Molybdenum zinc tetraoxide

Endpoint summary

Description of key information

Key study: OECD 442B. GLP study.The test item does not have to be classified as a skin sensitizer, under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 0.92, 0.88 and 0.82 at concentrations of test item 25%, 50% and 80%, respectively.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because the available in vitro test methods are not applicable for the substance and therefore an in vivo skin sensitisation study was conducted

Justification for type of information:

JUSTIFICATION FOR DATA WAIVING
The solubility of the test item in test specific solvents was determined in pre-tests. The test item was insoluble in all solvents indicated by the respective OECD guidelines at the required concentrations. Under these circumstances, in vitro skin sensitization testing in accordance with OECD 442D and OECD 442E of Zinc molybdate is not possible. Testing with precipitates is technically not feasible in case of h-CLAT, and it is not recommended in case of the LuSens test, since a significant result cannot be obtained. DPRA according to OECD 442C is not applicable as the test item is a metal compound. Other validated in vitro tests on skin sensitization are not available at present. Therefore, an in-vivo skin sensitization study was performed.

Reference 2

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 November 2018 - 19 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EU Method B.51 (Skin sensitisation. local lymph node assay: BrdU-ELISA)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Species:

mouse

Strain:

CBA:J

Remarks:

CBA/JRj

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle).
- Females (if applicable) nulliparous and non-pregnant: yes.
- Age at study initiation: 8 weeks old.
- Weight at study initiation: average weight 19.5-24.4 g (treated groups and control group)
- Housing: Individually (to avoid any test item absorption by oral route) in a suspended solid-floor polypropylene cages furnished with softwood woodflakes.
- Diet: Ad libitum (Enviogo 2016)
- Water: Ad libitum (tap water)
- Acclimation period: at least 5 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19ºC-25ºC.
- Humidity (%): 30%-70%.
- Air changes (per hr): 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12 h light (7.00 to 19.00) / 12 h darkness

Vehicle:

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

Concentration:

80%, 50% and 25%.

No. of animals per dose:

4

Details on study design:

PRE-SCREEN TESTS: A preliminary screening test was performed using one mouse. The mouse was treated by daily application of 25 μL of the test item diluted at 90% in Acetone/olive oil (4:1 v/v) (AOO) to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The mouse was observed daily from day 1 to day 6. The bodyweight of the mice were recorded on Day 1 (prior to dosing) and on Day 6.

- Compound solubility: A preliminary solubility test was performed and Acetone/olive oil (4:1 v/v) was chosen as vehicle since it produced the most suitable formulation at the required concentration.
- Irritation: No sign of excessive irritation was noted at the tested concentration of 80%.
- Systemic toxicity: No mortality and no signs of systemic toxicity were noted.
- Ear thickness measurements: values were within the acceptable range.
- Erythema scores: no signs of erithema were observed.

MAIN STUDY
Groups of four mice were treated with the test item diluted at 80%, 50% and 25% in Acetone/olive oil (4:1, v/v) based on the results of the pre-screen test. The mice were treated by daily application of 25 µL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3).
- Clinical observations: all animals were observed daily on Days 1, 2, 3 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.
- Body weight: the bodyweight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).
- Ear thickness: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer. Furthermore, on day 6, punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and weighted in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Skin sensitisation. Local Lymph Node Assay:BrdU-ELISA
- Criteria used to consider a positive response: BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 - Batch No. 29134900). 100 µL of the suspension of lymph node cells (LNC) was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 min, 30 µL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.
The BrdU labelling index was defined as: BrdU labelling index = (ABSem - ABS blankem) - (ABSref - ABS blankref)
The test item will be regarded as a sensitiser if at least one concentration of the test item results is greater than 1.6 compared to control values.
However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (SI value between 1.6 and 1.9) is declared positive. Any test item failing to produce a SI>1.6 will be classified as a "non-sensitiser".
The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was detemined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6 -fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 - d) / (b - d)] x (a - c)
Legend: a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c

According to Regulation (EC) No. 286/2011, the positive test item will be classified in subcategory 1A or 1B in accordance with:
If the EC value ≤ 2, the test item will be classified in "sub-category 1A".
If the EC value > 2, the test item will be classified in "sub-category 1B".

TREATMENT PREPARATION AND ADMINISTRATION:
The test item was freshly prepared in Acetone/olive oil (4:1, v/v). Groups of four mice were treated with the test item diluted at 80%, 50% and 25% in Acetone/olive oil (4:1, v/v). The mice were treated by daily application of 25 µL of the appropiate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1,2,3). 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.
On day 5 (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.
The Brdu solution was prepared by weighing 206.90 mg of 5-bromo-2'-deoxyuridine (SIGMA – Batch No. HMBF7815V) in a glass sample bottle adding 20.69 mL of physiological saline. The preparations were magnetically stirred during 10 min.
On day 6 (end of the test), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes from the four mice were excised.
From each mouse, a single-cell suspension through of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of PBS (Ca2 +/Mg2+ - free) into a well of a multi-well 6. The optimised volume was based on achieving a mean absorbance of the negative control group within 0.1 -0.2.

Positive control substance(s):

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

Positive control results:

EC1.6= 24.66%. The substance has to be classified in category 1 "Skin sensitisation".

Key result

Parameter:

SI

Value:

0.92

Test group / Remarks:

25%

Key result

Parameter:

SI

Value:

0.88

Test group / Remarks:

50%

Key result

Parameter:

SI

Value:

0.82

Test group / Remarks:

80%

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 80%.

DETAILS ON STIMULATION INDEX CALCULATION
No stimulation index of more than 1.6 was recorded whatever the tested concentration. The Stimulation Index (SI) calculated by individual approach was 0.92, 0.88 and 0.82 for the treated groups at 25%, 50% and 80%, respectively.

EC1.6 CALCULATION
The EC1.6 cannot be determined in this study.

CLINICAL OBSERVATIONS:
No mortality and no signs of systemic toxicity were noted in the test and control animals during the test.
Residual test item was noted in all animals treated at 50% between Days 2 and 5.
No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 90%.
Therefore, the test item has to be considered as not excessively irritant at these concentrations.

BODY WEIGHTS
Bodyweight changes of the test animals between Day 1 and Day 6 were comparable to those observed in the corresponding control group animals over the same period.

Table 1. Preliminary screening test: Clinical observation, bodyweight and mortality data

 

Concentration %	Animal	Bodyweight (g)		DAY
		Day 1	Day 6	1	2	3	4	5	6
80%	Sf1218	21.2	22.3	0	0	0	0	0	0

0: No sign of systemic toxicity and no sign of erythema

 

	Ear thickness (mm) on day 1	Ear thickness (mm) on day 3	Ear thickness (mm) on day 6	Ear weight (mg) on day 6	Weight Lymph nodes (mg)
Sf1218	0.21	0.21	0.21	28.4	5.3

 

The concentration of 80% was chosen as the highest concentration for the main study.

 Table 2. Main study: Individual clinical observation and mortality data

 

Groups	Test item	Amimals	Day 1	Day 2	Day 3	Day 4	Day 5	Day 6
1	AOO	Nº Sf 1284	0	0	0	0	0	0
		Nº Sf 1285	0	0	0	0	0	0
		Nº Sf 1286	0	0	0	0	0	0
		Nº Sf 1287	0	0	0	0	0	0
2	10%	Nº Sf 1289	0	0	0	0	0	0
		Nº Sf 1290	0	0	0	0	0	0
		Nº Sf 1291	0	0	0	0	0	0
		Nº Sf 1292	0	0	0	0	0	0
3	25%	Nº Sf 1294	0	0*	0*	0*	0*	0
		Nº Sf 1295	0	0*	0*	0*	0*	0
		Nº Sf 1296	0	0*	0*	0*	0*	0
		Nº Sf 1297	0	0*	0*	0*	0*	0
4	50%	Nº Sf 1299	0	0	0	0	0	0
		Nº Sf 1300	0	0	0	0	0	0
		Nº Sf 1301	0	0	0	0	0	0
		Nº Sf 1303	0	0	0	0	0	0

0: No sign of systemic toxicity

*: Residual test item

AOO: Acetone/olive oil (4:1 v/v)

 

 Table 3. Individual body weight and body weight gain

Groups	Test item	Animals No.	Body weight (g)		Body weight gain (g)
			Day 1	Day 6
1	AOO	Sf1284	20.8	21.4	0.6
		Sf1285	20.4	20.2	-0.2
		Sf1286	20.6	20.5	-0.1
		Sf1287	20.6	21.3	0.7
	MEAN		20.6	20.9	0.2
	Standard deviation		0.2	0.6	0.5
2	25%	Sf1289	20.8	21.6	0.8
		Sf1290	21.8	22.8	1.0
		Sf1291	21.2	22.0	0.8
		Sf1292	20.6	20.6	0.0
	MEAN		21.1	21.8	0.7
	Standard-deviation		0.5	0.9	0.4
3	50%	Sf1294	21.2	21.5	0.3
		Sf1295	21.7	21.6	-0.1
		Sf1296	24.4	22.4	-2.0
		Sf1297	22.8	23.3	0.5
	MEAN		22.5	22.2	-0.3
	Standard-deviation		1.4	0.8	1.1
4	80%	Sf1299	20.2	20.4	0.2
		Sf1300	22.1	22.0	-0.1
		Sf1301	19.8	19.9	0.1
		Sf1303	19.5	19.6	0.1
	MEAN		20.4	20.5	0.1
	Standard-deviation		1.2	1.1	0.1

AOO: Acetone/olive oil

 

Table 4. BrdU index & Stimulation index per group and calculation of EC1.6

 

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	AOO	0.589	n.a.	n.a.	n.a.
2	25%	0.540	0.92±0.10	negative	n.a
3	50%	0.520	0.88±0.04	negative
4	80%	0.482	0.82±0.03	negative

*: mean:Σindividual value / 4

AOO: Acetone/olive oil

  

Table 5. Individual Ear thickness and irritation level.

Groups	Test item	Animals No.	Day 1 ear thickness (mm)	Day 3 ear thickness (mm)	Day 6 ear thickness (mm)	Ear thickness increase D3/D1 %	Ear thickness increase D6/D1 %
1	AOO	Sf1284	0.21	0.21	0.21	0.0	0.0
		Sf1285	0.21	0.21	0.21	0.0	0.0
		Sf1286	0.21	0.21	0.21	0.0	0.0
		Sf1287	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.21	0.21	0.0	0.0
	Standard-deviation		0.00	0.00	0.00	0.00	0.00
2	25%	Sf1289	0.20	0.21	0.20	5.0	0.0
		Sf1290	0.20	0.21	0.21	5.0	5.0
		Sf1291	0.21	0.21	0.20	0.0	-4.8
		Sf1292	0.21	0.21	0.20	0.0	-4.8
	MEAN		0.21	0.21	0.20	2.5	-1.1
	Standard-deviation		0.01	0.00	0.00	2.9	4.7
3	50%	Sf1294	0.21	0.21	0.19	0.0	-9.5
		Sf1295	0.20	0.21	0.20	5.0	0.0
		Sf1296	0.21	0.21	0.21	0.0	-4.8
		Sf1297	0.21	0.21	0.21	0.0	0.0
	MEAN		0.21	0.21	0.20	1.0	-3.6
	Standard-deviation		0.00	0.00	0.01	2.5	4.6
4	80%	Sf1299	0.21	0.21	0.20	0.0	-4.8
		Sf1300	0.20	0.21	0.21	5.0	5.0
		Sf1301	0.21	0.21	0.21	0.0	0.0
		Sf1303	0.21	0.21	0.19	0.0	-9.5
	MEAN		0.21	0.21	0.20	1.3	-2.3
	Standard-deviation		0.00	0.00	0.01	2.5	6.2

AOO: Acetone/olive oil

 

  

Table 6. Individual Ear biopsy weight and lymph node weight.

Groups	Test item	Animals No.	ear weight Day 6 (mg)	% of ear weight increased/group1	Lymph nodes (mg)
1	AOO	Sf1284	28.1		5.7
		Sf1285	28.2		5.3
		Sf1286	29.2		4.9
		Sf1287	26.8		5.0
	MEAN		28.1		5.2
	Standard-deviation		1.0		0.4
2	25%	Sf1289	26.3	-0.6	6.1
		Sf1290	30.2		5.2
		Sf1291	29.0		5.4
		Sf1292	26.1		6.7
	MEAN		27.9		5.9
	Standard-deviation		2.0		0.7
3	50%	Sf1294	28.4	1.2	7.6
		Sf1295	30.1		5.2
		Sf1296	27.5		5.7
		Sf1297	27.6		5.8
	MEAN		28.4		6.1
	Standard-deviation		1.2		1.1
4	800%	Sf1299	28.2	-2.1	4.9
		Sf1300	26.0		5.0
		Sf1301	27.9		4.9
		Sf1303	27.8		5.5
	MEAN		27.5		5.1
	Standard-deviation		1.0		0.3

AOO: Acetone/olive oil

 

Table 7. Summary of result – skin irritation

 

Groups	Test item	Ear thickness increase D6/D1 (%)	Biopsy ear weight Increase (%)	Excessive irritation#
1	AOO	0.0	n.a	No
2	25%	-1.1	-0.6	No
3	50%	-3.6	1.2	No
4	80%	-2.3	-2.1	No

#: O.E.C.D. criteria: (% increase in ear thickness higher than 25%, score of erythema higher than 3)

AOO: Acetone/olive oil

 

Table 8. BrdU index & Stimulation index per group and calculation of EC1.6 of the positive control (study performed 04 July 2018 – 11 July 2018)

 

Groups	Test item	BrdU-index (mean*)	Stimulation Index SI (mean + standard deviation)	Result	EC1.6 value
1	AOO	0.637	n.a.	n.a.	n.a.
2	5%	0.614	0.96±0.10	negative	24.66%
3	10%	0.744	1.17±0.17	negative
4	25%	1.023	1.61±0.20	positive

AOO: Acetone/olive oil

 

 

 

 

 

 

Interpretation of results:

GHS criteria not met

Conclusions:

The test item does not have to be classified as a skin sensitizer, under the tested conditions in the LLNA assay (OECD 442B). The Stimulation Index (SI) calculated by individual approach were 0.92, 0.88 and 0.82 at concentrations of test item 25%, 50% and 80%, respectively.

Executive summary:

The skin sensitisation potential of the test item was tested in the LLNA assay according to OECD 442B and E.U. B.51 method, following GLP. Firstly, a preliminary screening test was performed using one mouse treated with 25 μL of the test item diluted at 90% in Acetone/olive oil (4:1 v/v) for three consecutive days. No mortality or signs of systemic toxicity and no signs of excessive irritation were noted in the preliminary study. In the main test, three groups of four mouse CBA/J were treated for three consecutive days with 50 µL (25 µL per ear) of the test item diluted at concentrations of 25%, 50% and 80% in Acetone/olive oil (4:1, v/v). A control group was treated with Acetone/olive oil (4:1, v/v). On day 5, 0.5 mL of BrdU solution (10mg/mL) was injected by intraperitoneal route. On day 6, the proliferation of lymphocytes in the draining auricular lymph nodes was deremined by measurement of BrdU content in DNA of lymphocyte using an ELISA kit. No mortality and no signs of systemic toxicity were noted in the test and control animals during the test. No increase in ear thickness and in ear weight was noted in animals treated at 25%, 50% and 80%. Therefore, the test item has to be considered as not excessively irritant at these concentrations.The Stimulation Index (SI) calculated by individual approach was 0.92, 0.88 and 0.82 for the treated groups at 25%, 50% and 80% respectively. Therefore, the EC1.6 cannot be determined due to the absence of SI value higher than 1.6. Under these experimental conditions, the test item does not have to be classified as a skin sensitizer in accordance with the CLP Regulation (EC) no. 1272/2008.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Justification for classification or non-classification

Based on available data, the substance is not classified for skin sensitization according to the CLP Regulation (EC) no. 1272/2008.