Zinc hydroxide

Endpoint summary

Description of key information

ZnSO₄:

Zinc sulphate was tested in Balb/c mice during a local lymph node assay (Ikarashi et al., 1992) at a concentration of 5% zinc sulphate solution in 20% ethanol. Zinc sulphate did not induce proliferative activity.

The skin sensitising potential of zinc sulphate was also investigated in female Dunkin Hartley guinea pigs at a concentration of 0.1% test item injected intradermally and epidermally exposed to a 50% concentration. The skin reactions were comparable among the experimental and control animals, and there was poor consistency of the skin reactions among individual experimental animals after the first and second challenge, therefore the observed skin reactions can be considered to be non-specific signs of irritation.

Hence, it can be concluded that zinc sulphate did not induce hypersensitivity in experimental animals (Van Huygevoort, 1999).

Zn(H₂PO₄)₂ :

Zinc bis(dihydrogen phosphate) was tested in a Balb/c mice during a local lymph node assay (Plodikova, 2010) at concentrations of 30%, 3%, 0.3% (w/v) in DAE 433. The test item showed a positive dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes and showed a tendency to increased ear weight which could also be a sign of irritation of the skin. There were no further clinical symtoms. The positive result could be due a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

The skin sensitising potential of zinc bis(dihydrogen phosphate) was also investigated in guinea pigs to rule out whether it has an allergenic effect or not (Slais, 2010). A maximisation test (OECD guideline 406) was done on albino guinea pigs at a dose of 500 mg/animal.

No sensitizing effect of the Test item Zinc bis(dihydrogen phosphate) was observed in any of the animals after the challenge exposure. Under the test conditions used the Test item Zinc bis(dihydrogen phosphate) was assessed to be not sensitizing.

ZnO:

The skin sensitising potential of zinc oxide (purity 99.69%) was investigated in female Dunkin Hartley guinea pigs in two maximisation tests, at concentrations of  20% (intradermal) and epidermally exposed to a 50% concentration.

In the first study, in response to the 50% test substance concentration skin reactions of grade 1 were observed in 4/10 experimental animals 24 hours after the challenge (40% sensitisation rate), while no skin reactions were evident in the controls.

In contrast, in the second study no skin reactions were evident in the experimental animals (0% sensitisation rate), while a skin reaction grade 1 was seen in one control animal. The skin reaction observed in one control animal is probably a sign of non specific irritation (Van Huygevoort, 1999b1, 1999b2).

In a third well-performed maximisation test, no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate. The only difference with the studies described above was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected.

ZnNO₃ :

Zinc nitrate was tested in a Balb/c mice during a local lymph node assay (Plodikova, 2010) at concentrations were 30%, 3%, 0.3% (w/v) in DAE 433. The test substance caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes. The test substance also showed a tendency to increased ear weight at the 30% concentration, which could also be a sign of irritation of the skin. There were no further pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The positive result could be due a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

The skin sensitising potential of zinc nitrate was also investigated in guinea pigs to rule out whether it has an allergenic effect or not (Slais, 2010). A maximisation test (OECD guideline 406) was done in albino guinea pigs at a dose of 500 mg/animal. 

No sensitizing effect of the Test item zinc nitrate was observed in any of the animals after the challenge exposure. Under the test conditions used the Test item zinc nitrate was assessed to be not sensitizing.

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:

2010

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

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.

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:

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

500mg/animal

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

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

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

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

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

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

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

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

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.

Reference 2

Endpoint:

skin sensitisation: in vivo (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

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 18.7-21.7g
- Housing: maximum six in macrolon cages (35X20X15cm) with sterilized softwood shavings
- Diet: pelleted standard diet for experimental animals ad libitum (ST1 BERGMAN)
- Water: drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23.4
- Humidity (%): 41-48
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol)

Concentration:

30%, 3%, 0.3% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
in pilot experiment, the highest concentration 30% (maximum technically practicable concentration) was administered to 3 animals to assess and/or discard a possible systemic toxicity or high irritation of skin. The route of administration was the same as in the main study. During the pilot experiment no test item related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: if the simulation index (SI) is ≥3 , and the response increases in dose-related manner (dose-response relationship)
- dosage volume: 25µl/ear /animal

TREATMENT PREPARATION AND ADMINISTRATION:
all suspensions were prepared by mixing an appropriate amount of zinc bis(dihydrogen phosphate) (1.5g, 0.15g or 0.015g) and the vehicle to obtain the application form (3X5mL) in concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on mangetic stirrer and then were still mixed during application. Suspensions were prepared on each day immediately before administration (10-20min)

Positive control substance(s):

other: dinitrochlorobenzene

Statistics:

Kruskal-Wallis test: for the comparision of the measured effect in all treatment groups with the vehicle control group, as global test
Mann-Whitney test: for all 2-group comparisions

Parameter:

SI

Value:

9.29

Test group / Remarks:

positive control

Remarks on result:

other: positive control

Parameter:

SI

Value:

1

Test group / Remarks:

negative control

Remarks on result:

other: negative control

Parameter:

SI

Value:

4.19

Test group / Remarks:

30% test substance

Remarks on result:

other: 30% test substance

Parameter:

SI

Value:

2.68

Test group / Remarks:

3% test substance

Remarks on result:

other: 3% test substance

Parameter:

SI

Value:

0.99

Test group / Remarks:

0.3% test substance

Remarks on result:

other: 0.3% test substance

group	radioisotope incorporation		ear weight
	mean disintegrations per minute (DPM)	SI	mean (mg)
negative control	799.43	1.00	23.66
positive control	7427.57	9.29+	38.88*
30%	3346.85	4.19 +	36.14*
3%	2138.49	2.68	24.18
0.3%	795.30	0.99	23.30

*= statistically significant p<0.05 (Mann-Whitney test)

+= values ≥3

Interpretation of results:

study cannot be used for classification

Remarks:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Conclusions:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Executive summary:

The test substance, zinc nitrate hexahydrate was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The local lymph node assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, skin sensitization: local lymph node assay. In this study the contact allergenic potential of zinc nitrate hexahydrate was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB: 0.5% (w/v) and zinc nitrate hexahydrate: 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control. The positive control substance DNCB elicted a reaction pattern with statistically significant increase in ear weight and stimulation index of cell proliferation 9.29, which was in congruence with his expected mode of action as a contact allergen.

The test substance showed a tendency to increased ear weight in the highest of concentrations tested. The result of skin irritation effect was considered as positive- it means the thest substance caused irritation of skin. Comparison of stimulation indexes between treated groups and control group revealed that the test substance caused significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent, with a significant ratio of 4.19 at 30%.

The test substance zinc nitrate hexahydrate had a positive result in LLNA test. Positive results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance zinc nitrate hexahydrate could be a contact allergen in mice but potential irriation effect does not rule out the possibility that it could be false positive result.

Reference 3

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

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.

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:

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

500mg/animal

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

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 .

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

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.

Reference 4

Endpoint:

skin sensitisation: in vivo (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

Qualifier:

according to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Qualifier:

according to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Breeding farm VELAZ s.r.o., Kolec u Kladna, Czech Republic
- Age at study initiation: 8-10 weeks
- Weight at study initiation: 18.7-21.7g
- Housing: maximum six in macrolon cages (35X20X15cm) with sterilized softwood shavings
- Diet: pelleted standard diet for experimental animals ad libitum (ST1 BERGMAN)
- Water: drinking tap water ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22.5-23.4
- Humidity (%): 41-48
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

other: DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol)

Concentration:

30%, 3%, 0.3% (w/v)

No. of animals per dose:

5

Details on study design:

RANGE FINDING TESTS:
in pilot experiment, the highest concentration 30% (maximum technically practicable concentration) was administered to 3 animals to assess and/or discard a possible systemic toxicity or high irritation of skin. The route of administration was the same as in the main study. During the pilot experiment no test item related effects were found in all three animals, respectively no clinical symptoms of systemic toxicity and no macroscopic changes (after necropsy) were detected.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: LLNA
- Criteria used to consider a positive response: if the simulation index (SI) is ≥3 , and the response increases in dose-related manner (dose-response relationship)
- dosage volume: 25µl/ear /animal

TREATMENT PREPARATION AND ADMINISTRATION:
all suspensions were prepared by mixing an appropriate amount of zinc bis(dihydrogen phosphate) (1.5g, 0.15g or 0.015g) and the vehicle to obtain the application form (3X5mL) in concentration of 30%, 3% or 0.3% (w/v). The suspensions were prepared before the start of application by mixing on mangetic stirrer and then were still mixed during application. Suspensions were prepared on each day immediately before administration (10-20min)

Positive control substance(s):

other: dinitrochlorobenzene

Statistics:

Kruskal-Wallis test: for the comparision of the measured effect in all treatment groups with the vehicle control group, as global test
Mann-Whitney test: for all 2-group comparisions

Parameter:

SI

Value:

9.29

Test group / Remarks:

positive control

Parameter:

SI

Value:

1

Test group / Remarks:

negative control

Parameter:

SI

Value:

3.13

Test group / Remarks:

30% test substance

Parameter:

SI

Value:

1.09

Test group / Remarks:

3% test substance

Parameter:

SI

Value:

0.69

Test group / Remarks:

0.3% test substance

group	radioisotope incorporation		ear weight
	mean DPM	SI	mean (mg)
negative control	799.43	1.00	23.66
positive control	7427.57	9.29+	38.88*
30%	2503.43	3.13+	26.18*
3%	869.99	1.09	23.52
0.3%	550.90	0.69	22.78

*= statistically significant p<0.05 (Mann-Whitney test)

+= values ≥3

Interpretation of results:

study cannot be used for classification

Remarks:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Conclusions:

Test substance had a positive result in LLNA test, but potential irriation effect does not rule out the possibility that it could be false positive result.

Executive summary:

The test substance, zinc bis(dihydrogen phosphate) was tested for the assessment of skin sensitisation potential with the murine local lymph node assay.

The local lymph node assay (LLNA) with radionuclides was used. The testing was conducted according to the EU Method B.42, skin sensitization: local lymph node assay. In this study the contact allergenic potential of zinc bis(dihydrogen phosphate) was evaluated after topical application to female BALB/c mice. Five mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Primary proliferation of lymphocytes in the lymph node draining the site of application was evaluated on the base on using radioactive labelling. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation index, was determined. Statistical evaluation of ear weight was performed for elimination of false positive findings with certain skin irritants.

Concentrations: positive control DNCB: 0.5% (w/v) and zinc bis(dihydrogen phosphate): 30%, 3%, 0.3% (w/v) in DAE 433.

The animals exposed to the test substance at all concentrations showed no pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. There was no significant difference in body weight increment of all groups in comparison to the vehicle control. The positive control substance DNCB elicted a reaction pattern with statistically significant increase in ear weight and stimulation index of cell proliferation 9.29, which was in congruence with his expected mode of action as a contact allergen.

The test substance showed a tendency to increased ear weight in the highest of concentrations tested. The result of skin irritation effect was considered as positive- it means the thest substance caused irritation of skin. Comparison of stimulation indexes between treated groups and control group revealed that the test substance caused significant increase in radioisotope incorporation into the DNA of dividing lymphocytes. This effect was dose dependent, with a significant ratio of 3.13 at 30%.

The test substance zinc bis(dihydrogen phosphate) had a positive result in LLNA test. Positive results in cell proliferation and no clinical symptoms of systemic toxicity revealed that the test substance zinc bis(dihydrogen phosphate) could be a contact allergen in mice but potential irriation effect does not rule out the possibility that it could be false positive result.

Reference 5

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

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.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

see reference

Route:

intradermal

Vehicle:

water

Concentration / amount:

2%

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Day(s)/duration:

24h

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Adequacy of challenge:

not specified

No. of animals per dose:

10 in each test in main study
5 controls in each test

Details on study design:

In a third well-performed maximisation test, conducted according to the same guidelines and with the same experimental design, another analytical grade zinc oxide was tested (Zincweiß Pharma A; purity 99.9%). The only difference with the studies described for purity ZnO 99.69% was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected. In this test no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate for Zincweiß Pharma A. White staining of the treated skin by the test substance was observed in some animals 24 and 48 hours after challenge

Challenge controls:

see details on study designs

Positive control substance(s):

yes

Positive control results:

The sensitivity and reliability of the experimental procedure is assessed regularly a year in the lab by use of items which are known to have moderate skin sensitisation properties

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

2 %

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

2%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

Interpretation of results:

GHS criteria not met

Conclusions:

Not sensitising

Executive summary:

In a third well-performed maximisation test, conducted according to the same guidelines and with the same experimental design, another analytical grade zinc oxide was tested (Zincweiß Pharma A; purity 99.9%). The only difference with the previous studies described for zinc oxide of purity 99.69% was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected. In this test no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate for Zincweiß Pharma A. White staining of the treated skin by the test substance was observed in some animals 24 and 48 hours after challenge.

Reference 6

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

GLP compliance:

yes

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.

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

see reference

Route:

intradermal

Vehicle:

water

Concentration / amount:

20%

Adequacy of induction:

not specified

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Day(s)/duration:

24h

Adequacy of induction:

highest technically applicable concentration used

No.:

#1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

50%

Adequacy of challenge:

not specified

No. of animals per dose:

10 in each test in main study
5 controls in each test

Details on study design:

Based on the results of a preliminary study, in the main studies experimental animals (10 in each test) were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration (i.e. the highest practically feasible concentration). Control animals (5 in each test) were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle.

Challenge controls:

see details on study designs

Positive control substance(s):

yes

Positive control results:

The sensitivity and reliability of the experimental procedure is assessed regularly a year in the lab by use of items which are known to have moderate skin sensitisation properties

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

Study 1

Remarks on result:

positive indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

5

Clinical observations:

Study 1

Remarks on result:

no indication of skin sensitisation

Group:

positive control

Remarks on result:

not measured/tested

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

Study 2

Remarks on result:

no indication of skin sensitisation

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 %

No. with + reactions:

1

Total no. in group:

5

Clinical observations:

Study 2

Remarks on result:

no indication of skin sensitisation

Interpretation of results:

study cannot be used for classification

Conclusions:

conflicting results

Executive summary:

The skin sensitising potential of zinc oxide (purity 99.69%) was investigated in female Dunkin Hartley guinea pigs in two well-performed maximisation tests, conducted according to Directive 96/54/EC B.6 and OECD guideline 406. Based on the results of a preliminary study, in the main studies experimental animals (10 in each test) were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration (i.e. the highest practically feasible concentration). Control animals (5 in each test) were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle.In the first study, in response to the 50% test substance concentration skin reactions of grade 1 were observed in 4/10 experimental animals 24 hours after the challenge (40% sensitisation rate), while no skin reactions were evident in the controls. In contrast, in the second study no skin reactions were evident in the experimental animals (0% sensitisation rate), while a skin reaction grade 1 was seen in one control animal. The skin reaction observed in one control animal is most likely sign of non-specific irritation.

Reference 7

Endpoint:

skin sensitisation: in vitro

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available

Reference 8

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Not reported

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Remarks:

Used in risk assessment report for ZnSO4, acceptable for assessment

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

not specified

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Deviations:

not specified

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

No information

Route:

intradermal and epicutaneous

Vehicle:

water

Concentration / amount:

Intradermal induction - 0.1%
Epicutaneous induction - 50%
Epicutaneous challenge - 50%

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

Intradermal induction - 0.1%
Epicutaneous induction - 50%
Epicutaneous challenge - 50%

No. of animals per dose:

10

Details on study design:

RANGE FINDING TESTS: No details provided


MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: Two, intradermal and epicutaneous
- Exposure period: 24 h
- Frequency of applications: Once
- Duration: 24 h
- Concentrations: 0.1% (intradermal) and 50% (epicutaneous)

10% of SDS applied at 24 h after induction exposure.


B. CHALLENGE EXPOSURE
- No. of exposures: Two
- Day(s) of challenge: Day 15 and Day 21
- Concentrations: 50% (epicutaneous)
- Evaluation (hr after challenge): 48 h

Challenge controls:

Five control animals were used. Induced and challenged with water alone.

Positive control substance(s):

no

Positive control results:

Not applicable

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

5

Total no. in group:

10

Clinical observations:

skin reactions of grade 1

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: skin reactions of grade 1 .

Reading:

rechallenge

Hours after challenge:

48

Group:

test chemical

Dose level:

50%

No. with + reactions:

4

Total no. in group:

10

Clinical observations:

skin reactions of grade 1

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: test group. Dose level: 50%. No with. + reactions: 4.0. Total no. in groups: 10.0. Clinical observations: skin reactions of grade 1 .

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

2

Total no. in group:

5

Clinical observations:

skin reactions of grade 1

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: skin reactions of grade 1 .

Reading:

rechallenge

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

2

Total no. in group:

5

Clinical observations:

skin reactions of grade 1

Remarks on result:

other: Reading: rechallenge. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: skin reactions of grade 1 .

As the skin reactions were comparable among the experimental and control animals, and as there was poor consistency of the skin reactions among individual experimental animals after the first and second challenge, the observed skin reactions can be considered to be non-specific signs of irritation.

Interpretation of results:

GHS criteria not met

Conclusions:

Hence, it can be concluded that zinc sulphate did not induce hypersensitivity in experimental animals

Executive summary:

The skin sensitising potential of zinc sulphate (ZnSO_4.7H₂O) was investigated in guinea pigs. A well-performed maximisation test, conducted according to Directive 96/54/EC B.6 and OECD guideline 406, was carried out in female Dunkin Hartley guinea pigs.

Based on the results of a preliminary study, in the main study 10 experimental animals were intradermally injected with a 0.1% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle (water) alone. Approximately 24 h before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. A second challenge followed one week after the first.

In response to the 50% test substance concentration, in some experimental animals and controls skin reactions of grade 1 were observed 48 hours after the first (5/10 and 2/5, respectively) and the second challenge (4/10 and 2/5, respectively).

As the skin reactions were comparable among the experimental and control animals, and as there was poor consistency of the skin reactions among individual experimental animals after the first and second challenge, the observed skin reactions can be considered to be non-specific signs of irritation.

Hence, it can be concluded that zinc sulphate did not induce hypersensitivity in experimental animals

Reference 9

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Not applicable

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

The basic principle underlying the LLNA method used is to induce a primary proliferation of lymphocytes in the lymph node draining the site of test material application. An ear abrasion technique is employed which enhances penetration of the metal ion by removing the horny layer of the epidermis. It enables the detection of ear-swelling responses of weak contact allergens by increasing their LNC proliferation. This proliferation is proportional to the dose applied (and to the potency of the allergen) and provides a simple means of obtaining an objective and quantitative measurement of sensitisation. The LLNA assesses this proliferation, wherein the proliferation in test groups is compared to that in vehicle treated controls. The ratio of the proliferation in treated groups to that in vehicle controls (Stimulation Index), is determined, and must be at least three. The methods described here are based on the use of radioactive labelling to measure the proliferation of the lymph node cells.

GLP compliance:

not specified

Type of study:

mouse local lymph node assay (LLNA)

Species:

mouse

Strain:

Balb/c

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 6-8 wk

Vehicle:

other: 20 % ethanol

Concentration:

10 % test material in 20 % ethanol solution

No. of animals per dose:

Three

Details on study design:

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: Stimulation index ≥ 3


TREATMENT PREPARATION AND ADMINISTRATION: 25 µL of test material was applied to the dorsum of both ears for three consecutive days. Prior to the test material treatment, the ears of each mouse were gently abraded using a 19 g needle. Four days following the initial application, draining lymph nodes were excised.

Preparation of cell suspension: A single cell suspension of LNC was prepared by mechanical disaggregation through sterile 200 mesh steel gauge. The cells were washed twice with an excess phosphate-buffered saline (PBS) and resuspended in RPMI-1640 culture medium supplemented with 10 % fetal calf serum (FCS), 25 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), 100 µg/mL penicillin and 100 U/mL streptomycin. The cell concentration was adjusted to give 5 x 10E6 cells/mL.
Lymphocyte suspensions were seeded into 96 well microtiter plates at a concentration of 1 x 10E6 cells/well (5 wells per animal), and cultured with 0.5 µCi [3H] methyl thymidine ([3H]TdR) for 18 h at 37 °C in a humidified atmosphere of 5 % CO2 in air.
The incorporation of [3H]TdR was measured using a liquid scintillation counter and expressed as mean counts per min (cpm) ± standard deviation per node of three animals for each test group.

Other: Compound solubility - Completely dissolved in 20 % ethanol solution

Positive control substance(s):

not specified

Statistics:

Not reported

Positive control results:

Not reported

Parameter:

SI

Value:

1.41

Variability:

na

Test group / Remarks:

3 animals per dose

Remarks on result:

other: 10 % zinc sulfate = 1.41

Parameter:

SI

Remarks on result:

other: not tested <10% zinc sulfate

Parameter:

SI

Remarks on result:

other: not tested >10% zinc sulfate

Parameter:

other: disintegrations per minute (DPM)

Value:

2.14

Variability:

0.77

Test group / Remarks:

3 animals per dose

Remarks on result:

other: Mean value cpm ± SD ( x 10-3) = 2.14 ± 0.77

Validity of ear abrasion technique: It does not affect the LNC response in the vehicle treated group.

Interpretation of results:

not sensitising

Conclusions:

Under the conditions of the test, the test material was determined to be non-sensitising to mice.

Executive summary:

A study was conducted to evaluate the skin sensitisation potential of the test material in mouse using a modified Local Lymph Node Assay. No guideline or GLP compliance was documented in the study report.

Groups of BALB/c mice (n=3) were treated with 10% concentration of test material or vehicle (20% ethanol solution) by applying 25 µL to the dorsum of both abraded ears for three consecutive days. Four days following the initial application, draining lymph nodes were excised. A single cell suspension of LNC was prepared and the incorporation of [3H]TdR was measured using a liquid scintillation counter.

[3H]TdR incorporation (expressed as mean counts per min (cpm) ± standard deviation per node x 10^-3) was 2.14 ± 0.77 and the ratio of the proliferation in treated group to that in vehicle control (stimulation index) was 1.41.

Hence, under the conditions of the test, the test material was determined to be non-sensitising to mice.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not sensitising)

Additional information:

ZnSO₄:

Zinc sulphate (ZnSO₄•7 H₂O) was tested in a mouse local lymph node assay (Ikarashiet al., 1992), according to the testing methods developed by Kimberet al.(1989 and 1990). After gentle dermal abrasion, 25ml of a 5% zinc sulphate solution in 20% ethanol was applied for three consecutive days at the dorsal side of both ears of 3 Balb/c mice. On the fourth day the animals were sacrificed and the ear-draining lymph nodes were collected. Lymph node lymphocyte proliferation was determined by tritiated thymidin incorporation. The results were compared to those of vehicle-treated controls. Zinc sulphate did not induce proliferative activity, whereas for potassium bichromate, nickel sulphate and cobalt chloride (known dermal sensitizers) positive results were obtained.

The skin sensitising potential of zinc sulphate (ZnSO₄•7 H₂O) was also investigated in guinea pigs. A well-performed maximisation test, conducted according to Directive 96/54/EC B.6 and OECD guideline 406, was carried out in female Dunkin Hartley guinea pigs. Based on the results of a preliminary study, in the main study 10 experimental animals were intradermally injected with a 0.1% concentration and epidermally exposed to a 50% concentration. Five control animals were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. A second challenge followed one week after the first. In response to the 50% test substance concentration, in some experimental animals and controls skin reactions of grade 1 were observed 48 hours after the first (5/10 and 2/5, respectively) and the second challenge (4/10 and 2/5, respectively). As the skin reactions were comparable among the experimental and control animals, and as there was poor consistency of the skin reactions among individual experimental animals after the first and second challenge, the observed skin reactions can be considered to be non-specific signs of irritation. Hence, it can be concluded that zinc sulphate did not induce hypersensitivity in experimental animals (Van Huygevoort, 1999).

 

Zn(H₂PO₄)₂ :

Zinc bis(dihydrogen phosphate) was tested in a mouse local lymph node assay (Plodikova, 2010). Five Balb/c mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Concentrations were 30%, 3%, 0.3% (w/v) in DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol). Lymph node lymphocyte proliferation was determined by tritiated thymidin incorporation.The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The test substance caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes, with a significant ratio of 3.13 at 30%. The test substance also showed a tendency to increased ear weight at the 30% concentration, which could also be a sign of irritation of the skin. The animals exposed to the test substance at all concentrations showed no further pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The positive result could be due a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

The skin sensitising potential of zinc bis(dihydrogen phosphate) was also investigated in guinea pigs to rule out whether it has an allergenic effect or not (Slais, 2010).A well-performed maximisation test, conducted according to OECD guideline 406, was carried out in albino guinea pigs.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. 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.

 

ZnNO₃ :

Zinc nitrate was tested in a mouse local lymph node assay (Plodikova, 2010). Five Balb/c mice per group were exposed on the dorsum of both ears once a day by test and control substances during 3 consecutive days. Concentrations were 30%, 3%, 0.3% (w/v) in DAE 433 (mixture of 40% dimethylacetamide, 30% acetone and 30% ethanol). Lymph node lymphocyte proliferation was determined by tritiated thymidin incorporation. The ratio of the proliferation in treated groups to that in vehicular controls, termed the Stimulation Index, was determined. The test substance caused a dose dependent increase in radioisotope incorporation into the DNA of dividing lymphocytes, with a significant ratio of 4.19 at 30%. The test substance also showed a tendency to increased ear weight at the 30% concentration, which could also be a sign of irritation of the skin. The animals exposed to the test substance at all concentrations showed no further pathological skin reactions and no other negative clinical symptoms of intoxication throughout the experiment. The positive result could be due a contact allergen in mice but potential irritation effect does not rule out the possibility that it could be false positive result.

The skin sensitising potential of zinc nitrate was also investigated in guinea pigs to rule out whether it has an allergenic effect or not (Slais, 2010). A well-performed maximisation test, conducted according to OECD guideline 406, was carried out in albino guinea pigs. 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. No sensitizing effect of the Test item zinc nitrate 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 was assessed to be not sensitizing.

 

ZnO:

The skin sensitising potential of zinc oxide (purity 99.69%) was investigated in female Dunkin Hartley guinea pigs in two well-performed maximisation tests, conducted according to Directive 96/54/EC B.6 and OECD guideline 406. Based on the results of a preliminary study, in the main studies experimental animals (10 in each test) were intradermally injected with a 20% concentration and epidermally exposed to a 50% concentration (i. e. the highest practically feasible concentration). Control animals (5 in each test) were similarly treated, but with vehicle (water) alone. Approximately 24 hours before the epidermal induction exposure all animals were treated with 10% SDS. Two weeks after the epidermal application all animals were challenged with a 50% test substance concentration and the vehicle. In the first study, in response to the 50% test substance concentration skin reactions of grade 1 were observed in 4/10 experimental animals 24 hours after the challenge (40% sensitisation rate), while no skin reactions were evident in the controls. In contrast, in the second study no skin reactions were evident in the experimental animals (0% sensitisation rate), while a skin reaction grade 1 was seen in one control animal. The skin reaction observed in one control animal is probably a sign of non specific irritation (Van Huygevoort, 1999b1, 1999b2).

In a third well-performed maximisation test, conducted according to the same guidelines and with the same experimental design, another analytical grade zinc oxide was tested (Zincweiß Pharma A; purity 99.9%). The only difference with the studies described above was the intradermal induction concentration, which was 2% as for Zincweiß Pharma A this was considered the highest concentration that could reproducibly be injected. In this test no skin reactions were evident in both experimental and control animals, hence a 0% sensitisation rate for Zincweiß Pharma A. White staining of the treated skin by the test substance was observed in some animals 24 and 48 hours after challenge (Van Huygevoort, 1999a).

Human data: In a human patch test performed with 100 selected leg-ulcer patients, 11/100 patients gave an allergic reaction with zinc ointment (60% ZnO and 40% sesame oil). However, 14/81 patients gave a positive response when treated with sesame oil alone. This study does not give any indication for a skin sensitizing potential of zinc oxide in humans (Malten and Kuiper, 1974). The effect of zinc oxide on contact allergy to colophony was investigated. With 14 patients with earlier history of moderate patch test reactions to colophony (a patch test) with 10% ZnO (2.3 mg Zinc/cm²) with and without colophony was performed. No positive response was observed in the 14 patients when only a 10% solution of zinc oxide was used. The addition of zinc oxide to colophony decreased the allergic reaction induced by colophony (Söderberg et al., 1990). 

Zinc oxide nanomaterial:   Repeat human insult patch tests performed specifically on nano-ZnO demonstrate that coated and uncoated ZnO (Zano 10 Plus and Zano 10, respectively) tested as 25% oily dispersion did not produce any skin irritation or skin sensitisation in human volunteers under the conditions of this test (Tatsene 2007). In conclusion, for nano-ZnO no nano-specific sensitization effects could be identified. Zn²⁺ion determines the toxicity of ZnO and read across between various forms of ZnO (micro-scale, nano, coated or not) is fully supported. 

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information:

While there is no particular study addressing respiratory sensitisation in experimental animals, there is no information suggesting zinc compounds to cause such effects animals.Taking into account the complete absence of skin sensitization potential of zinc compounds, respiratory sensitisation is not expected to be of concern for the zinc and zinc compounds.

Considering the absence of evidence of respiratory sensitization responses in humans, this endpoint is not expected to be of concern for zinc and zinc compounds.

Justification for classification or non-classification

Zn(H₂PO₄)₂: The data on soluble zinc bis(dihydrogen phosphate) indicates a positive skin sensitisation potential in a study done with the Mouse local lymph node assay but no skin sensitisation potential was indicated in another study done with the Guinea pig maximization test. Since this latter test is known to be more trustful in studies with metals, no skin sensitization is expected and therefore classification for skin sensitization is not required according to EC criteria.

ZnO : The data on slightly soluble zinc oxide indicated no skin sensitising potential (negative in animal and human studies) therefore classification for skin sensitisation is not required according to EC criteria.

ZnNO₃ : The data on soluble zinc nitrate indicates a positive skin sensitisation potential in a study done with the Mouse local lymph node assay but no skin sensitisation potential was indicated in another study done with the Guinea pig maximization test. Since this latter test is known to be more trustful in studies with metals, no skin sensitization is expected and therefore classification for skin sensitization is not required according to EC criteria.

ZnSO₄: The data on soluble zinc sulphate indicates no sensitizing potential and therefore no classification is required according to EC criteria.