Registration Dossier

Diss Factsheets

Administrative data

Description of key information

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Total amount of test material applied is not stated
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was conducted due to non-REACH regulatory requirements. With the existing data from this study not only being acceptable but of good quality (Klimisch Score 1), this study precludes the need for an additional LLNA study. In addition, a supplementary LLNA study would violate the ECHA objectives with regards to animal welfare.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 41-9660

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS
- pH (as 1% solution): 5
- Homogeneity : homogeneous by visual inspection
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-16s-Elbeuf, France
- Age at study initiation: 3 months
- Weight at study initiation: 374 ± 22 g
- Housing: individually in polycarbonate cages with stainless steel lid
- Diet: "106 pelleted diet" ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature : 21 ± 2°C
- Relative humidity : 30 to 70%
- Light/dark cycle: 12 h/12 h
- Ventilation: approximately 12 cycles/hour of filtered, non-recycled air
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
intradermal injections: test material at the concentration of 0.5 % (w/w) in corn oil,
topical application: test material at the concentration of 30 % (w/w) in corn oil
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
intradermal injections: test material at the concentration of 0.5 % (w/w) in corn oil,
topical application: test material at the concentration of 30 % (w/w) in corn oil
No. of animals per dose:
5 females/control group
10 females/treated group
Details on study design:
RANGE FINDING TESTS:
In order to determine the concentration of the test substance in the main study one range finding test were performed on two animals (1 male and 1 female).
By intradermal route (tested concentrations: 1 % and 0.1 % (w/w):
24 hours before treatment, the dorsal region of the animals was clipped, intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region, cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route (tested concentrations: 30 % and 10 % (w/w):
24 hours before treatment, both flank regions of the animals were clipped, the filter paper of a chamber was fully-loaded with one dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank) . The chamber was held in place by means of an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: once intradermal (day 1) and additionally once cutanenously (day 8)
- Test groups: 1
- Control group: 2

INTRADERMAL EXPOSURE
- Site: six injections as pairs in the interscapular area
- Treatment: Test group: A) front row: Freund's complete adjuvant (FCA) at 50 % (v/v) in 0.9% NaCl; B) middle row: 0.1 mL test material at 0.5 % (w/w) in corn oil; C) test substance at 0.5 % (w/w) in the mixture FCA/0.9 % NaCl (50/50)
Control groups: The animals were given the same injections (A, B, C) but without test substance, only with the formulating agent.

CUTANEOUS EXPOSURE
- Site: interscapular area
- Treatment: Test group: a pad of filter paper (approximately 8 cm²) was fully-loaded with the test substance at the concentration of 30 % (w/w) and was then applied to the interscapular region of the animals. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster
Control groups: received an application of the vehicle alone under the same experimental conditions.

B. CHALLENGE EXPOSURE
Challenge:
- Day of challenge: day 22 (21 days after intradermal induction)
- Exposure period: 24 h
- Site: interscapular
- Treatment: Test groups: received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chambero) was fully-loaded with the test substance at the concentration of 30 % (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive an allergenic waterproof plaster. On removal of the dressing, any residual test substance was removed by means of a moistened
gauze pad.
Control groups: Control group one was treated like the treatment group; control group 2 remained untreated
- Evaluation (hr after challenge): 24, 48 h

Rechallenge:
- Day of challenge: day 29
- Exposure period: 24 h
- Treatment: the animals of all groups received an application of the test substance at the concentration of 30% (w/w) to the anterior left flank and the vehicle to the anterior right flank, under the same experimental conditions as for the first challenge application.
- Evaluation (hr after challenge): 24, 48 h
Challenge controls:
Yes, 2 groups
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
3
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
other: Negative control group 1
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Negative control group 1
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Negative control group 1. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness of skin in 3/10 animals
Reading:
rechallenge
Hours after challenge:
24
Group:
other: Negative control group 1
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
48
Group:
other: 1egative control group 1
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Dryness of skin was observed in 1/5 animals
Reading:
rechallenge
Hours after challenge:
24
Group:
other: Negative control group 2
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
48
Group:
other: negative control group 2
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10

One animal of the control group 1 was found dead on day 13. Hypoactivity and dyspnea were observed prior to death. The authors stated that such spontaneous clinical signs and mortality are sometimes observed in this species.

Interpretation of results:
not sensitising
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be non-sensitizing only the EDTA-Ni complex must be regarded as dagerous. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their effects. In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-Ni(NH4)2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
Total amount of test material applied is not stated
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
This study was conducted due to non-REACH regulatory requirements. With the existing data from this study not only being acceptable but of good quality (Klimisch Score 1), this study precludes the need for an additional LLNA study. In addition, a supplementary LLNA study would violate the ECHA objectives with regards to animal welfare.
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Lot/batch No.of test material: 41-9660

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at room temperature

OTHER SPECIFICS
- pH (as 1% solution): 5
- Homogeneity : homogeneous by visual inspection
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River France, 76410 Saint-Aubin-16s-Elbeuf, France
- Age at study initiation: 3 months
- Weight at study initiation: 374 ± 22 g
- Housing: individually in polycarbonate cages with stainless steel lid
- Diet: "106 pelleted diet" ad libitum
- Water: ad libitum
- Acclimation period: at least 5 days


ENVIRONMENTAL CONDITIONS
- Temperature : 21 ± 2°C
- Relative humidity : 30 to 70%
- Light/dark cycle: 12 h/12 h
- Ventilation: approximately 12 cycles/hour of filtered, non-recycled air
Route:
intradermal and epicutaneous
Vehicle:
corn oil
Concentration / amount:
intradermal injections: test material at the concentration of 0.5 % (w/w) in corn oil,
topical application: test material at the concentration of 30 % (w/w) in corn oil
Route:
epicutaneous, occlusive
Vehicle:
corn oil
Concentration / amount:
intradermal injections: test material at the concentration of 0.5 % (w/w) in corn oil,
topical application: test material at the concentration of 30 % (w/w) in corn oil
No. of animals per dose:
5 females/control group
10 females/treated group
Details on study design:
RANGE FINDING TESTS:
In order to determine the concentration of the test substance in the main study one range finding test were performed on two animals (1 male and 1 female).
By intradermal route (tested concentrations: 1 % and 0.1 % (w/w):
24 hours before treatment, the dorsal region of the animals was clipped, intradermal injections of the dosage form preparations (0.1 mL) were performed in the interscapular region, cutaneous reactions were evaluated approximately 24, 48 hours and 6 days after the injections.
By cutaneous route (tested concentrations: 30 % and 10 % (w/w):
24 hours before treatment, both flank regions of the animals were clipped, the filter paper of a chamber was fully-loaded with one dosage form preparation. The chamber was then applied to the clipped area of the skin (one concentration per flank) . The chamber was held in place by means of an occlusive dressing for 24 hours, cutaneous reactions were evaluated approximately 24 and 48 hours after removal of the dressings.

MAIN STUDY
A. INDUCTION EXPOSURE
- No. of exposures: once intradermal (day 1) and additionally once cutanenously (day 8)
- Test groups: 1
- Control group: 2

INTRADERMAL EXPOSURE
- Site: six injections as pairs in the interscapular area
- Treatment: Test group: A) front row: Freund's complete adjuvant (FCA) at 50 % (v/v) in 0.9% NaCl; B) middle row: 0.1 mL test material at 0.5 % (w/w) in corn oil; C) test substance at 0.5 % (w/w) in the mixture FCA/0.9 % NaCl (50/50)
Control groups: The animals were given the same injections (A, B, C) but without test substance, only with the formulating agent.

CUTANEOUS EXPOSURE
- Site: interscapular area
- Treatment: Test group: a pad of filter paper (approximately 8 cm²) was fully-loaded with the test substance at the concentration of 30 % (w/w) and was then applied to the interscapular region of the animals. The pad was held in place for 48 hours by means of an adhesive hypoallergenic dressing and an adhesive anallergenic waterproof plaster
Control groups: received an application of the vehicle alone under the same experimental conditions.

B. CHALLENGE EXPOSURE
Challenge:
- Day of challenge: day 22 (21 days after intradermal induction)
- Exposure period: 24 h
- Site: interscapular
- Treatment: Test groups: received an application of the test substance and vehicle. The filter paper of a chamber (Finn Chambero) was fully-loaded with the test substance at the concentration of 30 % (w/w) and was then applied to a clipped area of the skin of the posterior right flank of all animals. The vehicle was applied under the same experimental conditions to the skin of the posterior left flank. The chambers were held in contact with the skin for 24 hours by means of an adhesive an allergenic waterproof plaster. On removal of the dressing, any residual test substance was removed by means of a moistened
gauze pad.
Control groups: Control group one was treated like the treatment group; control group 2 remained untreated
- Evaluation (hr after challenge): 24, 48 h

Rechallenge:
- Day of challenge: day 29
- Exposure period: 24 h
- Treatment: the animals of all groups received an application of the test substance at the concentration of 30% (w/w) to the anterior left flank and the vehicle to the anterior right flank, under the same experimental conditions as for the first challenge application.
- Evaluation (hr after challenge): 24, 48 h
Challenge controls:
Yes, 2 groups
Positive control substance(s):
yes
Remarks:
Mercaptobenzothiazole
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
3
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Reading:
1st reading
Hours after challenge:
24
Group:
other: Negative control group 1
Dose level:
0%
No. with + reactions:
0
Total no. in group:
5
Reading:
2nd reading
Hours after challenge:
48
Group:
other: Negative control group 1
Dose level:
0%
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: Negative control group 1. Dose level: 0%. No with. + reactions: 0.0. Total no. in groups: 10.0.
Reading:
rechallenge
Hours after challenge:
24
Group:
test chemical
Dose level:
30%
No. with + reactions:
1
Total no. in group:
10
Reading:
rechallenge
Hours after challenge:
48
Group:
test chemical
Dose level:
30%
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
Dryness of skin in 3/10 animals
Reading:
rechallenge
Hours after challenge:
24
Group:
other: Negative control group 1
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
48
Group:
other: 1egative control group 1
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Clinical observations:
Dryness of skin was observed in 1/5 animals
Reading:
rechallenge
Hours after challenge:
24
Group:
other: Negative control group 2
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
rechallenge
Hours after challenge:
48
Group:
other: negative control group 2
Dose level:
30%
No. with + reactions:
0
Total no. in group:
5
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
20%
No. with + reactions:
7
Total no. in group:
10

One animal of the control group 1 was found dead on day 13. Hypoactivity and dyspnea were observed prior to death. The authors stated that such spontaneous clinical signs and mortality are sometimes observed in this species.

Interpretation of results:
not sensitising
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The study is well-documented by the publication and thus acceptable for assessment.
Qualifier:
according to guideline
Guideline:
other: Guinea pig maximization test and open epicutaneous test
Principles of method if other than guideline:
Two different open epicutaneous sensitization tests (open epicutaneous sensitization with adjuvant (OE+FCA) and open epicutaneous sensitization (OE)) and the Magnusson-Kligman maximization method (Guinea pig maximization (GMP))
GLP compliance:
not specified
Type of study:
other: Guinea pig maximization test and open epicutaneous test
Justification for non-LLNA method:
At the time the study was performed the guinea pig maximization test and the open epicutaneous test were standard test methods for the skin sensitisation potential. Thus, the study is considered to be reliable.
Specific details on test material used for the study:
GPM: 5% NiSO4•6H2O aqueos solution was used for intracutaneous injections
OE: the animals were painted with 25% NiSO4•6H2O aqueous solution
Topical challenge was performed with 5% NiSO4 in pet. and systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 240-300 g
- Housing: 2-4 animals were housed together in plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
Route:
other: intracutaneous injection
Concentration / amount:
5% NiSO4•6H2O aqueos solution was used for intracutaneous injections
Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
The animals were painted with 25% NiSO4•6H2O aqueous solution. Topical challenge was performed with 5% NiSO4 in pet. and systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline.
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
The animals were painted with 25% NiSO4•6H2O aqueous solution. Topical challenge was performed with 5% NiSO4 in pet.
Route:
intradermal
Vehicle:
other: saline
Concentration / amount:
systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline (0.2 ml nickel solution)
Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
5% NiSO4
Day(s)/duration:
2 weeks after the sensitization
Route:
intradermal
Vehicle:
other: saline
Concentration / amount:
2% NiSO4•6H2O
Day(s)/duration:
2 weeks after the primary patch test
No. of animals per dose:
Guinea Pig Maximization Test: 31 test animals, 11 control animals.
Open Epicutaneous test: 22 test animals, 13 control animals.
Open Epicutaneous test with Freund's complete adjuvant: 13 test animals, 7 control animals.
Details on study design:
Guinea pis maximiziation test (GPM): The test anaimals were sensitized according to the Magnusson-Kligman method. It includes next to intradermal and closed epicutaneous allergen application also adjuvant injection. The adjuvant (with and without nickel sulphate solution) were injected intradermally.
Open epicutaneous sensitization with adjuvant (OE + FCA (Freund's complete adjuvant)): The test animals were treated with a modified technique of Klecak et al.. For this a 2 x 4 cm area on the back was painted with 0.1 mL allergen solution 5 days a week for 4 weeks. Before the 3rd allergen application, 0.1 mL FCA was injected intradermally into the painted area.
Open epicutaneous sensitization (OE): The test animals were sensitized as described in the OE + FCA method, but without the FCA injections.
Challenge controls:
none reported
Positive control substance(s):
not specified
Key result
Reading:
other: Skin reaction (GMP)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
16
Total no. in group:
31
Key result
Reading:
other: Skin reaction (OE + FCA)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
10
Total no. in group:
13
Key result
Reading:
other: Skin reaction (OE)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
14
Total no. in group:
22
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
5% NiSO4-6H20 causes sensitising reactions in guinea pigs tested with the Guinea Pig Maximization Test, Open Epicutaneous test and Open Epicutaneous test with Freund's complete adjuvant.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be non-sensitizing only the EDTA-Ni complex must be regarded as dagerous. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their effects. In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-Ni(NH4)2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: Guinea pig maximization test and open epicutaneous test
Principles of method if other than guideline:
Two different open epicutaneous sensitization tests (open epicutaneous sensitization with adjuvant (OE+FCA) and open epicutaneous sensitization (OE)) and the Magnusson-Kligman maximization method (Guinea pig maximization (GMP))
GLP compliance:
not specified
Type of study:
other: Guinea pig maximization test and open epicutaneous test
Justification for non-LLNA method:
At the time the study was performed the guinea pig maximization test and the open epicutaneous test were standard test methods for the skin sensitisation potential. Thus, the study is considered to be reliable.
Specific details on test material used for the study:
GPM: 5% NiSO4•6H2O aqueos solution was used for intracutaneous injections
OE: the animals were painted with 25% NiSO4•6H2O aqueous solution
Topical challenge was performed with 5% NiSO4 in pet. and systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline.
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
not specified
Details on test animals and environmental conditions:
TEST ANIMALS
- Weight at study initiation: 240-300 g
- Housing: 2-4 animals were housed together in plastic cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-23 °C
Route:
other: intracutaneous injection
Concentration / amount:
5% NiSO4•6H2O aqueos solution was used for intracutaneous injections
Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
The animals were painted with 25% NiSO4•6H2O aqueous solution. Topical challenge was performed with 5% NiSO4 in pet. and systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline.
Route:
epicutaneous, occlusive
Vehicle:
petrolatum
Concentration / amount:
The animals were painted with 25% NiSO4•6H2O aqueous solution. Topical challenge was performed with 5% NiSO4 in pet.
Route:
intradermal
Vehicle:
other: saline
Concentration / amount:
systemic (i.p.) challenge was carried out with 2% NiSO4•6H2O solution in saline (0.2 ml nickel solution)
Route:
epicutaneous, open
Vehicle:
petrolatum
Concentration / amount:
5% NiSO4
Day(s)/duration:
2 weeks after the sensitization
Route:
intradermal
Vehicle:
other: saline
Concentration / amount:
2% NiSO4•6H2O
Day(s)/duration:
2 weeks after the primary patch test
No. of animals per dose:
Guinea Pig Maximization Test: 31 test animals, 11 control animals.
Open Epicutaneous test: 22 test animals, 13 control animals.
Open Epicutaneous test with Freund's complete adjuvant: 13 test animals, 7 control animals.
Details on study design:
Guinea pis maximiziation test (GPM): The test anaimals were sensitized according to the Magnusson-Kligman method. It includes next to intradermal and closed epicutaneous allergen application also adjuvant injection. The adjuvant (with and without nickel sulphate solution) were injected intradermally.
Open epicutaneous sensitization with adjuvant (OE + FCA (Freund's complete adjuvant)): The test animals were treated with a modified technique of Klecak et al.. For this a 2 x 4 cm area on the back was painted with 0.1 mL allergen solution 5 days a week for 4 weeks. Before the 3rd allergen application, 0.1 mL FCA was injected intradermally into the painted area.
Open epicutaneous sensitization (OE): The test animals were sensitized as described in the OE + FCA method, but without the FCA injections.
Challenge controls:
none reported
Positive control substance(s):
not specified
Key result
Reading:
other: Skin reaction (GMP)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
16
Total no. in group:
31
Key result
Reading:
other: Skin reaction (OE + FCA)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
10
Total no. in group:
13
Key result
Reading:
other: Skin reaction (OE)
Hours after challenge:
48
Group:
test chemical
Dose level:
5% NiSO4•6H2O
No. with + reactions:
14
Total no. in group:
22
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
5% NiSO4-6H20 causes sensitising reactions in guinea pigs tested with the Guinea Pig Maximization Test, Open Epicutaneous test and Open Epicutaneous test with Freund's complete adjuvant.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with acceptable restrictions. Scoring results for Individual animals not provided.
Qualifier:
according to guideline
Guideline:
other: open epicutaneous skin painting (Zissu et al. 1987)
Principles of method if other than guideline:
Open epicutaneous skin painting (Zissu et al. 1987)
GLP compliance:
not specified
Type of study:
skin painting test
Justification for non-LLNA method:
At the time the study was performed the skin painting test was a standard test methods for the skin sensitisation potential. Thus, the study is considered to be reliable.
Specific details on test material used for the study:
The test item used was Nickel Sulfate Hexahydrate, CAS 10101-97-0. Nickel sulfate 1% and 3% (analytical grade (E. Merck, Darmstadt, Gcrmany)) were prepared at the hospital pharmacy in a 1% Ianolin cream. Nickel sulfate, 0.3 %, 1% and 3%, was prepared in hydroxypropyl cellulose
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dunkin-Hartley, Mollegaard, Ll, Skensved, Denmark
- Age at study initiation: not reported
- Weight at study initiation: 350-450 g
- Housing: housed in groups of 3
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 deg. C
- Humidity (%): 50-60%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hr light-dark cycle

Animals were allowed to adapt for one week before use.
Route:
epicutaneous, open
Vehicle:
other: lanolin and hydroxypropyl cellulose
Concentration / amount:
0.3, 1, 3% NiSO4
Route:
epicutaneous, open
Vehicle:
other: lanolin and hydroxypropyl cellulose
Concentration / amount:
2% nickel sulphate in distilled water and 1% nickel sulphate in petrolatum
No. of animals per dose:
12-15
Details on study design:
TEST GROUPS:
Group 1: control, 15 animals treated with both lanolin and hydroxypropyl cellulose (HPC)
Group 2: 14 animals treated with 1% nickel sulphate in lanolin cream
Group 3: 15 animals treated with 3% nickel sulphate in lanolin cream
Group 4: 12 animals treated with 0.3% nickel sulphate in HPC
Group 5: 12 animals treated with 1% nickel sulphate in HPC
Group 6: 12 animals treated with 3% nickel sulphate in HPC

TEST METHOD
Procedure of Zissu, et al. (1987) was followed in detail. Aliquot of nickel sulfate preparation (0.5 ml) were administered epicutaneously to a dippcd, shavcd and SLS pretreated skin area of 2x4 cm located on the upper back of the animals. Nickel sulphate (0.5 ml) treatments were painted on the shaved skin sites 5 times a week for 4 weeks. Once a week, animals were given an intradermal injection of aluminium potassium sulphate (1% in sterile water) as an adjuvant. Animals were weighed on days 0, 7, 14, 17, 21 and 26.

CHALLENGE TEST
Test animals were challenged on days 14 and 21 after the last day of treatment by an occlusive patch test, using Finn chambers on Scanpor fixed with Acyrlastic and Leucoflex on the flank of the animal. Challenge concentrations used were 2% nickel sulphate in distilled water on day 42 and 1% nickel sulphate in petrolatum on day 49. Challenge reactions were scored blind 2-3 days after administration.

SCORING
The following scoring system was used:
0 (no visible reaction), + (isolated or patchy erythema), ++ (moderate or confluent erythema), +++ (swelling and erythema).
10 weeks following the challenge test, 30 animals (9 control + 21 nickel sensitive animals) were rechallenged with 1% nickel sulphate in petrolatum.

STATISTICAL METHOD
Fisher's exact test.
Positive control results:
not applicable
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1% NiSO4 in lanolin cream
No. with + reactions:
9
Total no. in group:
14
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
3% NiSO4 in lanolin cream
No. with + reactions:
12
Total no. in group:
14
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.3% NiSO4 in HPC
No. with + reactions:
5
Total no. in group:
12
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1% NiSO4 in HPC
No. with + reactions:
7
Total no. in group:
12
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
3% NiSO4 in HPC
No. with + reactions:
4
Total no. in group:
12
Remarks on result:
other: Both challenges

Frequency of positive reactions (2% nickel sulphate in distilled water) 3 days following challenge, by group tested: 
Group 1 (control): 2/15 
Group 2: 12/14* 
Group 3: 9/15* 
Group 4: 3/12 
Group 5: 9/12* 
Group 6: 6/12 
* - significantly different from the control group  

Frequency of positive reactions (1% nickel sulphate in petrolatum) 3 days following challenge, by group tested: 
Group 1 (control): 1/15 
Group 2: 8/14* 
Group 3: 9/14* 
Group 4: 5/12 
Group 5: 7/12* 
Group 6: 1/12 
* - significantly different from the control group 

3% nickel sulphate in HPC was less irritating than 3% nickel sulphate in lanolin cream.  All test animals (n=21) were positive in the 10-week  

rechallenge test with 1% nickel sulphate in petrolatum.

The total weight increase during the 4 weeks of induction treatment was 42.6±5.1% for the control group, 32.3±7.3% for the I% Ianolin cream group, 23.7±8.6% for the 3% IanoIin cream group, and 35.5±7.2%, 31.6±10.5%, 31.9±7.2% for the 0.3 %, 1% and 3% HPC groups, respectively.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Open topical treatment with nickel Sulfate 1% in either a Ianolin cream or hydroxypropyl cellulose is suitable lor the induction of nicke! allergy in guinea and the contact allergy seems to be long Iasting. Repeated open application of nickel sulfate at the concentrations 1% and 3% in both vehicles were close to or above the irritation Iimit when administered on SLS~pretreated skin in combination with weekly adjuvant injections.
The lower weight gain of the animals in the test groups suggests that nickel toxicity affects the animals and that the degree is dose related.
3% nickel sulphate in HPC was less irritating than 3% nickel sulphate in lanolin cream.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Meets generally accepted scientific standards with acceptable restrictions. Scoring results for Individual animals not provided.
Justification for type of information:
The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be non-sensitizing only the EDTA-Ni complex must be regarded as dagerous. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their effects. In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-Ni(NH4)2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
other: open epicutaneous skin painting (Zissu et al. 1987)
Principles of method if other than guideline:
Open epicutaneous skin painting (Zissu et al. 1987)
GLP compliance:
not specified
Type of study:
skin painting test
Specific details on test material used for the study:
The test item used was Nickel Sulfate Hexahydrate, CAS 10101-97-0. Nickel sulfate 1% and 3% (analytical grade (E. Merck, Darmstadt, Gcrmany)) were prepared at the hospital pharmacy in a 1% Ianolin cream. Nickel sulfate, 0.3 %, 1% and 3%, was prepared in hydroxypropyl cellulose
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Dunkin-Hartley, Mollegaard, Ll, Skensved, Denmark
- Age at study initiation: not reported
- Weight at study initiation: 350-450 g
- Housing: housed in groups of 3
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not reported

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-22 deg. C
- Humidity (%): 50-60%
- Air changes (per hr): not reported
- Photoperiod (hrs dark / hrs light): 12 hr light-dark cycle

Animals were allowed to adapt for one week before use.
Route:
epicutaneous, open
Vehicle:
other: lanolin and hydroxypropyl cellulose
Concentration / amount:
0.3, 1, 3% NiSO4
Route:
epicutaneous, open
Vehicle:
other: lanolin and hydroxypropyl cellulose
Concentration / amount:
2% nickel sulphate in distilled water and 1% nickel sulphate in petrolatum
No. of animals per dose:
12-15
Details on study design:
TEST GROUPS:
Group 1: control, 15 animals treated with both lanolin and hydroxypropyl cellulose (HPC)
Group 2: 14 animals treated with 1% nickel sulphate in lanolin cream
Group 3: 15 animals treated with 3% nickel sulphate in lanolin cream
Group 4: 12 animals treated with 0.3% nickel sulphate in HPC
Group 5: 12 animals treated with 1% nickel sulphate in HPC
Group 6: 12 animals treated with 3% nickel sulphate in HPC

TEST METHOD
Procedure of Zissu, et al. (1987) was followed in detail. Aliquot of nickel sulfate preparation (0.5 ml) were administered epicutaneously to a dippcd, shavcd and SLS pretreated skin area of 2x4 cm located on the upper back of the animals. Nickel sulphate (0.5 ml) treatments were painted on the shaved skin sites 5 times a week for 4 weeks. Once a week, animals were given an intradermal injection of aluminium potassium sulphate (1% in sterile water) as an adjuvant. Animals were weighed on days 0, 7, 14, 17, 21 and 26.

CHALLENGE TEST
Test animals were challenged on days 14 and 21 after the last day of treatment by an occlusive patch test, using Finn chambers on Scanpor fixed with Acyrlastic and Leucoflex on the flank of the animal. Challenge concentrations used were 2% nickel sulphate in distilled water on day 42 and 1% nickel sulphate in petrolatum on day 49. Challenge reactions were scored blind 2-3 days after administration.

SCORING
The following scoring system was used:
0 (no visible reaction), + (isolated or patchy erythema), ++ (moderate or confluent erythema), +++ (swelling and erythema).
10 weeks following the challenge test, 30 animals (9 control + 21 nickel sensitive animals) were rechallenged with 1% nickel sulphate in petrolatum.

STATISTICAL METHOD
Fisher's exact test.
Positive control results:
not applicable
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1% NiSO4 in lanolin cream
No. with + reactions:
9
Total no. in group:
14
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
3% NiSO4 in lanolin cream
No. with + reactions:
12
Total no. in group:
14
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
0.3% NiSO4 in HPC
No. with + reactions:
5
Total no. in group:
12
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
1% NiSO4 in HPC
No. with + reactions:
7
Total no. in group:
12
Remarks on result:
other: Both challenges
Key result
Reading:
2nd reading
Hours after challenge:
72
Group:
test chemical
Dose level:
3% NiSO4 in HPC
No. with + reactions:
4
Total no. in group:
12
Remarks on result:
other: Both challenges

Frequency of positive reactions (2% nickel sulphate in distilled water) 3 days following challenge, by group tested: 
Group 1 (control): 2/15 
Group 2: 12/14* 
Group 3: 9/15* 
Group 4: 3/12 
Group 5: 9/12* 
Group 6: 6/12 
* - significantly different from the control group  

Frequency of positive reactions (1% nickel sulphate in petrolatum) 3 days following challenge, by group tested: 
Group 1 (control): 1/15 
Group 2: 8/14* 
Group 3: 9/14* 
Group 4: 5/12 
Group 5: 7/12* 
Group 6: 1/12 
* - significantly different from the control group 

3% nickel sulphate in HPC was less irritating than 3% nickel sulphate in lanolin cream.  All test animals (n=21) were positive in the 10-week  

rechallenge test with 1% nickel sulphate in petrolatum.

The total weight increase during the 4 weeks of induction treatment was 42.6±5.1% for the control group, 32.3±7.3% for the I% Ianolin cream group, 23.7±8.6% for the 3% IanoIin cream group, and 35.5±7.2%, 31.6±10.5%, 31.9±7.2% for the 0.3 %, 1% and 3% HPC groups, respectively.

Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Open topical treatment with nickel Sulfate 1% in either a Ianolin cream or hydroxypropyl cellulose is suitable lor the induction of nicke! allergy in guinea and the contact allergy seems to be long Iasting. Repeated open application of nickel sulfate at the concentrations 1% and 3% in both vehicles were close to or above the irritation Iimit when administered on SLS~pretreated skin in combination with weekly adjuvant injections.
The lower weight gain of the animals in the test groups suggests that nickel toxicity affects the animals and that the degree is dose related.
3% nickel sulphate in HPC was less irritating than 3% nickel sulphate in lanolin cream.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The study is well-documented by the publication and thus acceptable for assessment.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Principles of method if other than guideline:
The study was conducted similar to OECD Guideline 406.
GLP compliance:
not specified
Type of study:
other: adjuvant and patch test (APT)
Justification for non-LLNA method:
At the time the study was performed the guinea pig sensitization test was a standard test method for the skin sensitisation potential. Thus, the study is considered to be reliable.
Specific details on test material used for the study:
Test item was purchased from Sigma
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Inc.
- Weight at study initiation: 380 to 500 g
- Housing: The animals were housed individually in a stainless-steel cage.
- Diet: ad libitum; pellet diet
- Water: ad libitum; sterilised water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3
- Humidity (%): 55 +/- 15
- Photoperiod: 12 hours dark/light cycle
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
3 %
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
0.01%, 0.1%, 0.3%, 1% and 3%
No. of animals per dose:
5 animals for sensitizing group, 5 animals for control group
Details on study design:
main study:
intradermal injection of 0.1 mL emulsified FCA on day 1
The test material was applied occlusively for 24 hours
Two further occlusive applications at days 2 and 3
Occlusive applications of the test material on day 9
Exposure period from day 1 to 3

challenge exposure:
0.01 mL aliquots of various concentrations of the test material in the vehicle were applied for challenge on day 21.
duration of challenge: 1 day
Observations were conducted at day 22 and 23
Positive control substance(s):
not specified
Key result
Group:
test chemical
Dose level:
0.1 %
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: The reactions are expressed as 40 % at a concentration of 0.1 %.
Key result
Group:
test chemical
Dose level:
0.3 %
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: The reactions are expressed as 100 % at a concentration of 0.3 %.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Nickel sulphate was determined to be a skin sensitizer category 1 based on the test results.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
September 2000
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be non-sensitizing only the EDTA-Ni complex must be regarded as dagerous. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their effects. In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-Ni(NH4)2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.
Reason / purpose for cross-reference:
read-across source
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Principles of method if other than guideline:
The study was conducted similar to OECD Guideline 406.
GLP compliance:
not specified
Type of study:
other: adjuvant and patch test (APT)
Justification for non-LLNA method:
At the time the study was performed the guinea pig sensitization test was a standard test method for the skin sensitisation potential. Thus, the study is considered to be reliable.
Specific details on test material used for the study:
Test item was purchased from Sigma
Species:
guinea pig
Strain:
Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Japan SLC Inc.
- Weight at study initiation: 380 to 500 g
- Housing: The animals were housed individually in a stainless-steel cage.
- Diet: ad libitum; pellet diet
- Water: ad libitum; sterilised water

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 +/- 3
- Humidity (%): 55 +/- 15
- Photoperiod: 12 hours dark/light cycle
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
3 %
Route:
epicutaneous, open
Vehicle:
water
Concentration / amount:
0.01%, 0.1%, 0.3%, 1% and 3%
No. of animals per dose:
5 animals for sensitizing group, 5 animals for control group
Details on study design:
main study:
intradermal injection of 0.1 mL emulsified FCA on day 1
The test material was applied occlusively for 24 hours
Two further occlusive applications at days 2 and 3
Occlusive applications of the test material on day 9
Exposure period from day 1 to 3

challenge exposure:
0.01 mL aliquots of various concentrations of the test material in the vehicle were applied for challenge on day 21.
duration of challenge: 1 day
Observations were conducted at day 22 and 23
Positive control substance(s):
not specified
Key result
Group:
test chemical
Dose level:
0.1 %
No. with + reactions:
2
Total no. in group:
5
Remarks on result:
other: The reactions are expressed as 40 % at a concentration of 0.1 %.
Key result
Group:
test chemical
Dose level:
0.3 %
No. with + reactions:
5
Total no. in group:
5
Remarks on result:
other: The reactions are expressed as 100 % at a concentration of 0.3 %.
Interpretation of results:
Category 1 (skin sensitising) based on GHS criteria
Conclusions:
Nickel sulphate was determined to be a skin sensitizer category 1 based on the test results.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The study is well-documented by the publication and thus acceptable for assessment.
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is mentioned in the study. However, the methods and materials are well-described and follow general scientific principles. Thus, the study is considered to be reliable.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time the study was performed the guinea pig sensitization test was a standard test method for the skin sensitisation potential. Thus, the study is considered to be reliable.
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
10 male animals were obtained from Buckberg Lab Animals, Inc., New York, USA
Route:
other: topical treatments
Vehicle:
other: 9:1 solution of dipropylene glycol methyl ether and polyethylene sorbitan monooleate
Concentration / amount:
10% in the solution
Day(s)/duration:
4 times in 10 days
Route:
other: topical treatments
Vehicle:
other: 9:1 solution of dipropylene glycol methyl ether and polyethylene sorbitan monooleate
Concentration / amount:
10% in solution
Day(s)/duration:
2 weeks after the last treatment
No. of animals per dose:
10 for Na3HEDTA and 10 for the positive control group
Details on study design:
5 animals per cage
temperature 73 °F (ca. 23 °C)
relative humidity 45 %
light/dark cycle: 12 h/ 12 h
Positive control substance(s):
yes
Remarks:
diglycidyl ether of 2,2-di(p,p-hydroxyphenyl) propane was used for the positive control group
Positive control results:
The positive control substance sensitized 9 of 10 animals producing a slight to marked erythema (9 of 10 animals) and a slight to moderate edema (9 of 10 animals).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
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
Group:
positive control
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance was not sensitizing under the test conditions.
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The complex EDTA-Ni(NH4)2 consists of the organic moiety EDTA, the central atom Ni2+ and the ammonium ions. As the latter are known to be non-sensitizing only the EDTA-Ni complex must be regarded as dagerous. For both, free EDTA and Ni2+ compounds well-documented studies are available, which shows their effects. In general, the complex with the chelated Ni2+ ion should be less toxic than the free Ni2+ ion. Based on that and that the complex EDTA-Ni has an average stability, a worst-case assessment where the EDTA-Ni(NH4)2 complex dissociate in its components can be done and so a read-across is possible to free EDTA and other Ni2+ compounds.
Reason / purpose for cross-reference:
read-across source
Qualifier:
no guideline followed
Principles of method if other than guideline:
No guideline is mentioned in the study. However, the methods and materials are well-described and follow general scientific principles. Thus, the study is considered to be reliable.
GLP compliance:
not specified
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
At the time the study was performed the guinea pig sensitization test was a standard test method for the skin sensitisation potential. Thus, the study is considered to be reliable.
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
10 male animals were obtained from Buckberg Lab Animals, Inc., New York, USA
Route:
other: topical treatments
Vehicle:
other: 9:1 solution of dipropylene glycol methyl ether and polyethylene sorbitan monooleate
Concentration / amount:
10% in the solution
Day(s)/duration:
4 times in 10 days
Route:
other: topical treatments
Vehicle:
other: 9:1 solution of dipropylene glycol methyl ether and polyethylene sorbitan monooleate
Concentration / amount:
10% in solution
Day(s)/duration:
2 weeks after the last treatment
No. of animals per dose:
10 for Na3HEDTA and 10 for the positive control group
Details on study design:
5 animals per cage
temperature 73 °F (ca. 23 °C)
relative humidity 45 %
light/dark cycle: 12 h/ 12 h
Positive control substance(s):
yes
Remarks:
diglycidyl ether of 2,2-di(p,p-hydroxyphenyl) propane was used for the positive control group
Positive control results:
The positive control substance sensitized 9 of 10 animals producing a slight to marked erythema (9 of 10 animals) and a slight to moderate edema (9 of 10 animals).
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10 %
No. with + reactions:
0
Total no. in group:
10
Remarks on result:
no indication of skin sensitisation
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
Group:
positive control
No. with + reactions:
9
Total no. in group:
10
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
The test substance trisodium hydrogen ethylenediaminetetraacetate was not sensitizing under the test conditions.
Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The chelating effect of sodium EDTA in nickel-allergic patients was investigated. It could be shown how EDTA has a blocking effect for skin irritant reaction (eczema) on humans.
Principles of method if other than guideline:
Study on humans in order to investigate an EDTA effect which prevent the dermatitis-provoking potential of nickel.
GLP compliance:
not specified
Type of study:
patch test
Specific details on test material used for the study:
aqueous nickel sulphate (prepared by the hospital pharmacist)
Details on the study design:
human skin model - upper back of 17 nickel-allergic female patients
The right half of the upper back was lightly rubbed with a cream containing 10% EDTA (w/w) (disodium salt of ethylenediamine tetra acetate) in cetomacrogol cream FNA. After 15 min, when the cream was no longer visible, 3 concentrations of aqueous nickel sulphate were patch tested in the pretreated area. The left upper back was pretreated in a similar way with cetomacrogol cream FNA without EDTA. The same concentrations of nickel sulphate were patch tested in this area. Patch testing was performed with the Silver Patch Testers. The reactions were graded according to the ICDRG after 48 and 72 h.

Control samples:
Yes, concurrent positive control. Cetomacrogol cream without EDTA and with 10% EDTA. The cream with 10% EDTA did not induce irritant reactions in 20 control patients (before study).

Amount/concentration applied:
0.01% (24.4 ppm), 0.1% (244 ppm), 1% (2440 ppm)
Key result
Run / experiment:
other: 1-6, 8-14 and 16
Parameter:
other: penetration time (in minutes)
Remarks:
After 2880 and 4320 min
Positive controls validity:
not specified
Remarks on result:
no indication of skin sensitisation
Remarks:
at 2440 ppm nickel sulphate
Key result
Run / experiment:
other: 7, 15 and 17
Parameter:
other: penetration time (in minutes)
Remarks:
After 2880 and 4320 min
Positive controls validity:
not specified
Remarks on result:
positive indication of skin sensitisation
Remarks:
slight reaction was observed
Conclusions:
The blocking effect of the 10% EDTA cream appeared to be significant in comparison with that of the cream base only (p < 0.01). In the majority of patients, 10% EDTA does chelate nickel sulphate in 0.01% (24.2 ppm), 0.1% (244 ppm) and 1% (2440 ppm) by reducing the dermatitis-provoking potential of nickel. A barrier cream with 10% EDTA might be of help in nickel-allergic patients with eczema of the hands.
Executive summary:

In the area pretreated with 10% EDTA in cetomacrogol cream FNA, only 3 of 17 patients showed a + positive reaction to 1% (2440 ppm) nickel sulphate. 3 patients showed no reaction to any concentration of nickel sulphate in either area. The reactivity to nickel sulphate in the area pretreated with 10% EDTA appeared to be far less than in the control area (rank sum test p < 0.01). No skin sensitisation or irritation to the 10% EDTA cream were observed.

Endpoint:
skin sensitisation: in vitro
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Justification for type of information:
The chelating effect of sodium EDTA in nickel-allergic patients was investigated. It could be shown how EDTA has a blocking effect for skin sensitisation/irritation (eczema) on humans.
This blocking effect results due to the complexation of the irritant nickel. It is an important study which must be considered for the classification of the EDTA-Ni(NH4)2 complex.
Reason / purpose for cross-reference:
read-across source
Principles of method if other than guideline:
Study on humans in order to investigate an EDTA effect which prevent the dermatitis-provoking potential of nickel.
GLP compliance:
not specified
Type of study:
patch test
Specific details on test material used for the study:
aqueous nickel sulphate (prepared by the hospital pharmacist)
Details on the study design:
human skin model - upper back of 17 nickel-allergic female patients
The right half of the upper back was lightly rubbed with a cream containing 10% EDTA (w/w) (disodium salt of ethylenediamine tetra acetate) in cetomacrogol cream FNA. After 15 min, when the cream was no longer visible, 3 concentrations of aqueous nickel sulphate were patch tested in the pretreated area. The left upper back was pretreated in a similar way with cetomacrogol cream FNA without EDTA. The same concentrations of nickel sulphate were patch tested in this area. Patch testing was performed with the Silver Patch Testers. The reactions were graded according to the ICDRG after 48 and 72 h.

Control samples:
Yes, concurrent positive control. Cetomacrogol cream without EDTA and with 10% EDTA. The cream with 10% EDTA did not induce irritant reactions in 20 control patients (before study).

Amount/concentration applied:
0.01% (24.4 ppm), 0.1% (244 ppm), 1% (2440 ppm)
Key result
Run / experiment:
other: 1-6, 8-14 and 16
Parameter:
other: penetration time (in minutes)
Remarks:
After 2880 and 4320 min
Positive controls validity:
not specified
Remarks on result:
no indication of skin sensitisation
Remarks:
at 2440 ppm nickel sulphate
Key result
Run / experiment:
other: 7, 15 and 17
Parameter:
other: penetration time (in minutes)
Remarks:
After 2880 and 4320 min
Positive controls validity:
not specified
Remarks on result:
positive indication of skin sensitisation
Remarks:
slight reaction was observed
Conclusions:
The blocking effect of the 10% EDTA cream appeared to be significant in comparison with that of the cream base only (p < 0.01). In the majority of patients, 10% EDTA does chelate nickel sulphate in 0.01% (24.2 ppm), 0.1% (244 ppm) and 1% (2440 ppm) by reducing the dermatitis-provoking potential of nickel. A barrier cream with 10% EDTA might be of help in nickel-allergic patients with eczema of the hands.
Executive summary:

In the area pretreated with 10% EDTA in cetomacrogol cream FNA, only 3 of 17 patients showed a + positive reaction to 1% (2440 ppm) nickel sulphate. 3 patients showed no reaction to any concentration of nickel sulphate in either area. The reactivity to nickel sulphate in the area pretreated with 10% EDTA appeared to be far less than in the control area (rank sum test p < 0.01). No skin sensitisation or irritation to the 10% EDTA cream were observed.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The complex nickel diammonium ethylenediaminetetraacetate consists of the ethylenediaminetetraacetate moiety, the nickel(2 +) moiety and the ammonium(+) moiety. The latter is known to be non-sensitizing. The first can be stated as not sensitizing based on the provided studies. However, nickel(II) salts are known to be strong sensitizers as prooved by the studies on nickel(II) sulphate. The chelating effect of the EDTA moiety may decrease the sensitizing properties of the EDTA-Ni(NH4)2 complex but the sensitizing properties cannot be excluded. The EDTA-Ni(NH4)2 complex will classified as skin sensitizer.