Dimolybdenum trizinc nonaoxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Dimolybdenum trizinc nonaoxide

EC Number:

245-322-4

EC Name:

Dimolybdenum trizinc nonaoxide

Cas Number:

22914-58-5

Molecular formula:

Mo2O9Zn3

IUPAC Name:

dimolybdenum trizinc nonaoxide

impurity 1

Reference substance name:

Molybdenum zinc tetraoxide

EC Number:

237-377-8

EC Name:

Molybdenum zinc tetraoxide

Cas Number:

13767-32-3

Molecular formula:

MoO4Zn

IUPAC Name:

molybdenum zinc oxide

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA/Ca

Remarks:

CBA/CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo RMS B.V.
- Females (if applicable) nulliparous and non-pregnant: Yes
- Microbiological status of animals, when known: SPF quality
- Age at study initiation: 8 to 12 weeks old
- Weight at study initiation: 15 to 23 g
- Housing: Housed in suspended solid floor polypropylene cages furnised with softwood woodflakes.
- Diet (e.g. ad libitum): 2014C Teklad Global Rodent diet supplied ad libitum.
- Water (e.g. ad libitum): Mains tap water provided ad libitum
- Acclimation period: At least 5 days
- Indication of any skin lesions: None noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 30 to 70%
- Air changes (per hr): At least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle

Study design: in vivo (LLNA)

Vehicle:

dimethylformamide

Concentration:

Preliminary screening test: 25 µL of the test item as a 50% w/w suspension in DMF.
Main study: 25 µL of the test item as a 10, 25 or 50% w/w suspension in DMF.

No. of animals per dose:

Groups of 4 mice were treated

Details on study design:

The test item was prepared as a suspension in DMF. The test substance was formulated within 2 hours of being applied to the test system.

Prelininary screening test:
A mouse was treated by daily application of 25 µL of the test substance as a 50% w/w suspension in DMF to the dorsal surface of each ear for three consecutive days. Local skin irritation was socred daily. Any clinical signs of toxicity, if present, were also recorded. The thickness of each ear was measured using a gauge, pre-dose on Day 1 and post dose on Days 3 and 6. Any changes in ear thickness measurements were noted. A mean ear thickness increase of equal to or greater than 25% was considered to indicate excessive irritation and limited biological relevance to the endpoint of sensitisation.

Main test:
The preliminary screening test suggested that the test substance would not produce systemic toxicity or excessive local skin irritation at the highest suitable concentration. The mice were treated by daily application of 25 µL of the appropriate concentration of the test substance to the dorsal surface of each ear for three consecutive days. The test item formulation was administered using an automatic micropipette and spread over the dorsal surface of the ear using the tip of the pipette. A further group of four mice received the vehicle alone in the same manner.

3H-Methyl thymidine Administration: Five days following the first topical application of the test item or vehicle (Day 6) all mice were injected via the tail vein with 0.25 mL of PBS containing 3H-methyl thymidine giving a total of 20 µCi to each mouse.

Observations: All animals were observed twice daily on Days 1, 2 and 3 and on a daily basis on Days 4, 5 and 6. Any signs of toxicity or ill health during the test were recorded. bodyweights were recorded on Day 1 prior to dosing and Day 6 prior to termination.

Terminal procedures: Five hours following 3HTdR injection all mice were killed by CO2 asphyxiation followed by cervical seperation. The draining auricular lymph nodes from the 4 mice were excised and pooled for each experimental group. For each group 1 mL of PBS was added to the pooled lymph nodes.

Preparation of a single cell suspension: A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through a 200-mesh stainless steel gauze. The lymph node cells were rinsed through the gauze with 4 mL PBS into a petri dish labeled with the study number and dose concentration. The lymph node cell suspension was transferred to a centrifuge tube. The petri dish was washed with an additional 5 mL of PBS to remove all remaining lymph node cells and these were added to the centrifuge tube. The pooled lymph node cells were pelleted at 1400 rpm (approximately 190 g) for 10 minutes. The pellet was re-suspended in 10 mL of PBS and re-pelleted. To precipitate out the radioactive material, the pellet was re-suspended in 3 mL of 5% Trichloroacetic acid (TCA).

Determination of 3HTdR incorporation: After approximately 18 hours incubation at approximately 4°C, the precipitates were recovered by centrifugation at 2100 rpm (approximately 450 g) for 10 minutes, re-suspended in 1 mL of TCA and transferred to 10 mL of scintillation fluid. 3HTdR incorporation was measured by B-Scintillation counting. The vials containing the samples and scintillation fluid were placed in the sample changer of the scintillator and left to stand in darkness for approximately 20 minutes. The purpose of this period of time in darkness was to reduce the risk of luminescence, which has been shown to affect the reliability of the results. After approximately 20 minutes, the vials were shaken vigourously. The number of radioactive disintegrations per minute was then measured using the Beckman LS6500 scintillation system.

Data Evaluation: The proliferation response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (disintegrations per minute/node) and as the ratio of 3HTdR incorporation into lymph node cells of test nodes relative to that recorded for the control nodes (Stimulation Index). The test item will be regarded as a sensitiser if at least one concentration of the test item results in a threefold or greater increase in 3HTdR incorporation compared with control values. Any test item failing to produce a threefold or greater increase in 3HTdR incorporation will be classified as a 'non-sensitiser'.

Positive control substance(s):

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

Statistics:

Not required as all SI values < 3 and therefore EC3 not determined.

Results and discussion

Positive control results:

The SI index from the latest non-concurrent positive control study using the same vehicle was 7.28 (25% v/v solution) indicating a positive response for skin sensitisation under the test conditions.

In vivo (LLNA)

Results

Results 1

Parameter:

SI

Value:

1.03

Variability:

Not applicable (pooled method)

Test group / Remarks:

10 (% w/w)

Results 2

Parameter:

SI

Value:

1.24

Variability:

Not applicable (pooled method)

Test group / Remarks:

25 (% w/w)

Results 3

Parameter:

SI

Value:

1.17

Variability:

Not applicable (pooled method)

Test group / Remarks:

50 (% w/w)

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA : The SI index ranged from 1.03 to 1.24 with no dose dependancy. All results are less than 3 indicating a negative response.

DETAILS ON STIMULATION INDEX CALCULATION : Ratio of 3HTdR incorporation into lymph nodes of test nodes relative to that recorded for the control nodes.

EC3 CALCULATION: Not calculated as all SI values < 3.

CLINICAL OBSERVATIONS: There were no deaths and no signs of systemic toxicity were noted in the test or control animals during the test.

BODY WEIGHTS: Body weight change of the test animals betwen Day 1 and Day 6 was comparable to that observed in the corresponding control group animals over the same period.

Any other information on results incl. tables

Disintegrations per minute, disintegrations per minute/node and Stimulation Index

Concentration (% w/w) in dimethyl formamide	dpm	dpm/node	Stimulation Index	Result
Vehicle	10301.34	1287.67	NA	NA
10	10572.98	1321.62	1.03	Negative
25	12799.96	1600.00	1.24	Negative
50	12100.82	1512.60	1.17	Negative

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

As the SI Index at all doses was less that 3, the test item was considered to be a non-sensitiser under the conditions of the test.