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Description of key information

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:
20 August - 21 September 2012
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study conducted in compliance with OECD Guideline 406 with minor deviation: temperature in the animal room was slightly outside the range
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
according to
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
yes
Remarks:
temperature in the animal room was slightly outside the range
Principles of method if other than guideline:
Not applicable
GLP compliance:
yes (incl. certificate)
Type of study:
guinea pig maximisation test
Species:
guinea pig
Strain:
Dunkin-Hartley
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, L’Arbresle, France.
- Age at study initiation: 5 weeks
- Weight at study initiation: 227-277 g
- Housing: Animals were housed in groups of 2 (in case of odd number, the last animal was housed individually) in polycarbonate containers.
- Diet: Food (SDS, FD1 guinea pig breeding and maintenance diet), ad libitum
- Water: Drinking water (tap water from public distribution system), ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 19-25 °C
- Humidity: 30-70 %
- Air changes: Approximately 10-15 changes/h
- Photoperiod: 12 h dark / 12 h light
Route:
other: intradermal and topical, occlusive
Vehicle:
other: liquid paraffin for the intradermal injections and in olive oil for the topical applications
Concentration / amount:
Preliminary test:
- Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): 0.25, 0.5, 1, 2.5, 5 and 10 % in olive oil; due to necrosis observed at all concentrations, the same animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 3 concentrations: diluted at 0.05, 0.1 and 0.2 % in olive oil.
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): 10, 20, 35 and 60 % in liquid paraffin.
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC): 5, 10, 20 and 35 % in liquid paraffin.

Main test:
Induction phase:
First Intradermal (ID) Induction:
Group 1 (Negative control):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in olive oil.
2 ID: olive oil
2 ID: a mixture with equal volumes of: Freund’s Complete Adjuvant at 50 % and olive oil
Group 2 (Treated):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in olive oil,
2 ID: test item at 0.2 % in olive oil,
2 ID: a test mixture in equal volumes of: Freund’s Complete Adjuvant at 50 % and the test item at 0.4 % in olive oil.

Second Topical Induction:
Group 1 (Negative control): 0.5 mL of liquid paraffin.
Group 2 (treated): 0.5 mL of the test item at 60 % in liquid paraffin.

Challenge phase:
- Group 1 (Negative control) and Group 2 (Treated): 1 sample cup containing the test item at 10 % (MNIC) and 1 sample cup containing the vehicle (liquid paraffin).
Route:
other: topical, occlusive
Vehicle:
other: liquid paraffin for the intradermal injections and in olive oil for the topical applications
Concentration / amount:
Preliminary test:
- Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): 0.25, 0.5, 1, 2.5, 5 and 10 % in olive oil; due to necrosis observed at all concentrations, the same animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 3 concentrations: diluted at 0.05, 0.1 and 0.2 % in olive oil.
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): 10, 20, 35 and 60 % in liquid paraffin.
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC): 5, 10, 20 and 35 % in liquid paraffin.

Main test:
Induction phase:
First Intradermal (ID) Induction:
Group 1 (Negative control):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in olive oil.
2 ID: olive oil
2 ID: a mixture with equal volumes of: Freund’s Complete Adjuvant at 50 % and olive oil
Group 2 (Treated):
2 ID: Freund’s Complete Adjuvant diluted at 50 % in olive oil,
2 ID: test item at 0.2 % in olive oil,
2 ID: a test mixture in equal volumes of: Freund’s Complete Adjuvant at 50 % and the test item at 0.4 % in olive oil.

Second Topical Induction:
Group 1 (Negative control): 0.5 mL of liquid paraffin.
Group 2 (treated): 0.5 mL of the test item at 60 % in liquid paraffin.

Challenge phase:
- Group 1 (Negative control) and Group 2 (Treated): 1 sample cup containing the test item at 10 % (MNIC) and 1 sample cup containing the vehicle (liquid paraffin).
No. of animals per dose:
Preliminary test:
- Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): 2 animals
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): 2 animals
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC): 3 animals

Main test:
- Group 1 (negative control): 5 females
- Group 2 (treated): 10 females
Details on study design:
PRELIMINARY TEST:
- Determination by intradermal injection of the Maximal Non Necrotizing Concentration (MNNC): Two animals received on both sides of the spine, a volume of 0.1 mL of the test item, at 0.25, 0.5, 1, 2.5, 5 and 10 % in olive oil and cutaneous reactions were recorded 24 h after the injections; due to necrosis observed at all concentrations, the same animals received 0.1 mL of the test item at concentrations of 0.05, 0.1 and 0.2 % in olive oil. Cutaneous reactions were conducted 24 h after the injection.
- Determination by topical application of the Pre-Maximal Non Irritant Concentration (Pre-MNIC): Test item was applied on the shorn skin of dorso-lumbar zone of two guinea pigs with occlusive dressing for 24 h at concentrations of 10, 20, 35 and 60 % in liquid paraffin. Cutaneous reactions were recorded 24 h after removal of the dressing and rinsed with liquid paraffin.
- Determination by topical application of the Maximal Non Irritant Concentration (MNIC): Three guinea pigs were treated according to the same treatment as animals from Group 1 (negative control) for the induction phase (i.e. olive oil and liquid paraffin). During the challenge phase, the animals were treated with the test item placed onto the selected treatment sites and covered with an occlusive dressing for a period of 24 h at the concentrations of 5, 10, 20 and 35 % in liquid paraffin. Cutaneous reactions were recorded 24 and 48 hours after removal of the occlusive dressing.

MAIN STUDY
A. INDUCTION EXPOSURE: INTRADERMAL
- No. of exposures: One
- Exposure period: 7 days
- Test groups: On Day 0, three pairs of intradermal injections (ID) of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil (2 ID), test item at 0.2 % in olive oil (2 ID) and a test mixture in equal volumes of Freund’s Complete Adjuvant at 50 % and the test item at 0.4 % in olive oil (2 ID) were performed on the either side of the spine in the scapular region.
- Control group: On Day 0, three pairs of intradermal injections (ID) of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil (2 ID), olive oil (2 ID) and a mixture with equal volumes of Freund’s Complete Adjuvant at 50 % and olive oil (2 ID) were performed on the either side of the spine in the scapular region.
- Site: Scapular region
- Duration: Days 0-6

B. INDUCTION EXPOSURE: TOPICAL
- No. of exposures: One
- Exposure period: 48 h
- Test groups: Topical application of 0.5 mL of the test item at 60 % in liquid paraffin under occlusive dressing for 48 h was performed on the injection sites of each animal.
- Control group: Topical application of 0.5 mL of liquid paraffin under occlusive dressing for 48 h was performed on the injection sites of each animal.
- Site: Scapular region
- Frequency of applications: Single application
- Duration: Days 7-20

C. CHALLENGE EXPOSURE: TOPICAL
- No. of exposures: One
- Day(s) of challenge: Day 20
- Exposure period: 24 h
- Test groups: Topical application of 1 sample cup containing the test item at 10% (MNIC) on either side of the spine, under occlusive dressing for 24 h.
- Control group: Topical application of 1 sample cup containing the vehicle (liquid paraffin) on either side of the spine, under occlusive dressing for 24 h.
- Site: Shorn dorso-lumbar zone
- Evaluation (h after challenge): 24, 48 and 72 h

OTHER:
- Animals were observed for mortality and weighed individually on the day of beginning (Day 0) and end of the study (Day 23).
Challenge controls:
Topical application of 1 sample cup containing the vehicle (liquid paraffin) on either side of the spine on Day 20 under occlusive dressing for 24 h.
Positive control substance(s):
yes
Remarks:
α-Hexylcinnamaldehyde (Study no. SMK-2011-002, SMK-2011-003 and SMK-2012-001)
Concentration:
Not applicable
No. of animals per dose:
Not applicable
Details on study design:
Not applicable
Statistics:
Not applicable
Positive control results:
α-Hexylcinnamaldehyde induced positive skin sensitization reactions in guinea pigs.
Reading:
1st reading
Hours after challenge:
24
Group:
test group
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 10 %. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
48
Group:
test group
Dose level:
10 %
No. with + reactions:
5
Total no. in group:
10
Clinical observations:
none
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: test group. Dose level: 10 %. No with. + reactions: 5.0. Total no. in groups: 10.0. Clinical observations: none.
Reading:
1st reading
Hours after challenge:
72
Group:
test group
Dose level:
10 %
No. with + reactions:
7
Total no. in group:
10
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 72.0. Group: test group. Dose level: 10 %. No with. + reactions: 7.0. Total no. in groups: 10.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
24
Group:
negative control
Dose level:
Liquid paraffin
No. with + reactions:
2
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: Liquid paraffin. No with. + reactions: 2.0. Total no. in groups: 5.0. Clinical observations: None.
Reading:
1st reading
Hours after challenge:
48
Group:
negative control
Dose level:
Liquid paraffin
No. with + reactions:
1
Total no. in group:
5
Clinical observations:
None
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 48.0. Group: negative control. Dose level: Liquid paraffin. No with. + reactions: 1.0. Total no. in groups: 5.0. Clinical observations: None.
Parameter:
SI
Remarks on result:
other: Not applicable
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: Not applicable

Table 7.4.1/1: Results of skin sensitization – challenge phase

 

Groups

Reading time (h)

Concentrations (%)

Scores

% of

positive

responses

1

% of

animal

sensitized#

Negative

Control

Group

24

10

3

1

1

0

40

 

NA

48

10

4

1

0

0

20

72

10

5

0

0

0

0

Treated

Group

24

10

1

3

1

5

90

50

48

10

0

5

2

3

100

50

72

10

3

7

0

0

70

70

 

Grading scales:

Erythema

0: No visible modification

1: Slight or patches of erythema

2: Moderate confluent erythema

3: Internal erythema and swelling

Oedema

0: No visible modification

1: Slight oedema

2: Moderate oedema

3: important oedema  

 

# A comparison of the intensities and persistence of reactions at the test item challenge sites in the test and control animals permits identification

of sensitisation reactions.

Others:

- No mortality was observed during the study.

- No abnormality was recorded in the body weight gain of treated and control groups.

Interpretation of results:
sensitising
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Under these test conditions, Zinc dimethacrylate 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 Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, groups of 10 Dunkin-Hartley female guinea pigs were induced with three pairs of intradermal injections of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil (2 ID), test item at 0.2 % in olive oil (2 ID) and a test mixture in equal volumes of Freund’s Complete Adjuvant at 50 % and the test item at 0.4 % in olive oil on Day 0 on three different sites on either side of the spine in the scapular region. Control group of 5 females was intradermally induced with three pairs of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil, olive oil and a mixture with equal volumes of Freund’s Complete Adjuvant at 50 % and olive oil. After one week the same area was topically induced with 0.5 mL of the test item at 60 % in liquid paraffin under occlusive dressing for 48 h, whereas control group was applied with 0.5 mL of liquid paraffin under occlusive dressing for 48 h. After 2 weeks of rest period, a challenge application of vehicle and test item at 10 % was applied on either side of the spine on the dorso-lumbar region. The test concentrations for the main study were determined from a preliminary toxicity study.

No mortality was observed during the study. No abnormality was recorded in the body weight gain of treated and control groups. In the treatment group (treatment dose of 10%), a more severe reaction was observed in the treated site than in the control site (treated with liquid paraffin only) in 90% (9/10), 100% (10/10) and 70% (7/10) of the animals, 24, 48 and 72 hours respectively after the challenge phase. In the control group, a more severe reaction was observed in the treated site than in the control site (treated with liquid paraffin only) in 40% (2/5) and 20% (1/5) of the animals, 24 and 48 hours respectively after the challenge phase. As irritation was noted in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a sensitization reaction was noted in 50% (5/10), 50% (5/10) and 70% (7/10) of the animals from the treated group with the test item at 10%. Historical data on positive control (α-Hexylcinnamaldehyde) exhibited evidence of sensitization indicating the validity of the study.

 

Under these test conditions, Zinc dimethacrylate 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).
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

In a Magnusson & Kligman maximisation study (GPMT) performed according to OECD Guideline 406 and in compliance with GLP, groups of 10 Dunkin-Hartley female guinea pigs were induced with three pairs of intradermal injections of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil (2 ID), test item at 0.2 % in olive oil (2 ID) and a test mixture in equal volumes of Freund’s Complete Adjuvant at 50 % and the test item at 0.4 % in olive oil on Day 0 on three different sites on either side of the spine in the scapular region. Control group of 5 females was intradermally induced with three pairs of 0.1 mL of Freund’s Complete Adjuvant diluted at 50 % in olive oil, olive oil and a mixture with equal volumes of Freund’s Complete Adjuvant at 50 % and olive oil. After one week the same area was topically induced with 0.5 mL of the test item at 60 % in liquid paraffin under occlusive dressing for 48 h, whereas control group was applied with 0.5 mL of liquid paraffin under occlusive dressing for 48 h. After 2 weeks of rest period, a challenge application of vehicle and test item at 10 % was applied on either side of the spine on the dorso-lumbar region. The test concentrations for the main study were determined from a preliminary toxicity study.

No mortality was observed during the study. No abnormality was recorded in the body weight gain of treated and control groups. In the treatment group (treatment dose of 10%), a more severe reaction was observed in the treated site than in the control site (treated with liquid paraffin only) in 90% (9/10), 100% (10/10) and 70% (7/10) of the animals, 24, 48 and 72 hours respectively after the challenge phase. In the control group, a more severe reaction was observed in the treated site than in the control site (treated with liquid paraffin only) in 40% (2/5) and 20% (1/5) of the animals, 24 and 48 hours respectively after the challenge phase. As irritation was noted in the control group animals, only reactions in the test group animals that exceeded the most severe reactions seen in the control group animals were attributed to skin sensitization. Therefore, a sensitization reaction was noted in 50% (5/10), 50% (5/10) and 70% (7/10) of the animals from the treated group with the test item at 10%. Historical data on positive control (α-Hexylcinnamaldehyde) exhibited evidence of sensitization indicating the validity of the study.


Migrated from Short description of key information:
More than 30% positive reactions were observed in a guinea pigs 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 zinc methacrylate induced 50% of positive response following intradermal induction at 0.2% and challenge at 10%, 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.