Hydroxy(2-methylprop-2-enoato-O)zinc

Endpoint summary

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Reference

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16 August - 05 October 2012

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

GLP study conducted in compliance with OECD Guideline 406 with minor deviations: the temperature recorded in the animal room was sometimes outside of the target ranges; the application sites were shaved again after the 24 h scoring of cutaneous reactions during the induction phase of the preliminary test.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

yes

Remarks:

the temperature recorded in the animal room was sometimes outside of the target ranges; the application sites were shaved again after the 24 h scoring of cutaneous reactions during the induction phase of the preliminary test.

Qualifier:

according to guideline

Guideline:

EU Method B.6 (Skin Sensitisation)

Version / remarks:

Version of 30 May 2008

Deviations:

yes

Remarks:

the temperature recorded in the animal room was sometimes outside of the target ranges; the application sites were shaved again after the 24 h scoring of cutaneous reactions during the induction phase of the preliminary test.

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes (incl. QA statement)

Type of study:

guinea pig maximisation test

Species:

guinea pig

Strain:

Hartley

Sex:

male/female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, L’Arbresle, France.
- Age at study initiation: 1-2 months
- Weight at study initiation: Mean body weight was 298 g (range: 259-385 g) for the males and 283 g (range: 261-316 g) for the females.
- Housing: Animals were individually housed in polycarbonate cages with stainless steel lid (Tecniplast 2154, 940 cm²) containing autoclaved sawdust (SICSA, Alfortville, France).
- Diet: 106 pelleted maintenance diet (SAFE, Augy, France), ad libitum
- Water: Tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2 °C
- Humidity: 50 ± 20 %
- Air changes: Approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod: 12 h dark / 12 h light

Route:

other: intradermal and topical

Vehicle:

other: Induction - intradermal injection: corn oil; topical application: ethanol/ drinking water treated by reverse osmosis (80/20) using ELIX 5 (Millipore SA) and challenge application: Acetone

Concentration / amount:

Preliminary test:
- Intradermal injection: 2.5, 5 and 10 %
- Topical induction application: 5 and 10 %
- Topical challenge application: 5 and 10 %

Main test:
Induction phase:
- Intradermal injection: 1 %
- Cutaneous application: 10 %
Challenge phase:
- Topical application: 5 %

Route:

other: topical

Vehicle:

other: Induction - intradermal injection: corn oil; topical application: ethanol/ drinking water treated by reverse osmosis (80/20) using ELIX 5 (Millipore SA) and challenge application: Acetone

Concentration / amount:

Preliminary test:
- Intradermal injection: 2.5, 5 and 10 %
- Topical induction application: 5 and 10 %
- Topical challenge application: 5 and 10 %

Main test:
Induction phase:
- Intradermal injection: 1 %
- Cutaneous application: 10 %
Challenge phase:
- Topical application: 5 %

No. of animals per dose:

Preliminary test: 4 animals (2 males and 2 females),
Main test: 30 animals (15 males and 15 females).
- Negative control: 5 males and 5 females
- Test item: 10 males and 10 females

See table 7.4.1/1 and 7.4.1/2 for further details

Details on study design:

PRELIMINARY TEST:
- Intradermal injection: Dose formulations were administered with or without Freund’s Complete Adjuvant (FCA) by intradermal injection in the clipped interscapular region. Cutaneous reactions were recorded before treatment, 24 and 48 h and 6 days after injection.
- Topical induction application: A filter paper (approximately 8 cm^2) was fully-loaded with the dose formulations, and then applied to the clipped interscapular region. The filter paper was held in place by an occlusive dressing for 48 h. Upon dressing removal, the residual dose formulation was removed using a cotton pad moistened with drinking water treated by reverse osmosis in female. No residual test item was observed in male. Cutaneous reactions were evaluated before treatment, 24 and 48 h after removal of the dressing.
- Topical challenge application: A Finn Chamber filter paper was fully-loaded with the dosage forms, and then applied to the shaved posterior right or left flank. The chamber was held in place by an occlusive dressing for 24 h. Upon dressing removal, any residual dose formulation was removed using a cotton pad moistened with drinking water treated by reverse osmosis. Cutaneous reactions were evaluated before treatment, 24 and 48 h post dressing removal.

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Exposure period: 7 days
- Test groups: On Day 1, three pairs of intradermal injections of 0.1 mL of FCA/0.9% NaCl (50/50,v/v), test item in vehicle and test item (w/v) in FCA/0.9% NaCl (50/50,v/v) were performed on the top, middle and down site of the interscapular region, respectively.
- Control group: On Day 1, three pairs of intradermal injections of 0.1 mL of FCA/0.9% NaCl (50/50,v/v), vehicle and vehicle at 50% (w/v) in FCA/0.9% NaCl (50/50,v/v) were performed on the top, middle and down sites of the interscapular region, respectively.
- Site: Interscapular region
- Duration: Days 1-7

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One
- Day of exposures: Day 8
- Exposure period: 48 h
- Test groups: A filter paper (approximately 8 cm^2) was fully-loaded with the dose formulations, and then applied to the clipped interscapular region, over the intradermal injection sites under occlusive dressings.
- Control group: Control animals received the vehicle only.
- Site: Interscapular region
- Frequency of applications: Single application
- Duration: Days 8-21

C. CHALLENGE EXPOSURE: TOPICAL
- No. of exposures: One
- Day(s) of challenge: Day 22
- Exposure period: 24 h
- Test groups: A Finn Chamber filter paper was fully-loaded with the dose formulations. The test item dose formulations were applied to the shaved posterior right flank of animals and the vehicle was applied to the shaved posterior left flank. The chamber was held in contact with the skin by an occlusive dressing for 24 h.
- Evaluation (h after challenge): Before treatment, 24 and 48 h after removal of the dressing.

OTHER:
- Morbidity and mortality: Each animal was checked for mortality and morbidity at least once a day during the treatment and observation periods, including weekends and public holidays.
- Clinical signs: Each animal was observed at least once a day, at approximately the same time, for the recording of clinical signs.
- Body weight: Body weight of each animal was recorded on the day of group allocation, then on the first day of treatment and at sacrifice.
- Pathology: On completion of the observation period, all animals were sacrificed by an intraperitoneal injection of pentobarbital sodium then by cervical dislocation. No macroscopic post-mortem examination was performed in any animals. For all animals, skin samples of the challenged application sites were preserved in 10 % buffered formalin. No microscopic examination was performed.

Challenge controls:

The test item dose formulations were applied to the shaved posterior right flank of animals and the vehicle was applied to the shaved posterior left flank. The chamber was held in contact with the skin by an occlusive dressing for 24 h.

Positive control substance(s):

no

Positive control results:

Not applicable

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

5 %

No. with + reactions:

10

Total no. in group:

20

Clinical observations:

At the 24-h reading, a discrete and moderate erythema (grade 1 and 2, respectively) were noted on the right flank (treated with the test item) of 10/20 and 1/20 animals, respectively.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 5 %. No with. + reactions: 10.0. Total no. in groups: 20.0. Clinical observations: At the 24-h reading, a discrete and moderate erythema (grade 1 and 2, respectively) were noted on the right flank (treated with the test item) of 10/20 and 1/20 animals, respectively..

Reading:

1st reading

Hours after challenge:

48

Group:

test chemical

Dose level:

5 %

No. with + reactions:

7

Total no. in group:

20

Clinical observations:

At 48-h reading, a discrete erythema (grade 1) was noted on the right flank (test item) of 7/20 animals with dryness of the skin in 2/10 females. A moderate erythema (grade 2) was noted on the right flank (test item) of 1/20 animals with dryness of skin.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 5 %. No with. + reactions: 7.0. Total no. in groups: 20.0. Clinical observations: At 48-h reading, a discrete erythema (grade 1) was noted on the right flank (test item) of 7/20 animals with dryness of the skin in 2/10 females. A moderate erythema (grade 2) was noted on the right flank (test item) of 1/20 animals with dryness of skin..

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

0

No. with + reactions:

0

Total no. in group:

10

Clinical observations:

No cutaneous reactions were observed on the left flank (treated with the vehicle) and on the right flank (treated with the test item) of the animals.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 0. No with. + reactions: 0.0. Total no. in groups: 10.0. Clinical observations: No cutaneous reactions were observed on the left flank (treated with the vehicle) and on the right flank (treated with the test item) of the animals. .

Reading:

1st reading

Hours after challenge:

48

Group:

negative control

Dose level:

0

No. with + reactions:

1

Total no. in group:

10

Clinical observations:

A discrete erythema (grade 1) associated with dryness of the skin were observed on the right flank (treated with the test item) of 1/10 animals. No cutaneous reactions were noted in the other animals of the control group.

Remarks on result:

other: see Remark

Remarks:

Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 0. No with. + reactions: 1.0. Total no. in groups: 10.0. Clinical observations: A discrete erythema (grade 1) associated with dryness of the skin were observed on the right flank (treated with the test item) of 1/10 animals. No cutaneous reactions were noted in the other animals of the control group..

Cutaneous reactions:

- In the control group, at the 24-h reading, no cutaneous reactions were observed on the left flank (treated with the vehicle) and on the right flank (treated with the test item) of the animals. At the 48-h reading, a discrete erythema (grade 1) associated with dryness of the skin were observed on the right flank (treated with the test item) of 1/10 animals. No cutaneous reactions were noted in the other animals of the control group. 

- In the test item-treated group, at the 24-h reading, a discrete and moderate erythema (grade 1 and 2, respectively) were noted on the right flank (treated with the test item) of 10/20 and 1/20 animals, respectively. At the 48-h reading, a discrete erythema (grade 1) was noted on the right flank (treated with the test item) of 7/20 animals associated with dryness of the skin in 2/10 females. A moderate erythema (grade 2) was noted on the right flank (treated with the test item) of 1/20 animals associated with dryness of the skin.

- As the dermal reactions observed on the right flank (treated with the test item) of the test item-treated animals were of higher incidence and severity than those recorded in the control animals, they were considered attributable to delayed contact hypersensitivity.

Others:

- No unscheduled deaths occurred during the study.

- No clinical signs indicative of systemic toxicity were observed in any animals. However, scabs and cracks were observed at the interscapular region of all animals during the observation period, associated with wound in one of them.

- Body weight of the animals was unaffected by the test item treatment.

Interpretation of results:

sensitising

Remarks:

Migrated information Criteria used for interpretation of results: EU

Conclusions:

Under these test conditions, Hydroxy(2-methylprop-2-enoato-O)zinc is classified as R43 “May cause sensitisation by skin contact” according to the Annex VI to the Directive 67/548/EEC and “Category 1B” according to the CLP Regulation (EC) N° (1272-2008).

Executive summary:

In a GLP-compliant Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406, groups (10 animals/sex) of Hartley guinea pigs were induced with three pairs of intradermal injections of 0.1 mL of FCA/0.9% NaCl (50/50,v/v), test item (Hydroxy (2 -methylprop-2 -enoato-O)zinc in vehicle and test item (w/v) in FCA/0.9% NaCl (50/50,v/v) that were performed on the top, middle and bottom sites of interscapular region, respectively. The control group (5 animals/sex) was intradermally induced with three pairs of 0.1 mL of FCA/0.9% NaCl (50/50,v/v), vehicle and vehicle at 50% (w/v) in FCA/0.9% NaCl (50/50,v/v) that were performed on the top, middle and bottom sites of the interscapular region, respectively. On Day 8, a filter paper (approximately 8 cm^2) was fully-loaded with the dose formulations, and then applied to the clipped interscapular region, over the intradermal injection sites under occlusive dressings for 48 h. Control animals received the vehicle only. After a 2 week rest period, a challenge application of test item dose formulations were applied to the shaved posterior right flank of animals and the vehicle was applied to the shaved posterior left flank, under occlusive dressings for 24 h. The test concentrations for the main study were determined from a preliminary toxicity study.

No mortality was observed during the study. No clinical signs indicative of systemic toxicity were observed in any animals. However, scabs and cracks were observed at the interscapular region of all animals during the observation period, associated with wound in one of them. Body weight of the animals was unaffected by the test item treatment. In the control group, at the 24-h reading, no cutaneous reactions were observed on the left flank (treated with the vehicle) and on the right flank (treated with the test item) of the animals. At the 48-h reading, a discrete erythema (grade 1) associated with dryness of the skin were observed on the right flank (treated with the test item) of 1/10 animals. No cutaneous reactions were noted in the other animals of the control group. In the test item-treated group, at the 24-h reading, a discrete and moderate erythema (grade 1 and 2, respectively) were noted on the right flank (treated with the test item) of 10/20 and 1/20 animals, respectively. At the 48-h reading, a discrete erythema (grade 1) was noted on the right flank (treated with the test item) of 7/20 animals associated with dryness of the skin in 2/10 females. A moderate erythema (grade 2) was noted on the right flank (treated with the test item) of 1/20 animals associated with dryness of the skin. As the dermal reactions observed on the right flank (treated with the test item) of the test item-treated animals were of higher incidence (8/20) and severity than those recorded in the control animals, they were considered attributable to delayed contact hypersensitivity.

 

Under these test conditions, Hydroxy(2-methylprop-2-enoato-O)zinc is classified as R43 “May cause sensitisation by skin contact” according to the Annex VI to the Directive 67/548/EEC and “Skin sensitization - Category 1B” according to the CLP Regulation (EC) N° (1272-2008).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (sensitising)

Additional information:

In a GLP-compliant Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406, 20 Hartley guinea pigs (10/sex) were induced with intradermal injections of a mixture of FCA/0.9% NaCl (50/50, v/v), the submission substance 1% (w/v) in corn oil, and the submission 1% (w/v) substance in a mixture of FCA/0.9% NaCl (50/50, v/v) and 7 days later by a topical application of the submission substance 10% in a mixture ethanol/drinking water treated by reverse osmosis (80/20). They were then challenged with the submission substance 5% (w/v) in acetone.

 

Scabs and cracks were observed at the interscapular region of all animals during the observation period, associated with wound in one of them.

In the control group, a discrete erythema was observed in 1/10 animals at the 48-hr reading. No other skin reactions were observed in this group.

In the test item-treated group, at the 24-h reading, a discrete and moderate erythema (grade 1 and 2, respectively) were noted on the right flank (treated with the test item) of 10/20 and 1/20 animals, respectively. At the 48-h reading, a discrete erythema (grade 1) was noted on the right flank (treated with the test item) of 7/20 animals associated with dryness of the skin in 2/10 females. A moderate erythema (grade 2) was noted on the right flank (treated with the test item) of 1/20 animals associated with dryness of the skin.

As the dermal reactions observed on the right flank (treated with the test item) of the test item-treated animals were of higher incidence (8/20) and severity than those recorded in the control animals, they were considered attributable to delayed contact hypersensitivity.

Migrated from Short description of key information:
>30% positive reactions were observed in a Guinea Pig Maximisation Test

Justification for selection of skin sensitisation endpoint:
Only one study available

Respiratory sensitisation

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

As Hydroxy(2-methylprop-2-enoato-O)zinc induced delayed contact sensitization in 40% of the guinea pigs under these test conditions, it is classified as R43 “May cause sensitisation by skin contact” according to the Annex VI to the Directive 67/548/EEC and “Skin sensitization - Category 1B” according to the CLP Regulation (EC) No 1272-2008.