Registration Dossier

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-referenceopen allclose all
Reason / purpose:
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 2018-07-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
17 July 1992
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.6 (Skin Sensitisation)
Version / remarks:
30 May 2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Version / remarks:
March 2003
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 (study code: 17/232-001P) 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.

FORM AS APPLIED IN THE TEST
The formulations were prepared with mortar and pestle. The concentrations were applied as suspensions. The test item was weighed and formulations were prepared daily on a weight by volume basis (as % (w/v).
No correction for the 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, ~ 8 weeks old
- Weight at study initiation: 499 – 611 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 28 days before the 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 (%): 22–89
- Air changes (per hr): 15-20 air exchange/hour
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light
Route:
intradermal
Vehicle:
other: 1% methylcellulose
Concentration / amount:
concentration: 2.5% (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
Concentration / amount:
concentration: 100% (w/v)
amount: 0.5 mL
Day(s)/duration:
day 8 of treatment; 48 hours of exposure
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: 1% methylcellulose
Concentration / amount:
50% (w/v)
Day(s)/duration:
day 22 of treatment; 24 hours of exposure
Adequacy of challenge:
other: highest non irritant concentration based on preliminary study results
No.:
#2
Route:
epicutaneous, occlusive
Vehicle:
other: 1% methylcellulose
Concentration / amount:
100% (w/v)
Day(s)/duration:
day 38 of treatment; 24 hours of exposure
Adequacy of challenge:
other: highest non irritant concentration based on dermal induction and 1st challenge results (main test)
No. of animals per dose:
Preliminary studies: 8 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 25, 50, 75 and 100% (w/v) in 1% MC for dermal application. Local effects were examined and scored 1, 24, 48 and 72 hours after the 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. 1 hour to 72 hours after intradermal injections of concentrations of 5% (w/v) with and without FCA:saline, very slight to well defined erythema was observed. The erythema was high enough and persistent for the main study. However, the injections of the 5% (w/v) concentrations with and without FCA were feasible but difficult, therefore the use of 5% (w/v) concentration was not advisable for the main study, as there was a risk that these formulations could not be injected as a whole. A concentration of 2.5% (w/v) caused very slight erythema which was observed from 1 hour to 48 hours after injection and 1% (w/v) caused only very slight erythema at 24 hours, therefore 2.5 % (w/v) was considered appropriate for 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 topical application was approximately 48 hours. One concentration was used on the right side and another concentration on the left side of animals. Two animals per concentrations were used. Very slight erythema was observed in the 100% (w/v) concentration groups at the 1-hour and 24-hour observation and 75% (w/v) at 1 hour after patch removal. No effects were observed at concentrations of 25 and 50% (w/v).

MAIN STUDY
On the basis of results of the Preliminary Dose Range Finding Study, the following treatments were chosen for the main study:
- Intradermal induction: 2.5% (w/v) test item formulated in 1% MC was used in the test group. For the control group, injections of the vehicle (1% MC) were chosen.
- Dermal induction: 100% (w/v) test item formulated in 1% MC. For control animals, 1% MC. Considering the irritation observed in the preliminary study at the chosen concentration, sodium dodecyl sulphate was considered to be unnecessary before topical treatment.
- Challenge phase: All animals of the treatment and control groups were treated with 50% (w/v) test item in 1% MC on the left side (highest concentration which caused no irritation) and with 1% MC on the right side.

A. INDUCTION EXPOSURE
- No. of exposures: once
- Test groups:
2 injections of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
2 injections of 2.5% (w/v) test item in 1% MC,
2 injections of 2.5% (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 of Freund's Complete Adjuvant and physiological saline solution in a 1:1 (v/v) mixture,
2 injections of 1% MC,
2 injections of vehicle (1% MC), formulated in a 1:1 mixture (v/v) of Freund's Complete Adjuvant and physiological saline solution.

- Site: scapular region
- Time of observation: 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) test item formulated in 1% methylcellulose
control group: 1% methylcellulose

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: 50% w/v (in 1% methylcellulose) applied to the left side of animals and 1% methylcellulose applied to the right side

D. RECHALLENGE EXPOSURE
- No. of exposures: once
- Day(s) of challenge: on day 38
- 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% methylcellulose) applied to an untreated area of the left flank of each animal animals and 1% methylcellulose applied to an untreated area of the right flank
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.
No naive control animals were used for the rechallenge, as the animals were symptom-free during the challenge phase.
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 were 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 group
Dose level:
50% (w/v) (in 1% methylcellulose)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test group
Dose level:
50% (w/v) (in 1% methylcellulose)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of systemic toxicity observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
50% (w/v) (in 1% methylcellulose)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of systemic toxicity observed
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
50% (w/v) (in 1% methylcellulose)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of systemic toxicity 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.
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
negative control
Dose level:
100 % (w/v) in 1% methylcellulose
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
discrete or patchy erythema (score 1)
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
negative control
Dose level:
100 % (w/v) in 1% methylcellulose
No. with + reactions:
5
Total no. in group:
5
Clinical observations:
discrete or patchy erythema (score 1)
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
24
Group:
test group
Dose level:
100 % (w/v) in 1% methylcellulose
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
discrete or patchy erythema (score 1)
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
rechallenge
Hours after challenge:
48
Group:
test group
Dose level:
100 % (w/v) in 1% methylcellulose
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
discrete or patchy erythema (score 1)
Remarks on result:
no indication of skin sensitisation

Skin effect during induction phase

Intradermal

No test item-related signs of erythema, local reactions or systemic toxicity were observed in any animal during the intradermal induction phase.

 

Topical

No test item-related signs of systemic toxicity were observed in any animal during the topical induction phase. Slight erythema (score 1) was observed on 2 out of 10 animals after 24 hours in the test group. The animals were symptom-free at 48- and 72-hour observations. No erythema was observed in the control group during the topical induction phase.

Skin effects after the challenge exposure

Test group

After the challenge with the test item at a concentration of 50% (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.

 

After the rechallenge with the test item at a concentration of 100% (w/v) formulated in 1% MC, positive responses were observed (erythema score 1) in 10 out of 10 treated animals on the left flank. The mean of the score was 1.00 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 50% (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 was treated with 1% MC and no reaction was noted.

 

After the rechallenge with the test item at a concentration of 100% (w/v) formulated in 1% MC, positive responses were observed (erythema score 1) in 5 out of 5 animals on the left flank. The mean of the scores was 1.00 according to the 24- and 48-hour results. The right shaved side of control animals was treated with 1% MC and no reaction was noted.

Scores of erythema after treatment of challenge and rechallenge exposure

 

Groups

Animal
Number

Day 24

Day 25

Day 40

Day 41

Left side

Right side

Left side

Right side

Left side

Right side

Left side

Right side

1. Control group

11

0

0

0

0

1

0

1

0

8

0

0

0

0

1

0

1

0

3

0

0

0

0

1

0

1

0

15

0

0

0

0

1

0

1

0

4

0

0

0

0

1

0

1

0

2. Dosed group

5

0

0

0

0

1

0

1

0

12

0

0

0

0

1

0

1

0

1

0

0

0

0

1

0

1

0

20

0

0

0

0

1

0

1

0

19

0

0

0

0

1

0

1

0

14

0

0

0

0

1

0

1

0

16

0

0

0

0

1

0

1

0

17

0

0

0

0

1

0

1

0

13

0

0

0

0

1

0

1

0

2

0

0

0

0

1

0

1

0

0 = no visible change

1 = discrete or patchy erythema

2 = moderate and confluent erythema

3 = intense erythema and swelling

Based on the scores measured after challenge and rechallenge, the dermal reactions of the test group animals were not different from the reaction in the animals of the control group, therefore the test result was considered to be negative.

Clinical observations/mortality

No signs of systemic toxicity were observed during induction, challenge or rechallenge.

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 the test item (tungsten zirconium hydroxide oxide) evoked no positive responses in the test animals previously sensitised with the test item or in the control group. In the control and treated animals, the mean of the scores were 0.00 according to the 24- and 48-hour results after the first challenge.

Rechallenge with the test item (tungsten zirconium hydroxide oxide) evoked erythema score 1 in the test animals previously sensitised with the test item, however the control group produced the same reaction. In the control and treated animals, the mean of the scores were 1.00 according to the 24- and 48-hour results after the rechallenge.

The dermal reactions of the test group animals were not different from the reaction in the animals of the control group, therefore the test result was considered to be negative.

Under the conditions of the present assay the test item tungsten zirconium hydroxide oxide was shown to have no sensitisation potential and is classified as a non- sensitiser, according to current GHS criteria and EU-regulations.
Reason / purpose:
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:
OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)
Version / remarks:
17 Dec 2001
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)
Version / remarks:
30 May 2008
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 (15-25°C, 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 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: 205 - 232 g. Body weight variation did not exceed +/- 20% of the sex mean.
- Fasting period before study: overnight
- Housing: Type II. polypropylene/polycarbonate cages, with 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 – 23.9 °C
- Humidity (%): 32 – 71 %
- Air changes (per hr): 15-20 air exchanges/hour
- Photoperiod (hrs dark / hrs light): 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: 200 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg
- 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

DOSAGE PREPARATION (if unusual): 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:
A single dose of 2000 mg/kg bw.
No. of animals per sex per dose:
3 females per group, 2 groups
Control animals:
no
Details on study design:
- Procedure: A single dose of 2000 mg/kg bw was administered by oral gavage (using a stainless steel bulb tipped gastric feeding tube attached to a syringe) in the morning. - Food was withheld overnight for a maximum of 16 hours before treatment, 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. Individual observations were performed on the skin, fur, eyes, mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern. Particular attention was directed to observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. 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:
All animals were symptom-free during the observation period at a dose level of 2000 mg/kg bw.
Body weight:
There were no treatment related body weight changes. Body weights were within the range commonly recorded for this strain and age.
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 tungsten zirconium hydroxide oxide was found to be above 2000 mg/kg bw in female Crl:(WI) rats.
The substance is not classified according to the CLP Regulation.

Data source

Materials and methods

Results and discussion

Applicant's summary and conclusion