Zinc oxide

Endpoint summary

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

dermal absorption in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 428 (Skin Absorption: In Vitro Method)

Qualifier:

according to guideline

Guideline:

other: SCCNFP/O75O/O3 (Oct. 2003)

GLP compliance:

yes (incl. QA statement)

Radiolabelling:

no

Species:

pig

Strain:

other: Pietrain-Deutsche Landrasse-Hybrid

Sex:

not specified

Details on test animals or test system and environmental conditions:

Supplier: BASF Agricultural Station, Offenbach, Germany

Type of coverage:

semiocclusive

Vehicle:

other: Oil/water formulation without zinc oxid.

Duration of exposure:

24 h

Doses:

4 mg/cm² of the test formulation (corresponding to approx. 400 µg/cm² ZnO).

No. of animals per group:

8 skin preparations per pig; in total 3 animals

Control animals:

yes

Remarks:

Skin preparations without treatment

Details on study design:

MORPHOLOGY OF TEST MATERIAL
Cryosections of the test material in the cosmetic formulation were analyzed by Transmission Electron Microscopy at different magnifications.

VEHICLE
Basic cosmetic formulation of the following composition: Phase A (Caprylic/Capric Triglycerides 8%, Polyglyceryl-3 Dioleate 1%, Ceteareth-25 0.2%, Cetyl Alcohol 0.5%); Phase B (Hydroxyethyl Acrylate, Acryloyldimethyl Taurate Copolymer, Squalane, Polysorbate 60 1.5%, Xanthan Gum 0.5%); Phase C (Phenoxyethanol, Methylparaben, Ethylparaben, Butylparaben, Propylparaben, Isobutylparaben 0.5%); water ad 100%.

Details on in vitro test system (if applicable):

SKIN PREPARATION
- Source of skin: 5-month old domestic pigs of the Pietrain—Deutsche Landrasse—Hybrid strain
- Type of skin: visually intact skin from the lateral abdominal region
- Preparative technique: dermatomed skin was prepared using a dermatome (Accu-Dermatome GA630, Aesculap, Germany).
- Thickness of skin (in mm): around 0.5
- Membrane integrity check: by measuring its electrical resistance with a LCR bridge (LCR 400, Thurlby Thandar Instruments, England)
- Storage conditions: mounted in modified Franz static dermal penetration cells (Laboratory Glass Apparatus Inc., USA or BASF AG), storage at -20°C


PRINCIPLES OF ASSAY
- Diffusion cell: mounted in modiWed Franz static dermal penetration cells (Laboratory Glass Apparatus Inc., USA or BASF AG)
- Receptor fluid: physiological saline containing 5% bovine serum albumin
- Solubility of test substance in receptor fluid: 1.6 µg/ml
- Test temperature: 31-33 °C.
- Flow rate: approx. 0.3 ml/30 sec.

Absorption in different matrices:

Small amounts of Zn were present in the receptor fluid samples gathered before application. This finding is due to the equilibration period of the diffusion cells, during which Zn from the natural pool in the skin diffused into the receptor fluid.

There was a slight increase of the cumulative absorbed amount of Zn in the receptor fluid over time in both, diffusion cells treated with the vehicle or those treated with the test substance.

As the absorption curves of all test groups run in parallel, no difference in the absorption rates is obvious between vehicle treated and substance treated cells. The increase thus is due to further diffusion of physiological Zn from the skin preparations into the receptor fluid. This also means that no Zn ions or ZnO particles from the applied test substance penetrated through the skin preparations.
Therefore calculation of the absorption rate is not meaningful and was not performed.

Total recovery:

The mean total recoveries of Zn measured in diffusion cells equipped with skin of all 3 pigs were in the range of 102-107% and thus fulfill the SCCNFP and OECD quality criteria. The values of individual diffusion cells ranged between 97.6 and 108.8%.

The major amount of test substance was recovered from the pooled first 5 tape strips (about 99%, 98% and 102% in pig 1, 2 and 3, respectively). Minute amounts were also present in the 3 further pools of tape strips, decreasing with their number.

About 1 % of Zn was found in the treated skin membranes. From the absolute values and when setting in relation the natural Zinc content of untreated or vehicle treated skin to the applied dose of Zn it is obvious that there is no difference of Zn content between untreated, vehicle treated or test substance treated skin.

The mean physiological absolute amounts of Zn found in the skin preparations without zinc application were in the range of 3-5 µl/preparation, which is slightly above 1% of the applied dose. This narrow range showed that the skin punches used were of very similar size.
The mean absolute amounts of Zn found in the receptor compartment was comparable in vehicle and substance treated skin in the range of 5- 9 µg corresponding to around 2% of the applied Zn dose.

Remarks on result:

other:

MORPHOLOGY OF TEST MATERIAL

Representative Transmission Electron Micrographs are shown in the attached document.

ABSORPTION IN DIFFERENT MATRICES

There was no difference of Zn content when comparing untreated skin preparations or those treated with either vehicle or test substance. No increased Zn concentrations were observed in the receptor fluid of substance treated skin as compared to vehicle treated.

Thus, no penetration of Zn or ZnO particles into or through the skin occurred.

Conclusions:

The results indicated that no Zn2+ from the Z-COTE containing formulation penetrated into or through the skin of domestic pigs under the conditions of this study.

Executive summary:

In an in vitro dermal absorption study according to OECD 428 under GLP conditions, dermatomed pig skin mounted on Franz-type diffusion cells was treated with nominal doses of 4 mg/cm2 of a 10% oil/water formulation of Z-COTE (corresponding to approx. 400 μg/cm2 ZnO or 320 μg/cm2 Zn2+) for 24 hours. The pig skin used as diffusion barrier between the donor compartment of a diffusion cell and the receptor compartment filled with the receptor medium. At the end of exposure, the test substance was removed from skin preparations by tape stripping and was also recovered from all other relevant compartment of each diffusion cell (considered as non-absorbed). Fractions present in the remaining skin after tape stripping and receptor chamber fraction are considered as recovery. Zinc analyses were carried out by using the Flame Atomic Absorption Spectrometry. The results indicated that no detectable amounts of Zn2+ from the Z-COTE containing formulation penetrated into or through the skin of domestic pigs under the conditions of this study.

Reference 2

Endpoint:

basic toxicokinetics in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

The study is comparable to a guideline study with acceptable restrictions. The study was not performed under GLP conditions.

Objective of study:

other: dissolution

Principles of method if other than guideline:

The dissolution behaviour in lysosomal pH4.5 conditions was studied to simulate the
clearance by dissolution. The method is compliant with a draft OECD guidance (WPMN
Task 1.5), and was previously validated against in-vivo clearance (Koltermann-Jülly et al.
2018, Koltermann-Jülly et al. 2019, Keller et al. 2020).

GLP compliance:

no

Radiolabelling:

no

Details on test animals or test system and environmental conditions:

not applicable

Details on exposure:

not applicable

No. of animals per sex per dose / concentration:

not applicable

Control animals:

not relevant

Positive control reference chemical:

not applicable

Details on study design:

The flow-through setup was described in detail by several publications as an implementation of a
Continuous Flow System (CFS) according to ISO TR 19057 (ISO/TR19057 2017, Koltermann-Jülly et al. 2018, Keller et al. 2020, Keller et al. 2020). CFS is established as a screening method of the dissolution kinetics of mineral fibres (Guldberg 1995, IARC 2002, Wohlleben et al. 2017). A ZnO mass of M0 = 1 mg was weighed onto a membrane (cellulose triacetate, Sartorius Stedim Biotech GmbH, Goettingen, Germany: 47 mm diameter, 5 kDa pore size), topped by another membrane, and enclosed in flow through cells. The flow through cells at 37 ± 0.5 °C were kept upright to ensure that emerging air bubbles can leave the system and do not accumulate within the cell. The flow rate (V) was 48 mL/d; this corresponds to a ratio, SA/V around 0.02 h/cm, which was independently found to lead to correct predictions.(Keller et al. 2020) The PSF medium – previously validated for the purpose of particle dissolution (Stefaniak et al. 2005) and recommended by the ISO 19057– was employed to simulate the lysosomal compartment at pH 4.5. The programmable sampler drew 10 mL eluates twice per day. These were stabilised by 0.1N HNO3, and analysed by ICP-MS (Perkin Elmer Nexion 2000b).

Details on dosing and sampling:

A ZnO mass of M0 = 1 mg was weighed onto a membrane (cellulose triacetate, Sartorius Stedim Biotech GmbH, Goettingen, Germany: 47 mm diameter, 5 kDa pore size), topped by another membrane, and enclosed in flowthrough cells. The flow rate (V) was 48mL/d; this corresponds to a ratio, SA/V around 0.02 h/cm. The programmable sampler drew 10 mL eluates twice per day.

Statistics:

not specified

Preliminary studies:

not applicable

Type:

other: dissolution

Results:

k min 378ng/cm2/h

Type:

other: dissolution

Results:

k max 323ng/cm2/h

Details on absorption:

not applicable

Details on distribution in tissues:

not applicable

Details on excretion:

not applicable

The dissolution of test material T0420 was tested in lysosomal conditions (pH4.5). The fitted halftime from exponential decay was 0.19 days. The dissolution rate obtained from the conversion of the fitted halftime (kmin) was 378ng/cm2/h.
The highest observed dissolution rate in a single sampling interval (kmax) was 323ng/cm2/h.

Conclusions:

The dissolution of test material T0420 was tested in lysosomal conditions (pH4.5). The dissolution rate obtained from the conversion of the fitted halftime (kmin) was 378ng/cm2/h.
The highest observed dissolution rate in a single sampling interval (kmax) was 323ng/cm2/h.

Executive summary:

ZnO test item T0420 was tested in a continuous flow through system in lysosomal conditions at pH4.5 to simulate clearance by dissolution.

Description of key information

Zinc oxide nanomaterial:

In the framework of the evaluation decision and to select the nanoforms for toxicity testing, the dissolution rate in biological fluid was measured on all nano samples on the EU market. 

The dissolution behaviour of ZnO nanoforms was assessed as part of a ZnO nanoform characterization study. The method used is compliant with a draft OECD guidance (WPMN Task 1.5)  which was previously validated against in-vivo clearance (Koltermann-Jülly et al. 2018, Koltermann-Jülly et al. 2019, Keller et al. 2020). The nanoforms were tested in a continuous flow through system in lysosomal conditions at pH 4.5 to simulate clearance by dissolution. All ZnO materials dissolved in the lysosomal pH 4.5 conditions with half-times of less than 1 day. The dissolution kinetics were overall compatible with mono-exponential decays. 

 

Toxicokinetics according to OECD 417 under non-GLP conditions were examined as part of repeated Dose Inhalation Toxicity Studies. Overall, no relevant amounts of increased ZCOTE-HP1 (NM-111) were detected in any body compartment demonstrating the rapid elimination.

 

In the 90-day repeated dose inhalation toxicity study with male Wistar rats exposed (nose only) to coated nanoscaled ZnO (Z-COTE HP1) and non-coated microscaled ZnO, the toxicokinetics analytical results revealed practically complete dissolution of the retained test substance. Overall, no relevant amounts of increased nanoscaled ZnO were detected in any body compartment demonstrating the rapid elimination (Creutzenberg, 2013).

 

 

Overall, there was no indication of nano-specific toxicity. The observed biological effects are mainly caused by dissolved Zn-ions.

Additional information

Zinc oxide nanomaterial:

In the framework of the evaluation decision and to select the nanoforms for toxicity testing, the dissolution rate in biological fluid was measured on all nano samples on the EU market. We have documented in this Lead Registrant dossier, the 2 materials that were further selected for the toxicity tests.

The dissolution behaviour of ZnO nanoforms was assessed as part of a ZnO nanoform characterization study. The method used is compliant with a draft OECD guidance (WPMN Task 1.5)  which was previously validated against in-vivo clearance (Koltermann-Jülly et al. 2018, Koltermann-Jülly et al. 2019, Keller et al. 2020). The nanoforms were tested in a continuous flow through system in lysosomal conditions at pH 4.5 to simulate clearance by dissolution. All ZnO materials dissolved in the lysosomal pH 4.5 conditions with half-times of less than 1 day. The dissolution kinetics were overall compatible with mono-exponential decays. For ZnO nanomaterials T0420 and T0421 (selected nanomaterials, see section 13.2 "ZnO Nano Particle Selection Process for Human Tox Testing"): The dissolution rate obtained from the conversion of the fitted halftime (kmin) was 97.5 ng/cm2/h for nanomaterial T0421 and 378 ng/cm2/h for nanomaterial T0420. The highest observed dissolution rate in a single sampling interval (kmax) was 246 ng/cm2/h for nanomaterial T0421 and 323 ng/cm2/h for nanomaterial T0420. 

 

Toxicokinetics according to OECD 417 under non-GLP conditions were examined as part of repeated Dose Inhalation Toxicity Studies. In the 14-day-inhalation-study (Creutzenberg, 2013) one day after end of exposure, in the high dose group of Z-COTE® HP1 the absolute Zn content was slightly increased (statistically significant) to 365% in the lung as compared to the clean air control group. In all other organs the Zn levels were very close to the control values. The deposited mass of Z-COTE®HP1 in the 10-day exposure period was approx. 290 μg/lung, the analytical results thus demonstrating a practically complete dissolution of the retained test item. After the 14-day recovery, some statistivally significant increases were detected, however, as these values were close to controls and not dosedependent, they are considered as incidental findings. Overall, no relevant amounts of increased ZCOTE-HP1 (NM-111) were detected in any body compartment demonstrating the rapid elimination. The absolute Zn content following exposure to NM-110 and NM-113 in lung were in the same order but not significantly increased.

 

In the 90-day repeated dose inhalation toxicity study with male Wistar rats exposed (nose only) to coated nanoscaled ZnO (Z-COTE HP1) and non-coated microscaled ZnO, the toxicokinetics analytical results revealed practically complete dissolution of the retained test substance. Overall, no relevant amounts of increased nanoscaled ZnO were detected in any body compartment demonstrating the rapid elimination (Creutzenberg, 2013).

 

ZnO nanoparticles of approximately 50 nm in size (TEM evaluation) were compared to ZnO microparticles showing at least one diameter >100 nm (TEM evaluation). After oral and intraperitoneal administration for both ZnO nanoparticles and microparticles, Zn could be observed in serum indicating uptake from the GI–tract, either as particulate materials or as dissolved Zn ions. For ZnO nanoparticles the systemic availability was somewhat higher compared to that of ZnO microparticles as indicated by Zn measurements by ICP-MS. Zn showed a higher distribution in the liver, spleen and lung after treatment with ZnO nanoparticles compared to treatment with ZnO microparticles (Li et al 2012).

 

There are no indications for significant, if any, penetration of nanoparticles through the skin, most likely only a minimal amount of Zn ions released from the nanoparticles may be available for systemic exposure.

The in vivo study according to current standards OECD Guideline 427 (Creutzenberg, 2011) shows that coated ZnO nanoparticles (Z-COTE® HP1) are not absorbed after dermal application in rats. This result is supported by other literature data. Studies in volunteers with ZnO nanoparticles in sun-blocking formulations or in vitro experiments using coated or uncoated nanoparticles have shown that ZnO nanoparticles are not able to penetrate the stratum corneum (Zvyagin et al. 2008).There isno evidence that ZnO nanoparticles penetrate through intact or sunburned skin.In view of the discussion above, it is assumed that penetration of the skin, if any, is caused by Zn ions released from ZnO nanoparticles.

 

Overall, there was no indication of nano-specific toxicity. The observed biological effects are mainly caused by dissolved Zn-ions.