Olivine, cobalt silicate blue

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-04-26 to 2017-05-18

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Remarks:

In accordance with the method according to Ehling et al (2005): NMRI mice were used instead of CBA mice, mice were sacrificed on day 4, two days without treatment were skipped

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Test guideline 1

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2010-07-22

Deviations:

yes

Remarks:

modified OECD 429, method according to Ehling et al. 2005

Test guideline 2

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)

Version / remarks:

2008-05-31

Deviations:

yes

Remarks:

modified B.42, method according to Ehling et al. 2005

Principles of method if other than guideline:

The test was performed in accordance with the method according to Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: first round, Toxicology 212 (2005) 60-68 and Ehling et al (2005): An european inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round, Toxicology 212 (2005) 69-79.

Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive
(these values were fixed empirically during the inter-laboratory validation of this method). In addition, the lymph node weights were determined for concentration related properties.

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2014-05-14

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Olivine, cobalt silicate blue

EC Number:

269-093-5

EC Name:

Olivine, cobalt silicate blue

Cas Number:

68187-40-6

Molecular formula:

Co2SiO4

IUPAC Name:

silicon(4+) bis(λ²-cobalt(2+)) tetraoxidandiide

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Olivine, cobalt silicate blue (Pigment Blue 73)
- Substance type: inorganic pigment
- Storage condition of test material: Keep dry and container tightly closed. Keep container in an adequately ventilated storage

In vivo test system

Test animals

Species:

mouse

Strain:

NMRI

Remarks:

Crl:NMRI

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 62 days
- Weight at study initiation: 27 - 33 g
- Housing: housed separately in order to prevent their licking off the test item from the ears of the other animals, in MAKROLON cages (type II) with a basal surface of approx. 360 cm² and a height of approx. 14 cm, granulated textured wood (Granulat A2, J. Brandenburg, 49424 Goldenstedt, Germany) was used as bedding material for the cages, cages were changed and cleaned once a week, periodic analysis of the bedding material for contaminants based on EPA/USA is conducted
- Diet (ad libitum): Commercial diet ssniff® R/M-H V1534 (ssniff Spezialdiäten GmbH, 59494 Soest, Germany), periodic analysis of the food for contaminants based on EPA/USA4 is conducted
- Water (ad libitum): tap water, examined according to German Regulations on drinking water 2001 is conducted at least four times a year
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 °C ± 3 °C (maximum range)
- Humidity (%): 55 % ± 10 % (maximum range)
- Air changes (per hr): 12 - 18/ hr
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

acetone/olive oil (4:1 v/v)

Concentration:

10 %, 25% and 50% (w/w) (concentrations based on preliminary experiment)

No. of animals per dose:

6 female mice

Details on study design:

PRE-SCREEN TESTS:
A preliminary experiment was carried out in 3 animals to examine the irritating potential and handling/application of the test item in order to select the appropriate concentrations. Three concentrations of 10, 25 and 50% of olivine, cobalt silicate blue (Pigment blue 73) in acetone/olive oil (4:1, v/v) were examined.
No irritating properties were observed in this preliminary experiment at concentrations of 10%, 25% or 50%, i.e. no erythema score ≥ 3 and/or no increase in ear thickness of ≥ 25 %.
- Compound solubility: a 50% suspension was the highest feasible concentration of Olivine, cobalt silicate blue (Pigment Blue 73) in acetone/olive oil (4:1, v/v)
- Erythema scores: 0 in all animals

MAIN STUDY

TREATMENT PREPARATION AND ADMINISTRATION:
The experimental schedule of the assay was as follows:

- Day 1:
The weight and any clinical observations of each animal were individually identified and recorded. In addition, ear swelling measurements were carried out at the helical edge of both ears using an Oditest micrometer.
Open application of 25 μL of the appropriate dilution of the test item, the vehicle alone or the positive control (as appropriate) were administered to the dorsum of each ear.
- Days 2 and 3:
The application procedure carried out on day 1 was repeated.
On all administration days possible clinical signs were noted.
- Day 4 (24 hours after the last application):
Ear swelling measurements (immediately before sacrificing the mice) were carried out at the helical edge of both ears using an Oditest micrometer.
The animals were sacrificed under ether anaesthesia.
Punch biopsies of 8 mm in diameter of the apical area of both ears were prepared and immediately weighed on an analytical balance.
Lateral pairs of auricular lymph nodes draining the ear tissue were excised, carefully separated from remaining fatty tissue and weighed on an analytical balance immediately following preparation. The lymph nodes were then stored on ice in PBS/0.5% BSA and subjected to the preparation of single cell suspensions by mechanical tissue disaggregation. The cells were counted automatically in a cell counter.

OBSERVATIONS
The following observations were made during the course of the study:

- Clinical signs: Animals were observed once daily for any clinical signs of local systemic irritation at the application site or of systemic toxicity. Observations were recorded for each individual animal.
- Cageside observations: included skin/fur, eyes, mucous mem-branes, respiratory and circulatory systems, somatomotor activity and behaviour patterns. The onset, intensity and duration of any signs observed were recorded. In addition, animals were checked regularly throughout the working day from 7:30 a.m. to 4:30 p.m. On Saturdays and Sundays animals were checked regularly from 8:00 a.m. to 12:00 noon with a final check performed at approximately 4:00 p.m., if applicable.
- Body weight: The weight of each mouse was recorded at the time of allocation of animals to groups (test day 1) and at the time of necropsy (test day 4).

ANALYSIS OF RESULTS
The so-called stimulation (or LLN-) indices to determine the sensitising potential (this value was fixed empirically during the interlaboratory validation of this method, for details see Ehling et al. 2005a and 2005b), were calculated by dividing the average absolute lymph node weight or lymph node cell counts per group of the test item treated animals by the vehicle treated ones.
Thus, in case of no stimulating effect the index for the lymph node cell count is always below 1.4 (cut-off value). An index above 1.4 is considered positive.
The stimulation indices were calculated by dividing the average ear weight and average ear thickness on test day 4 per group of the test item treated animals by the vehicle treated ones. The cut-off threshold value for ear weight was set at 1.1.


References:

Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: First round; Toxicology 212, 60-68 (2005a)

Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b)

Positive control substance(s):

hexyl cinnamic aldehyde (CAS No 101-86-0)

Statistics:

For lymph node weight significance at p ≤ 0.01 is considered positive (U-test according to MANN and WHITNEY) for possible irritating properties of the test item (Ehling, G., M. et al. (2005b) and Vohr, H.-W. and Ahr, H.-J. (2005)). A possible concentration-response-relationship for the lymph node weight was examined by linear regression analysis employing PEARSON's correlation coefficient. An U-test was also performed for the cell count.

The acute inflammatory skin reaction (irritating potential) was measured by ear weight determination of circular biopsies of the ears and ear thickness measurements on test day 1 and test day 4 to identify skin irritation properties of the test item employing the U-test according to MANN and WHITNEY by comparing the test groups to the vehicle control.

References:

Vohr, H.-W. and Ahr, H.-J.: The local lymph node assay too sensitive? Arch. Toxicol. 79: 721-728 (2005)

Ehling, G., M. Hecht, A. Heusener, J. Huesler, A. O. Gamer, H. van Loveren, T. Maurer, K. Riecke, L. Ullmann, P. Ulrich, R. Vandebriel, H.-W. Vohr: An European inter-laboratory validation of alternative endpoints of the murine local lymph node assay: 2nd round; Toxicology 212, 69-79 (2005b).

Results and discussion

Positive control results:

The positive control group caused the expected increases in lymph node cell count and lymph node weight (statistically significant at p ≤ 0.01). The values for the stimulation index of lymph node cell count and lymph node weight were 1.748 and 1.545 , respectively.Therefore, the study can be regarded as valid. The slightly higher than 1.1 value of the stimulation index of the ear weight (1.146) is of no relevance for the acceptance criteria of the study.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.701

Test group / Remarks:

10 % w/w test item

Remarks on result:

other: SI (lymph node weight) = 1.273

Results 2

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.029

Test group / Remarks:

10 % w/w test item

Remarks on result:

other: SI (ear thickness) = 1.028

Results 3

Key result

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.624

Test group / Remarks:

25 % w/w test item

Remarks on result:

other: SI (lymph node weight) = 1.236

Results 4

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.041

Test group / Remarks:

25 % w/w test item

Remarks on result:

other: SI (ear thickness) = 1.036

Results 5

Key result

Parameter:

SI

Remarks:

lymph node cell count

Value:

1.547

Test group / Remarks:

50 % w/w test item

Remarks on result:

other: SI (lymph node weight) = 1.218

Results 6

Key result

Parameter:

SI

Remarks:

ear weight

Value:

1.082

Test group / Remarks:

50 % w/w test item

Remarks on result:

other: SI (ear thickness) = 1.056

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
Threshold values of the stimulation indices of lymph node cell count and ear weight were calculated by dividing the average values per group of the test item treated animals by the vehicle treated ones. Values above 1.4 (cell count) or 1.1 (ear weight) are considered positive (these values were fixed empirically during the inter-laboratory validation of this method).

RESULTS ON SKIN SENSITISATION
In the main study treatment with Olivine, cobalt silicate blue (Pigment Blue 73) at concentrations of 10%, 25% or 50% revealed increased values for the lymph node cell count. The stimulation indices of the lymph node cell count exceeded the threshold level of 1.4 (statistically significant at p ≤ 0.01 for the 10% and 50% concentrations).
Thee lymph node weights were increased at all 3 concentrations tested (statistically significant at p ≤ 0.01 for the concentration of 10%), pointing to possible irritating properties of the test item.
The threshold level for the ear weight of 1.1 was not exceeded and no increase of ear thickness was observed, i.e. no irritating properties were noted for Olivine, cobalt silicate blue (Pigment Blue 73).

CLINICAL OBSERVATIONS:
No signs of local or systemic intolerance were recorded.

BODY WEIGHTS:
The animal body weight was not affected by the treatment.

Any other information on results incl. tables

Stimulation indices (SI):

 

Parameter	Group 1, negative control	Group 2, 10%	Group 3, 25%	Group 4, 50%	Group 5, positive control
Lymph node cell count	1.000	1.701 *	1.624	1.547 *	1.748 *
Lymph node weight	1.000	1.273 *	1.236	1.218	1.545 *
Ear weight	1.000	1.029	1.041	1.082	1.146 *
Ear thickness, TD4	1.000	1.028	1.036	1.056	1.113

* significantly different from control at p ≤ 0.01

Applicant's summary and conclusion

Interpretation of results:

Category 1 (skin sensitising) based on GHS criteria

Conclusions:

Under the present test conditions, olivine, cobalt silicate blue (Pigment blue 73) at concentrations of 10%, 25% or 50% (w/w) in acetone/olive oil (4:1 v/v) revealed sensitising properties in the local lymph node assay and therefore should be classified and labelled as Category 1 (H317 "May cause an allergic skin reaction ") according to Regulation (EC) No. 1272/2008 and its subsequent regulations.