Bis(5-oxo-L-prolinato-N1,O2)zinc

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 April 2003 to 30 June 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2008

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Principles of method if other than guideline:

A study was conducted regarding the test material in accordance with the Maximization Test. The control group was administered a physiological saline solution (intradermal induction) and distilled water for injection (epidermal induction) which were used as vehicles in the same manner as for the test material sensitisation group.

GLP compliance:

no

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

An adequate skin sensitisation study using the guinea pig maximisation test method was already available and therefore further testing was not required.

Test material

Test material information

Constituent 1

Reference substance name:

Bis(5-oxo-L-prolinato-N1,O2)zinc

EC Number:

239-473-5

EC Name:

Bis(5-oxo-L-prolinato-N1,O2)zinc

Cas Number:

15454-75-8

Molecular formula:

C10H12N2O6Zn

IUPAC Name:

(3'aR)-3,3',6,6'-tetraoxo-3a,3'a,4,4',5,5',6,6'-octahydro-3H,3'H-1,1'-spirobi[pyrrolo[1,2-c]1-oxa-3-aza-2-zincacyclopentane]-1,1-bis(ylium)-2,2'-diuide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Details on test material:

- Appearance: Powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Hartley

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5 weeks
- Weight at study initiation: 301.7 g to 339.0 g
- Housing: Bracket cages; 2 animals/cage.
- Diet: Ad libitum
- Water: Ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 3 °C
- Humidity (%): 50 ± 20 %
- Air changes (per hr): 10 to 15 cycles per hour
- Photoperiod (hrs dark / hrs light): 12 hours, from 7 A.M. until 7 P.M., between 200 and 600 lux.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal

Vehicle:

physiological saline

Concentration / amount:

The test material was prepared as 0.1 % and 0.2 % (w/w) solutions using physiological saline solution as the vehicle. The 0.2 % solution was mixed with an equal volume of FCA to prepare an FCA/emulsion fluid.

Induction 2

Route:

other: Epidermal

Vehicle:

water

Concentration / amount:

The test material was prepared as a 10.0 % (w/w) solution using injection solvent as the vehicle.

Challenge

Route:

epicutaneous, occlusive

Vehicle:

other: Physiological saline and distilled water

Concentration / amount:

The test material was prepared at 1.0 % and 0.5 % (w/w) solutions.

No. of animals per dose:

- 10 animals for the test material group; 5 animals for the control group.

Details on study design:

RANGE FINDING TESTS:
APPLICATION OF TEST MATERIAL AND CONTROL SUBSTANCE - PRELIMINARY STUDIES
- Application route: Intradermal induction and occlusive adhesion
- Reason for selecting application route: Consideration was given to the usage method of the test article as a product, and the application method for the Maximum Test was followed.
- Reason for setting application dosages: Giving consideration to the concentration of the test material during use as well as the amount that can be physically applied, preliminary studies were conducted. Based on the results of the preliminary studies, a concentration that does not cause necrosis during intradermal induction, the lowest concentration for epidermal induction, and the highest concentration for which irritation is not observed for challenge exposure were set as the dosages to be applied.

PREPARATION OF VEHICLE AND SUBSTANCE TO BE ADMINISTERED – PRELIMINARY STUDIES
- Method for preparing substances to be administered: All were prepared immediately before administration.
For intradermal induction, 4 concentrations were prepared using physiological saline solution as the vehicle. A 20 % (w/w) solution was prepared first, which was further diluted and prepared as 10 %, 5 %, and 1 % (v/v) solutions.
For epidermal induction, 4 concentrations were prepared using distilled water for injection as the vehicle. A 25 % (w/w) solution was prepared first, which was further diluted and prepared as 10 %, 5 %, and 1 % (v/v) solutions.
The test article was prepared in the same manner for challenge exposure. A10 % (w/w) solution was prepared first, which was further diluted and prepared as 5 %, 1 %, and 0.5 % (v/v) solutions for a total of 4 concentrations.
- White petrolatum containing 10.0 % SLS: SLS was mixed into white petrolatum at a concentration of 10.0 % (W/W) and an ointment was prepared.
- FCA emulsion: FCA and physiological saline solution were mixed in equal volumes, and an emulsion fluid was prepared.

PRELIMINARY STUDY FOR INTRADERMAL INDUCTION
Before intradermal induction, a preliminary study was conducted based on the procedure below using 2 untreated animals. From the results of this test, the concentration of the test material to use for intradermal induction was determined.
- The fur on the back side of the neck of the guinea pigs was shaved over an area of 6 cm x 6 cm using clippers and an electric shaver.
- 0.1 mL of the test article was intra-dermally induced into a 4 cm x 4 cm patch inside the shaved area. The condition of the administration site was observed immediately after induction and 4 hours after induction, and was graded according to the grading standards below.
Assessment as to whether there was necrosis was also conducted.
0: No visible change
1: Discrete or patchy erythema
2: Moderate and confluent erythema
3: Intense erythema and swelling

PRELIMINARY STUDY FOR EPIDERMAL INDUCTION
Before epidermal induction, a preliminary study was conducted 2 untreated animals using the procedure below. Based on the results of this test, the concentration of the test material to use for epidermal induction was determined.
- The fur on both sides of the abdomen of the animals was shaved over an area of 5 cm x 10 cm using clippers and an electric shaver.
- Pieces of cotton lint (2 cm x 2 cm) that were immersed in 0.2 mL or 0.2 g of the test article were applied in order from 1 to 4 onto the shaved area and fixed in place using tape. Occlusive adhesion was carried out for 48 hours.
- After occlusive adhesion of 48 hours, the cotton lint was removed, and skin irritation of the application site was graded based on the following grading standards immediately after removal and 24 hours after removal.
0: No visible change
1: Discrete or patchy erythema
2: Moderate and confluent erythema
3: Intense erythema and swelling

PRELIMINARY STUDY FOR CHALLENGE EXPOSURE
Before challenge exposure, a preliminary study was conducted based on the procedure below using 4 animals for which intradermal induction and epidermal induction had been carried out in the same manner as those in the control group. Based on the results of this test, the concentrations of the test material to use for challenge exposure were determined.
- The fur on both sides of the abdomen of the animals was shaved over an area of 5 cm x 10 cm using clippers and an electric shaver.
- The next day, pieces of cotton lint (2 cm x 2 cm) that were immersed in 0.2 mL of the test article were applied in order from 1 to 4 onto the shaved area and fixed in place using tape. Occlusive adhesion was carried out for 24 hours.
- After occlusive adhesion of 24 hours, the cotton lint was removed, and skin irritation of the application site was graded based on the following grading standards immediately after removal and 24 hours after removal.
0: No visible change
1: Discrete or patchy erythema
2: Moderate and confluent erythema
3: Intense erythema and swelling

COUNTING OF TEST DATES
During the animal testing period, observations and tests were conducted on the dates that are stated in this report. Dates for tests are counted as follows.
Day that sensitization induction was initiated (day of intradermal induction): Day 1
Day after intradermal induction and subsequent days: Day 2, Day 3, etc.


MAIN STUDY
A. INDUCTION EXPOSURE
- Site: The fur on the necks of the animals in the test article sensitization group and the control group was shaved over an area of approximately 6 cm x 4 cm using electric clippers and an electric shaver.
- Intradermal induction: 0.1 mL of the FCA emulsion fluid, 0.1 mL of the test material solution, and 0.1 mL of the test material/FCA emulsion fluid (or vehicle/FCA emulsion fluid) were intradermally induced in each pair over an area of 2 cm x 4 cm inside the shaved site.
- Epidermal induction: On Day 7, the fur on the administration sites of the animals in the test material sensitization group and the control group was shaved, and white petrolatum containing 10.0 % SLS was applied onto the skin. The next day, pieces of cotton lint (2 cm x 4 cm) that were coated with 0.5 mL of the test material (or vehicle) of the specified concentration were applied onto the same site, and fixed in place using tape.
- Duration: For epidermal induction, occlusive adhesion was carried out for 48 hours

B. CHALLENGE EXPOSURE
- Site: On Day 21, the fur on the right side of the abdomen of the animals in the test article sensitization group and the control group was shaved over an area of approximately 5 cm x 10 cm.
- Challenge exposure: The next day, 3 pieces of cotton lint (2 cm x 2 cm each) that were immersed in the 0.2 mL each of the 2 different concentrations of the test material and 0.2 mL of the vehicle were applied in locations 1 to 3 inside the shaved site, and fixed in place using tape.
- Exposure period: Occlusive adhesion was carried out for 24 hours.
- Evaluation (hr after challenge): Observations of the application sites were made 24 hours and 48 hours after removal of the application, and sensitivity was assessed. In order to eliminate bias resulting from the application site, the location for applying the material was made different for each animal.


OTHER:
Observations:
- Clinical observations: Observations were made for clinical signs once a day from Day 1 to Day 25 for all of the animals.
- Body weight: The body weight of all of the animals was measured on the day of intradermal induction (Day 1), epidermal induction (Day 8), challenge exposure (Day 22), and days that observations were made after challenge exposure (Day 24 and Day 25).
- Sensitisation: 24 hours and 48 hours after removal of the application for challenge exposure, the extent and frequency of erythema and oedema on the application site were graded based on the following grading criteria.
0: No visiable change
1: Discreate or pathcy erythema
2: Moderate and confluent erythema
3: Intense erythema and swelling

Challenge controls:

- No. of animals per dose: 5 animals.
- Concentrations: Test material sensitization group for 1.0 % challenge exposure and 0.5 % challenge exposure sites
Distilled water was used as the vehicle control substance.
Physiological saline solution was used for intradermal induction, distilled water was used for epidermal induction and the same vehicle was used for challenge exposure as the the test material sensitisation group.

Positive control substance(s):

no

Results and discussion

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0.5 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No reactions

Remarks on result:

no indication of skin sensitisation

Results 2

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

1.0 %

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No reactions

Remarks on result:

no indication of skin sensitisation

Any other information on results incl. tables

Observations

- Clinical observations: During the study period, there were no abnormalities in clinical signs observed.

- Body weight: A decrease in body weight was observed in 9/10 animals in the study group and 4/5 animals in the control group on Day 24, and in 3/10 animals in the study group and 1/5 animals in the control group on Day 25. The cause is not clear, but since a similar phenomenon was observed in the control group as well, it was determined that this decrease in body weight did not result from the test material.

- Challenge exposure: Skin reaction was not observed in the test material sensitization group for 1.0 % challenge exposure and 0.5 % challenge exposure sites. Skin reaction was also not observed in the control group as well as the area where distilled water for injection was applied.

Preliminary Test Resuts

Extreme necrosis was observed for all concentrations. As a result, the concentration of the solution used for intradermal induction was lowered to 0.1 %.

Skin irritation was observed for all concentrations. With the 25 % concentration, moderate skin irritation was observed, and with concentrations of 10 % and lower, slight skin irritation was observed.

As a result, the concentration for epidermal induction was set to 10 %, which is the highest concentration with which skin irritation was observed.

Immediately after removal of the application, slight to moderate erythema was observed with the 10 % and 5 % solutions, and slight erythema was observed with the 1 % and 0.5 % solutions. 24 hours after removal of the application, there were no skin reactions with regard to all of the concentrations.

The concentrations of the solutions for challenge exposure were set to 1 % and 0.5 %, for which only slight skin irritation was observed immediately after removal of the application.

Applicant's summary and conclusion

Interpretation of results:

other: Not classified according to EU criteria.

Conclusions:

Under the conditions of this study, it was concluded that the test material has no sensitisation potential.

Executive summary:

The potential for the test material to cause skin sensitization was assessed in a Guinea pig maximisation test. The study was conducted in accordance with generally accepted scientific principles, possibly with incomplete reporting or methodological deficiencies, which do not affect the quality of the relevant results. Ten Guinea pigs were exposed to the test material and 5 guinea pigs were used in the control group. During the ionduction exposure, 0.1 mL of the FCA emulsion fluid, 0.1 mL of the test article solution, and 0,1 mL of the test material/FCA emulsion fluid (or vehicle/FCA emulsion fluid) were intradermally induced in each pair over an area of 2 cm x 4 cm inside the shaved site. On the other hand, in the epidermal induction on Day 8, pieces of cotton lint (2 cm x 4 cm) that were coated with 0.5 mL of the test material (or vehicle) of the specified concentration were applied onto the same site, and fixed in place using tape. Occlusive adhesion was carried out for 48 hours.

Skin reaction was not observed in the test material sensitization group for 1.0 % challenge exposure and 0.5 % challenge exposure sites. Skin reaction was also not observed in the control group as well as the area where distilled water for injection was applied.

Under the conditions of this study, it was concluded that the test material has no sensitisation potential.