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Diss Factsheets

Administrative data

Description of key information

Discussion of skin sensitization:

In determining the skin sensitivity of the Copper Chelate Complex (LZ 60102), two of the three standard in-vitro studies KeratoSens (OECD 442D) and U-SENS/ h-CLAT (OECD 442E) were conducted. The third DPRA (OECD 442C) was not conducted because of its false reading with metal compounds. Also noted is that KeratoSens (OECD 442D) and U-SENS/ h-CLAT (OECD 442E) are not recommended, in their separate guidelines, for use with chemicals that are not soluble or do not form a stable dispersion. 

LZ 60102 hydrolyses immediately forming metal ions and the starting material LZ 399. This lack of stability, prone at any contact with water, lead to poor test results based on non-stability as noted in the test guidelines. This bring uncertainty to the results of the KeratoSens (OECD 442D) and U-SENS/ h-CLAT (OECD 442E), which were positive because the chemicals are not in the scope of their guidelines. Therefore, the in-vitro tests were considered to be supporting studies only.

LZ 60102 has not been tested in-vivo, but copper hydroxide and copper sulfate pentahydrate did not produce a delayed contact sensitization response in the maximization tests and are not considered as a dermal sensitizers. In addition the testing of the hydrolysis product LZ 399 in a maximization test was negative for dermal sensitization. A similar compound (Zinc Chelate Complex) is also not a dermal sensitizer in an in-vivo Buehler study. The Buehler study was selected instead of the LLNA study design as the LLNA has shown to have false reading when used with metal compounds (as described in literature) and the Buehler is an acceptable substitute for the LLNA.

In-vivo studies carry more weight than in-vitro studies and are at the end of the key events sequence required to cause the presentation of allergic contact dermatitis according to the adverse outcome pathway (AOP) model. With the result of the in-vitro studies in conflict with the negative results for the in-vivo studies of the components, the weight of evidence leads to the conclusion that the Copper Chelate Complex is not a dermal sensitizer, as the result of the component studies were all negative. 

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation, other
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Study period:
September - November 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Reason / purpose for cross-reference:
read-across source
Reason / purpose for cross-reference:
read-across source
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Species:
guinea pig
Key result
Remarks on result:
no indication of skin sensitisation
Remarks:
test item itself could not be tested as such, therefore, read.accross approach to decomposition product and analogeous substance
Interpretation of results:
not sensitising
Conclusions:
It is concluded that, under the conditions of the two in-vivo studies on the decomposition product and on the analogeous substance, repeated applications of the Copper Chelate Complex did not cause delayed contact hypersensitivity in the guinea-pig.
Executive summary:

LZ 60102 has not been tested in-vivo, but copper hydroxide and copper sulfate pentahydrate did not produce a delayed contact sensitization response in the maximization tests and are not considered as a dermal sensitizers. In addition the testing of the hydrolysis product LZ 399 in a maximization test was negative for dermal sensitization. A similar compound (Zinc Chelate Complex) is also not a dermal sensitizer in an in-vivo Buehler study. The Buehler study was selected instead of the LLNA study design as the LLNA has shown to have false reading when used with metal compounds (as described in literature) and the Buehler is an acceptable substitute for the LLNA.

In-vivo studies carry more weight than in-vitro studies and are at the end of the key events sequence required to cause the presentation of allergic contact dermatitis according to the adverse outcome pathway (AOP) model. With the result of the in-vitro studies in conflict with the negative results for the in-vivo studies of the components, the weight of evidence leads to the conclusion that the Copper Chelate Complex is not a dermal sensitizer, as the result of the component studies were all negative. 

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from similar mixture/product
Adequacy of study:
key study
Study period:
March 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
Buehler test
Justification for non-LLNA method:
The LLNA test is not the method of choice to test metal compounds, as false positive results can be obtained. The Buehler study was conducted for US-Premanufacture Notice (PMN), as in-vitro sensitization studies alone are not sufficient for PMN-submission.
Specific details on test material used for the study:
- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)
Species:
guinea pig
Strain:
Hartley
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Elm Hill Breeding Labs, USA
- Females: males used
- Age at study initiation: young adults
- Weight at study initiation: 275.8-389.3 gr
- Housing: g roup housing in in suspended stainless steel perforated bottom caging
- Diet (e.g. ad libitum): PMI Guinea Pig Lab Diet" #5025 (approximately 20 grams/day)
- Water (e.g. ad libitum): Filtered tap water
- Acclimation period: 7 or 21 days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-27°C
- Humidity (%): 29-58%
- Air changes (per hr): 12 changes
- Photoperiod (hrs dark / hrs light): 12-hour light/dark cycle
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
60%
Day(s)/duration:
6 hours (once a week for three weeks)
Adequacy of induction:
highest technically applicable concentration used
No.:
#1
Route:
epicutaneous, occlusive
Vehicle:
other: mineral oil
Concentration / amount:
60%
Day(s)/duration:
24 and 48 hours
Adequacy of challenge:
highest non-irritant concentration
No. of animals per dose:
- Number of Animals: 34
- Number of Groups: 3
- Number of Animals per Group: Preliminary Irritation Group: 4 / Test Group: 20 / Naive Control Group: 10
Details on study design:
RANGE FINDING TESTS:
A group of four animals was used to determine the highest non-irritating concentration of the test substance prior to the challenge dose. The fur was removed by clipping the flanks of each guinea pig. This area was divided into four test sites (two sites on each side of the midline) on each animal. The test substance was mixed with mineral oil to yield w/w concentrations of 60%, 45%, 30% and 15%. Each concentration was applied (0.4 g or mL each) to a test site using an occlusive 25 mm Hill Top Chamber. The sites were wrapped with non-allergenic Durapore adhesive tape. After 6 hours of exposure, the chambers were removed and the test sites were
gently cleansed with a 3% soap solution followed by tap water and a clean paper towel to remove any residual test substance. Approximately 24 hours after application, each site was evaluated for local reactions (erythema). From these results, the the highest concentration that produced responses in 4 guinea pigs no more severe than two scores of 0.5 and two scores of zero) was established and used for challenge. The HNIC selected for the challenge phase was a 60% w/w mixture in mineral oil.

MAIN STUDY
A. INDUCTION EXPOSURE
Once each week for three weeks, four-tenths of a gram of a 60% w/w mixture of the test substance in mineral oil was applied to the left side of each test animal using an occlusive 25 mm Hill Top Chamber. The chambers were secured in place and wrapped with non-allergenic Durapore adhesive tape to avoid dislocation of the chambers and to minimize loss of the test substance for six hours. After the 6-hour exposure period, the chambers were removed and the test sites were gently cleansed with a 3% soap solution followed by tap water and a clean paper towel to remove any residual test substance. Approximately 24 and 48 hours after each induction application, readings were made of local reactions (erythema) according to the following scoring system ( 0 - no reaction / 0.5 - very faint erythema, usually non-confluent* / 1 - faint erythema, usually confluent / 2 - moderate erythema / 3 - severe erythema with or without edema // *Very faint erythema is not considered a positive reaction).

B. CHALLENGE EXPOSURE
Twenty-seven days after the first induction dose, four-tenths of a gram of a 60% w/w mixture of the test substance in mineral oil (HNIC) was applied to a naive site on the right side of each animal as a challenge dose, using the procedures described above. These sites were evaluated for a sensitization response (erythema) approximately 24 and 48 hours after the challenge application according to the system described in Section 5 .F.
In addition to the test animals, I 0 guinea pigs from the same shipment were maintained under identical environmental conditions and were treated with the HNIC of the test substance at challenge only. These animals constituted the "naive control" group.
Challenge controls:
60% w/w mixture of the test substance in mineral oil (HNIC)
Positive control substance(s):
yes
Positive control results:
The procedures used in this study were validated using Hexyl Cinnamic Aldehyde (HCA) as a positive control substance. The most recent validation and testing was performed by the test lab between January 12 - February 11, 2021. This test was conducted with Hartley strain albino guinea pigs from Elm Hill Breeding Labs following induction and challenge procedures similar to those described in this study. The results obtained from this testing are presented below:

Induction Phase:
Historical Positive Control Animals (100% HCA): Very faint erythema (0.5) was noted at three positive control sites during the induction phase.

Challenge Phase:
Historical Positive Control Animals (100% HCA): Four of ten positive control animals exhibited signs of a sensitization response (1 - faint erythema 24 hours after the challenge application. Positive responses persisted at two positive control sites through 48 hours. Very faint erythema (0.5) was noted at six sites 24 hours after application, which persisted in three sites through 48 hours.
Historical Naive Control Animals: (100% HCA): Very faint erythema (0.5) was noted at three of five naive control sites 24 hours after the challenge application, which cleared by 48 hours.
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
60% test item in mineral oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
60% test item in mineral oil
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
60% test item in mineral oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
60% test item in mineral oil
No. with + reactions:
0
Total no. in group:
10
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
100% Hexyl Cinnamic Aldehyde (HCA)
No. with + reactions:
10
Total no. in group:
10
Clinical observations:
faint erythema in 4 animals; very faint erythema in 6 animals
Remarks on result:
positive indication of skin sensitisation
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Dose level:
100% Hexyl Cinnamic Aldehyde (HCA)
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
very faint erythema in 5 animals
Remarks on result:
positive indication of skin sensitisation
Interpretation of results:
GHS criteria not met
Conclusions:
Based on the results of this study, the test substance is not considered to be a contact sensitizer.
Executive summary:

A dermal sensitization test was conducted with guinea pigs to determine the potential for LZ 61000 to produce sensitization after repeated topical applications. A 60% w/w mixture of the test substance in mineral oil was topically applied to twenty healthy test guinea pigs, once each week for a three-week induction period. Twenty-seven days after the first induction dose, a challenge dose of the test substance at its highest non-irritating concentration (HNIC, determined in the preliminary irritation screen to be a 60% w/w mixture in mineral oil) was applied to a naive site on each guinea pig. A naive control group (ten animals) was maintained under the same environmental conditions and treated with the test substance at challenge only. Approximately 24 and 48 hours after each induction and challenge dose, the animals were scored for erythema. The incidence and severity of the sensitization response noted after challenge is found below:

- Test Animals 0/20 at 24 h and 48 h reading (all scores = 0)

- Naive Control Animals: 0/10 at 24 h and 48 h reading (all scores = 0)

Based on the results of this study, the test substance is not considered to be a contact sensitizer. The positive response observed in the historical positive control validation study with Hexyl Cinnamic Aldehyde (HCA) validates the test system used in this study.

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from similar mixture/product
Adequacy of study:
key study
Study period:
September - November 1995
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-compliant guideline study
Reason / purpose for cross-reference:
read-across: supporting information
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.6 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
the maximization test was performed before the LLNA test methods was available
Specific details on test material used for the study:
- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: David Hall, Darley Oaks, Newchurch, Burton on Trent, Staffordshire, England
- Age at study initiation: 6-8 weeks
- Housing: gang housing; 5 animals of same sex per cage
- Diet (e.g. ad libitum): Guinea-pig F.D.1., from Special Diets Services Limited, Witham, Essex, England
- Water (e.g. ad libitum): Tap water
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18°C
- Humidity (%): 55%
- Air changes (per hr): 10 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours artificial light
Route:
intradermal and epicutaneous
Vehicle:
water
Concentration / amount:
After consideration of the primary irritation screen results and the criteria for selection of treatment concentrations, the following regime was adopted:

First induction*:
- 50% v/v P5II7 in purified water
- 50% v/v P5II7 in FCA

Second induction*
- 10% v/v PSII7 in purified water

Challenge*
- 10% v/v P5II7 in purified water
- 3% v/v P5II7 in purified water

* Initially the first induction was undertaken employing 50% v/v formulations, however, due to ulceration of the administration sites the study was stopped. The study was restarted, with additional animals, using 5% v/v formulations
Route:
epicutaneous, occlusive
Vehicle:
water
Concentration / amount:
After consideration of the primary irritation screen results and the criteria for selection of treatment concentrations, the following regime was adopted:

First induction*:
- 50% v/v P5II7 in purified water
- 50% v/v P5II7 in FCA

Second induction*
- 10% v/v PSII7 in purified water

Challenge*
- 10% v/v P5II7 in purified water
- 3% v/v P5II7 in purified water

* Initially the first induction was undertaken employing 50% v/v formulations, however, due to ulceration of the administration sites the study was stopped. The study was restarted, with additional animals, using 5% v/v formulations
No. of animals per dose:
- Control group: 5 animals per sex and group
- Test group: 10 animals per sex and group
Details on study design:
INDUCTION:
1. Primary induction
Three pairs of injections (0.1 m!) were made deep into the dermis, such that on either side of the dorsal median line there were three injection sites in a row parallel to the spinal column. AIl injection sites lay near the periphery of a dermal test site 2 cm wide x 4 cm long, overlying the scapulae. The anterior and middle sites were positioned close together and distant from the posterior sites.

Injection sites Test group treatment Control group treatment
Anterior sites (A) FCA FCA
Middle sites (B) Test material in vehicle Vehicle
Posterior sites (C) Test material in FCA Vehicle in FCA

2. Secondary induction
On Day 8, the dermal site overlying the scapulae were treated by topical application of 0.6 ml ofa test material formulation to test animals, while controls received 0.6 ml of the vehicle. Each dose was applied to a 4 x 2.5 cm absorbent patch (Whatman No. 3 filter paper) which was applied to the skin and covered by an occlusive dressing (Blenderm and Elastoplast) for 48 hours. The application site was wiped with a paper tissue moistened with the vehicle immediately after removal of the bandage.

CHALLENGE:
Both flanks of all animals were clipped on Day 21. On Day 22 these areas were wet shaven to reveal a 5 x 5 cm area on the left flank and a 10 x 5 cm area on the right flank. Approximately one hour later the left site was treated by topical application of 0.03 ml of the vehicle while the right side received 0.03 ml of the maximum non-irritant concentration (as determined by Phase 3 of the primary skin irritation screen) to one site and a dilution to a second site. The doses were applied to 1 cm diameter absorbent patches (AI-test) and cover ed by an occlusive dressing (Blenderm and Elastoplast) for 24 hours. The test site was wiped with a paper tissue moistened with vehicle immediately after removal of the bandage. Reactions to challenge were assessed approximately 24 and 48 hours after removal of the occlusive dressings. The observations were made without knowledge of the number or group identity of the animal under examination.
Positive control substance(s):
not specified
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
3% test item in purified water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
10% test item in purified water
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
these responses reflected primary irritation rather than dermal sensitization.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
3% test item in purified water
No. with + reactions:
0
Total no. in group:
20
Clinical observations:
none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
10% test item in purified water
No. with + reactions:
8
Total no. in group:
20
Clinical observations:
these responses reflected primary irritation rather than dermal sensitization.none
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
purified water
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
these responses reflected primary irritation rather than dermal sensitization.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
negative control
Dose level:
purified water
No. with + reactions:
4
Total no. in group:
10
Clinical observations:
these responses reflected primary irritation rather than dermal sensitization.
Remarks on result:
no indication of skin sensitisation
Key result
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Remarks on result:
not measured/tested
Key result
Reading:
2nd reading
Hours after challenge:
48
Group:
positive control
Remarks on result:
not measured/tested

Slight erythema or a more marked reaction were observed in eight of twenty test and four often control animals following challenge with 10% P5117 in purified water and in no animal following challenge application of 3% P5117 in purified water (the maximum non-irritant concentration). No animal showed a reaction to challenge application of purified water alone.

Since the incidence and severity of responses to challenge with the 10% formulation was the same in the test and control animals and none of the responders showed a reaction after challenging with 3% P5117, it is considered that they reflected primary irritation rather than dermal sensitization. No positive responses to challenge with 3% P5117 were found.

Interpretation of results:
not sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
It is concluded that, under the conditions of this study, repeated applications of LZ399 did not cause delayed contact hypersensitivity in the guinea-pig.
Executive summary:

The skin sensitisation potential of LZ399 was assessed in maximization test (Magnusson & Kligman) according to methds OECD no. 406 and EU B.6 on guinea pigs. 20 animals were used in the test group and 10 animals in the control group. Concentrations of 3% and 10% test item in purified water were tested. Challenge application of 10% test item in purified water gave rise to slight erythema or a more marked reaction in eight of twenty test and four of ten control animals. It is considered that these responses reflected primary irritation rather than dermal sensitization. Challenge application of 3% test item caused no positive response in test or control animals. Challenge application of purified water alone caused no significant response. It was concluded that, under the conditions of this study and the criteria of the EC, repeated administration of LZ399 did not cause delayed contact hypersensitivity in guinea-pigs.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
July - August 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442D (In Vitro Skin Sensitisation: ARE-Nrf2 Luciferase Test Method)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EURL ECVAM DB-ALM Protocol n° 155: KeratinoSens™. (Adopted March, 2018).
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
activation of keratinocytes
Justification for non-LLNA method:
The derived data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. According to the current REACH guidance document, skin sensitization needs to be tested in in-vitro test systems first (OECD 442 C, D and E). A LLNA is required in case of unclear in-vitro study results only.
Specific details on test material used for the study:
- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)
Details of test system:
Keratinoses transgenic cell line [442D]
Details on the study design:
The test item was suspended in dimethyl sulfoxide (DMSO) at 100 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 – 1000 μM (2-fold dilution series) in the first experiment and in test concentrations of 12 – 1000 μM (1.5-fold dilution series) in the second experiment. The highest test concentration was considered to be the limit of solubility. The test item precipitated at the highest dose level tested. Two independent experiments were performed.
Vehicle / solvent control:
DMSO
Negative control:
other: DMSO (vehicle)
Positive control:
other: Ethylene dimethacrylate glycol
Positive control results:
- The luciferase activity induction obtained with the positive control, Ethylene dimethacrylate glycol, was statistically significant above the threshold of 1.5-fold in at least one concentration.
- The EC1.5 of the positive control was within two standard deviations of the historical mean (42 μM and 29 μM in experiment 1 and 2, respectively). A dose response was observed and the induction at 250 μM was higher than 2-fold.
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
IC30 [442D]
Value:
142 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
IC50 [442D]
Value:
173 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
Imax [442D]
Value:
7.36 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC 1.5 [442D]
Value:
74
At concentration:
1 000
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC30 [442D]
Value:
441 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
IC50 [442D]
Value:
505 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
Imax [442D]
Value:
24.06 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC 1.5 [442D]
Value:
15 µM
At concentration:
1 000 mM
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Outcome of the prediction model:
positive [in vitro/in chemico]
Other effects / acceptance of results:
The test item showed toxicity (IC30 values of 142 μM and 441 μM and IC50 values of 173 μM and 505 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 74 μM and 15 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 7.36-fold and 24.06-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control. Although a biphasic response was observed in the second experiment, inductions > 1.5-fold were observed at several dose levels and the first experiment already showed a positive response, and therefore the test item is classified as positive.
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item is classified as positive (biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this study.
Executive summary:

The objective of this study was to evaluate the ability of the test item to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study was performed in accordance with OECD test method 442D. The test item was suspended in dimethyl sulfoxide (DMSO) at 100 mM. From this stock 11 spike solutions in DMSO were prepared. The stock and spike solutions were diluted 100-fold in the assay resulting in test concentrations of 0.49 – 1000 μM (2-fold dilution series) in the first experiment and in test concentrations of 12 – 1000 μM (1.5-fold dilution series) in the second experiment. The highest test concentration was considered to be the limit of solubility. The test item precipitated at the highest dose level tested. Two independent experiments were performed.

The test item showed toxicity (IC30 values of 142 μM and 441 μM and IC50 values of 173 μM and 505 μM in experiment 1 and 2, respectively). A biologically relevant, dose-related induction of the luciferase activity (EC1.5 values of 74 μM and 15 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 7.36-fold and 24.06-fold in experiment 1 and 2 respectively. The test item is classified as positive in the KeratinoSensTM assay since positive results (>1.5-fold induction) were observed at test concentrations < 1000 μM with a cell viability of >70% compared to the vehicle control. Although a biphasic response was observed in the second experiment, the first experiment already showed a positive response, and therefore the test item is classified as positive.

In conclusion, the test item is classified as positive (biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this test.

Further testing (OECD 442E) is necessary to confirm the positive result in this study.

Endpoint:
skin sensitisation: in vitro
Type of information:
experimental study
Adequacy of study:
key study
Study period:
September - November 2020
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 442E (In Vitro Skin Sensitisation assays addressing the key event on activation of dendritic cells on the Adverse Outcome Pathway for skin sensitisation)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
U937 cell line activation test (U-SENS™)
Justification for non-LLNA method:
The derived data may be used as part of an integrated approach to testing and assessment (IATA) to support the discrimination between skin sensitisers and non-sensitisers for the purpose of hazard classification and labelling. According to the current REACH guidance document, skin sensitization needs to be tested in in-vitro test systems first (OECD 442 C, D and E). A LLNA is required in case of unclear in-vitro study results only.
Specific details on test material used for the study:
- Storage conditions: Controlled room temperature (15-25°C, ≤70% relative humidity), protected from light and humidity (stored in a tightly closed container)
Details of test system:
U-937 cell line [442E]
Details on the study design:
The test item was dissolved in complete medium at 0.4 and 0.2 mg/mL in the first ad second experiment, respectively. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment the stock was diluted to five test concentrations (1, 10, 15, 20 and 50 μg/mL). The test item precipitated at the dose level of 200 μg/mL tested.
Vehicle / solvent control:
cell culture medium
Negative control:
DL-Lactic acid
Positive control:
picrylsulfonic acid/2,4,6-trinitro-benzene-sulfonic acid (TNBS) [442E]
Positive control results:
Experiment 1: The positive control (TNBS) showed a S.I. ≥ 557% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Experiment 2: The positive control (TNBS) showed a S.I. ≥ 1237% in all wells and was non-cytotoxic at all concentrations (cell viability ≥ 70%).
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
CV70 [442E]
Value:
39 µg/mL
Cell viability:
cell viability of >70% compared to the vehicle control
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 1
Parameter:
EC150, CD86 [442E]
Value:
4.9 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
CV70 [442E]
Value:
29 µg/mL
Cell viability:
cell viability of >70% compared to the vehicle control
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Key result
Group:
test chemical
Run / experiment:
run/experiment 2
Parameter:
EC150, CD86 [442E]
Value:
0.81 µg/mL
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
positive indication of skin sensitisation
Outcome of the prediction model:
positive [in vitro/in chemico]
Other effects / acceptance of results:
The test item showed toxicity (CV70 values of 39 μg/mL and 29 μg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 4.9 μg/mL and 0.81 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70%
compared to the vehicle control.
Interpretation of results:
study cannot be used for classification
Conclusions:
The test item is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.
Executive summary:

The objective of this study was to evaluate the ability of the test item to increase the expression levels of CD86 cell surface marker in the U937 cell line activation Test (U-Sens™) assay. The study procedures described in this report were based on the most recent OECD guideline. The test item was dissolved in complete medium at 0.4 and 0.2 mg/mL in the first ad second experiment, respectively. In the first experiment the stock was diluted to six test concentrations (1, 10, 20, 50, 100 and 200 μg/mL). In the second experiment the stock was diluted to five test concentrations (1, 10, 15, 20 and 50 μg/mL). The test item precipitated at the dose level of 200 μg/mL tested.

The test item showed toxicity (CV70 values of 39 μg/mL and 29 μg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 4.9 μg/mL and 0.81 μg/mL in experiment 1 and 2, respectively) was measured in both experiments. The test item is classified as Positive in the U-Sens™ assay since positive results (> 150% increase) were observed at test concentrations with a cell viability of >70%

compared to the vehicle control. In conclusion, LZ 60102 is classified as positive (increase in the expression levels of CD86 cell surface marker in the U937 cell line) under the experimental conditions described in this report.

Endpoint:
skin sensitisation: in vivo (LLNA)
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint:
skin sensitisation: in vitro
Data waiving:
study technically not feasible
Justification for data waiving:
other:
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)
Additional information:

Guideline studies; Klimisch 1

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

In-vivo studies carry more weight than in-vitro studies and are at the end of the key events sequence required to cause the presentation of allergic contact dermatitis according to the adverse outcome pathway (AOP) model. With the result of the in-vitro studies in conflict with the negative results for the in-vivo studies of the components, the weight of evidence leads to the conclusion that the Copper Chelate Complex is not a dermal sensitizer, as the result of the component studies were all negative.