Bis(glycinato-N,O)zinc

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

- Principle of test: A zinc glycinate skin sensitization test based on the Adjuvant and Patch Test was conducted using 30 male Hartley guinea pigs.

- Short description of test conditions:
For the primary sensitization a mixture of Freund's complete adjuvant and water for injection was first intradermally injected into the neck of the guinea pig. Afterwards, an abrasion in a criss cross lattice was created at the injection site and a test sample was applied in an occlusive manner for 24 hours. The abrasion treatment and occlusive application of the sample was repeated once per day for three consecutive days. For the secondary sensitization petroleum jelly containing 10% sodium lauryl sulphate was applied to the skin at the injection site 7 days after the beginning of the sensitization. On the following day an occlusive application of the sample was carried out for 48 hours at the same location. The elicitation phase started 21 days after the beginning of the sensitization and the sample was applied openly. The reaction of the skin was observed 24 as well as 48 hours after the application. The test substance was prepared as a 50 w/v% suspension with Japanese Pharmacopeia water for injection (hereafter referred to as water for injection). Additionally, 2,4-dinitrochlorobenzene (DNCB) was used as a positive control.

- Parameters analysed / observed: Skin reaction was observed. Erythema criteria and edema formation were assessed with standardized criteria.

GLP compliance:

yes (incl. QA statement)

Type of study:

other: Skin sensitization test based on the Adjuvant and Patch Test (Sato, Y., Katsumura, Y., lchikawa, H., Kobayashi,T., Kozuka,T., Morikawa, F. and Ohta, S. (1981): A modified technique of guinea pig testing to identify delayed hypersensitivity allergens.)

Justification for non-LLNA method:

The present test was performed in 2006, and the LLNA method (OECD 429, adopted in July 2010) was not available yet by the time the study was conducted. Thus, an additional test according to OECD method was not performed in order to limit animal testings.

Test material

Test material information

Constituent 1

Reference substance name:

Bis(glycinato-N,O)zinc

EC Number:

238-173-1

EC Name:

Bis(glycinato-N,O)zinc

Cas Number:

14281-83-5

Molecular formula:

C4H8N2O4Zn

IUPAC Name:

Zinc bis(aminoacetate)

Test material form:

solid: particulate/powder

Details on test material:

Batch number: 4002
Pourcentage of Zinc: 28.2%
Molecular Weight: 231.52

Specific details on test material used for the study:

- Batch number of test material: 4002
- Percentage of Zinc: 28.2%
- Appearance: White powder
- Storage Conditions: Room temperature (permissible range between 10-30°C), dark location, temperature:13.8-25.2°C (actual measurement)

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Japan SLC, Inc.
- Microbiological status of animals, when known: Clean Animal
- Age at study initiation: 5 weeks
- Weight at study initiation: 382-411 g
- Housing: Suspension type aluminium cage (32W x 48D x 33H cm, with wire mesh cage floor, Tokiwa Kagaku Kikai Co., Ltd.). Material was sterilized using steam and exchanged once every 5 to 7 days.
- Diet (e.g. ad libitum): Solid feed for laboratory animals (Labo G Standard, Nosan Corporation) - Free feeding - Leftovers were disposed of when exchanging the feeder and new feed was provided.
- Water (e.g. ad libitum): Tap water filtered with a 5μm filter and subsequent ultraviolet irradiation treatment - Free water feeding - Inspection of water quality was carried out periodically (twice a year) by the Diamond Analysis Center Co., Ltd. (now Mitsubishi Chemical Analytech Co., Ltd.). Conformation to the criteria of the standard operating procedure manual, which were based on water supply regulations, was assured.
- Acclimation period: The general condition of each animal was observed once per day for 5 days. The weight was measured on the day of arrival and the end of quarantine. A good general condition without unusual weight gain was confirmed for all animals.
The acclimatization was continued for a day in the time between the end of quarantine and the beginning of the administration and the general condition was checked once per day.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): Actual temperature 21.0-22.9°C (permissible range 19.0-25.0°C)
- Humidity (%): Actual humidity 56.6-77.4% (permissible range 35.0-75.0%)
- Air changes (per hr): 6-20 air changes per hour, “All Fresh Air” supply
- Photoperiod (hrs dark / hrs light): 12 hours light per day, between 7:00 and 19:00

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

epicutaneous, occlusive

Vehicle:

water

Concentration / amount:

Concentration: 50 w/v% Zinc Glycinate

Day(s)/duration:

8 days after the intradermal injection (day 9) 0.2 ml of the sample for administration during the sensitization phase ; 48-hour occluded application

Adequacy of induction:

not specified

Induction 2

Route:

intradermal

Vehicle:

water

Concentration / amount:

Concentration: 50 w/v%
A suspension was made using the test substance (powder) and water for injection.
Note that water for Injection was prepared as follow: an emulsion with a 1:1 (v/v) mixture of FCA (Freund’s Complete Adjuvant) and water.

Day(s)/duration:

1 administration (day 1)

Adequacy of induction:

not specified

Challenge

No.:

#1

Route:

epicutaneous, open

Vehicle:

water

Concentration / amount:

Concentration: 50 w/v% Zinc Glycinate ;
0.01 ml of the sample for administration.

Day(s)/duration:

Started 21 days after the beginning of the sensitization (day 22) ; One application.

Adequacy of challenge:

not specified

No. of animals per dose:

TEST SUBSTANCE
Test substance: 10 animals
Test substance control: 10

POSITIVE CONTROL (DNCB)
DNCB Administration: 5
DNCB control: 5

Details on study design:

CONCENTRATION OF TEST SUBSTANCE
The concentration of the test substance was based on the results of a preliminary test conducted at the test facility (test number B050676). The concentration used was 50 w/v% for both the sensitization and the elicitation.
The skin irritation of guinea pigs was examined for the case of occlusive as well as open application in the preliminary test. For the occlusive application 0.1 ml each of a 100, 50 and 10 w/v% test substance suspension were applied to the skin of two guinea pigs. As a result, a mild skin reaction at the location of the application was observed in one of the two animals for concentrations of 100 and 50 w/v%.
There were operational difficulties during the stability validation for the 100 w/v% suspension. Due to this, the test substance was administered 0.01 ml each as a 50, 10 and 1 w/v% suspension for the open application onto the skin of two guinea pigs. This resulted in no skin reaction at the location of the application.
Concluding from these results, the highest concentration that was possible to be analysed, 50 w/v%, was chosen. This provoked a mild skin reaction for the occlusive application and no skin reaction for the open application.

CONCENTRATION OF DNCB
The concentration of DNCB was set to 0.05 w/v%, for which a positive skin reaction was confirmed at the test facility for the sensitization as well as the elicitation phase.

CONFIRMATION OF STABILITY AND HOMOGENEITY OF THE LIQUID FOR ADMINISTRATION
According to a different test conducted at the test facility, the stability and homogeneity, when storing a 0.1 and 50 w/v% suspension of the test substance at a cool place for 8 days, was confirmed. An analysis to confirm the stability of DNCB was not conducted.

CONFIRMATION OF THE CONCENTRATION AND HOMOGENEITY OF THE LIQUID FOR ADMINISTRATION
Before the first administration, a sample was extracted from the container of the prepared test substance liquid from each the top, middle and top layer. It was confirmed that the average value resulting from the analysis was within ±10% of the nominal concentration value (ratio to the nominal concentration 100.8%). Further, it was confirmed that the analysis value of the C.V. was within 10% (0.1%). An analysis to confirm the concentration and homogeneity of DNCB was not conducted.

ANALYTICAL METHOD FOR THE LIQUID ADMINISTRATION
The analysis of the test substance liquid for the administration was conducted in agreement with the descriptions in the analytical method validation.

GROUP FORMATION AND ANIMALS
Test Substance: Sensitization with 50 w/v% Zinc Glycinate / Elicitation with 50 w/v% Zinc Glycinate and Water for Injection (10 animals)
Test Substance Control: Sensitization with Water for Injection / Elicitation with 50 w/v% Zinc Glycinate and Water for Injection (10 animals)
DNCB administration : Sensitization with 0.05 w/v% DNCB / Elicitation with 0.05 w/v% DNCB and Acetone (5 animals)
DNCB COntrol : Sensitization with acetone / Elicitation with 0.05 w/v% DNCB and Acetone (5 animals)

SENSITIZATION
(1) Primary Sensitization (Intradermal Injection and Application)
The day before the intradermal injection, a 4x6 cm area of the neck of the animal was clipped using electric clippers and electric blades to form the area for the sensitization. On the following day (day 1), 0.1 ml of water for injection/ FCA was injected intradermally into each of the four corner areas. An injection needle was used to create a criss cross lattice (#) of abrasions through the stratum corneum of each injection location. Afterwards, 0.1 ml of the respective sample for administration in the sensitization phase, was spread on an adhesive bandage for patch tests with a diameter of 1.5 cm and applied to the skin. Additionally, the area of application was covered with an adhesive bandage of approximately 5x40 cm for the 24-hour occluded application. The intradermal injection was carried out only on the first day of the administration, while the criss cross lattice abrasions and the application of the sample were repeated once per day for three consecutive days.

(2) Secondary Sensitization (Topical Application)
7 days after the intradermal injection (day 8) the sensitization area was once again clipped using electric clippers and electric blades. 0.2 ml of petroleum jelly containing 10% SLS was applied to the 2x4 cm inner injection site. 8 days after the intradermal injection (day 9) 0.2 ml of the sample for administration during the sensitization phase was spread on a 2x4 cm cotton flannel pad (patch) and affixed to the inner injection site. After covering the patch with a 5x5 cm adhesive bandage backed with vinyl tape, it was fixed with a 5x40 cm adhesive bandage for the 48-hour occluded application.

ELICITATION
The elicitation phase started 21 days after the beginning of the sensitization (day 22).
One day before the elicitation the back area was clipped using the same method as before. The day after the clipping 0.01 ml of the sample for administration during the elicitation was openly applied in a circle shape with a diameter of 1.5 cm. The test substance or DNCB was applied on the left side of the median line and water for injection or acetone on the right.

REASON FOR THE CHOOSING THIS METHOD OF ADMINISTRATION
In a preliminary test conducted at the test facility 0.1 ml each of a 50, 25, 10 and 1 w/v% suspension of the test substance were intradermally administered into the back of two guinea pigs. The result was that the intradermal administration was not possible for a concentration of 25 w/v% and higher and even the other suspensions induced at least a moderate skin reaction. It was thus concluded that a Maximization Test would be difficult, as it necessitates intradermal administration during the primary sensitization treatment. The Adjuvant and Patch Test was chosen, which is widely used in skin sensitization testing.

OBSERVATION OF SKIN REACTION AND GRADING CRITERIA
24 and 48 hours after the elicitation treatment the skin at the area of administration was observed with the naked eye. The skin reaction was graded according to the grading criteria of Sato and Co. Furthermore, a photography was taken of a representative example at each observation. Approximately 3 hours before the observation the back of the animal, including all administration areas, was clipped using electric blades.

(a) Erythema formation Evaluation Points
No erythema: 0
Very slight erythema (barely perceptible): 1
Well defined erythema: 2
Moderate to severe erythema: 3
Severe erythema (beet redness to slight eschar formation-injuries in depth): 4
(b) Edema formation
No edema: 0
Slight edema: 1
Moderate edema: 2
Severe edema (raised more than 1mm and extending beyond area of exposure): 3

OBSERVATION OF THE GENERAL CONDITION
The general condition of all of the animals was observed once per day from the beginning of the sensitization to the end of the observation.

BODY WEIGHT MEASUREMENT
The weight of the animals was measured directly before the primary sensitization treatment, the secondary sensitization treatment, and the elicitation treatment, as well as 48 hours after the end of the elicitation treatment. An animal scale (EB-16KS, Shimadzu Corporation or PG12001S, Mettler Toledo) was used for measuring.

Challenge controls:

Challenge controls were performed.

Positive control substance(s):

yes

Remarks:

DNCB was dissolved in acetone and the 0,05 w/v% solution was prepared extemporaneously.

Results and discussion

Positive control results:

2,4-dinitrochlorobenzene (DNCB) was used as a positive control.
The group elicitated with DNCB and DNCB administration during the sensitization (DNCB group) showed a positive reaction of the skin.
No skin reaction was observed in the group treated with acetone during the sensitization (DNCB control group).

In vivo (non-LLNA)

Results

Results 1

Key result

Reading:

other: Sensitization

Hours after challenge:

24

Group:

test chemical

Dose level:

50 w/v% Zinc Glycinate

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Induction substances: Zinc Glycinate and Vehicle (Water)

Results 2

Key result

Reading:

other: Sensitization

Hours after challenge:

24

Group:

negative control

Dose level:

Water for Injection

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Induction substances: Zinc Glycinate and Vehicle (Water)

Results 3

Key result

Reading:

other: Sensitization

Hours after challenge:

24

Group:

positive control

Dose level:

0.05% w/v DNCB

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Remarks:

Induction substance: DNCB

Results 4

Key result

Reading:

other: Sensitization

Hours after challenge:

48

Group:

negative control

Dose level:

Water for injection

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Induction substances: Zinc Glycinate and Vehicle (Water)

Results 5

Key result

Reading:

other: Sensitization

Hours after challenge:

48

Group:

test chemical

Dose level:

50 w/v% zinc glycinate

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Remarks:

Induction substances: Zinc Glycinate and Vehicle (Water)

Results 6

Key result

Reading:

other: Sensitization

Hours after challenge:

48

Group:

positive control

Dose level:

0.05 w/v% DNCB

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Remarks:

Induction substance: DNCB

Any other information on results incl. tables

SKIN REACTION

- Test Substance Group: No skin reaction was observed in any animal at the area of administration of the test substance as well as water for injection.

- Test Substance Control Group: No skin reaction was observed in any animal at the area of administration of the test substance as well as water for injection.

- DNCB Group: A skin reaction could be confirmed in all animals at the area of the DNCB administration. The confirmed skin reaction at the area of the DNCB administration reached 3 evaluation points for erythema and oedema 24 hours after the elicitation. The same was confirmed 48 hours after the elicitation, so that the evaluation points of the skin reaction at the area of DNCB administration reached a mean of 6.0 points for 24 hours and 48 hours after the elicitation. The positivity rate was 100% 24 hours as well as 48 hours after the elicitation. At the area of acetone administration no skin reaction could be found in any animal.

GENERAL CONDITION

No abnormalities were observed in any animal from the first sensitization treatment until the end of the observation of the skin reaction.

BODY WEIGHT

A regular body weight increase was confirmed for each animal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The skin sensitization of zinc glycinate was examined using the Adjuvant and Patch Test.
Concluding from the results, zinc glycinate did not have a skin sensitizing effect in guinea pigs under the conditions of this test.

Executive summary:

The skin sensitization of zinc glycinate was examined using the Adjuvant and Patch Test.

After the elicitation with the test substance, no skin reaction was found in the test substance group as well as the test substance control group. Further, after the elicitation with injection water, no skin reaction was found in the test substance group as well as the test substance control group.

For the DNCB group, as a result of the elicitation with DNCB, a decidedly positive skin reaction at the area of DNCB administration could be confirmed for all animals. There was no skin reaction at the area of acetone administration and it can therefore be assumed that the procedure of this test was appropriate.

Concluding from the results above, zinc glycinate did not have a skin sensitizing effect in guinea pigs under the conditions of this test.