Registration Dossier

Administrative data

Description of key information

LLNA:

In the study the test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide formulated in PG (propylene glycol) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.13, 1.10, and 1.14 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG, respectively.

The test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was not a skin sensitiser under the test conditions of this study.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records
Reference
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
The study was conducted between 16 January 2019 and 05 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
GLP compliance:
yes (incl. certificate)
Type of study:
mouse local lymph node assay (LLNA)
Specific details on test material used for the study:
Identification: Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide
Batch Number: Y180510A
Purity: 95.0%
Physical state/Appearance: Black powder
Expiry Date: 31 May 2019
Storage Conditions: At room temperature, light protected
Species:
mouse
Strain:
CBA
Remarks:
CBA/CaOlaHsd
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS :
Source: Envigo RMS B.V., Inc Postbus 6174 5960 AD Horst / The Netherlands
Number of animals for the pre-test: 2 females
Number of animals for the main study: 16 females
Number of animals per group: 4 females (mulliparous and non-pregnant)
Number of test groups: 3
Number of control (vehicle) groups: 1
Age (beginning of treatment): Pre-test: 9 - 10 weeks; Main study: 8 - 9 weeks
Bodyweight:-
Pre-test (prior to 1st application): 19.3 - 19.6 g
Main experiment (at start of the experiment): 15.9 - 21.2 g
Acclimation: At least 5 days prior to the start of dosing under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS/HUSBANDRY:
Housing: group
Cage Type: Makrolon Type II (pre-test) / III (main study), with wire mesh top
Bedding: granulated soft wood bedding
Feed: 2018C Teklad Global 18% protein rodent diet (certified), ad libitum
Water: tap water, ad libitum
Environment:-
temperature 22 ± 2°C
relative humidity approx. 45-65% (except for deviation)
artificial light 6.00 a.m. - 6.00 p.m.

Vehicle:
propylene glycol
Concentration:
Main test: 5, 10, 25%
No. of animals per dose:
Main test: 4
Details on study design:
VEHICLE AND DOSE SELECTION:
The highest test item concentration, which could be technically used was a 25% suspension in PG. Grinding of the test item in a mortar was used to formulate the test item.

To determine the highest non-irritant test concentration that at the same time did not induce signs of systemic toxicity, a pre-test was performed in two animals and stated in raw data and report. Two mice were treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 10 and 25% once daily each on three consecutive days. Prior to the first application of the test item and before sacrifice the body weight was determined. Clinical signs were recorded at least once daily. Eventual signs of local irritation were documented and a score was used to grade a possible erythema of the ear skin. Furthermore, prior to the first application of the test item (day 1), on day 3 and before sacrifice (day 6) the ear thickness was determined using a micrometer. Additionally, for both animals, the ears were punched after sacrifice (day 6) at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2) and were immediately pooled per animal and weighed using an analytical balance. Eventual ear irritation was considered to be excessive if an erythema of the ear skin of a score value ≥3 was observed at any observation time and/or if an increase in ear thickness of ≥25% was recorded on day 3 or day 6

At the tested concentrations the animals did not show any signs of systemic toxicity. On day 3 (1h after the third application only), the animal treated with 10% test item concentration transiently showed a very slight erythema of the ear skin (Score 1), whereas redness of ear skin the animal treated with 25% test item concentration could not be detected at this observation time point only, due to the inherent colour of the test item.

The test item in the main study was assayed at 5, 10, and 25%. The highest concentration tested was the highest level that could be achieved whilst avoiding systemic toxicity and excessive local skin irritation as confirmed in the pre-experiment.

TEST ITEM PREPARATION:
The test item was placed into an appropriate container on a tared balance and PG was added (weight per weight).
The different test item concentrations were prepared individually. Homogeneity of the test item in vehicle was maintained during treatment using a magnetic stirrer.
The preparations were made freshly before each dosing occasion.

EXPERIMENTAL DESIGN AND STUDY CONDUCT:
TEST ITEM ADMINISTRATION:
Each test group of mice was treated by (epidermal) topical application to the dorsal surface of each ear with test item concentrations of 5, 10, and 25% in PG. The application volume, 25 μL/ear/day, was spread over the entire dorsal surface (∅ ∼ 8 mm) of each ear once daily for three consecutive days. A further group of mice (control animals) was treated with an equivalent volume of the relevant vehicle alone (control animals).

ADMINISTRATION OF 3H-METHYL-THYMIDINE:
Five days after the first topical application (day 6) 250 μL of phosphate-buffered saline containing 19.8 μCi of 3H-methyl thymidine (equivalent to 79.3 μCi/mL 3HTdR) were injected into each test and control mouse via the tail vein.

TERMINAL PROCEDURE:
Approximately five hours after treatment with 3HTdR all mice were euthanized by using CO2, which was, after harvesting of the lymph nodes, followed by cervical dislocation to ensure death.

PREPARATION OF SINGLE CELL SUSPENSIONS:
The draining lymph nodes were rapidly excised and pooled for each experimental group (8 nodes per group). Single cell suspensions (in phosphate buffered saline) of pooled lymph node cells were prepared by gentle mechanical disaggregation through stainless steel gauze (200 μm mesh size). After washing two times with phosphate buffered saline (approx. 10 mL) the lymph node cells were resuspended in 5 % trichloroacetic acid (approx. 3 mL) and incubated at approximately +4 °C for at least 18 hours for precipitation of macromolecules.

DETERMINATION OF CELLULAR PROLIFERATION (INCORPORATION OF 3HTdR):
The precipitates were then resuspended in 5% trichloroacetic acid (1 mL) and transferred to scintillation vials with 10 mL of scintillation liquid and thoroughly mixed. The level of 3HTdR incorporation was then measured in a β-scintillation counter. Similarly, background 3HTdR levels were also measured in two 1 mL-aliquots of 5 % trichloroacetic acid. The β-scintillation counter expresses 3HTdR incorporation as the number of radioactive disintegrations per minute (DPM).

OBSERVATIONS:
CLINICAL OBSERVATIONS:
All animals were observed on a daily basis, including pre- and post-dose observations on days 1, 2 and 3. Any clinical signs of systemic toxicity, local skin irritation or signs of ill health during the study were recorded.

DETERMINATION OF EAR THICKNESS:
In the pre-test, the ear thickness was determined prior to the first application of the test item (day 1), on day 3, and on day 6 prior to sacrifice using a micrometer.

DETERMINATION OF EAR WEIGHTS:
In the pre-test, after the lymph nodes have been excised, both ears of mice were punched at the apical area using a biopsy punch (Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed per animal using an analytical balance. The values obtained were taken down manually.

DETERMINATION OF BODY WEIGHTS:
The body weights were recorded on day 1 (prior to dosing) and prior to sacrifice (pre-test) or prior to treatment with 3HTdR (main experiment).

DATA EVALUATION:
INTERPRETATION OF RAW DATA:
The proliferative response of the lymph node cells is expressed as the number of radioactive disintegrations per minute per lymph node (DPM/lymph node) and as the ratio of 3HTdR incorporated into lymph node cells of test animals relative to that recorded for lymph nodes of control animals (Stimulation Index; S.I.). Before DPM/lymph node values were determined, mean scintillation-background DPM was subtracted from test and control raw data.

A test item is regarded as a sensitiser in the LLNA if the following criteria are fulfilled:
• First, that exposure to at least one concentration of the test item resulted in an incorporation of 3HTdR at least 3-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
• Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.





















Positive control substance(s):
other: α-hexyl cinnamaldehyde
Positive control results:
Positive control substance: α-Hexylcinnamaldehyde
Vehicle: acetone:olive oil (4:1 v/v)

Test item concentration: 5%
S.I: 1.41

Test item concentraiton: 10%
S.I: 2.70

Test item concentration: 25%
S.I: 10.33

EC3 value: 10.6%
Parameter:
SI
Value:
1
Test group / Remarks:
Control group
Parameter:
SI
Value:
1.13
Test group / Remarks:
5% test item concentration
Remarks on result:
other: non-sensitiser
Parameter:
SI
Value:
1.1
Test group / Remarks:
10% test item concentration
Remarks on result:
other: non-sensitiser
Parameter:
SI
Value:
1.14
Test group / Remarks:
25% test item concentration
Remarks on result:
other: non-sensitiser
Parameter:
EC3
Remarks on result:
not determinable
Remarks:
none of the tested concentrations induced a S.I. greater than the threshold value of 3.
Cellular proliferation data / Observations:
Calculation of the EC3 value:
The EC3 value could not be calculated, since all S.I.´s are below the threshold value of 3.

Viability/Mortality:
No deaths occurred during the study period.

Clinical Signs:
No symptoms of local skin irritation at the ears of the animals and no signs of systemic toxicity were observed during the study period.

Body Weights:
The body weight of the animals, recorded prior to the first application and prior to treatment with 3HTdR, was within the range commonly recorded for animals of this strain and age.


Calculation and Results of Individual Data:

Vehicle: PG

Test item concentration %

Group

Measurement DPM

Calculation

Result

DPM-BGa

Number of lymph nodes

DPM per lymph nodeb

S.I.

-

BG I

19

-

-

-

-

-

BG II

9

-

-

-

-

0

1

5884

5870

8

733.8

1.00

5

2

6624

6610

8

826.2

1.13

10

3

6488

6474

8

809.2

1.10

25

4

6712

6698

8

837.2

1.14

1 = Control group

2 -4 = Test group

a = The mean value was taken from the figures BG I and BG II

b = Since the lymph nodes of the animals of a dose group were pooled, DPM/node was determined by dividing the measured value by the number of lymph nodes pooled.

Interpretation of results:
GHS criteria not met
Remarks:
EU CLP
Conclusions:
The test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was not a skin sensitiser under the test conditions of this study.
Executive summary:

In the study the test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide formulated in PG (propylene glycol) was assessed for its possible skin sensitising potential.

For this purpose a local lymph node assay was performed using test item concentrations of 5, 10, and 25% (w/w). The highest concentration tested was the highest concentration that could technically be achieved.

The animals did not show any signs of systemic toxicity or local skin irritation during the course of the study and no cases of mortality were observed.

In this study Stimulation Indices (S.I.) of 1.13, 1.10, and 1.14 were determined with the test item at concentrations of 5, 10, and 25% (w/w) in PG, respectively.

The test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was not a skin sensitiser under the test conditions of this study.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (not sensitising)

Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

The test item Reaction mass of crystalline magnesium silicate and crystalline silicon and synthetic amorphous silicon dioxide was not a skin sensitiser under the test conditions of an LLNA study.

Based on this result the substance does not need to be classified for skin sensitisation according to EU Classification, Labeling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.