oxozirconium; silane

Acute Toxicity: dermal

Administrative data

Endpoint:

acute toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

the study does not need to be conducted because the substance does not meet the criteria for classification as acute toxicity or STOT SE by the oral route and no systemic effects have been observed in in vivo studies with dermal exposure (e.g. skin irritation, skin sensitisation)

Cross-reference

Cross-reference 1

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-08-22 to 2017-09-06

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Version / remarks:

December 17, 2001

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Version / remarks:

30 May 2008,

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled room temperature (15-25ºC, below 70 RH%)
- Stability under test conditions: Analysis of stability, homogeneity and concentration of the test item under test conditions was not performed as part of this study.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The formulation was homogenised to visually acceptable levels and was stirred up to finishing the treatment to ensure sufficient homogeneity. No correction was made for the purity/composition of the test item.

Species:

rat

Strain:

other: Crl:(WI)

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, D-97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 weeks old
- Weight at study initiation: 197 - 223 g. Body weight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: overnight
- Housing: Type II. polypropylene/polycarbonate cages, 3 animals per cage, Lignocel 3/4-S Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH + CO.KG (D-73494 Rosenberg, Germany) was available to animals during the study. Arbocel crinklets natural produced by J.Rettenmaier & Söhne GmbH + CO.KG (D-73494 Rosenberg, Germany) was available to animals during the study.
- Diet (e.g. ad libitum): Animals received ssniff® SM R/M "Autoclavable complete diet for rats and mice – breeding and maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest, Germany (Batch number: 262 21592, Expiry date: 31 January 2018), ad libitum.
- Water (e.g. ad libitum): Tap water from the municipal supply, as for human consumption from a 500-mL bottle, ad libitum.
- Acclimation period: At least 12 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.1 – 24.0°C
- Humidity (%): 30 – 71%
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 12 hours daily, from 6.00 a.m. to 6.00 p.m.

Route of administration:

oral: gavage

Vehicle:

methylcellulose

Remarks:

1%

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 2000 mg/kg bw
- Amount of vehicle (if gavage): 200 mg/mL
- Justification for choice of vehicle: The selection of the vehicle was based on trial formulations with the test item. The test item was found to be insoluble in distilled water (the formulation separated immediately to different phases) at a concentration of 200 mg/mL. With 1% methylcellulose aqueous solution the formulation (suspension) was found to be acceptable for oral gavage.
- Lot/batch no. (if required): 5115851
The test item was freshly formulated at a concentration of 200 mg/mL in the vehicle, in the Pharmacy of Citoxlab Hungary Ltd. on the day of administration. The formulation container was magnetically stirred continuously up to the end of dose administration procedures. Formulations were used within 4 hours after preparation.

Doses:

Single dose of 2000 mg/kg bw per female.

No. of animals per sex per dose:

3 females per group, 2 groups

Control animals:

no

Details on study design:

- Procedure: A single dose at 2000 mg/kg bw was administered by oral gavage (stainless steel bulb tipped gastric feeding tube attached to syringe). Food was withheld overnight for a maximum of 16 hours and returned 3 hours after treatment.
- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Clinical observations were performed on all animals at 30 minutes, 1, 2, 3, 4 and 6 hours after dosing and daily for 14 days thereafter. The body weight was recorded on the day before treatment (Day -1), on the day of the treatment (Day 0), weekly thereafter (Day 7) and at necropsy (Day 14).
- Necropsy of survivors performed: yes
- Other examinations performed: Clinical signs, body weight, macroscopic observations.

Statistics:

No statistical analysis was performed (the method used is not intended to allow the calculation of a precise LD50 value).

Preliminary study:

Initially, three female animals were treated with 2000 mg/kg bw of the test item. No mortality was observed, therefore further 3 animals were treated at the dose level of 2000 mg/kg bw. As no mortality was observed in this second dose group, further testing was not required according to the test guidelines.

Key result

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

The test item did not cause mortality at a dose level of 2000 mg/kg bw in any animal.

Clinical signs:

other: All animals were symptom-free during the observation period at a dose level of 2000 mg/kg bw.

Gross pathology:

There was no evidence of macroscopic changes at a dose level of 2000 mg/kg bw in any animal.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of this study, the acute oral LD50 value of the test item was found to be above 2000 mg/kg bw in female Crl:(WI) rats.
The study result triggers the following classification/labelling:
- Regulation (EC) No 1272/2008 (CLP): “No category”

Cross-reference 2

Reason / purpose for cross-reference:

data waiving: supporting information

Reference

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.