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Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report date:
2010

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The metals industry has historical data to indicate that metals can induce false positives/negatives in LLNA studies; this is confirmed from experiences in test labs.

Test material

Constituent 1
Chemical structure
Reference substance name:
Zinc bis(dihydrogen phosphate)
EC Number:
237-067-2
EC Name:
Zinc bis(dihydrogen phosphate)
Cas Number:
13598-37-3
Molecular formula:
Zn(H2PO4)2
IUPAC Name:
zinc bis(dihydrogen phosphate)
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
- Name of test material (as cited in study report): Zinc bis(dihydrogen phosphate)
- Physical state: white crystalline solid powder
- Analytical purity: ZnO(%): 25-27 P2O5(%): 43-49.5
- Composition of test material, percentage of components: Zinc bis(dihydroogen phosphate)*2H2O : phosphate (as P2O5) 44.6% ±5% rel.; Zinc: 21.5%±0.5 % rel.
- Lot/batch No.: Z 11-81
- Stability under test conditions: stable at room temperature
- Storage condition of test material: dark , ambient temperature, original container
- Expiration date of the lot/batch: 31.8.2011

In vivo test system

Test animals

Species:
guinea pig
Strain:
other: albino
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Biotest s.r.o.
- Age at study initiation: 15-20 weeks
- Weight at study initiation: M: 492-1012g, F: 564-844g
- Housing: individually in environmentally monitored and ventilated experimental room No2, part 1, building No2
- Diet : standard pelletized diet diet KKK/O ad libitum
- Water: water of monitored quality ad libitum
- Acclimation period: 5days for the pilot study and 13 days for the main study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: To:

Study design: in vivo (non-LLNA)

Induction
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
500mg/animal
Challenge
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
500mg/animal
No. of animals per dose:
in main study: 20 tested, 10 positive control, 10 negative control
in pilot study: 3
Details on study design:
RANGE FINDING TESTS:
The pilot study with three animals was performed before the main test to provide data for establishing of the dose of the Test item to be used in the main study.
Three guinea pigs were administered topically on shaved skin of area 25×25 mm with three different doses of the Test item. The doses were 10 mg, 70 mg and 500 mg/animal. The Test item was moisturized with water to obtain a paste before the application. After the administration, the test area was covered with porous gauze dressing. The exposure lasted 4 hours. After the exposure, the test area was cleaned with water and the animals were observed for skin reaction 24, 48 and 72 hours after the exposure. Since no skin irritation was observed in any of the observation intervals, the dose of 500 mg/animal was used for induction and challenge exposures.

MAIN STUDY
A. INDUCTION EXPOSURE
Phases I. (Day 1), II.(Day 8) and III. (Day 15):
500mg/animal of the test item was applied on the shaved area of skin and covered with porous gauze dressing. The duration of induction exposures was 6 hours. After 6 hours, the Test item was removed and the application area was washed with water. The shaved skin area of control group animals was only covered with the 4-stratums of Sterilux ES (25×25 mm) and covered with adhesive non irritating tape. The shaved skin area of the positive control group animals was administered with the reference item in the dose of 0.5 mL/animal and covered with the 4-stratums Sterilux ES (25×25 mm) and adhesive non irritating tape.

B. CHALLENGE EXPOSURE
Provocation – challenge was performed two weeks after the last induction (Day 29). After shaving, a dose of 500 mg/animal of the Test item was moisturized with 0.5 mL water for injections to obtain a paste and applied on the shaved area (25×25 mm) of skin of all the tested and control (negative only) animals and covered with a porous gauze dressing. The challenge exposure was performed on the left hip of all the animals. The duration of
challenge exposure was 6 hours. After 6 hours, the Test item was removed and the application sites were cleaned with water.
The positive control group animals were administered in the same manner, but with the reference item in the dose of 0.5 mL/animal.
Twenty-one hours after the end of challenge exposure, the rests of fur on test area were shaved to ensure clear skin for skin reaction evaluation. In the intervals of 24 and 48 hours after the challenge, the exposure skin reaction of all the animals was evaluated.

OTHER: Clinical observations
-clinical observations and mortality:
once a day during acclimationand study period. Clinical Observations included: Signs of toxicity, changes in the skin and fur, eyes and mucous membranes, respiratory, circulatory, autonomic and central nervous system, somatomotor activity and behaviour pattern, changes in gait, posture and response to handling, the presence of clonic or tonic movements and stereotypes.
-body weight: All guinea pigs were individually weighed at the delivery, immediately before the first administration and than weekly.
-skin reaction:
21 hours after removing the patch the challenge area was cleaned and closely-clipped
3 hours later (approximately 30 hours from the start of the challenge application) the skin reaction was observed and recorded according to the grades shown below.
24 hours after this observation a second observation (54 hours after the start) was made and once again recorded.
The skin reaction for erythema and oedema was graded according to following Magnusson and Kligman scale:

Reaction Numerical grading
No visible changes 0
Non-continuous or patchy erythema 1
Moderate and merged erythema 2
Severe erythema and oedema 3
The test system is considered reliable if the positive control group comprises min. 15% of the animals with positive reaction. This condition was fulfilled as 2/10 (20%) of the animals reacted positively.
Challenge controls:
positive control: 2-mercaptobenzothiazole, negative control: sterilux
Positive control substance(s):
yes
Remarks:
2-mercaptobenzothiazole

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
500mg/animal
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 500mg/animal. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
500mg/animal
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 500mg/animal. No with. + reactions: 0.0. Total no. in groups: 20.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
0.5mL/animal
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: see Remark
Remarks:
Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: 0.5mL/animal. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
0.5mL/animal
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
no pathological changes. No changes in the gait, somatomotor activity or behavior pattern
Remarks on result:
other: see Remark
Remarks:
Reading: 2nd reading. . Hours after challenge: 48.0. Group: positive control. Dose level: 0.5mL/animal. No with. + reactions: 2.0. Total no. in groups: 10.0. Clinical observations: no pathological changes. No changes in the gait, somatomotor activity or behavior pattern .

Any other information on results incl. tables

The body weight gains of all the animals during the study corresponded to their age and were without any abnormalities.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
No sensitizing effect of the Test item Zinc bis(dihydrogen phosphate) was observed in any of the animals after the challenge exposure. The clinical observations of the animals were without any pathological findings. The body weight values of all the animals increased during the study without abnormalities and corresponded to the age of the animals.
Non-continuous or patchy erythema was observed in two positive control animals after the challenge exposure; no reactions were observed in the negative control group. Based on these results the test system can be considered to be reliable.
Under the test conditions used the Test item Zinc bis(dihydrogen phosphate) was assessed to be not sensitizing.
Executive summary:

SUMMARY

The aim of the present study was to estimate a potential sensitization effect of the Test item Zinc bis(dihydrogen phosphate) on intact skin of the guinea pig (according to OECD 406 and EN ISO 10993-10 ).

METHOD

Three animals for the pilot study and 20 test animals, 10 negative control and 10 positive control animals were used for the study. The Buehler test was chosen according to properties of the Test item.

A pilot study for establishing of the dose for the main study was conducted first. Three guinea pigs were exposed to three doses (10, 70 and 500 mg/animal) of the Test item for 4 hours. The animals were observed for skin reaction 24, 48 and 72 hours after the exposure. Since no skin reaction was observed, a dose of 500 mg/animal was chosen for the main study.

The tested animals in the main study were treated by three inductions – topic application with one week intervals. The negative control animals were treated only with Sterilux in the same manner. The positive control animals were treated with the reference item (2 -mercaptobenzothiazole) in the dose of 0.5 mL/animal. Two weeks after the last induction, a topical challenge was performed in the treated and negative control animals. The positive control animals were treated with the reference item. At the intervals of 24 and 48 hours after the challenge exposure, the skin reaction of all the animals was evaluated. Daily clinical observations and weekly body weight values were recorded.

RESULTS

No sensitizing effect of the Test item Zinc bis(dihydrogen phosphate) was observed in any of the animals after the challenge exposure. The clinical observations of the animals were without any pathological findings related to the treatment. The body weight values of all the animals increased during the study without abnormalities and corresponded to the age of the animals.

Non-continuous or patchy erythema was observed in two positive control animals after the challenge exposure; no reactions were observed in the negative control animals. Based on these results the test system was considered to be reliable.

Under the test conditions used the Test item Zinc bis(dihydrogen phosphate) was assessed to be not sensitizing.

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