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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation, other
Remarks:
Academic research - 2 sensitization tests: Guinea-pig sensitization test and Sensitive mouse lymph node assay
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1995 - 1996
Reliability:
1 (reliable without restriction)
Justification for type of information:
To see details about read-across rationale, refer to summary endpoint of sensitization.
Cross-referenceopen allclose all
Reason / purpose:
read-across source
Reference
Endpoint:
transformation / dissolution of metals and inorganic metal compounds
Type of information:
experimental study
Adequacy of study:
key study
Study period:
- Start of the test period: 02.08.2016 - End of the test period: 17.11.2016
Reliability:
1 (reliable without restriction)
Qualifier:
according to
Guideline:
OECD Series on Testing and Assessment No. 29 (23-Jul-2001): Guidance document on transformation/dissolution of metals and metal compounds in aqueous media
Deviations:
yes
Principles of method if other than guideline:
1) Preliminary test
In order to determine an appropriate pH of the dissolution medium a preliminary test according to paragraph 26 of OECD 29 was performed. 100 ml glass bottles were filled with 50 ml of aqueous buffer solution at pH 5,5, pH 7 and pH 8,5, each in duplicate. A sample amount of approx. 5 mg was weighed under Argon atmosphere and suspended in the buffer solution at 23 °C. After 2 days a filtered aliquot of each bottle was analyzed for Zirconium content by ICP-OES according to EN ISO 11885.
2) Full test (7 and 28 days)
- For the full test according to paragraphs 34 and 35 of OECD 29 a buffered aqueous solution with pH 6 was chosen in order to maximize the potential of dissolution. 600 ml glass bottles were filled with 500 ml of the buffer medium. After a half-hour equilibrium phase pH, temperature and dissolved oxygen concentration of each vessel were measured. Duplicate aliquots of each vessel were taken in order to determine the background metal concentration of the medium by ICP-OES according to EN ISO 11885.
- Three different loadings of the test substance were prepared (1 mg/l, 10 mg/l, 100 mg/l), each in triplicate. An appropriate sample amount was weighed under Argon atmosphere into the buffer. The closed glass bottles were gently moved in a temperature controlled cabinet in the dark at 23 °C for the entire test duration of 29 days.
- At established time intervals the bottles were visually inspected and duplicate sample aliquots were taken for quantitative analysis of the Zirconium concentration. In order to prevent sample particles from being transferred to the analytical process all aliquots were filtered through 0,2 μm regenerated Cellulose membrane syringe filter. For each aliquot temperature, pH and dissolved oxygen were determined as well.
- The agitation period startet on 16.08.2016, 12:30. Sampling at the short term endpoint was on 23.08.2016, 13:30. Sampling at the long term endpoint was on 14.09.2016, 11:30. Undissolved substance was still visible at the bottom of each bottle at the end of the test period.

Note:
- pH was determined by an electronic pH-meter, device Metrohm 781
- Dissolved oxygen concentration was determined according to DIN EN 25813.
GLP compliance:
no
Other quality assurance:
ISO/IEC 17025 (General requirements for the competence of testing and calibration laboratories)
Type of method:
other: See above
Specific details on test material used for the study:
Sample : Zirconium hydride G, Batch no. 73311
Appearance : Grey powder
Container : Metal can, 250 ml
ASG-ID : 2404379_002
Key result
Water solubility:
< 0.08 mg/L
Conc. based on:
element
Remarks:
Zirconium
Loading of aqueous phase:
1 mg/L
Incubation duration:
ca. 29 d
Temp.:
23 °C
pH:
5.8
Water solubility:
< 0.08 mg/L
Conc. based on:
element
Remarks:
Zirconium
Loading of aqueous phase:
10 mg/L
Incubation duration:
ca. 29 d
Temp.:
23 °C
pH:
5.8
Water solubility:
< 0.08 mg/L
Conc. based on:
element
Remarks:
Zirconium
Loading of aqueous phase:
100 mg/L
Incubation duration:
ca. 29 d
Temp.:
23 °C
pH:
5.8
Conclusions:
The water solubility of zirconium hydride (ZrH2) following the OECD guideline n°29: "transformation/dissolution of metals and metal compounds in aquaeous media" was found below the detection limit (<0.08 mgZr/L).
As a final conclusion of all data it can be stated that the test substance Zirconium hydride is insoluble in water.
Reason / purpose:
read-across source
Reference

Metal hydride – Zirconium hydride (CAS 7704-99-6; EC 231-727-3)

Sensitization

Background:To induce sensitization, metal ions need to penetrate through the outer stratum corneum barrier layer of the skin and reach the underlying viable epidermis. It is generally believed that to be immunologically reactive, metal ions must bind to macromolecules such as proteins to form a hapten complex. Antigen presenting cells display this hapten complex on their cell surfaces and when the hapten is recognized as foreign by naïve T-lymphocyte cells, these cells undergo differentiation to form hapten-specific effector and memory helper T-cells (i.e., a person becomes sensitized). Upon repeated contact with the offending metal, at exposure levels that result in sufficient metal ion release and stratum corneum penetration, memory T-cells are recruited to the site of skin contact and elicit an inflammatory reaction (Stefaniak et al., 2014; Gibbs et al., 2018).

Regarding skin sensitization of zirconium hydride as requested under REACH regulation1907/2006, no data are currently available. To meet the skin sensitization endpoint requirement, read-across strategy with zirconium salts has been used.

Zirconium salts:

Testing of potential sensitizers of metals is traditionally carried out by applying the metal test chemical in the form of salt to the skin of animals or reconstructed skin under standard conditions. Preferably the salt should dissolve to form metal ions. In that respect, a couple of papers publicly available have been found.

Ikarashi et al., 1996

Sensitization potency of a zirconium salt (ZrCl4) was studied using the guinea-pig maximization test (GPMT) and adjuvant and patch test (APT). In addition, a sensitive mouse lymph node assay was also ran (SLNA). As result, ZrCl4 did not show any sensitization responses in all tests made.

Turk and Parker, 1977

Guinea pigs (Outbred Hartley-strain) were injected intradermally with 5 mg, 0.5 mg, and 0.05 mg of zirconium carbonate (ZrC03). No increase in skin thickness was detected. While ZrCO3 induced no measurable granulomas, the injection site usually contained a small collection of macrophages that had ingested crystalline material. There was no evidence of fibrosis and the macrophages remained undifferentiated with no other cellular infiltrate. At no time epithelioid cells were seen.

 

Conclusion: According to the papers found in the literature and publicly available, zirconium salts would not be classified as dermal sensitizer. On this basis, the non-sensitization of zirconium hydride has been extrapolated. In addition, water solubility of zirconium hydride at various pH was assessed and showed that the test item is insoluble (<0.08 mg/L) in water at pH which maximises the solubilisation (pH 5.8). This argument strengthens the non-allergic property of zirconium dihydride.

Reason / purpose:
read-across source
Reference
Endpoint:
skin sensitisation, other
Remarks:
Academic research - Intradermal injection to Guinea pigs
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
1976-1977
Reliability:
2 (reliable with restrictions)
Justification for type of information:
To see details about read-across rationale, refer to summary endpoint of sensitization.
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Reason / purpose:
read-across source
Qualifier:
equivalent or similar to
Guideline:
other: Academic study for sensitization
Version / remarks:
Academic study for sensitization - Lab: Department of Pathology. Royal College of Surgeons of England, London. England
Deviations:
not applicable
Principles of method if other than guideline:
Guinea pigs were injected intradermally at 3 sites into the shaved skin with 0.1 ml of 3 10-fold dilutions of the metal compound in physiologic saline.
The skin thickness fold around the injection site was measured using a Schnelltii.ster (Kroplin A02T) at 1 day and then weekly for 4 months.
Five animals were killed at 1, 2, 3, 4, and 6 weeks and skin of the 3 injection sites was taken for histologic examination.
ZrCO3 suspension was injected in a dose of 5 mg, 0.5 mg, and 0 .05 mg.
GLP compliance:
no
Type of study:
intracutaneous test
Specific details on test material used for the study:
Zirconium carbonate was obtained from Magnesium Electron Ltd., Clifton Junction, Manchester.
Note: Zirconium carbonate was analyzed and confirmed for metal content by Dr. A. Wolstenholme of British Aluminum Company, Research Laborator ies, Chalfont Park, England.
Species:
guinea pig
Strain:
Hartley
Sex:
not specified
Details on test animals and environmental conditions:
Outbred Hartley-strain guinea pigs (400 gm initially) were used from a colony kept at the Royal College of Surgeons or purchased from Messrs. A. Tuck and Sons Ltd .. Rayleigh, Essex. Animals were fed on a pelleted diet RGP (E. Dixon and Sons. Ware. Herts) and given water and cabbage ad libitum.
Route:
intradermal
Vehicle:
other: Physiologic saline
Details on study design:
Experiment contained initially at least 35 animals which were injected intradermally at 3 sites into the shaved skin with 0.1 ml of 3 10-fold dilutions of the metal compound in physiologic
saline.

The skin thickness fold around the injection site was measured using a Schnelltii.ster (Kroplin A02T) at 1 day and then weekly for 4 months.
Five animals were killed at 1, 2, 3, 4, and 6 weeks and skin of the 3 injection sites was taken for histologic examination.
ZrCO3 suspension was injected in a dose of 5 mg, 0.5 mg, and 0 .05 mg.

Tissues taken for histologic examination were fixed in Bouin's solution and stained with hematoxylin and eosin , Weigert's elastic tissue stain counterstained with VanGieson's stain, phosphotungstic acid hematoxylin, and reticulin stains.
Challenge controls:
No controls
Positive control substance(s):
no
Statistics:
Not reported in the published study
Key result
Reading:
other: Skin Tickness and Histological examination
Group:
test group
Dose level:
0.05 mg; 0.5 mg, 5 mg
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation

* Time course of granuloma formation: At no time was it possible to detect any increase in skin thickness with the 3 conrentrations of ZrCO3 used.

* Histolologic appearances: The granulomas induced by ZrCO3 that could not be observed macroscopically consisted of a small collection of macrophages that had ingested cystalline material. An occasional giant cell was seen. However, there was no evidence of fibrosis and the macrophages remained undifferentiated with no other cellular infiltrate. At no time were epithelioid cells seen. At 28 days, the only macrophages remaining were those that had ingested crystals of the material. As well as the quantitative difference in the size of the granulomas, the main difference in histologic appearance

between these two granulomas had a particularly swollen appearance, whereas those in the zirconium lesions did not show this effect.

Conclusions:
Following an intradermal injection in Guinea pigs with 5 mg, 0.5 mg, and 0.05 mg of zirconium carbonate (ZrCO3), no increase in skin thickness was detected. In addition,while ZrCO3 induced no measurable granulomas, the injection site usually contained a small collection of macrophages that had ingested crystalline material.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
1996

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
other: Guinea-pig maximization test (GPMT)
Version / remarks:
methods described by Magnusson and Kligman
Deviations:
yes
Remarks:
Nakamura A, et al. A new protocol and criteria for quantitative determination of sensitization potencies of chemicals by guinea pig maximization test, Contact Dermatitis 1994; 31: 72-85.
Qualifier:
according to
Guideline:
other: Patch test (APT)
Version / remarks:
Sato Y, Katsumura Y, Ichikawa H, Kobayashi T, Kozuka T, Morikawa F, Ohta S. A modified technique of guinea pig testing to identify delayed
hypersensitivity allergens. Contact Dermatitis 1981; 7: 225-237.
Deviations:
no
Qualifier:
according to
Guideline:
other: Sensitive mouse lymph node assay
Version / remarks:
Ikarashi Y, Tsuchiya T, Nakamura A. A sensitive mouse lymph node assay with two application phases to detection of contact allergens. Arch Toxicol 1993; 67:
629-636.
Deviations:
no
Principles of method if other than guideline:
1) Guinea-pig maximization test (GPMT)

Test guinea-pigs received a series of 6 intradermal injections of the test chemical at 1% in FCA emulsion into the shoulder region to induce sensitization. Seven days later, the animals received a 48 h occluded patch containing the chemical at the same site to boost sensitization.

Patch test (APT)

On the first day, 0.1 ml of saline-FCA emulsion was injected intradermally into the nuchal skin, at the four corners of a 2 x 4 cm2 area, and then a criss-cross lattice of abrasions was made at the site of injection. At the same time, 0.1 ml of test sample was applied. Four patches were applied occlusively to the abraded skin for 24 h. Similar abrasions and applications of patches were repeated the following two days. At the second stage of induction, an occluded patch was applied a.

Challenge
The animals were challenged on flank with 20 µl of various concentrations of test chemical on the 14 days following the induction. Skin reaction was determined by visual assessment 48 h after challenge. The intensity of the reaction was scored according to the criteria: 0 = no visible change, 1 = slight or discrete erythema,
2 = moderate and confluent erythema, 3 = intense erythema and swelling. The sensitization rate and mean skin response were also calculated. Rechallenge was performed on the 7 days after the first challenge.

Note for 1): Control guinea-pigs were treated with the appropriate vehicle under identical conditions. Animals: Adult female Hartley strain guinea-pigs (350400 g) purchased from Japan SLC Inc. (Shizuoka, Japan).

2) Sensitive mouse lymph node assay

Groups of mice (n = 3) were initially injected intradermally, totalling 50 µl of test chemical-FCA emulsion into two sites of the abdominal skin. Five days after injection, the mice received 25 µl of test chemical in vehicle on both sides of each ear for three consecutive days. Control mice were treated by intradermal injection of vehicle-FCA emulsion into the abdomen and, after 5 days, the mice were exposed to vehicle alone on the ears for three consecutive days. The day after the final application, auricular lymph nodes were excised, pooled for each experimental group and weighed. A single cell suspension of lymph node cell (LNC) was prepared by mechanical disaggregation through sterile 200-mesh gauge, and washed once with Hanks’ balanced salt
solution. The LNCs were resuspended in RPMI-1640 culture medium supplemented with 25mM HEPES, 100 units/ml penicillin, 100 pg/ml streptomycin and
10% fetal calf serum, and total LNC number was determined using an automated cell counter. The increase in LNC number relative to vehicle-treated
controls was derived for each experimental group and recorded as a stimulation index of LNC number (SI,).
The LNC suspensions (1 x 10^6 cells) were seeded into 96-well culture plates (five wells per group) and cultured with 0.5 µCi (3^H)lmethyl thymidine (3^HTdR)
for 24 h at 37°C in a humidified atmosphere of 5% CO2, in air. Culture was terminated by using a semiautomatic cell harvester, and the 3^HTdR incorporation
was determined by liquid scintillation counting. The increase in 3^HTdR incorporation relative to controls was derived for each experimental group and recorded
as a stimulation index of LNC proliferation (SIp). SItotal was also defined as SIn x SIp, and it indicates the total lymph node activation induced by test chemicals. A chemical was regarded as positive (a sensitizer) if it showed an SItotai value of 3 or greater.

Note for 2): young adult female BALB/c strain mice (6-8 weeks old) were purchased from Japan SLC Inc. (Shizuoka, Japan).




GLP compliance:
no
Remarks:
Academic study
Type of study:
other: 1) GPMT and 2) sensitive mouse lymph node assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
not specified
Specific details on test material used for the study:
Zirconium chloride (ZrCl4) were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).

In vivo test system

Test animals

Species:
other: Guinea pig
Strain:
other: Guinea pig (Hartley)
Sex:
female
Details on test animals and environmental conditions:
No data reported

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
other: Saline
Remarks:
Freund's complete adjuvant (FDA)
Concentration / amount:
1%
Day(s)/duration:
GPMT (7 days)
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
5%
Day(s)/duration:
24 h and repeated the following two days
Challenge
Route:
other: on flank
Vehicle:
other: 70% Ethanol
Concentration / amount:
5%
Day(s)/duration:
48 hours - Note: rechallenge was performed on the 7 days after the first challenge.
No. of animals per dose:
GPMT:
- Intradermal injection: ZrCl4: Concentration: 1% - Vehicle: saline
- Topical application: ZrCl4: Concentration 5% - vehicle Petrolatum
- Challenge: ZrCl4: Concentration 5% - vehicle: 70% Ethanol

Details on study design:
Two sensitization methods:
- guinea-pig maximization test (GPMT) - procedure Magnusson and Kligman
- adjuvant and patch test (APT) - procedure Sato et al. (Sato Y, Katsumura Y, Ichikawa H, Kobayashi T, Kozuka T, Morikawa F, Ohta S. A modified technique of guinea pig testing to identify delayed hypersensitivity allergens. Contact Dermatitis 1981;7: 225-237).
Positive control substance(s):
no

Study design: in vivo (LLNA)

Vehicle:
other: chemical-FCA emulsion
Concentration:
Sensitive mouse lymph node assay (SLNA)
- Intradermal injection: ZrCl4: Concentration (vehicle): 0 (saline), 0.02, 0.2, 2%
- Topical application: ZrCl4: Concentration (vehicle): 0 (70% DMSO), 5, 5, 5%
No. of animals per dose:
n=3
Details on study design:
Groups of mice (n = 3) were Injected lntradermally with 50 µml of various concentrations of test chemical in saline or with saline alone in Freund’s complete adjuvant emulsion into the abdomen. Five days later, 25 µL of 5% test chemical in 70% DMSO or 70% DMSO alone was applied to both ears daily for 3 consecutive days. The day following the final application. draining auricular lymph nodes were excised, pooled for each experimental group and weighed. A single cell suspension of lymph node cells (LNCs) was prepared. Total cell number was determined, and LNC proliferation was measured. The data are mean 3HTdR incorporation from five culture wells. Increases in LNC number and 3HTdR relative to vehicle-treated controls were calculated for each test group and expressed as SIn, SIp respectwely. SItotal was obtained from SIn x SIp

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
336
Group:
test group
Dose level:
challenge conc. % : 5
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no reaction
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
336
Group:
negative control
No. with + reactions:
0
Total no. in group:
3
Clinical observations:
no reaction
Remarks on result:
no indication of skin sensitisation

In vivo (LLNA)

Resultsopen allclose all
Key result
Parameter:
SI
Remarks:
stimulation index of LNC proliferation (SItotal). SItotal was also defined as SIn x SIp and it indicates the total lymph node activation induced by test chemicals.
Value:
ca. 0.94
Test group / Remarks:
0.02
Parameter:
SI
Value:
ca. 0.9
Test group / Remarks:
0.2
Parameter:
SI
Value:
ca. 1.09
Test group / Remarks:
2

Any other information on results incl. tables

Results of a sensitive mouse lymph node assay (SLNA)

Concentration (%)    

       

 Intradermal

injection

(vehicle)

 Topical

application

(vehicle)

 

Lymph

node

weight

(mg)

 
 

Lymph

node cell

nber (x10^6)

 
 SIn

 3HTdR

Incorporation

SIp  SItotal 
 0 (saline) 0 (70% DMSO) 28.5   22.93  -  2313  -  -
 0.02  5  29.8  26.92  1.17  1859  0.80  0.94
 0.2  5  26.7  22.52  0.98  2135  0.92  0.90
 2  5  30.0  26.75  1.17  2150  0.93  1.09

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Sensitization was tested following guinea-pig sensitization test and sensitive mouse lymph node assay on zirconium chloride (ZrCl4). Sensitized animals were challenged with various concentrations of the chemical. However, ZrCl4-treated animals produced no reaction. ZrCl4 did not show any sensitization response.