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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:
August 2, 1994 - October 7, 1994
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1995
Report date:
1995

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
yes
Remarks:
(No positive control was performed).
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
(No positive control was performed).
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
The study was performed in 1994.

Test material

Constituent 1
Chemical structure
Reference substance name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
EC Number:
418-570-8
EC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Cas Number:
25383-07-7
Molecular formula:
C11H20NO5P
IUPAC Name:
(R)-α-phenylethylammonium (-)-(1R, 2S)-(1,2-epoxypropyl)phosphonate monohydrate
Details on test material:
- Name of test material (as cited in study report): Fosfomycin PEA salt
- Physical state: white crystalline powder
- Analytical purity: 99.8 %
- Lot/batch No.: 4177
- Storage condition of test material: screw capped plastic container, at room temperature protected from light.
- Other: manufacturing date: June 10, 1994

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A
- Age at study initiation: 6 weeks
- Weight at study initiation: 364-465 g
- Housing: 2 or 3 animals/cage in an air-conditioned room. Cage: wire cages (40.5x38.5x18h cm) with a stainless steel feeder.
- Diet (e.g. ad libitum): Ad libitum (GLP diet - certificate coded 8 GP 22)
- Water (e.g. ad libitum): Ad libitum (from the municipal water main, filtered)
- Acclimation period: Ten days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 ºC
- Humidity (%): 55 ± 10 %
- Air changes (per hr): 20 per hour (filtered on HEPA 99.97%)
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h light (7 a.m. - 7 p.m.)

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal and epicutaneous
Vehicle:
other: Intraderma: water. Epicutaneous: vaseline oil.
Concentration / amount:
Induction: intradermal: 55 and 27.5 mg/ml
Booster: epicutaneous: 55 mg/ml
Challenge: epicutaneous, occlusive: 55 mg/ml
Challengeopen allclose all
Route:
epicutaneous, occlusive
Vehicle:
other: Intraderma: water. Epicutaneous: vaseline oil.
Concentration / amount:
Induction: intradermal: 55 and 27.5 mg/ml
Booster: epicutaneous: 55 mg/ml
Challenge: epicutaneous, occlusive: 55 mg/ml
No. of animals per dose:
10 animals for the treated group.
5 animals for the control group.
2 animals for the preliminary test.
Details on study design:
RANGE FINDING TESTS: Four concentrations 55, 27.5, 13.75 and 6.875 mg/ml, were assayed for intradermal injection and two concentrations 55 and 13.75 mg/ml, for the patch application. Each test article concentration was tested on 2 animals. 0.1 ml of each test article concentration was injected intradermally, while occlusive patches (2x2 cm) loaded with two test article concentrations were applied to the skin. Twenty-four hours after the administration, the patches were removed and the animals observed for up to 48 h for local reactions on the skin areas. All concentrations tested resulted well tolerated and non-irritating for both applications. Therefore, in the experiment the test article was used at the concentration of 55 mg/ml (corresponding to the maximum solubility in the vehicle).

MAIN STUDY
A. INDUCTION EXPOSURE
INTRADERMAL:
(Day 0) After clipping the fur from an area of 4x6 cm (day -1), each animal was given three pairs of intradermal injections so that there were two rows of three injections, one on each side of the midline.
- Test groups: 1 (10 animals)
1) 0.1 ml FCA emulsion (1:1 mixture (v/v) FCA/water)
2) 0.1 ml test article alone
3) 0.1 ml test article in a 1:1 mixture (v/v) FCA/water (test article solubilized in water for injection prior to mixing with FCA).
- Control group: 1 (5 animals)
1) 0.1 ml FCA emulsion (1:1 mixture (v/v) FCA/water)
2) 0.1 ml vehicle alone
3) 0.1 ml at 50% w/v formulation of the vehicle in a 1:1 mixture (v/v) FCA/water
- Site: shoulder region
- Frequency of applications: single
- Concentrations: 55 mg/ml (test article) and 27.5 mg/ml (test article/FCA/water)
- Doses: 16.5 mg/animal
- Evaluation (hr after injection): 24 hours later the injection sites were observed.

EPICUTANEOUS (Booster):
(Day 6) The area destined to receive the booster (that used for the induction), was clipped and treated with 0.5 ml of 10% sodium lauryl sulfate in vaseline oil, in order to create a local irritation and therefore to enhance skin permeability to the compound. In the following day (day 7) a filter paper (2x4 cm) was fully-loaded with the test article or vehicle and applied to the skin areas. The patch was covered by an overlapping, impermeable, hypoallergenic plastic adhesive tape. This in turn was occluded by use of a thin film of polyethylene and held in place by Poroplast. The dressing was left in place for 48 h.
- Test groups: 1 (10 animals)
- Control group: 1 (5 animals)
- Site: shoulder region
- Exposure period: 48 h
- Concentrations: 55 mg/ml (test article)
- Doses: 16.5 mg/animal (0.3 ml/patch)
- Evaluation (hr after booster): 24 hours after removal of the patches.

B. CHALLENGE EXPOSURE (epicutaneous)
(Day 21) after clipping an area about 5x5 cm on both flanks of the animals, occlusive patches (2x2 cm) loaded with the test article and the vehicle respectively were applied to the animals of the two groups.
- Test groups: 1 (10 animals)
- Control group: 1 (5 animals)
- Site: Left flank (patch with the test article) and right flank (patch with the vehicle alone).
- Exposure period: 24 hours
- Concentrations: 55 mg/ml
- Doses: 16.5 mg/ animal (0.3 ml/patch)
- Evaluation (hr after challenge): 21 h after removing the patches the challenge areas were clipped and 3 h later (48 h from the start of the challenge application) the skin reaction was observed (day 23). 24 h after, a second observation (72 h) was made (day 24).
Positive control substance(s):
no

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
16.5 mg/animal
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 16.5 mg/animal. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None..
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
16.5 mg/animal
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
None.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 16.5 mg/animal. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: None..
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
0 mg/animal
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None.
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0 mg/animal. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None..
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
0 mg/animal
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
None.
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0 mg/animal. No with. + reactions: 0.0. Total no. in groups: 5.0. Clinical observations: None..

Any other information on results incl. tables

The body weight gain of the animals throughout the test was normal.

24 h after the intradermal injections all animals were in good health and, as expected, at each injection site of FCA emulsion (FCA/water), FCA/vehicle and FCA/test article a swollen reddish area was seen, while no reaction was seen after injection of the test article or vehicle alone.

Piloerection was observed in animals of the treated group 24 h after the removal of the 48 h closed patch (booster), associated in some of them with a slight body weight loss, which was recorded after 4 days. No clinical sign was observed in animals of negative control group.

Signs of irritation such as redness and thickening were seen in the area of the skin where the test article or vehicle was applied due to the sodium lauryl sulfate application.

No animals showed positive reactions at the challenge performed using an occlusive patch.

No skin reactivity was observed in the negative control group.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Remarks:
EU criteria.
Conclusions:
Under the experimental conditions applied, Fosfomycin PEA salt did not appear to possess sensitizing capacity.
Executive summary:

The contact sensitivity is a T-lymphocyte-mediated delayed hypersensitivity reaction. The immunological events in skin sensitisation can be separated into two main phases: development of sensitization and elicitation of clinical effects following subsequent exposure to the same chemical.

The sensitizing potential of the test article Fosfomycin PEA salt was assessed in guinea-pigs using the maximization test of Magnusson and Kligman according to EU Method B.6. and OECD Guideline 406. The test animals were initially exposed to the test substance by intradermal injection (day 0) and epidermal application (day 7). Following a rest period, during which an immune response may developed, the animals were exposed to a challenge dose (55 mg/ml, day 21) for a 24 h period of time. The extent and degree of skin reaction to the challenged exposure (at 48 and 72 h of the start of the challenge) was observed and compared with those demonstrated by control animals.

No animal treated with the test article concentration applied showed positive reaction at the challenge.

No skin reactivity was observed in the negative control group.

On the basis of this result, under the experimental conditions applied, Fosfomycin PEA salt did not appear to possess sensitizing capacity.