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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Remarks:
read-across from hydrolysis products
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to guidelines
Justification for type of information:
Magnesium ethanolate decomposes within 1 minute at pH 4; pH 7, and pH 9 at 10°C and at room temperature in water. Based on the hydrolysis products ethanol, 90% of the hydrolysis products could already be detected after 1 minute.
In contact with water, magnesium ethanolate reacts very rapidly, quantitatively and exothermically to ethanol and magnesium hydroxide: Mg++-(O-C2H5)2+ 2 H2O ---> 2 C2H5OH + Mg(OH)2.

Therefore, conclusions on the skin sensitizing potential of Magnesium ethanolate are drawn from the skin sensitizing potential of the hydrolysis products. This read-across is based on ethanol and, mainly referring to a Skin sensitisation statement, Magnesium hydroxide.
Cross-referenceopen allclose all
Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Although an appropriate guideline was followed, the study was not designed specifically to assess the sensitizing potential of ethanol. Ethanol was used as a vehicle control only; there was thus no adequate concurrent negative control for comparison.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
yes
Remarks:
. OECD Guideline 429 recommends that animals are housed individually, and that females are used.
Principles of method if other than guideline:
The study was designed to evaluate the effect of vehicles (ethanol or diethyl phthalate) for use in the mouse local lymph node assay.
GLP compliance:
not specified
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Interfauna UK, Shaw's Farm, Blackthorne, Bicester, Oxon, UK
- Age at study initiation: 8 - 12 wk
- Weight at study initiation: not stated
- Housing: Groups of four animals per cage under standard conditions
- Diet (e.g. ad libitum): Pelleted diet ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: not stated


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): not stated
- Photoperiod (hrs dark / hrs light): Fluorescent light - 12-hr dark/12-hr light cycle
Vehicle:
unchanged (no vehicle)
No. of animals per dose:
4
Details on study design:
Four alternative vehicles (ethanol; 3:1 ethanol:diethyl phthalate; 1:3 ethanol:diethyl phthalate; and diethyl phthalate) were evaluated for their utility in the LLNA, and their influence on the skin sensitization potential of four test fragrance materials (p-t-butyl-alpha-methylhydrocinnamic aldehyde; geraniol; eugenol; and hydroxycitronellal). Groups of four male mice were treated with each test fragrance, at one of five concentrations, in one of the four vehicles or to the same volume of the vehicle (ethanol or diethyl phthalate) alone (see Table 1 for further details). Stimulation Index (SI), as a measure of T-lymphocyte proliferation, was calculated for each concentration of test fragrance relative to the concurrent vehicle-treated animals.
Positive control substance(s):
other: The test fragrances used in the study (p-t-butyl-alpha-methylhydrocinnamic aldehyde; geraniol; eugenol; and hydroxycitronellal) effectively acted as positive controls; they are all known to be mild to moderate sensitizers.
Statistics:
SI values for the test fragrances were compared using an analysis of variance. Estimated concentrations of test fragrances required to elicit as SI of 3 or more (EC3 value) were also calculated using dose-response data. As ethanol was tested as a vehicle control in this study, there was no appropriate comparison with untreated animals (or with the alternative vehicle used, diethyl phthalate).
Positive control results:
Although not true positive controls, the test fragrances clearly induced dose-related stimulation of T-lymphocyte proliferation (see Table 1).
Parameter:
SI
Remarks on result:
other: Not applicable
Parameter:
other: disintegrations per minute (DPM)
Remarks on result:
other: see Remark
Remarks:
When tested alone, ethanol resulted in mean counts per minute per lymph node (pooled from four mice) in the range 133 - 175. Although there was no true control data for camparison, the other vehicle tested as a control in this study, diethyl phthalate, resulted in mean counts per minute/node in the range 185 - 267.

Although the study was not specifically designed to assess the sensitizing potential of ethanol, it is clear from the results (Table 1) that the level of induced T-lymphocyte proliferation (a necessary component of contact sensitization) in the lymph node draining the site of topical chemical application was low (as measured by the incorporation of radiolabelled thymidine into the dividing cells) for ethanol when compared with that for fragrance materials known to be mild to moderate skin sensitizers, and comparable to that for the other (negative) control vehicle tested, diethyl phthalate. In addition, there was no discernable induction (increase in cpm/node) across the four test fragrance control series when the ethanol concentration (in diethyl phthalate) was 0% [100% diethyl phthalate], 33% [1:3 ethanol:diethyl phthalate], 67% [3:1 ethanol:diethyl phthalate], and 100%, which would be expected were ethanol a significant skin sensitizer.

Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
The investigators concluded that ethanol is an appropriate vehicle for use in the LLNA and is a suitable alternative to the current guideline recommended vehicles. A prerequisite for use as a vehicle would be a lack of sensitizing potential itself.
Executive summary:

A study was carried out to evaluate the effect of vehicles (ethanol or diethyl phthalate) for use in the mouse local lymph node assay (LLNA), and their influence on the skin sensitization potential of four test fragrance materials (p-t-butyl-alpha-methylhydrocinnamic aldehyde; geraniol; eugenol; and hydroxycitronellal). Groups of 4 mice were treated with each test fragrance, at one of five concentrations, either in ethanol or diethyl phthalate (and 1:3 or 3:1 mixtures of the two), or with ethanol (or diethyl phthalate) alone. Although there were no true control data for comparison with the ethanol-alone treated animals, the level of induced T-lymphocyte proliferation was low for ethanol when compared with that for fragrance materials known to be mild to moderate skin sensitizers, and comparable to that for the other (negative) control vehicle tested, diethyl phthalate.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP study conducted according to guidelines
Qualifier:
according to guideline
Guideline:
OECD Guideline 406 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EC No. 440/2008, L 142
Deviations:
no
GLP compliance:
yes
Type of study:
guinea pig maximisation test
Justification for non-LLNA method:
Existing study on an anolog substance.
Species:
guinea pig
Strain:
other: Crl:HA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles RIver, 97633 Sulzfeld, Germany
- Age at study initiation: Approximately 5 weeks old
- Weight at study initiation:354 - 408 g
- Housing: The animals were kept in groups inTerluran- cages on Altromin saw fibre bedding in an air-conditioned room.
- Diet: Free access to autoclaved hay and to Altromin 3122 maintenance diet for guinea pigs
- Water: Free access to tap water
- Acclimation period: At least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 55 ± 10%
- Air changes (per hr): At least 10 x/ hour

IN-LIFE DATES: From: 25 January 2010 To: 25 February 2010
Route:
intradermal
Vehicle:
physiological saline
Concentration / amount:
in the preliminary test one animal was treated intradermally with concentrations of 5% and 2.5% of the test item. Two animals were treated topically with concentrations of 100% and 50% of the test item for 24 as well as 48 hours.
Based on the results of this preliminary test, a concentration of 5% was chosen for the intradermal application of the main test and a concentration of 50% was selected for the dermal induction.
A concentration of 50% was found to be the highest dose suspended in vehicle which did not cause any signs of irritation after topical treatment over 24 hours and therefore was chosen for the challenge application in the main test.
Route:
other: topical
Vehicle:
physiological saline
Concentration / amount:
in the preliminary test one animal was treated intradermally with concentrations of 5% and 2.5% of the test item. Two animals were treated topically with concentrations of 100% and 50% of the test item for 24 as well as 48 hours.
Based on the results of this preliminary test, a concentration of 5% was chosen for the intradermal application of the main test and a concentration of 50% was selected for the dermal induction.
A concentration of 50% was found to be the highest dose suspended in vehicle which did not cause any signs of irritation after topical treatment over 24 hours and therefore was chosen for the challenge application in the main test.
No. of animals per dose:
Number of animals in the test group: 10
Number of animals in the negative control group: 5
NUmber of animals in the dose range finding study: 3
Details on study design:
RANGE FINDING TESTS:

MAIN STUDY
A. INDUCTION EXPOSURE

Three pairs of intradermal injections of 0.1 mL volume were given in the shoulder region which was cleared of hair by clipping so that one of each pairs lies on each side of the midline.
Test group: day 0:
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl
Injection 2: a 5% concentration of the test item in physiological saline 0.9% naCl.
Injection 3: a 5% concentration of the test item formulated in a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl.

Control group: day 0
Injection 1: a 1:1 mixture (v/v) FCA/physiological saline 0.9% NaCl.
Injection 2: 100% physiological saline 0.9% NaCl
Injection 3: a 50% (v/v) formulation of NaCl 0.9% in a 1:1 (v/v) mixture FCA/physiological saline 0.9% NaCl.

Injections 1 and 2 were given close to each other and nearest to the head, while injection 3 is given toward the caudal part of the test area.

Induction: Second stage, Topical Application

Test and control group: day 6

Approximately 24 hours before the topical application the test area was painted with 0.5 mL of 10% sodium lauryl sulphate in vaseline after close clipping, in order to create a local irritation.
Test group: day 7
The test item was suspended in vaseline at a concentration of 50%. A patch was fully loaded with 0.5g of the prepared test item,applied to the test area and held in contact by an occlusive dressing for 48 hours.
Control group: day 7
A patch was fully loaded with 0.5 g of the vaseline and applied to the test area and held in contact by an occlusive dressing for 48 hours.


B. CHALLENGE EXPOSURE
Challenge:Topical application
The flanks of treated and control animals were cleared of hair by close-clipping.

Test and control group: day 20
The test item was suspended in vaseline at a concentration of 50%. A patch, loaded with 0.5 g of the prepared test item was applied to the left flank of the animals and a patch loaded with 0.5ml of the vehicle to the right flank (intraspecific control), respectively. The patches were held in contact by an occlusive dressing for 24 hours. The application area was not rinsed with water.

Observation period:
Test and control group:
Approximately 21 hours after removing the patch, the challenge area was cleared of hair by the use of a depilatory cream. Approximately 2 and 48 hours after removing the patch the skin reaction was obseved and recorded according to the classification in Table 1.
Additionally all animals were observed for signs of toxicity at least once daily during the test period.
Challenge controls:
A patch loaded with 0.5ml of the vehicle
Positive control substance(s):
yes
Remarks:
NaCl
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group and control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 2 in 5/5 control and 10/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test group and control. Dose level: Injection site 1 . Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 5/5 control and 10/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group and control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 2 in 4/5 control and 10/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test group and control. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 4/5 control and 10/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
erythema grade 1 in 9/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 9/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
eschar in 8/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: eschar in 8/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 2 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 1/5 control and 1/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 1 in 1/5 control and 9/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 1/5 control and 9/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 2 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control. Dose level: Injection site 3 . Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 1/5 control and 1/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control and 2/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control and 2/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
eschar in 1/5 control and 6/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: eschar in 1/5 control and 6/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 2 in 1/5 control and 3/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 1/5 control and 3/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 1 in 4/5 control and 7/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1 . Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 4/5 control and 7/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 2 in 1/5 control and 3/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 1/5 control and 3/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 1 in 4/5 control and 7/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 4/5 control and 7/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
necrosis in 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: necrosis in 1/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
eschar in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: eschar in 1/5 control and 1/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
erythema grade 1 in 4/5 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 4/5 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 1 in 2/5 control and 9/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 2/5 control and 9/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control and 1/10 test animals.
Reading:
other: Immediately after removing the patch
Hours after challenge:
48
Group:
other: test and control group
Total no. in group:
15
Clinical observations:
Desquamation in 1/5 control animals, erythema grade 1 in 2/10 test animals
Remarks on result:
other: Reading: other: Immediately after removing the patch. . Hours after challenge: 48.0. Group: other: test and control group. Total no. in groups: 15.0. Clinical observations: Desquamation in 1/5 control animals, erythema grade 1 in 2/10 test animals.

Preliminary Test:

One animal was treated intradermally with concentrations of 5% and 2.5% of the test item. Two animals were treated topically with concentrations of 100% and 50% of the test item for 24 as well as 48 hours.

Based on the results of this preliminary test, a concentration of 5% was chosen for the intradermal application of the main test and a concentration of 50% was selected for the dermal induction.A concentration of 50% was found to be the highest dose suspended in vehicle which did not cause any signs of irritation after a topical treatment over 24 hours and therefore was chosen for the challenge application in the main test.

Main test:

Signs of irritation during the induction:

Intradermal Induction I (24 hours reading)

Injection site 1: erythema grade 2 in 5/5 control and 10/10 test,oedema grade 2 in 4/5 control and 10/10 test animals and oedema grade 1 in 1/5 control animals.

Injection site 2: erythema grade 1 in 9/10 test animal

eschar in 8/10 test animals.

Injection site 3: erythema grade 2 in 1/5 control and 1/10 test animals, erythema grade 1 in 1/5 control and 9/10 test animals, oedema grade 2 in 1/5 control and 1/10 test animals, oedema grade 1 in 1/5 control and 2/10 test animals and eschar in 1/5 control and 6/10 test animals.

Intradermal Induction I (48 hours reading):

Injection site 1: erythema grade 2 in 1/5 control and 3/10 test animals, erythema grade 1 in 4/5 control and 7/10 test animals, oedema grade 2 in 1/5 control and 3/10 test animals, oedema grade 1 in 4/5 control and 7/10 test animals, necrosis in 1/10 animals and eschar in 1/5 control and 1/10 test animals.

Injection site 2: erythema grade 1 in 4/5 test animals

injection site 3: erythema grade 1 in 2/5 control and 9/10 test animals, oedema grade 1 in 1/5 control and 1/10 test animals.

Dermal Induction II (48 hours exposure, occlusive):

Immediately after removing the patch: Desquamation in 1/5 control animals, erythema grade 1 in 2/10 test animasl.

24 hours after removing the patch: Eschar in 5/5 control and 10/10 test animals, erythema grade 1 in 1/10 test animals, desquamation in 1/10 test animals.

Challenge exposure:

No erythema was observed in any of the test animals at any time. Erythema grade 1 was observed after 24 hours in one animal of the control group. No oedema was observed in any animal at any time. There was no evidence of sensitisation in the test item group at the challenge and the percentageof animals sensitised was 0%.

The animals of the test group showed no reduced weight gain compared to historical data.

Table 1: Classification System:

Patch test reaction

 

Grade

No visible change

0

Discrete or patchy erythema

1

Moderate and confluent erythema

2

Intense erythema and swelling

3
Interpretation of results:
not sensitising
Remarks:
Migrated information
Conclusions:
Under the conditions of the present study it can be stated that the test item Magnesium chloride hexahydrate caused no reactions identified as sensitisating at the tested concentration.
Executive summary:

Under the conditions of the present study it can be stated that the test item Magnesium chloride hexahydrate caused no reactions identified as sensitisating at the tested concentration.

According to the EC criteria for classification and labelling requirements for dangerous substances and preparations (Guidelines in Comission Directive 2001/59/EC) as well as Annex I of regulation (EC) 1272/2008 classification and labelling is not necessary as the sensitisation rate is less than 30%.

Reason / purpose for cross-reference:
read-across source
Reference
Endpoint:
skin sensitisation: in vivo (non-LLNA)
Remarks:
statement
Data waiving:
other justification
Justification for data waiving:
other:
Justification for type of information:
Although the results of the local lymph node assay (LLNA) test on magnesium hydroxide were apparently positive it is proposed that the positive result should be interpreted as a false positive and that magnesium hydroxide thus should not be classified as a skin sensitizer.   There are a number of reasons to suggest that it is highly unlikely that magnesium hydroxide is a skin sensitizer. An expert opinion has been provided from the Informationsverbund Dermatologischer Kliniken (IVDK; Schnuch, A. 2010). The IVDK is a network of over 50 dermatological hospitals, and was formed to monitor and survey the development of contact allergies. Despite 20 years of observations in 192,421 patients they have yet to observe a case of a contact allergy with magnesium hydroxide. Furthermore, no case of contact allergy to magnesium hydroxide has been reported in the world literature, despite extensive exposure of the general population to the substance. Some reports on flame retardants address magnesium hydroxide toxicity, and no sensitizing effects are reported (DFE (EPA), 2008; NAP, 2000).   Furthermore, the results of a guinea pig maximisation test (GPMT) performed with magnesium chloride were obtained from the magnesium chloride consortium. The results presented show that magnesium chloride was negative in the GPMT. As any possible skin sensitizing potential of magnesium hydroxide is likely to stem from the cation rather than the hydroxide anion, the outcome of this test provides additional evidence that magnesium ions should not be regarded as a skin sensitizer. Regarding the hydroxide portion of the compound, there is ample evidence in the literature that hydroxides of other metals are not skin sensitizers (NICNAS, 2003; Banerjee et. al., 2003; DFE (EPA), 2008).   False positives in the LLNA assay are not uncommon, and indeed the LLNA was judged to be no more than 90 % accurate during its validation (Basketter et. al., 2010). On this basis, certain substances have not been classified as contact sensitizers despite giving positive responses in the LLNA test. These include copper chloride (Basketter and Scholes, 1992), sodium lauryl sulphate (Loveless et. al., 1996; Montelius et. al., 1994), benzalkonium chloride (Basketter et. al., 2004) and ethanol (Basketter et. al., 2010).   Given the combination of the epidemiological data, the negative magnesium chloride guinea pig maximisation test and the history of false positives using the LLNA test, the weight of evidence suggests that the positive results obtained in the LLNA test are inaccurate and, thus, a skin sensitizing effect of magnesium hydroxide is unlikely. Therefore, magnesium hydroxide should not be classified as a skin sensitizer.    

Data source

Materials and methods

Test material

Constituent 1
Chemical structure
Reference substance name:
Magnesium ethanolate
EC Number:
219-323-5
EC Name:
Magnesium ethanolate
Cas Number:
2414-98-4
Molecular formula:
C2H6O.1/2Mg
IUPAC Name:
magnesium ethanolate
Test material form:
other: granular powder

Results and discussion

In vivo (non-LLNA)

Resultsopen allclose all
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group and control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 2 in 5/5 control and 10/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test group and control. Dose level: Injection site 1 . Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 5/5 control and 10/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test group and control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 2 in 4/5 control and 10/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test group and control. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 4/5 control and 10/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
positive control
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: positive control. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
erythema grade 1 in 9/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 9/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
eschar in 8/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: eschar in 8/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 2 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 1/5 control and 1/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 1 in 1/5 control and 9/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 1/5 control and 9/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 2 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control. Dose level: Injection site 3 . Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 1/5 control and 1/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control and 2/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control and 2/10 test animals.
Reading:
1st reading
Hours after challenge:
24
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
eschar in 1/5 control and 6/10 test animals
Remarks on result:
other: Reading: 1st reading. . Hours after challenge: 24.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: eschar in 1/5 control and 6/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 2 in 1/5 control and 3/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: erythema grade 2 in 1/5 control and 3/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
erythema grade 1 in 4/5 control and 7/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1 . Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 4/5 control and 7/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 2 in 1/5 control and 3/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 2 in 1/5 control and 3/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
oedema grade 1 in 4/5 control and 7/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 4/5 control and 7/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
necrosis in 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: necrosis in 1/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 1
Total no. in group:
15
Clinical observations:
eschar in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 1. Total no. in groups: 15.0. Clinical observations: eschar in 1/5 control and 1/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
test chemical
Dose level:
Injection site 2
Total no. in group:
15
Clinical observations:
erythema grade 1 in 4/5 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: Injection site 2. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 4/5 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
erythema grade 1 in 2/5 control and 9/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: erythema grade 1 in 2/5 control and 9/10 test animals.
Reading:
2nd reading
Hours after challenge:
48
Group:
other: test and control group
Dose level:
Injection site 3
Total no. in group:
15
Clinical observations:
oedema grade 1 in 1/5 control and 1/10 test animals
Remarks on result:
other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: other: test and control group. Dose level: Injection site 3. Total no. in groups: 15.0. Clinical observations: oedema grade 1 in 1/5 control and 1/10 test animals.
Reading:
other: Immediately after removing the patch
Hours after challenge:
48
Group:
other: test and control group
Total no. in group:
15
Clinical observations:
Desquamation in 1/5 control animals, erythema grade 1 in 2/10 test animals
Remarks on result:
other: Reading: other: Immediately after removing the patch. . Hours after challenge: 48.0. Group: other: test and control group. Total no. in groups: 15.0. Clinical observations: Desquamation in 1/5 control animals, erythema grade 1 in 2/10 test animals.

Applicant's summary and conclusion

Conclusions:
Under the conditions of the study on Magnesium chloride hexahydrate it can be stated that the test item caused no reactions identified as sensitisating at the tested concentration.
The Skin sensitisation Statement further elaborates why it is concluded that magnesium hydroxide should not be classified as a skin sensitizer.

Ethanol was tested as a possible vehicle for use in the LLNA and was indeed found to be a suitable alternative to the current guideline recommended vehicles. A prerequisite for use as a vehicle would be a lack of sensitizing potential itself. The study concluded that ethanol is not sensitising.
Executive summary:

Magnesium hydroxide:


Although the results of the local lymph node assay (LLNA) test on magnesium hydroxide were apparently positive it is proposed that the positive result should be interpreted as a false positive and that magnesium hydroxide thus should not be classified as a skin sensitizer. There are a number of reasons to suggest that it is highly unlikely that magnesium hydroxide is a skin sensitizer. An expert opinion has been provided from the Informationsverbund Dermatologischer Kliniken (IVDK; Schnuch, A. 2010). The IVDK is a network of over 50 dermatological hospitals, and was formed to monitor and survey the development of contact allergies. Despite 20 years of observations in 192,421 patients they have yet to observe a case of a contact allergy with magnesium hydroxide. Furthermore, no case of contact allergy to magnesium hydroxide has been reported in the world literature, despite extensive exposure of the general population to the substance. Some reports on flame retardants address magnesium hydroxide toxicity, and no sensitizing effects are reported (DFE (EPA), 2008; NAP, 2000).   Furthermore, the results of a guinea pig maximisation test (GPMT) performed with magnesium chloride were obtained from the magnesium chloride consortium. The results presented show that magnesium chloride was negative in the GPMT. As any possible skin sensitizing potential of magnesium hydroxide is likely to stem from the cation rather than the hydroxide anion, the outcome of this test provides additional evidence that magnesium ions should not be regarded as a skin sensitizer. Regarding the hydroxide portion of the compound, there is ample evidence in the literature that hydroxides of other metals are not skin sensitizers (NICNAS, 2003; Banerjee et. al., 2003; DFE (EPA), 2008).   False positives in the LLNA assay are not uncommon, and indeed the LLNA was judged to be no more than 90 % accurate during its validation (Basketter et. al., 2010). On this basis, certain substances have not been classified as contact sensitizers despite giving positive responses in the LLNA test. These include copper chloride (Basketter and Scholes, 1992), sodium lauryl sulphate (Loveless et. al., 1996; Montelius et. al., 1994), benzalkonium chloride (Basketter et. al., 2004) and ethanol (Basketter et. al., 2010). 


Given the combination of the epidemiological data, the negative magnesium chloride guinea pig maximisation test and the history of false positives using the LLNA test, the weight of evidence suggests that the positive results obtained in the LLNA test are inaccurate and, thus, a skin sensitizing effect of magnesium hydroxide is unlikely. Therefore, magnesium hydroxide should not be classified as a skin sensitizer.


 


Ethanol:


A study was carried out to evaluate the effect of vehicles (ethanol or diethyl phthalate) for use in the mouse local lymph node assay (LLNA), and their influence on the skin sensitization potential of four test fragrance materials (p-t-butyl-alpha-methylhydrocinnamic aldehyde; geraniol; eugenol; and hydroxycitronellal). Groups of 4 mice were treated with each test fragrance, at one of five concentrations, either in ethanol or diethyl phthalate (and 1:3 or 3:1 mixtures of the two), or with ethanol (or diethyl phthalate) alone. Although there were no true control data for comparison with the ethanol-alone treated animals, the level of induced T-lymphocyte proliferation was low for ethanol when compared with that for fragrance materials known to be mild to moderate skin sensitizers, and comparable to that for the other (negative) control vehicle tested, diethyl phthalate.