Calcium, carbonate hydroxy aluminum complexes

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (non-LLNA)

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Study period:

From 23 November 2009 to 18 December 2009; preliminary dose range finding study was from 16 November 2009 to 21 November 2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP - Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2010

Report date:

2010

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 406 (Skin Sensitisation)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 440/2008 of 30 May 2008; B.6

GLP compliance:

yes

Type of study:

guinea pig maximisation test

Test material

Test material information

Constituent 1

Reference substance name:

Aluminium hydroxide

EC Number:

244-492-7

EC Name:

Aluminium hydroxide

Cas Number:

21645-51-2

IUPAC Name:

aluminum trihydroxide

Details on test material:

- Name of test material (as cited in study report): Aluminium hydroxide
- Physical state: Powder/white or light grey, odorless
- Analytical purity: “suitable chemical purity”
- Lot/batch No.: 40/2009
- Expiration date of the lot/batch: unlimited
- Storage condition of test material: Room temperature (15-25°C)
- Other:
Safety precautions: “Routine safety and hygienic procedures (lab coat, safety glasses, gloves and mask) sufficient to ensure personnel health and safety”.

In vivo test system

Test animals

Species:

guinea pig

Strain:

Dunkin-Hartley

Sex:

male

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: LAB-ÁLL Bt. Budapest, 1174 Hunyadi u. 7.
- Age at study initiation: young adult
- Weight at study initiation: 307-315 g
- Housing: The animals were housed in macrolon cages, size III (42Χ42Χ19 cm). Two or three animals were kept in each cage.
- Diet (e.g. ad libitum): PURINA Base – Lap gr. diet for rabbits produced by AGRIBRANDS Europe Hungary PLC, H-5300 Karcag, Madarasi road, Hungary, ad libitum. The composition of the diet was provided in Appendix 8 of the report.
- Water (e.g. ad libitum): Animals were provided ad libitum with tap water from the municipal supply as for human consumption, containing 50 mg/100 ml ascorbic acid. The water was routinely analyzed and was considered not to contain contaminants that could affect the purpose or integrity of the study.
- Acclimation period: Animals were allowed to acclimate for 6-7 days
- Other:
- Bedding: The bedding used was Lignocel 3-4 Fasern (produced by J. Rettenmaier & Söhne GmbH+CO.KG, D-73494 Rosenberg, Germany).
- Animal health: Only animals in acceptable health condition were used for the test as certified by the veterinarian
- Identification: The animals were individually marked using ear punching and the cages were also marked with individual identity cards.
- Randomization: Animals were randomly assigned to treatment groups with verification that the actual body weights showed an “acceptable homogeneity and variability among the groups”.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): The temperate was maintained at 20±3C
- Humidity (%): and the relative humidity 30-70%
- Air changes (per hr): Air flow allowed 15-20 air exchanges/hour.
- Photoperiod (hrs dark / hrs light): Artificial light was used 12 hours daily from 6 a.m. to 6 p.m.

Study design: in vivo (non-LLNA)

Induction

Induction 1

Route:

intradermal and epicutaneous

Vehicle:

other: The vehicle used was 1 % Methylcellulose in Humaqua. This vehicle was selected based on results from a Preliminary Compatibility Test. The methyl cellulose (1%) was obtained from Molar Chemicals Ltd (Batch #: K93935287). The Humaqua was manufactured by

Induction 2

Concentration / amount:

Justification for selected concentrations: The selected concentrations were based on the results of a preliminary dose range finding study:

Preliminary dose range finding study:
Two animals were used per concentration:
- For intra-dermal application, concentrations 5.0, 1.0, 0.1 and 0.01% (w/v) were tested (volume 0.1 ml); none produced skin reactions.
- For dermal application, 25% and 50% (for 24 hours), 75% and 100 % (for 48 hours) w/v concentrations were tested (volume 0.5 ml). Concentrations 25, 50 and 75 % (w/v) produced no skin reaction (scores 0-0) and 100% w/v concentration produced slight erythema (score 1-1).

The next concentrations were considered for the main study for:
Induction exposure:
- Intra-dermal – 1% (w/v) was used.
- Dermal – 100% (w/v) was used.

Challenge exposure:
- 75% (w/v) concentration and its 50% dilution (37.5% concentration - as a safeguard dose) were used.

Challenge

Challenge 1

Route:

epicutaneous, occlusive

Vehicle:

other: The vehicle used was 1 % Methylcellulose in Humaqua. This vehicle was selected based on results from a Preliminary Compatibility Test. The methyl cellulose (1%) was obtained from Molar Chemicals Ltd (Batch #: K93935287). The Humaqua was manufactured by

Challenge 2

Concentration / amount:

Justification for selected concentrations: The selected concentrations were based on the results of a preliminary dose range finding study:

Preliminary dose range finding study:
Two animals were used per concentration:
- For intra-dermal application, concentrations 5.0, 1.0, 0.1 and 0.01% (w/v) were tested (volume 0.1 ml); none produced skin reactions.
- For dermal application, 25% and 50% (for 24 hours), 75% and 100 % (for 48 hours) w/v concentrations were tested (volume 0.5 ml). Concentrations 25, 50 and 75 % (w/v) produced no skin reaction (scores 0-0) and 100% w/v concentration produced slight erythema (score 1-1).

The next concentrations were considered for the main study for:
Induction exposure:
- Intra-dermal – 1% (w/v) was used.
- Dermal – 100% (w/v) was used.

Challenge exposure:
- 75% (w/v) concentration and its 50% dilution (37.5% concentration - as a safeguard dose) were used.

No. of animals per dose:

Test groups: 10 animals
Control group: 5 animals

Details on study design:

About 24 hours before the induction treatments, 5 x 5 cm on the scapular region of the animals was clipped free of hair and shaved.

Intra-dermal induction exposure
The animals in the test groups received three injections to each side:
- 1 injection with 0.10 ml of Freund's Complete Adjuvant mixed with physiological saline (1:1 v/v),
- 1 injection with 0.10 ml of the test item in 1 % Methylcellulose at the selected concentration,
- 1 injection with 0.10 ml of test item at the appropriate concentration in a 1:1 (v/v) mixture of Freund's Adjuvant and physiological saline.

The animals in the control group were treated similarly with the omission of the test item from the injections to each side:
- 1 injection with 0.1 ml mix of Freund's Complete Adjuvant and physiological saline (1:1 v/v)
- 1 injection with 0.1 ml of 1 % Methylcellulose
- 1 injection with 0.1 ml of 50 w/v% formulation of the vehicle in a 1:1 mixture (v/v) Freund's Adjuvant and physiological saline.

Dermal induction exposure
For dermal induction exposure, the animals in the test group were treated with 0.5mL of 100% concentration of the test item. The control group was treated with 0.5mL of vehicle.

To apply the solutions, the exposed areas were covered for 48 hours with 4 layers of porous gauze pads soaked in the formulations and fully occlusive foil. After removal of the patch, a swab was used to remove the remaining test item.

Challenge exposure
Timing: two weeks after the dermal induction exposure and 3 weeks after the intra-dermal induction exposure
Details: Twenty-four hours before the treatment a 6x8cm area on each flank of the animals had the hair clipped and was shaved. The test item was applied to the left flank of the test and control animals using a 5x5 cm sterile gauze patch saturated with the test item (75 % (w/v) concentration). Right flank areas of all animals were treated with a 50 % dilution of the dermal challenge dose (i.e., 37.5 % w/v concentration). The volumes applied were approximately 0.5 ml and the patches remained in place, occluded, for 24 hours. After patch removal, residual test item was removed with a swab.

Observations:
Body weights were recorded at the beginning and at the end of the experiment
Mortality was monitored daily from delivery of the animals to the termination of the test
Clinical signs were monitored daily during the test.

Skin reactions:
- In the preliminary dose range finding study, irritation was observed at 1, 24, 48 and 72 hours after the patch removal;
- Intra-dermal induction exposure: 24 hours after the treatment
- Dermal induction exposure: 1, 24, 48 and 72 hours after the patch removal
- For the challenge exposure, observations were made 24 and 48 hours after the patch removal

Scoring of skin reactions:
Dermal irritation (preliminary dose range finding study and induction dermal exposure) was evaluated according to scoring system by Draize (1977):
A. Erythema and eschar formation
- No erythema 0
- Very slight erythema (barely perceptible) 1
- Well defined erythema 2
- Moderate to severe Erythema 3
- Severe erythema (beet redness) to slight eschar formation (injuries in depth) 4
B. Oedema formation
- No oedema 0
- Very slight oedema (barely perceptible) 1
- Slight oedema (edges of area well defined by definite raising) 2
- Moderate oedema (raised app. 1 mm) 3
- Severe oedema (raised more than 1 mm and extending beyond area of exposure) 4

Classification of skin irritation
0 = non irritant
1 = slightly irritant
2-3 = mildly to moderately irritant
4 = severely irritant

Scoring of skin sensitisation
0 = no visible change
1 = discrete or patchy erythema
2 = moderate and confluent erythema
3 = intense erythema and swelling

The percentage of animals showing positive reactions was calculated for both treatment and control groups. The percentage of control animals responding to the challenge was subtracted from the percentage of responding treatment animals.

Challenge controls:

The “safeguard dose” (50% dilution of the maximum dermal challenge dose (37.5% w/v)) was applied to the right flank area of the animals.

Positive control substance(s):

yes

Remarks:

The sensitivity and reliability of the experimental procedure is assessed in the testing laboratory twice a year. The results of the latest reliability check (described below) do not raise any concerns. hexyl cinnamic aldehyde (CAS No 101-86-0); mercapt

Results and discussion

Positive control results:

In the test group, 10 animals were treated with the reference item. Challenge with the test item 2-Mercaptobenzothiazole resulted in a positive response in 50 % of the test animals sensitized previously. No visible changes were found in the control animals. The net score value was 0.50. On the basis of the results of the reliability check study, the test item was classified as a skin sensitizer. This demonstrated that the reliability checking for this method was successful.

In vivo (non-LLNA)

Results

Results 1

Reading:

1st reading

Hours after challenge:

24

Group:

negative control

Dose level:

50 and 75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: negative control. Dose level: 50 and 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 2

Reading:

1st reading

Hours after challenge:

24

Group:

test chemical

Dose level:

50 and 75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 1st reading. . Hours after challenge: 24.0. Group: test group. Dose level: 50 and 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Results 3

Reading:

2nd reading

Hours after challenge:

48

Group:

negative control

Dose level:

50 and 75%

No. with + reactions:

0

Total no. in group:

5

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: negative control. Dose level: 50 and 75%. No with. + reactions: 0.0. Total no. in groups: 5.0.

Results 4

Reading:

2nd reading

Hours after challenge:

48

Group:

test chemical

Dose level:

50 and 75%

No. with + reactions:

0

Total no. in group:

10

Remarks on result:

other: Reading: 2nd reading. . Hours after challenge: 48.0. Group: test group. Dose level: 50 and 75%. No with. + reactions: 0.0. Total no. in groups: 10.0.

Any other information on results incl. tables

Preliminary dose range finding study (2 animals per concentration)

- Intra-dermal treatment: Scores 0 for erythema (0-E) and 0 for oedema (0-O) for both animals for all the tested concentrations (5.0%, 1.0%, 0.1%, 0.01%) at 1, 24, 48 and 72 hours after exposure

- Dermal treatment: 1 hour after the application of 100% concentration, scores 1-E 0-O were recorded for both animals; scores 0-E and 0-0 were recorded for both animals at this concentration 24, 48 and 72 hours after the patch removal. Scores 0-E and 0-0 were recorded for both animals at concentrations 25%, 50% and 75% 1, 24, 48 and 72 hours after the patch removal

E-erythema; O-oedema

 

Intra-dermal induction exposure: no visible changes were observed in any animal in either test or control group 24 hours after the treatment

“The detailed description of FCA [Freund’s complete adjuvant] treatments is not announced in the report, as these FCA effects are well known.”

 

Dermal induction exposure:

- Control group: no visible changes were observed in any animal 1, 24, 48 and 72 hours after the patch removal.

- Test group. “Very slight erythema” was observed in one animal 1 hour after the patch removal. No visible changes were observed in this animal 24, 48 or 72 hours after the patch removal. No visible changes were observed in the other nine animals at 1, 24, 48 or 72 hours after the patch removal

 

Challenge exposure:

Skin sensitisation score 0 was recorded on left and right side of all control and tested animals 24 and 48 hours after the patch removal.

Dermal response scores after the challenge exposure (Appendix 5)

-----------------------------------------------------------------------------------

                                   Test animals          Control animals

                             24 h           48 h            24 h         48 h

------------------------------------------------------------------------------------

number of positive/      0/10          0/10            0/5            0/5

number of tested

All health effects/lesions/outcome examined:

Body weight

Individual data on body weight (with group means) were provided in the report. There were no notable differences in body weight between the test and the control groups.

 

Clinical observations

No overt signs of an adverse clinical response to treatment were observed during the course of the study.

 

Mortality

There was no mortality during the study

Positive control results

In the test group, 10 animals were treated with the reference item. Challenge with the test item 2-Mercaptobenzothiazoleresulted in a positive response in 50 % of the test animals sensitized previously. No visible changes were found in the control animals. The net score value was 0.50.On the basis of the results of the reliability check study, the test item was classified as a skin sensitizer. This demonstrated that the reliability checking for this method was successful.

Applicant's summary and conclusion

Interpretation of results:

not sensitising

Remarks:

Migrated information

Conclusions:

In this study, the Guinea-Pig Maximisation Test was used to determine the skin sensitisation potential of the test item aluminium hydroxide. In summary, the test item produced no positive responses in the previously sensitized test animals or in the control animals. The incidence rate of 0% and the net score 0.00 show that under the conditions of this test, aluminium hydroxide has no sensitisation potential.

Executive summary:

This study was performed in Guinea pigs (Dunkin Hartley (LAL/HA/BR) using the Magnusson and Kligman method (LAB Research Inc., 2010). The study design was based on OECD TG # 406 (17 July 1992), Commission Regulations (EC) No 440/2008 of 30 May 2008; B.6; and the US EPA OPPTS 870.2600 (EPA 712-C-03-197, March 2003). Methylcellulose (1%), selected based on results from a Preliminary Compatibility Test, was used as the vehicle in this study. Based on the preliminary dose range finding study, 1% (w/v) was used for a first induction stage by intradermal administration. This consisted of three injections to both left and right flanks: an injection with 0.10 mL of Freund's Complete Adjuvant mixed with physiological saline (1:1 v/v); an injection with 0.10 mL of the test item in 1% methylcellulose at the selected concentration; and an injection with 0.10 mL of test item at the appropriate concentration in a 1:1 (v/v) mixture of Freund's Adjuvant and physiological saline. The animals in the control group received three similar injections to each side with the omission of the test item. Again based on the results of a dose range finding study, 100% (w/v) was used for a second induction stage by dermal application. 0.5 mL of the suspension was applied with occlusion for 48 hours. Two weeks after the last induction exposure, two concentrations were used for the occlusive epicutaneous challenge exposure: 0.5 mL of 75% (w/v) suspension was applied to the left flank of the animals and 0.5 mL of 37.5% (w/v) suspension was applied to the right flank. The test item was applied to the flanks of the test and control animals using a 5x5 cm sterile gauze patch saturated with the test item. The patches remained in place, occluded, for 24 hours. After patch removal, residual test item was removed with a swab and observations were made at 24 and 48 hours. No irritation effects (scored according to Draize, 1977) were observed during the dose-range finding study or the induction exposures. In the test group, no positive responses were observed in the treated animal (n=10) with either the 75% (w/v) or 37.5% (w/v) formulations. No positive responses were observed on challenge exposure in the control animals (n=5). In summary, the Guinea-Pig Maximisation test was used to determine the skin sensitisation potential of the test item, aluminium hydroxide. Challenge with the test item produced no positive responses in the previously sensitized test animals or in the control animals. The incidence rate was 0% and the net score 0.00. Thus, it was shown that, under the conditions of this test, aluminium hydroxide had no detectable sensitisation potential and does not meet EU criteria for classification for sensitisation.