Zinc Chelate Complex

Endpoint summary

Description of key information

Discussion of skin sensitization:

In determining the skin sensitivity of the Zinc Chelate Complex (LZ 61000), 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 with chemicals that are not soluble or do not form a stable dispersion. LZ 61000 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. The Zinc Chelate complex tested in a Buehler study showed the test item was negative which agrees with the separate negative maximization test and human patch studies for Zn and a negative maximization test for the hydrolysis product LZ 399. The Buehler study was selected instead of the LLNA study design as the LLNA has show to have false reading when used with metal compounds and the Buehler test is an acceptable substitute.

The KeratoSens resulted in a positive finding and both the U-SENS and in-vivo Buehler studies were negative. In-vivo studies carry more weight than in-vitro studies and being at the end of the sensitization key events required to cause presentation of allergic contact dermatitis according to the Adverse Outcome Pathway (AOP) model. With the split result of the in-vitro studies and the negative for the in-vivo study, the weight of evidence leads to the conclusion that the Zinc Chelate complex is not a dermal sensitizer.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Reference 1

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 dissolved in dimethyl sulfoxide (DMSO) at 200 mM and 100 mM in the first and second experiment, respectively. 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 0.98 – 2000 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 the highest dose required in the current guideline. The test item precipitated at dose levels of 1000 μM and upwards in the first experiment. No precipitate was observed at any dose level tested in the second experiment. 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:

Imax [442D]

Value:

79.25 µM

At concentration:

2 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC30 [442D]

Value:

77 µM

At concentration:

2 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

IC50 [442D]

Value:

94 µM

At concentration:

2 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 1

Parameter:

EC 1.5 [442D]

Value:

63 µM

At concentration:

2 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC30 [442D]

Value:

389 µM

At concentration:

1 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

IC50 [442D]

Value:

444 µM

At concentration:

1 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

Imax [442D]

Value:

134.222 µM

At concentration:

1 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Key result

Group:

test chemical

Run / experiment:

run/experiment 2

Parameter:

EC 1.5 [442D]

Value:

297 µM

At concentration:

1 000 mM

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of skin sensitisation

Outcome of the prediction model:

negative [in vitro/in chemico]

Other effects / acceptance of results:

The test item precipitated at dose levels of 1000 μM and upwards in the first experiment. No precipitate was observed at any dose level tested in the second experiment. Two independent experiments were performed.
The test item showed toxicity (IC30 values of 77 μM and 389 μM and IC50 values of 94 μM and 444 μM in experiment 1 and 2, respectively). An induction of the luciferase activity (EC1.5 values of 63 μM and 297 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 79.25-fold and 134.22-fold in experiment 1 and 2 respectively. However, since positive results (>1.5-fold induction) were only observed at toxic test concentrations with a cell viability of <70% compared to the vehicle control, the results are considered not biologically relevant in accordance with the OECD test guideline 442D for interpretation of results.

Interpretation of results:

GHS criteria not met

Conclusions:

The test item is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Executive summary:

The objective of this study was to evaluate the ability of LZ 61000 to activate the antioxidant/electrophile responsive element (ARE)-dependent pathway in the KeratinoSens assay. The study was performed in accordance with OECD method 442D. The test item was dissolved in dimethyl sulfoxide (DMSO) at 200 mM and 100 mM in the first and second experiment, respectively. 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 0.98 – 2000 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 the highest dose required in the current guideline. The test item precipitated at dose levels of 1000 μM and upwards in the first experiment. No precipitate was observed at any dose level tested in the second experiment. Two independent experiments were performed.

The test item showed toxicity (IC30 values of 77 μM and 389 μM and IC50 values of 94 μM and 444 μM in experiment 1 and 2, respectively). An induction of the luciferase activity (EC1.5 values of 63 μM and 297 μM in experiment 1 and 2, respectively) was measured in both experiments. The maximum luciferase activity induction (Imax) was 79.25-fold and 134.22-fold in experiment 1 and 2 respectively. However, since positive results (>1.5-fold induction) were only observed at toxic test concentrations with a cell viability of <70% compared to the vehicle control, the results are considered not biologically relevant in accordance with the OECD test guideline 442D for interpretation of results.

In conclusion, LZ 61000 is classified as negative (no biologically relevant activation of the antioxidant/electrophile responsive element (ARE)-dependent pathway in keratinocytes) under the experimental conditions described in this report.

Reference 2

Endpoint:

skin sensitisation: in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

September - October 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 dimethyl sulfoxide at 50 and 12.5 mg/mL in the first and 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, 5, 10, 20 and 50 μg/mL). No precipitate was observed at any dose level 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:

58 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 1

Parameter:

EC150, CD86 [442E]

Value:

3.8 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:

42 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:

EC150, CD86 [442E]

Value:

7 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 58 μg/mL and 42 μg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 3.8 μg/mL and 7.0 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:

Category 1B (indication of skin sensitising potential) based on GHS criteria

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 dimethyl sulfoxide at 50 and 12.5 mg/mL in the first and 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, 5, 10, 20 and 50 μg/mL). No precipitate was observed at any dose level tested.

The test item showed toxicity (CV70 values of 58 μg/mL and 42 μg/mL in experiment 1 and 2, respectively). A biologically relevant, induction of the CD86 activity (EC150 values of 3.8 μg/mL and 7.0 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 61000 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.

Reference 3

Endpoint:

skin sensitisation: in vitro

Data waiving:

study technically not feasible

Justification for data waiving:

other:

Reference 4

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

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, detennined 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 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 being at the end of the sensitization key events required to cause presentation of allergic contact dermatitis acording to the Adverse Outcome Pathway (AOP) model. With the split result of the in-vitro studies and the negative for the in-vivo study, the weight of evidence leads to the conclusion that the Zinc Chelate complex is not a dermal sensitizer.