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Toxicological information

Skin sensitisation

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Administrative data

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of study:
mouse local lymph node assay (LLNA)

Test material

Constituent 1
Reference substance name:
oxocalcium; oxomagnesium; silanedione
EC Number:
610-130-5
Cas Number:
436083-99-7
Molecular formula:
Amorphous glass consisting of SinO(3n-1)2(n-1) Polymeric anions ionically bonded to Ca2+ and Mg2+ cations or other alkaline earth cations
IUPAC Name:
oxocalcium; oxomagnesium; silanedione
Details on test material:
- Name of test material (as cited in study report): Isofrax, Carbawool(sic)

In vivo test system

Test animals

Species:
mouse
Strain:
CBA/Ca
Remarks:
CBA/CaCrl strain mice
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 9 to 10 weeks old
- Weight at study initiation: 18 to 23 g
- Housing: The animals were housed in groups of up to five during acclimatisation and housed in pairs from Day 1. Bedding was provided on a weekly basis to each cage by use of clean European softwood bedding.
- Diet (e.g. ad libitum): 5LF2 EU Rodent Diet 14%, was freely available to the animals at all times. Each batch of diet had been analysed for specific constituents and contaminants by the manufacturer.
- Water (e.g. ad libitum): Mains water was provided, ad libitum, via cage-mounted water bottles.
- Acclimation period: At least 7 days
- Indication of any skin lesions: None noted

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25°C
- Humidity (%): 40 to 70%. On one occasion the minimum humidity was 34%. As this deviation was minimal and only occurred for a short length of time it was considered not to have affected the overall interpretation of study findings, nor compromised the integrity of the study.
- Air changes (per hr): At least 15 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Study design: in vivo (LLNA)

Vehicle:
propylene glycol
Concentration:
10, 25 and 50% w/v
No. of animals per dose:
4 females per dose
Details on study design:
PRE-SCREEN TESTS:
- Compound solubility: The vehicle was chosen because it was the first of the vehicles listed in the protocol that produced an overtly stable solution, emulsion or dispersion incorporating 50% w/v of the test article.
- Irritation: None noted.
- Systemic toxicity: Death or signs of systemic toxicity were not noted.
- Ear thickness measurements: No major changes noted
- Erythema scores: No erythema was observed.

MAIN STUDY

ANIMAL ASSIGNMENT AND TREATMENT
- Criteria used to consider a positive response: The test article is regarded as a sensitiser when the maximum value of the SI is 3.0 or above. The test article is classified as a non-sensitiser when the maximum value of the SI is less than 3.0. (This result is unchanged by observations of irritation at sites of application of the test formulation).

TREATMENT PREPARATION AND ADMINISTRATION: Formulations were freshly prepared as required using propylene glycol on Days 1, 2 and 3. The formulations were stored at room temperature, in sealed, air-tight containers prior to dosing and were used within two hours of preparation. The formulations were mixed by multiple inversion of the containers prior to administration to ensure homogeneity. Concentrations of test article were expressed gravimetrically and in terms of test article received (without regard to purity or active content).

Each mouse was manually restrained with both auditory pinnae left free. The outer aspect of both pinnae of each mouse was treated by direct application of the appropriate vehicle control or test formulation (0.025 mL/pinna) dispensed from an automatic micro pipette.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
No statistical analysis required

Results and discussion

Positive control results:
The sensitivity and reliability of the test system has been checked using α hexylcinnamaldehyde (CAS Number 101 86 0). A Stimulation Index of 3.0 or greater is expected from this substance. An SI of 5.54 was obtained in the latest positive control test (10 Feb 2021) at 25% in AOO (vehicle mean DPM 1276 and test group DPM 7073) indicating acceptability.

In vivo (LLNA)

Resultsopen allclose all
Parameter:
SI
Value:
1.32
Test group / Remarks:
10% w/v
Parameter:
SI
Value:
1.15
Test group / Remarks:
25% w/v
Parameter:
SI
Value:
1.54
Test group / Remarks:
50% w/v
Cellular proliferation data / Observations:
CELLULAR PROLIFERATION DATA

DETAILS ON STIMULATION INDEX CALCULATION: The scintillation counter provided data including the DPM value (disintegrations per minute during a ten minute period). The DPM value for each test group was divided by the DPM for the control group to provide the Stimulation Index (SI) value for each test group.

EC3 CALCULATION: As the SI index was less than 3, no EC3 value could be calculated.

CLINICAL OBSERVATIONS: There were no clinical signs indicative of a systemic effect of treatment among mice treated with the vehicle or with 10, 25 or 50% w/v formulations of the test article. The vehicle and test formulation application sites remained free of irritation.

BODY WEIGHTS: There was no indication of a treatment related effect on body weight.

SIGNS OF TOXICITY (including dermal irritation at the site of administration, if any, e.g. increased ear thickness): None noted

Any other information on results incl. tables

Table: Group DPM and SI




































Concentration (%w/v) in Propylene glycol



Group number



Group DPM



Simulation Index (SI)



Vehicle



1



172



NA



10



2



227



1.32



25



3



198



1.15



50



4



265



1.54


Applicant's summary and conclusion

Conclusions:
None of the Stimulation Index values crossed the threshold level to be considered a positive indicator and there was no evidence of a dose response relationship, therefore the Local Lymph Node Assay demonstrated that the substance does not have the potential to cause skin sensitisation under the conditions of the study.