Zinc nitrate

Skin sensitisation

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

Cross-reference

Reason / purpose for cross-reference:

reference to other 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.
Zinc nitrate hexahydrate was tested for the sensitisation potential first in the murine local lymph node assay (LLNA). Zinc nitrate has a positive result in the LLNA test. Positive results in cell proliferation revealed that the test substance could be a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.
The current study (guinea pig maximisation test) should rule out whether the test material has an allergenic effect or not.

Test material

Test material information

Constituent 1

Reference substance name:

Zinc nitrate

EC Number:

231-943-8

EC Name:

Zinc nitrate

Cas Number:

7779-88-6

Molecular formula:

HNO3.1/2Zn

IUPAC Name:

zinc nitrate

Test material form:

solid: crystalline

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:

500 mg/animal

No. of animals per dose:

in main study: 20 tested, 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)

Results

Results 1

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:

no indication of skin sensitisation

Results 2

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:

no indication of skin sensitisation

Results 3

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:

no indication of skin sensitisation

Results 4

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:

no indication of skin sensitisation

Results 5

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:

positive indication of skin sensitisation

Results 6

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:

positive indication of skin sensitisation

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 nitrate hexahydrate 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 nitrate hexahydrate 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 nitrate hexahydrate 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 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 nitrate hexahydrate 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 nitrate hexahydrate was assessed to be not sensitizing.