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Diss Factsheets

Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 Jan - 06 May 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2002
Report date:
2002

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
An in vivo skin sensitisation study according to OECD test guideline 406 was carried out in 1999, before the entry into force of the amendments to Annex VII.

Test material

Constituent 1
Chemical structure
Reference substance name:
Trimorpholinophosphine oxide
EC Number:
224-662-7
EC Name:
Trimorpholinophosphine oxide
Cas Number:
4441-12-7
Molecular formula:
C12H24N3O4P
IUPAC Name:
trimorpholinophosphine oxide
Test material form:
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 319 g (range from 301 to 339 g).

Identification and adaption
------------------------
Healthy young animals were allocated to the study groups at least 7 days before dosing to allow them to acclimatize. The guinea pigs were identified by earmark and colour.

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 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 (Art. 3418 for rabbits and guinea pigs, Provimi Kliba AG) and community tap water from Makrolon drinking bottles were available to the guinea pigs ad libitum.

The diet 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 21 to 22 °C and the relative atmospheric humidity 45 to 75%.

Study design: in vivo (non-LLNA)

Induction
Route:
intradermal and epicutaneous
Vehicle:
other: sodium chloride solution
Concentration / amount:
Induction (intradermal): 25 g/L
Induction (topical); 100 g/L
Day(s)/duration:
intradermal induction: 1 week; topical induction: 1 week
Adequacy of induction:
highest concentration used causing mild-to-moderate skin irritation and well-tolerated systemically
Challenge
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: sodium chloride solution
Concentration / amount:
Challenge: 100 g/L
Day(s)/duration:
1 day, 2 weeks after epicutaneouss induction; investigation for reactions 48 hours after start of the challenge
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
Total: 20 females
Pre-test: 5 females
Control group: 5 females
Test group 10 females
Details on study design:
The test material was prepared using sodium chloride solution or an FCA preparation (Freund´s complete adjuvant) directly prior to administration. No stability data of the test material in the vehicle is available.

Pretest:
To determine the suitable concemtrations for the study, a pretest was conducted with single intradermal or topical administration of the vehicle and of the test material. Intradermal injections were given to one animal. 4 further animals were used to determine the topical concentrations for the main study. 2 of the animals were exposed for 48 h without pretreatment to determine the concentration for the dermal induction. The 2 other animals were treated according to the control group incl. intradermal injections with FCA. One week after topical induction the 2 animals of the pretest group were exposed for 24 h to different test material concentratios to find out the challenge concentration.

RANGE FINDING TESTS:
- intradermal injection: 0, 1, 10, 25, 50 g/L (preparation in NaCl solution)
- topical applications, no pretreatment: 10, 100, 200, 400 g/L (preparation in NaCl solution)
- topical applications, with FCA: 10, 50, 100, 200 g/L (preparation in NaCl solution)

Main study:
Induction was performed in two stages. At first, the test material was intradermally injected with and without Freund´s complete adjuvant. Then, after one week, the test material was topically applied under occlusive patch conditions for 48 h. The challenge was performed 2 weeks later by topical application under the same conditions. The patches were then applied for 24 h on the shaven side of the animals.

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 in a total area of 2x4 cm)
- Test group: 0.1 ml FCA + NaCl solution (cranial), 25g/L test item in NaCl solution (medial), FCA + 25 g/L test item in NaCl solution (caudal)
- Control group: 0.1 ml FCA + NaCl solution (cranial), 0.1 ml NaCl solution (medial), 0.1 ml FCA + NaCl solution (caudal)
- Frequency of applications: single

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; 1 ml was applied
- Test groups: Test item (100 g/L) in NaCl solution
- Control group: NaCl solution
- Frequency of applications: single
- Duration: 48 h, occlusive
B. CHALLENGE EXPOSURE
- No. of exposures: two weeks after topical application; 0.5 ml was applied
- Day(s) of challenge: 24 h exposure duration (occlusive conditions)
- Test groups: 1g/L test item ad NaCl
- Control group: NaCl solution
- 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 MAGNUSSON and KLIGMAN scheme (0 = no reaction; 1 = scattered mild redness; 2 = moderate and diffuse redness; 3 = intense redness and swelling).
Positive control substance(s):
yes
Remarks:
alpha-Hexylcinnamaldehyde in PEG-400 (vehicle)

Results and discussion

Positive control results:
After induction (25 g/L) and challenge with alpha-Hexylcinnamaldehyde (10 g/L) positive reactions in 30 % of the animals in the first reading and 10 % of the animals in the 2ng reading were observed.

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
100 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
test chemical
Dose level:
vehicle (sodium chloride solution)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
100 g/L (challenge)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
vehicle (sodium chloride solution)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
100 g/L (challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
vehicle (sodium chloride solution)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
100 g/L (challenge)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
negative control
Dose level:
vehicle (sodium chloride solution)
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
vehicle (Polyethylene glycol 400)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
48
Group:
positive control
Dose level:
alpha-Hexylcinnanaldehyde, 10 g/L
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
vehicle (Polyethylene glycol 400)
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
72
Group:
positive control
Dose level:
alpha-Hexylcinnanaldehyde, 10 g/L
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
no signs of toxicity
Remarks on result:
positive indication of skin sensitisation

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
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 the 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 vehicle sodium chloride solution (group 1) and 10 females were treated with the test material (group 2).

Induction included intradermal injection of test material preparation (25 g/L with and without Freund's complete adjuvant) on experimental day 1, and topical application of test material preparation (400 g/L) for 48 hours on experimental day 8.

Challenge by topical application of the test material preparation (100 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

The animals did not show any clinical signs during the course of the study and no cases of mortality were observed. After challange no positive reaction was observed.

Conclusion

According to EU Regulation No. 1272/2008 (CLP), the test material is not classified as a skin sensitizer.