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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:
not specified
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions

Data source

Reference
Reference Type:
publication
Title:
Evaluation of the skin sensitizing potential of biodegradable magnesium alloys
Author:
Witte, F. et al.
Year:
2007
Bibliographic source:
Journal of Biomedical Materials Research Part A 86A: 1041 - 1047.

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Version / remarks:
1992-07-17
Deviations:
yes
Remarks:
purity/stability missing; negative control group treated with a skin irritant instead of the test items during challenge; topical induction lasted only 24 h; observation 48 h after patch removal missing; slightly modified Magnusson & Kligman scale used
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
not specified

Test material

Constituent 1
Chemical structure
Reference substance name:
Magnesium
EC Number:
231-104-6
EC Name:
Magnesium
Cas Number:
7439-95-4
Molecular formula:
Mg
IUPAC Name:
magnesium
Test material form:
solid
Details on test material:
- Name of test materials:
1) AZ31 (content: MgAl (2.4 - 3.6); Zn (0.5 - 1.5); Mn (0.15 - 1.0); Si (0.1); Cu (0.1))
2) AZ91 (content: MgAl (8.1 - 9.3); Zn (0.4 - 1.0); Mn (0.17 - 0.35); Si (0.2))
3) LAE442 (content: MgLi4Al (3.4 - 4.6); RE (1.8 - 3.0); Mn (min. 0.25); Zn (max. 0.22))
4) WE43 (content: MgY (3.7 - 4.3); RE (2.4 - 4.4); Zr (min: 0.4 - 1.0); Li (0.2); Mn (0.15))
Numbers in parenthese represent the percentage of the element content in the magnesium alloy displayed in weight percentage (wt %). The balance in each magnesium alloy consists of pure magnesium (Mg). RE stands for rare earth elements

Supplier
AZ31: Dead Sea Magnesium, Beer Sheva, Isreal
AZ91, WE43, and AE42: Magnesium Elektron, Manchester, UK
The magnesium alloy LAE442 was produced in the laboratory by adding 4 weight percent of lithium to magnesium alloy AE42.
Specific details on test material used for the study:
TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance were produced by carefully machining the alloys to small chips. Only chips of less than 0.8 x 0.8 x 0.2 mm³ were selected as substances for epicutanoeus tests.

FORM AS APPLIED IN THE TEST
All test materials were tested both in a dissolved and in a solid state. All materials were dissolved by boiling in 2M hydrochloride acid and buffered with 1M sodium hydroxide at pH 5.5. The supernatants were microfiltrated (22 µm) and the ional metal content was confirmed by inductively coupled plasma atomic emission spectrometry (ICP-AES).

In vivo test system

Test animals

Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Females (if applicable) nulliparous and non-pregnant: not specified
- Weight at study initiation: between 400 and 550 g
- Housing: housed in pairs in clear plastic cages (55 x 32.8 x 19 cm³) on standard bedding
- Diet (ad libitum): standard pellet diet
- Water (ad libitum)

ENVIRONMENTAL CONDITIONS
- Temperature: 20 ± 3 °C
- Relative humidity: 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (non-LLNA)

Inductionopen allclose all
Route:
intradermal
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % of dissolved test item
Day(s)/duration:
day 0
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % of dissolved test item
Day(s)/duration:
day 7 (duration: 24 hours)
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Route:
intradermal
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % test item
Day(s)/duration:
Day 0
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
100 % of test item
Day(s)/duration:
Day 7 (duration: 24 hours
Adequacy of induction:
non-irritant substance, but skin pre-treated with 10% SDS
Challengeopen allclose all
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % of dissolved test item
Day(s)/duration:
day 22 (duration: 24 hours)
Adequacy of challenge:
other: non-irritant concentration
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
unchanged (no vehicle)
Concentration / amount:
100 % of test item
Day(s)/duration:
Day 22 (duration: 24 hours)
Adequacy of challenge:
other: non-irritant concentration
No. of animals per dose:
20 guinea pigs/ alloy (10 animals were tested with test substance in a solid state and a dissolved state)
Details on study design:
All test materials were tested both in a dissolved and in a solid state.

RANGE FINDING TESTS:
Preliminary patch tests were conducted to establish a metal concentration of the tested solutions demonstrating no systemic or local skin toxicity in 4 guinea pigs (one animal/test item).

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: 2 (intradermal injection and topical application)
- Site: an area of 4 x 6 cm² was clipped between shoulders for intradermal induction. Seven days later the area was again clipped and shaved for topical induction.
- Frequency of applications: a total of six intradermal injections of 0.1 mL each were made in two rows of three injections each. The two cranial injections contained FCA (Freund's Complete AdjuvantTM) and physiological saline solution of equal volume parts. The pair of caudal injections contained FCA and test substance in equal volume parts.Seven days later, topical induction was performed. To get a deeper skin infiltration of the test substance, the shaved area was treated with 10 % sodium.lauryl-sulfate in petrolatum 24 hours before topical induction.
- Exposure period: 24 hours (dermal application)

B. CHALLENGE EXPOSURE
- No. of exposures: 1 (topical challenge)
- Exposure period: 24 hours
- Site: the epicutanoues challenge was performed by clipping and shaving another area of 5 x 5 cm² on the left flank.
- Evaluation (hr after challenge): immediately and 24 hours after patch removal
Grading of the skin reaction (erythema classification) according to Magnusson-Kligman test:
0 = no reaction
1 = mild redness, no swelling
2 = moderate and diffuse redness, no swelling
3 = intensive redness and swelling
4 = necrosis
A skin reaction graded greater than zero was defined as erythema.

OTHER
Additional skin biopsies were harvested 24 hours after patch removal for histomorphologic analysis. Epidermis and dermis were included in cell counting and histopmorphological analysis (Hunziker & Olmos, 1976)*. The criteria for positive reaction were oedema, dermal spongiosis, perivascular-, diffuse dermal-, and epidermal-mononuclear cell infiltration, especially basophile cells (Magnusson & Ligman, 1969; Hunziker & Olmos, 1976; Lindberg et al., 1988; Turk & Parker, 1977; Zissu et al., 1996)*. To be considered as a positive skin reaction, all morphological criteria had to be present . The number of basophile cells was counted 24 hours after patch-removal in visual fields of the dermal-epidermal border zone until 400 leucocyte cells had been counted on paraffin sections, which were stained (Robinson et al., 1990; Anderson, 1985; Groth & Skoog, 1979; Sjogren & Anderson, 2000)

STATISTICAL
Pearson coefficient has been used to estimate the correlation of variables. Differences between groups were detected using an ANOVA and the Tukey HSD post hoc test. Statistically significant differences were defined as p < 0.05.

*References:
- Hunziker N, Olmos L. Basophils in the infiltrate of guinea pigs experimental contact dermatitis. Dinitrochlorobenzene and citraconic anhydrife. Dermatologica 1976; 152: 270 - 280.
- Magnusson B, Ligman AM. The identification of conact allergens by animal assay. The guinea pig maximization test. J Invest Dermatol 1969; 52: 268 - 276.
- Lindberg LR, Johnell O, Linder L. Reactions in rat gluteal muscle to titanium implants. Biomaterials 1988, 9: 547 - 549.
- Turk JL, Parker D. Sensitization with Cr, Ni and Zr salts and allergic type granuloma formation in the guinea pig. J Invest Dermatol 1977; 68: 341 - 345.
- Zissu D, Binet S, Cavelier C. Patch testing with beryllium alloy samples in guinea pigs. Contact Dermatitis 1996; 34: 196 - 200.
- Robinson MK, Fletcher ER, Johnson GR, WyderWE, Maurer JK. Value of the cutaneous basophil hypersensitivity (CBH) response for distinuishing weak contact sensitization from irritation reactions in the guinea pig. J. Invest Dermatol 1990; 94: 636 - 643.
- Anderson C. The effect of selected immunomodulating agents on experimental contact reactions. Acta Derm Venereol Suppl (Stockh) 1985; 116: 1 - 48.
- Groth O, Skoog ML. Measurement and differentiation of the cellular infiltrate in experimental allergic contact dermatitis. Acta Derm Venereol 1979; 59: 129 - 134.
- Sjogren F, Anderson C. The spectrum of inflammatory cell response to dimethyl sulfoxide. Contact Dermatitis 2000; 42: 216 - 221.
Challenge controls:
15 guinea pigs were used as negative control animals.
The negative control group was treated with the solutions in which the test substances were dissolved together with sodium-lauryl-sulfate (SLS).
Positive control substance(s):
yes
Remarks:
hydroxy-cinnamon-aldehyde (HCA); number of treated animals: 15

Results and discussion

Positive control results:
NOTE: the positive skin reaction induced by the positive control is confirmed by an erythema that persists for 24 hours after patch removal.

- all guinea pigs exposed to the positive control showed persisting erythema for more than 24 hours after patch removal.
- one animal died during the challenge phase for reasons not related to the test and one skin biopsy was lost.
- histological analysis showed in 12 (92.3 %) of the remaining 13 biopsies all four criteria of allergy such as spongiosis, oedema, and diffuse as well as perivascular mononuclear infiltrates.
- positive control biopsies contained a mean of 52.7 basophile cells/400 leucocyte cells.
- in biopsies of the positive control group, a signficiant higher number of basophile cells was found compared to the negative conrol group and to all tested substnaces (p = 0.001, ANOVA).

In vivo (non-LLNA)

Resultsopen allclose all
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
negative control
Dose level:
sodium lauryl sulfate
No. with + reactions:
0
Total no. in group:
15
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
positive control
Dose level:
HCA
No. with + reactions:
15
Total no. in group:
15
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group AZ91
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
3
Total no. in group:
10
Clinical observations:
Mild skin reaction was observed. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ91)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Mild clinical skin response was observed. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ31)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
All erythema had faded. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ31)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (WE43)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (WE43)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (LAE442)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of dissolved test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (LAE442)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ91)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
2
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ91)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
10 % of solid test item
No. with + reactions:
9
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (AZ31)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (AZ31)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (WE43)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Animals gained body weight during the study.
Remarks on result:
other: test group (WE43)
Key result
Reading:
1st reading
Hours after challenge:
0
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (LAE442)
Key result
Reading:
2nd reading
Hours after challenge:
24
Group:
test chemical
Dose level:
100 % of solid test item
No. with + reactions:
1
Total no. in group:
10
Clinical observations:
Skin areas displayed erythema. Animals gained body weight during the study.
Remarks on result:
other: test group (LAE442)

Any other information on results incl. tables

Negative control

- no erythema was observed in all guinea pigs exposed to the standard irritant sodium-lauryl-sulfate.

- in 80 % of all histological sections, none or only one histomorphological criterion for contact dermatitis could be found.

- no skin biopsy displayed all four histomorphological criteria of allergy.

- mean number of basophile cells in the negative control was 6.33/400 leucocyte cells.

Solid test substances

- to identiy allergic erythema after 24 hours, dermal biopsies were taken.

- all biopsies exhibited significantly (p = 0.001) less histomorphological criteria of allergenicity compared to the positive control group.

- in all biopsies, no significant differences were found for basophil cells compared to the negative control.

- LAE442 group: one animal died unrelated to the study conditions.

- animals treated with AZ91 and LAE442 which still had an erythema 24 hours after patch removal, showed no criteria of allergenicity in histomorphological analysis.

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
The substances were not skin sensitisers.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.