oxozirconium; silane

Endpoint summary

Description of key information

Skin sensitisation - in vitro

Compatibility testing indicated that the test item silicon zirconium oxide is not compatible with the in vitro test methods. Therefore, an in vivo test was performed.

Skin sensitisation - in vivo

A reliable skin sensitisation study is available for silicon zirconium oxide (Tarcai, 2018, GPMT study performed according to OECD guideline 406, scored Klimisch 1). In this study, no signs of contact sensitisation were detected after the challenge phase in the 10 guinea pigs previously exposed to the test item. In the control and treatment group, the mean of the scores for erythema and oedema was 0.00 according to the 24- and 48-hour observations following the challenge phase. Under the conditions of the assay the test item silicon zirconium oxide was shown to have no skin sensitisation potential.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

from 2018-05-03 to 2018-08-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Version / remarks:

17 July 1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

30 May 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

March 2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

The GPMT method (OECD 406) was preferred above LLNA (OECD 429) since previous experience learned that this method is the most suitable method for testing insoluble inorganic substances, which are often not able to penetrate the skin.

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature.
- Stability under test conditions: The formulations were freshly formulated at appropriate concentrations in the vehicle on the day of administration and used within 4 hours after adding the vehicle to the test item.
- Solubility and stability of the test substance in the solvent/vehicle: The selection of the vehicle was based on trial formulations with the test item. It is preferred to use aqueous vehicles whenever possible, therefore physiological saline solution (saline) should be the first choice whenever possible (as distilled water is not compatible with intradermal treatments). However, saline was not tested as previous information from a different study showed that the test item was insoluble in distilled water (the formulation separated immediately to different phases). 1% methylcellulose (1% MC) was tested as a second option. During the Preliminary Compatibility Test, 1% MC was found to be suitable as a vehicle for the study.
The stability and homogeneity were assessed visually; chemical analysis was not performed.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Each formulation was prepared with mortar and pestle. The concentrations were applied as suspensions.
No correction for purity of the test item was applied.

Species:

guinea pig

Strain:

other: LAL/HA/BR

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: LAB-ÁLL Bt., Budapest, 1174 Hunyadi u. 7., Hungary
- Age at study initiation: Young adults, ~ 5 weeks old
- Weight at study initiation: 243 – 354 g
- Housing: Animals were housed in Macrolon cages size IV, with up to 3 animals/cage to allow socialisation. The bedding used was Lignocel® 3/4-S Hygienic Animal Bedding.
- Diet (e.g. ad libitum): Animals in the first preliminary test received Cunigra Diet for Rabbits ad libitum. Animals in the second preliminary test and main study received ssniff® "Complete feed for Guinea pigs – maintenance", ad libitum.
- Water (e.g. ad libitum): Animals in the first preliminary test received tap water from municipal supply as for human consumption, containing at least 50 mg/100 mL ascorbic acid, ad libitum. Animals in the second preliminary test and main study received tap water from municipal supply without addition of ascorbic acid, as the food contained the necessary level of ascorbic acid for guinea pigs.
- Acclimation period: At least 7 days before start of treatment under laboratory conditions.
- Indication of any skin lesions: Health inspection was performed at arrival of the animals. Only healthy animals were used for the test. The health status was certified by the staff Veterinarian.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.0–25.9
- Humidity (%): 22–76
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

Route:

intradermal

Vehicle:

other: 1% methylcellulose

Remarks:

suspension

Concentration / amount:

concentration: 1% (w/v)
volume: 0.1 mL

Day(s)/duration:

day 1 of treatment

Adequacy of induction:

highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically

Route:

epicutaneous, occlusive

Vehicle:

other: 1% methylcellulose

Remarks:

suspension

Concentration / amount:

concentration: 100% (w/v)
amount: 0.5 mL

Day(s)/duration:

day 8 of treatment; 48 hours of exposure

Adequacy of induction:

non-irritant substance, but skin pre-treated with 10% SDS

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

other: 1% methylcellulose

Concentration / amount:

Concentration: 100% (w/v)
Amount: 0.5 mL

Day(s)/duration:

day 22 of treatment; 24 hours of exposure

Adequacy of challenge:

highest non-irritant concentration

No. of animals per dose:

Preliminary studies: 7 animals
Main study: 10 in the test group, 5 in the control group.

Details on study design:

RANGE FINDING TESTS:
- A day prior to the test, the hair was removed from the right and left sides of the animals (approximately 5x5 cm). The hair removal was performed carefully to ensure animals are closely shaven.
- A series of test item concentrations was tested to identify the primary irritation following intradermal injection and dermal application: 1, 2.5 and 5% (w/v) concentrations in 1% MC were used for intradermal injection and 75% and 100% (w/v) for dermal application. Local effects were examined and scored at 1, 24, 48 and 72 hours after treatment (in case of intradermal injection) or after patch removal (in case of topical application). Skin effects were scored for erythema and oedema, any other observations of changes to the skin were recorded if present.
- For the intradermal application, 0.1 mL per concentration was injected intradermally into the hair free skin of the animals where the formulation properties made it possible. Two concentrations were injected on the right side and another two concentrations on the left side of the animals. The highest concentration of 5% (w/v) for intradermal treatment was also tested in a 1:1 mixture (v/v) of Freund's Complete Adjuvant (FCA) and physiological saline solution (saline). Each concentration was injected in duplicate where applicable. Two animals were used per concentration.
- The concentration of 5% (w/v) in 1% MC or in 1:1 FCA:saline formulation was not injectable intradermally, therefore this concentration was considered not suitable for treatment. The concentrations of 2.5% and 1% (w/v) in 1% MC were injectable and caused only very slight erythema (score 1) up to 48 hours after injection. Since the 5% (w/v) concentration was not injectable, a second preliminary study was considered appropriate to assess the applicability of lower concentrations in a formulation with FCA:saline and FCA:1% MC.
- A second preliminary test was performed at concentrations of 2.5% and 1% (w/v) in 1:1 mixture (v/v) of FCA:saline. This test was limited to test the easy passage of the formulation through a needle and if the formulation is suitable to inject into the animal's skin (possible to inject). Earlier experience has shown that in some cases 1% MC can enhance the applicability of the FCA, therefore, the concentrations of 2.5% and 1% (w/v) were also tested in a 1:1 mixture (v/v) of FCA:1% MC. Signs of systemic toxicity and/or irritation were not measured in the second preliminary test.
- The concentrations of 2.5% (w/v) in FCA:saline or in FCA:1% MC were not injectable intradermally. The concentrations of 1% in FCA:saline or in FCA:1% MC were both injectable.
- Since the highest injectable formulation with FCA:saline was at a concentration of 1% (w/v) and the erythema scores measured after intradermal injection of 1% (w/v) in 1% MC were acceptable, the 1% (w/v) formulation was used for intradermal application in the main study.
- For the topical application, the volume of the concentrations was 0.5 mL. A closed patch exposure was performed by means of an occlusive bandage using similar treatment procedures as for the main study. The time of exposure for the dermal application was 48 hours. One concentration was used on the right side and another concentration on the left side of animals. Two animals per concentration were used.

MAIN STUDY
- Control animals were treated similarly to test animals, except that during the induction phase, the test item was omitted.
- Induction involved two main procedures: intradermal treatment (Main Study I) and dermal exposure (Main Study II) with closed patch technique.
On the basis of results of the Preliminary Dose Range Finding Study, the following treatments were chosen in the main study:
• Intradermal induction: 1% (w/v) test item formulated in 1% MC was used in the test group. Injections of 1% MC only were used in the control group.
• Dermal induction: 100% (w/v) test item formulated in 1% MC in the test group. 1% MC only was applied in the control group.
Since the 100% (w/v) test item formulation was not a skin irritant in the dermal dose range finding study, the test area was painted with 0.5 mL of 10% sodium dodecyl sulphate (SDS) in vaseline 24 hours prior to the topical induction, in order to create a local irritation.
• Challenge phase: All animals of the treatment and control group were treated with 100% (w/v) test item in 1% MC (highest concentration which caused no irritation) on the left flank and with 1% MC on the right flank, respectively as a challenge exposure.

A. INTRADERMAL INDUCTION EXPOSURE
- No. of exposures: once
- Test groups:
2 injections (0.1 mL/site) of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
2 injections (0.1 mL/site) of 1% (w/v) test item in 1% MC,
2 injections (0.1 mL/site) of 1% (w/v) test item, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.

- Control group:
2 injections (0.1 mL/site) of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
2 injections (0.1 mL/site) of 1% MC,
2 injections (0.1 mL/site) of 1% MC, formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.

- Site: scapular region
- Time of observations: 24 (± 2) hours after treatment

B. DERMAL INDUCTION EXPOSURE
- No. of exposures: once
- Exposure period: 48 +/- 2 hours
- Site: same scapular region which received the intradermal injections
- Time of observations: 1(+/- 5 min), 24(+/- 2), 48(+/- 2) and 72 (+/- 2) hours after the patch removal
- Concentrations:
Test group: 100% (w/v) (in 1% MC)
Control group: 1% MC

C. CHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: on day 22
- Exposure period: 24 +/- 2 hours
- Site: left and right sides
- Evaluation (hr after challenge): 24 (+/- 2) and 48 (+/- 2) hours after the patch removal
- Concentrations: 100% w/v (in 1% MC) applied to the left side of animals and 1% MC applied to the right side

Challenge controls:

Control animals were treated with the test item at a concentration of 100% (w/v) formulated in 1% MC on the left side during challenge. The right side was treated with the vehicle only.

Positive control substance(s):

yes

Remarks:

2-mercaptobenzothiazole

Positive control results:

Challenge with the test item 2-mercaptobenzothiazole elicited discrete erythema (score 1) on the skin surface of previously sensitised guinea pigs. The mean of the scores was 0.80 (80% of animals) at the 24-hour observation and 0.70 (70% of animals) at the 48-hour observation. In the control group the mean of the scores was 0.00. On the basis of the results of the present study, the test item 2-mercaptobenzothiazole was classified as a skin sensitiser. This demonstrates that the Magnusson and Kligman method (OECD 406) in this laboratory is considered to be reliable.

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

100% w/v (in 1% MC)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

100% w/v (in 1% MC)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

100% w/v (in 1% MC)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

100% w/v (in 1% MC)

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

No signs of systemic or local toxicity were observed.

Remarks on result:

no indication of skin sensitisation

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

50% w/v (2-mercaptobenzothiazole)

No. with + reactions:

8

Total no. in group:

10

Clinical observations:

discrete erythema (score 1)

Remarks on result:

positive indication of skin sensitisation

Remarks:

In the control group of this study the mean of the scores was 0.00

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

50% w/v (2-mercaptobenzothiazole)

No. with + reactions:

7

Total no. in group:

10

Clinical observations:

discrete erythema (score 1)

Remarks on result:

positive indication of skin sensitisation

Remarks:

In the control group of this study the mean of the scores was 0.00

Skin Effects after the Challenge Exposure

Test group

After the challenge with the test item at a concentration of 100% (w/v) formulated in 1% MC, no positive response was observed in the treated animals on the left flank. The mean of the scores was 0.00 according to the 24- and 48-hour results. The right shaved side of test animals was treated with 1% MC and no reaction was noted.

Control group

After the challenge with the test item at a concentration of 100% (w/v) formulated in 1% MC, no visible changes were found at the 24- and 48-hour examinations on the left flank. The right shaved side of control animals were treated with 1% MC and no reaction was noted.

Clinical observations /mortality: No signs of systemic or local toxicity were observed. No mortality was observed during the study.

Body weight: There were no notable differences between the test animal group and the control group.

Interpretation of results:

GHS criteria not met

Conclusions:

Challenge with test item (silicon zirconium oxide) evoked no positive responses in the test animals previously sensitised with the test item or in the control group. The net response value represented an incidence rate of 0% and the net score value was 0.00.
Under the conditions of the present assay the test item silicon zirconium oxide was shown to have no skin sensitisation potential and is classified as non-sensitiser, according to current GHS criteria and EU-regulations.
 

Reference 2

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

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

A skin sensitisation study was performed with silicon zirconium oxide in the guinea pig according to the Magnusson and Kligman method, using a maximisation method with Freund's Complete Adjuvant to evaluate the sensitisation potential of the test item. The study was performed according to OECD Guideline 406 and in compliance with GLP guidelines (Tarcai, 2018, scored Klimisch 1). The GPMT method was preferred above the LLNA (OECD 429) since previous experience learned that this method is the most suitable method for testing this kind of substances, since insoluble inorganic compounds are often not able to penetrate the skin.

Based on the results of preliminary studies, 10 test animals were subjected to sensitisation procedures in a two-stage process incorporating an induction phase using an intradermal treatment followed by a 48-hour topical application (dermal treatment under an occlusive dressing). The test item was used at a concentration of 1% (w/v) for intradermal injections and at a concentration of 100% (w/v) for topical sensitisation treatment, both suspended in 1% methylcellulose (abbreviation: 1% MC). Five control guinea pigs were simultaneously exposed to 1% MC during the sensitisation phase (intradermal and dermal treatment). Since the 100% (w/v) test item concentration was not skin irritant in the dermal dose range finding study, the induction area was painted with 0.5 mL of 10% (w/w) sodium dodecyl sulphate in vaseline 24 hours prior to the topical induction application, in order to create a local irritation.

Two weeks after the last induction exposure, a challenge dose at a concentration of 100% (w/v) in 1% MC was administered on the left side of all animals. The right side of the animals was treated with 1% MC. Challenge was performed by dermal application of the test item for 24 hours with a fully occlusive foil (Closed Patch Test). Skin reactions were measured 24 and 48 hours after patch removal.

No signs of contact sensitisation were detected after the challenge phase in the 10 guinea pigs previously exposed to the test item. In the control and treatment group, the mean of the scores for erythema and oedema was 0.00 according to the 24- and 48-hour observations following the challenge phase.

In conclusion, under the conditions of the present assay the test item silicon zirconium oxide was shown to have no skin sensitisation potential and is classified as a non-sensitiser, according to current GHS criteria and EU-regulations.

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Skin sensitisation

Based on the test results of a guinea pig maximisation test performed with silicon zirconium oxide, the substance was concluded not to be classified as a skin sensitiser.

Respiratory sensitisation

No reliable study is available.