.
- Validation criteria: The positive control substance should produce a positive LLNA response shown by SI > 3 over the negative control group without causing excessive skin irritation (i.e. SI > 20) or systemic toxicity.

TREATMENT PREPARATION AND ADMINISTRATION:
25 µL of the test item was applied on the entire dorsal surface of each ear of each dose daily over 3 consecutive days. On Day 6, an injection of 250 µL phosphate buffered saline containing 20.5 µCi of 3H-methyl thymidine was made into the tail vein of each experimental mouse. Approx. 5 h later, animals were sacrificed and the draining auricular lymph node of each ear was excised and pooled per group. Single cell suspension of pooled lymph nodes were prepared. After repeated washings, macromolecules were precipitated with 5% trichloracetic acid at 4 °C for at least 18 h. The 3H-methyl thymidine incorporation was measured in a ß-counter and expressed as the number of disintegrations per minute per lymph node (DPM/lymph node) for each treatment group. Further, the ratio of 3H-methyl thymidine incorporation between test lymph nodes and control lymph nodes was calculated as Stimulation Index (SI).

FURTHER OBSERVATIONS:
- Mortality, viability and clinical signs (including systemic toxicity and local skin irritation) (at least once daily)
- body weights (prior to first application and treatment with 3H-methyl thymidine)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

Mean values and standard deviations were calculated.

Positive control results:

The positive control substance induced a significant, dose-dependent lymphoproliferative response as shown by stimulation index values of 0.88. 1.51 and 3.73 after topical application of 5, 10 and 25% resulting in an EC3 value of 20.1% (w/v).
Historical control data for the last 10 periodically checked experiments with 25% hexyl cinnamic aldehyde range from 3.73 - 10.77.

Parameter:

SI

Remarks on result:

other: see Remark

Remarks:

No significant lymphoproliferative response exceeding the threshold level (SI ≥ 3) was determined for the test substance at the tested concentrations. The observed stimulation index values were 0.8, 0.85 and 1.51 at test item concentrations of 2.5, 5 and 10%, respectively.

Parameter:

other: disintegrations per minute (DPM)

Remarks on result:

other: A slight, dose-dependent increase in DPM/lymph node values was observed in test animals (377.1, 303.5, 321.1 and 568.8 for control, low- mid- and high-dose animals, respectively).

Other Effects:

No significant effects on body weight were observed in any treatment group.

No mortality or symptoms of systemic toxicity were determined. On Day 2, animals treated with 5 and 10% test item solutions showed slight erythema of the ear skin scored with 1 which was still visible on Day 4 in test animals treated with 10% (score 1).

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

CLP: not classified
DSD: not classified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

Sensitisation

Sensitising properties of 2-Butenedioic acid (2Z)-, reaction products with ammonium di-µ3-hydroxyhexacosa-µ-oxododecaoxododecatungstate(6-) (6:1), ammonium octa-µ-oxodi-µ3-oxo-µ4-oxododecaoxoheptamolybdate(6-) (6:1), nickel(2+) nitrate (1:2) and nickel(2+) sulfate (1:1) were evaluated in a local lymph node assay according to OECD 429 in female CBA/CaOlaHsD mice (4 animals/group) (Bohnenberger, 2012). According to the performed pre-screen test, which revealed signs of skin irritation and systemic toxicity after topical application of 25%, animals were treated with 2.5, 5 and 10% test item solutions (w/v). Neither mortality or clinical signs of toxicity were observed. No significant effects on body weight were determined in any treatment group. Topical application of 5 and 10% test item solutions induced slight erythema of the ear skin scored with grade 1 on Day 2, which lasted until Day 4 in test animals treated with 10% (score 1). No lymphoproliferative response was noted for the test substance. Thus, 2-Butenedioic acid (2Z)-, reaction products with ammonium di-µ3-hydroxyhexacosa-µ-oxododecaoxododecatungstate(6-) (6:1), ammonium octa-µ-oxodi-µ3-oxo-µ4-oxododecaoxoheptamolybdate(6-) (6:1), nickel(2+) nitrate (1:2) and nickel(2+) sulfate (1:1) did not exhibit skin sensitising properties in the conducted study. However, it must be noted that nickel compounds including nickel sulfate and nickel chloride are known to produce false-negative results in the Local Lymph Node Assay.

Migrated from Short description of key information:
Skin sensitisation (OECD 429): not sensitising (Bohnenberger, 2012)

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 do not meet the classification criteria according to Regulation (EC) 1272/2008 or Directive 67/548/EEC, and are therefore conclusive but not sufficient for classification.