Diphosphoric acid, compd. with piperazine (1:1)

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

4 March to 28 March 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2003

Report date:

2003

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Test guideline 2

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:

This skin sensitisation study was performed before the testing guideline of the LLNA test was adopted.

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

457-330-7

EC Name:

-

Cas Number:

66034-17-1

Molecular formula:

Hill formula: C4H10N2:H4O7P2 CAS formula: C4H10N2:H4O7P2

IUPAC Name:

(phosphonooxy)phosphonic acid; piperazine

Test material form:

solid: particulate/powder

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

female

Details on test animals and environmental conditions:

Species: SPF -bred albino guinea pigs (Dunkin Hartley)
Supplier: Harlan, The Netherlands
Sex and age: females, ca 5 weeks at the start of the test
Date of arrival: 19 February 2003
Quarantine/acclimatization: 13 days
Start of study: 4 March 2003
Termination of study: 28 March 2003
Caging: in a mobile battery, containing 6 cages; maximal 10 animals per cage.
Lighting: 12 hours light/12 hours dark cycle
Temperature during testing: 20 ± 3 °C.
Relative humidity during testing: 30-70%. Upper limit incidentally up to 99.9%, because of meteorological circumstances and/or wet cleaning of the animal room.
Ventilation: ca 10 air changes/hour
Diet/water: standard laboratory diet ad libitum. Each batch of this diet is analysed by the supplier (SDS Special Diets services, Whitham, England) for the nutrients and contaminants and the results are kept available in the archives. Tap water (N.V. Hydron MiddenNederland) ad libitum. Results of routine physical, chemical and microbiological examination of drinking water as conducted by the supplier are kept available in the archives. The results of diet and water analyses were considered acceptable for this study.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

maize oil

Induction 2

Concentration / amount:

Intradermal treatment: 10, 3, 1 and 0.3%
Topical treatment: 30% and 10%
Challenge: 10%

Challenge

Challenge 1

Route:

epicutaneous, semiocclusive

Vehicle:

maize oil

Challenge 2

Concentration / amount:

Intradermal treatment: 10, 3, 1 and 0.3%
Topical treatment: 30% and 10%
Challenge: 10%

No. of animals per dose:

Preliminary study: 4 animals
Main study: 15 animals

Details on study design:

Preliminary experiments: The irritation response to intradermal injection of various concentrations of the test substance was examined in 2 guinea pigs. A sufficiently large area of the flanks was clipped free from hair with electric clippers. Amounts of 0.1 ml of the selected concentrations were applied by intradermal injection. Circa 24 hours after injection, the animals were examined for signs of irritation. A concentration causing slight to moderate irritation but otherwise well-tolerated by the animals, is usually taken for intradermal injection of the test substance in the induction phase of the main study. The irritation response to topical treatment of various concentrations of the test substance was examined in 2 other guinea pigs. The flanks of each of the animals were clipped free from hair with electric clippers. Patches (Silverpatch, v.d. Bend B.V., Brielle, the Netherlands) were loaded with the test material and placed on the clipped skin of each animal, and covered with a piece of hypoallergenic paper bandage (Leukopor) that was secured by elastic adhesive bandage (Tensoplast), wound around the torso of the animal. The dressing was left in place for ca 24 hours. Circa twenty four and 48 hours after removal of the dressing, the animals were examined for signs of skin irritation. A concentration causing slight to moderate skin irritation is usually chosen for topical induction and a non-irritant concentration for topical challenge.

Main study: Fifteen guinea pigs were randomly divided into two groups, viz. one test group of 10 animals and one control group of 5 animals. The animals were weighed one day before the study was initiated and at the completion of the study.
Induction was effected in two different ways, firstly by intradermal injections and secondly, one week later, by topical application over the injection sites.
Intradermal injections: For this purpose an area of about 24 cm2 of dorsal skin in the scapular region was clipped free from hair with electric clippers. Pairs of intradermal injections (0.1 ml each) were made simultaneously in the clipped area.
The following preparations were injected:
test animals
- two injections with Freund's Complete Adjuvant (FCA)/physiological saline (1: 1 ),
-two injections with the selected test concentration,
- two injections with the selected test concentration in FCA /vehicle ( 1: 1 ),
control animals
-two injections with FCA/physiological saline (1:1),
- two injections with the vehicle,
-two injections with FCA/vehicle (1:1).
Skin readings were made at ca 24 hours after the treatment.

Topical application: Six days after the intradermal injections, the dorsal skin in the scapular region of all test and control animals was closely clipped. On the following day, the induction by topical application was made in this region. The test animals were treated as follows:
A circa 2 x 4 cm patch of Whatman No. 3 MM filter paper was loaded with the selected concentration of the test substance. The loaded patch was placed over the sites of the intradermal injections and was secured as described above. The dressing was left in place for ca 48 hours. The controls were similarly treated with a patch loaded with the vehicle. Skin readings were made directly after removal of the patches.

The topical challenge with the test substance was carried out 14 days after the topical induction as follows:
An area of circa 5 x 5 cm on both flanks of each test and control animal was clipped free from hair. Patches were loaded with the test concentration selected and with the vehicle alone. Subsequently, the loaded patches were placed on the clipped area of the flanks of each test and control animal. The patches were covered with Leukopor bandage, and held in place by Tensoplast for ca 24 hours. Skin readings were made at ca 24 and 48 hours after removal of the patches.

Challenge controls:

The controls should not show any skin reaction. If so, the severity and type of reaction was taken into account upon evaluation of effects of the skin reactions exhibited by the test animals. In absence of skin reactions on the test site challenged with the test substance of the controls, the observed skin reactions on the challenged test site of the test animals were considered as signs of sensitization. If skin reactions are observed on the test site of the controls challenged with the test substance or with the vehicle alone or if skin reactions occur on the test site of the test animals challenged with the vehicle alone, the difference in type, severity, and persistence of these reactions were evaluated in order to establish whether or not the skin reactions of the test animals were indeed signs of sensitization.

Positive control substance(s):

yes

Remarks:

a-hexylcinnamaldehyde

Results and discussion

Positive control results:

The sensitivity of this test system was checked by means of a positive control study with α-hexylcinnamaldehyde.
The challenge treatment with a 20% and a 10% test dilution of α-hexylcinnamaldehyde in saline induced positive reactions in all test animals. Therefore, it was concluded that the experimental design and the strain of guinea pigs used for routine sensitization testing are suitable to detect possible sensitization potential of test materials.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

10%

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: slight erythema

Results 2

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

10%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

no indication of skin sensitisation

Results 3

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

0% - vehicle only

No. with + reactions:

4

Total no. in group:

10

Remarks on result:

other: slight erythema

Results 4

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

0% - vehicle only

No. with + reactions:

0

Total no. in group:

10

Results 5

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

10%

No. with + reactions:

3

Total no. in group:

4

Remarks on result:

other: slight erythema

Results 6

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

10%

No. with + reactions:

0

Total no. in group:

4

Results 7

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0% - vehicle only

No. with + reactions:

3

Total no. in group:

4

Remarks on result:

other: slight erythema

Results 8

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

0% - vehicle only

No. with + reactions:

0

Total no. in group:

4

Results 9

Key result

Reading:

1st reading

Hours after challenge:

24

Group:

positive control

Dose level:

both dose levels 10% and 20%

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Results 10

Key result

Reading:

2nd reading

Hours after challenge:

48

Group:

positive control

Dose level:

both dose levels 10% and 20%

No. with + reactions:

5

Total no. in group:

5

Remarks on result:

positive indication of skin sensitisation

Any other information on results incl. tables

Preliminary experiments: The degree of irritation observed after intradermal treatment with a 0.3% test concentration was considered acceptable for intradermal treatment during the induction phase. A 30% and a 10% test concentration were considered acceptable for topical treatment during the induction and the challenge phase, respectively. A concentration higher than 30% was considered unsuitable for topical application in this test. Since the 30% test concentration was irritating, it was decided not to pre-treat the induction test site with a 10% sodium lauryl sulfate solution (SLS) in vase line.

 

Main study: One control animal (no. 51) was found with a prolapse of the rectum on 13 March 2003. The animal was humanely sacrificed. All other animals remained in good health and gained body weight in a normal way during the experimental period.

Induction: The intradermal injections generallycaused the following skin reactions:

test animals

- FCA/physiological saline (1: 1 ): moderate erythema,

- selected test concentration: moderate erythema and abscesses,

- selected test concentration in FCA/vehicle (1:1): moderate erythema and abscesses,

control animals

- FCA/physiological saline (1:1): moderate erythema,

- vehicle: no skin reactions,

- FCA/vehicle (1:1): moderate erythema.

After the topical application of the vehicle alone, no skin reactions were observed in controls. After the 48-hour topical application of the selected test concentration, no skin reactions were observed in the test animals.

Challenge: At 24 hours after the challenge treatment with the vehicle alone, 4 test animals and 3 controls showed slight erythema.

At 24 hours after the challenge treatment with the test substance, also 4 test animals and 3 controls showed slight erythema.

At 48 hours after challenge with the test substance or with the vehicle alone, none of the controls or test animals showed any skin reaction.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Criteria used for interpretation of results: EU

Conclusions:

Since none of the test animals showed positive signs of sensitization, it can be concluded that T-1063FM is not a sensitizer.