Pyrazino[2,1-a]isochinolin-4-on, 4H-, 1,2,3,6,7,11b-hexahydr o-, Diphosphat

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 10, 2002 - January 24, 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in compliance with the Good Laboratory Practice (GLP) regulations (revised in 1997, ENV/MC/CHEM(98)17). The method followed that described in the OECD Guidelines for Testing of Chemicals, Updated Guideline No 406 Skin Sensitisation (adopted 17 Julyl 1992).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Principles of method if other than guideline:

None

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Justification for non-LLNA method:

This assay was performed before OECD 429 was default requirement fort his endpoint. However, the data are adequate and reliable to fill the endpoint with sufficient information.

Test material

Test material information

Constituent 1

Reference substance name:

Automatically generated during migration to IUCLID 6, no data available

IUPAC Name:

Automatically generated during migration to IUCLID 6, no data available

Test material form:

other: solid

In vivo test system

Test animals

Species:

guinea pig

Strain:

other: HsdPoc:DH

Sex:

female

Details on test animals and environmental conditions:

Test System

Guinea pig, HsdPoc:DH, females
Breeder: Harlan Winkelman GmbH, Borchen Age: about 5 weeks
The mean initial body weight at the start of the study was 328 g (range from 302 to 372 g).
In a large number of tests, with a variety of test materials, the guinea pig and the specific strain used has proven to be a species well suited for skin sensitization studies.

Identification and adaption
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow for acclimatization. The guinea pigs were identified by earmark and color.


Assignment
20 guinea pigs were used in this study.

Pretest: 5 females
Group 1: negative control group (5 females)
Group 2: test material group (10 females)


- Housing:
Five guinea pigs were housed in type GMI5 (EBECO) Makrolon cages with a shelter and placed on mobile racks. The animals were kept on conventional softwood granulate as the bedding. The bedding was changed two times a week.

- Diet (e.g. ad libitum):
Diet and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum.

The diet, Provimi Kliba 3418.0, was checked according to the specifications of the manufacturer by independent laboratories. Analysis included both qualitative and quantitative evaluation for heavy metals, aflatoxins, pesticides and antibiotics. The tap water was analyzed periodically according to the German regulations for human drinking water. The softwood granulate was analytically checked by independent laboratories.



ENVIRONMENTAL CONDITIONS
Temperature and atmospheric humidity were measured by a thermohygrograph. The room temperature within the study period was 20 to 21 °C and the relative atmospheric humidity 53 to 63%.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

other: intradermal and topical

Vehicle:

other: NaCl isotonic

Induction 2

Concentration / amount:

PRE TEST
intradermal: 50, 25, 10, 5 and 1 g/L
topical: 100, 50, 10, and 1 g/L
topical with FCA: 50, 25, 10, and 5 g/L

MAIN STUDY
100 mL of 5 g/L solution

Challenge

Challenge 1

Route:

other: topical

Vehicle:

other: NaCl isotonic

Challenge 2

Concentration / amount:

PRE TEST
intradermal: 50, 25, 10, 5 and 1 g/L
topical: 100, 50, 10, and 1 g/L
topical with FCA: 50, 25, 10, and 5 g/L

MAIN STUDY
100 mL of 5 g/L solution

No. of animals per dose:

Total: 20 females
Pre-test: 5 females
Control group: 5 females
Test group 10 females

Details on study design:

RANGE FINDING TESTS:
- intradermal: 50, 25, 10, 5 and 1 g/L
- topical: 100, 50, 10, and 1 g/L
- topical with FCA: 50, 25, 10, and 5 g/L



MAIN STUDY
A1. INDUCTION EXPOSURE (intradermal injection)
- No. of exposures: 6 injections of 0.1 ml each, three on each side of the spinal column)
- Test groups: 0.1 ml FCA + NaCl solution, 5g/L test item ad NaCl solution, FCA + 5 g/L test item + NaCl solution
- Control group: 0.1 ml FCA + NaCl solution, 0.1 ml NaCl solution, 0.1 ml FCA + NaCl solution
- Site: cranial, medial, caudal
- Frequency of applications: single
- Duration:
- Concentrations: 5 g/L

A2. INDUCTION EXPOSURE (topical application)
- No. of exposures: one week after injection (A1) single topical application of a filter plate soaked with either test solution, or vehicle
- Test groups: Test item (5 g/L) ad NaCl solution
- Control group: NaCl solution
- Site: cranial, medial, caudal
- Frequency of applications: single
- Duration: 48 h, occlusive
- Concentrations: 5 g/L

B. CHALLENGE EXPOSURE
- No. of exposures: single
- Day(s) of challenge: 1d
- Exposure period: 2 weeks after topical application
- Test groups: 25g/L test item ad NaCl solution
- Control group: naCl solution
- Site: cranial, medial, caudal
- Concentrations: 25 g/L
- Evaluation (hr after challenge): 48 and 72 h

-- Observations
During the induction phase, the guinea pigs were examined on days 2, 3, 7, 11, 12, 15, and 22 of the experimental part for local skin reactions and the results were documented.
The behavior and general condition of all animals were monitored daily. The challenge sites were investigated for reactions 48 hours after start of the challenge. Further inspections followed 72 hours after start of the challenge, to detect weak or slowly developing reactions.
After the challenge, skin changes at the application sites were evaluated according to the following MAGNUSSON and KLIGMAN scheme.

Positive control substance(s):

yes

Remarks:

Hexylcinnamaldehyde

Results and discussion

Positive control results:

50% positive reactions with control substance

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 g/L (induction I), 5 g/L (induction II), 25 g/L (challenge)

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 g/L (induction I), 5 g/L (induction II), 25 g/L (challenge). No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: no signs of toxicity.

Results 2

Reading:

2nd reading

Hours after challenge:

72

Group:

test chemical

Dose level:

5 g/L (induction I), 5 g/L (induction II), 25 g/L (challenge)

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

no signs of toxicity

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 72.0. Group: test group. Dose level: 5 g/L (induction I), 5 g/L (induction II), 25 g/L (challenge). No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: no signs of toxicity.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information Criteria used for interpretation of results: other: CLP REGULATION (EC) No 1272/2008

Conclusions:

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.

Executive summary:

Purpose

The purpose of this GPMT assay was to identify the contact allergenic potential of teh test material. This study should provide a rational basis for risk assessment to the sensitising potential of the test item in man.

Study Design

The test material was investigated for skin sensitizing properties in the guinea pig maximization test according to MAGNUSSON and KLIGMAN (1969). 5 females were treated with the vehicles NaCl solution (100 g/L) (group 1) and 10 females were treated with the test material (group 2). Induction included intradermal injection of test material preparation (5 g/L) in NaCl solution with and without Freund's complete adjuvant on experimental day 1, and topical application of test material preparation for 48 hours on experimental day 8. Challenge by topical application of the test material preparation (25 g/L) for 24 hours was performed two weeks after topical induction and readings were taken at 48 hours and 72 hours after start of treatment.

Results

After challenge, no positive reactions in the test material treated skin sites at the first reading were seen. At the second reading one animal showed positive reaction. This results in 10 % positive reactions after challenge.

Conclusion

Under the given experimental conditions, the test material induced no reactions. According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.