Manganese antimony titanium buff rutile

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2020-09-02 to 2020-09-22

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2020

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)

Version / remarks:

2018-06-25

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

GLP certificate signed 2019-10-14

Type of study:

mouse local lymph node assay (LLNA): BrdU-ELISA

Test material

Test material information

Constituent 1

Reference substance name:

Chrome tungsten titanium buff rutile

EC Number:

269-054-2

EC Name:

Chrome tungsten titanium buff rutile

Cas Number:

68186-92-5

IUPAC Name:

Chromium tungsten titanium rutile

Test material form:

solid: particulate/powder

Details on test material:

- Test item identification: Chrome tungsten titanium buff rutile
- C.I. name: Pigment Yellow 163
- Substance type: inorganic pigment
- Storage conditions: At room temperature, under moisture protection

Specific details on test material used for the study:

not specified

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Elevage Janvier Labs (F-53941 Le Genest Saint Isle)
- Females nulliparous and non-pregnant: yes
- Age at study initiation (main study): 8 weeks
- Weight at study initiation (main study): 21.0 - 24.4 g
- Housing: individually housed in suspended solid-floor polypropylene cages furnished with softwood woodflakes (individual housing is used to avoid ingestion of test item by licking of the ear of the other animals); enrichment item (Tunnel) was provided, which was considered not to contain any contaminant of a level that might have affected the purpose or integrity of the study.
- Diet (ad libitum): ENVIGO 2016
- Water (ad libitum): tap water from public distribution system
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature: 19°C to 25°C
- Humidity: 30% to 70%,
- Air changes (per hr): at least 10 changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

propylene glycol

Concentration:

10 %, 35 % and 70 % concentrations

No. of animals per dose:

4 female mice

Details on study design:

PRE-SCREEN TESTS:
A preliminary screening test was performed using one mouse with the highest technically possible concentration. The mouse was treated by daily application of 25 μL of the test item diluted at 70% in propylene glycol to the dorsal surface of each ear for three consecutive days (Days 1, 2, 3). The vehicle was chosen as it produced the most suitable formulation at the required concentration.

The mouse was observed daily from Day 1 to Day 6. Body weights were recorded pre-test and prior to termination (Day 6). One ear was observed for erythema and scored according to OECD guideline scale (Draize scale). Ear thickness was recorded using a thickness gauge (digital micrometer) on Day 1 (pre-dose), on Day 3 (approximately 48 hours after the first dose) and on Day 6. Any signs of toxicity or excessive local irritation noted during this period were recorded.

Additionally, on Day 6, ear thickness was determined by ear punch weight determinations, which was performed after the animal was humanely killed. Exessive local irritation was indicated by an erythema score ≥ 3 and/or ear thickness of ≥ 25% on any day of measurement. The highest dose selected for the main LLNA:BrdU-Elisa study was the next dose in the pre-screen concentration series that did not induce systemic toxicity and/or excessive local skin irritation.

Results:
No mortality was noted at the tested concentration of 70%.
No signs of systemic toxicity were observed at the tested concentration of 70%.
No signs of excessive irritation was noted at the tested concentration of 70%.
Therefore, 70% was chosen as the highest concentration for the main study.

MAIN STUDY
- Days 1, 2 and 3:
The mice were treated by daily application of 25 μL of the appropriate concentration of the test item to the dorsal surface of each ear for three consecutive days (Days 1, 2 and 3). A further group of four mice received the vehicle alone in the same manner.

- Day 5: 0.5 mL (5 mg/mouse) of BrdU (10 mg/mL) solution was injected by intra-peritoneal route.

- Day 6: on day 6 (end of the test, approximately 24 hours after BrdU injection), the animals were euthanized with sodium pentobarbital (Dolethal®). The draining auricular lymph nodes of each mouse were excised and processed separately on phosphate buffered saline (PBS). for each mouse.

From each mouse, a single-cell suspension of lymph node cells (LNC) excised bilaterally was prepared by gentle mechanical disaggregation through a disposable plastic pestle to crush the lymph nodes followed by passage through a #70 nylon mesh in 15 mL of DPBS (Ca2+ / Mg2+ - free) into a well of a multi-well 6. The optimized volume was based on achieving a mean absorbance of the negative control group within 0.1- 0.2.

BrdU was measured by ELISA using a commercial kit (Roche Applied Science, Mannheim, Germany, Catalogue Number 11 647 229 001 – Batch No. 43507600). Briefly, 100 μL of the LNC suspension was added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the LNC, anti-BrdU antibody was added to each well and allowed to react. Subsequently the anti-BrdU antibody was removed by washing and the substrate solution was then added and allowed to produce chromogen. After 5 to 30 minutes, 30 μL of 1 M H2SO4 was added in each well, then shaken for one minute. Absorbance at 450 nm with a reference wavelength of 690 nm was then measured.

The BrdU labelling index was defined as:
BrdU labelling index = (ABS em – ABS blank em) – (ABS ref – ABS blank ref)
(em = emission wavelength; and ref = reference wavelength)

Results were expressed as the Stimulation Index (SI).
Results for each treatment group were expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group.

The EC1.6 value (theoretical concentration resulting in a SI value of 1.6) was determined by linear interpolation of points on the dose-response curve, immediately above and below the 1.6-fold threshold. The equation used for calculation of EC1.6 was:
EC1.6 = c + [(1.6 – d) / (b – d)] x (a – c)
a = the lowest concentration giving stimulation index > 1.6
b = the actual stimulation index caused by a
c = the highest concentration failing to produce a stimulation index of 1.6
d = the actual stimulation index caused by c

DATA ANALYSIS:
The test item will be regarded as a sensitiser if at least one concentration of the test item results is equal or greater than 1.6 compared to control values. However, the strength of the dose-response relationship, the statistical significance and the consistency of the solvent/vehicle and positive control responses may also be used when determining whether a borderline result (i.e. SI value between 1.6 and 1.9) is declared positive.

Any test item failing to produce a SI > 1.6 will be classified as a "non-sensitiser".

OBSERVATIONS:
- clinical signs: all animals were observed daily on Days 1, 2, 3, 4, 5 and 6. Any signs of toxicity or signs of ill health during the test were recorded.

- body weights: body weight of each mouse was recorded on Day 1 (prior to dosing) and Day 6 (prior to termination).

- skin irritation assessment: On day 1 and on day 3 (before application) as well as on day 6 (after sacrifice) of each experiment, the thickness of the right ear of each animal of the vehicle control and treated groups was measured by a micrometer in order to determine the iritant effect of the test item. Furthermore, on day 6, punch biopsies (8 mm) in diameter of the apical area of both ears were prepared and weighed in order to assess the irritation potential of the test item and the two lymph nodes per mouse were weighed.

Any irritation reaction (erythema and oedema scored using the Draize scale as stated in the OECD guideline) was recorded in parallel. Any other observation (dryness, presence of residual test item…) was noted.

The test item should be considered as an excessive irritant if the score of erythema is higher or equal to 3. Furthermore, if ear thickness is increased by equal to or greater than 25% between day 1 and day 3 and/or between day 1 and day 6, the test item is considered to be an excessive irritant.

Positive control substance(s):

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

Results and discussion

Positive control results:

The positive control substance produced SI values (mean ± SD) of 1.40 ± 0.26, 1.51 ± 0.33 and 2.14 ± 0.36 for 5 %, 10 % and 25 % concentrations, respectively. Therefore, the positive control substance is considered to be a skin sensitiser, since one dose had a SI value above 1.6.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

0.95

Variability:

Standard deviation: 0.09

Test group / Remarks:

10 % concentration of test item

Results 2

Key result

Parameter:

SI

Value:

0.87

Variability:

Standard deviation: 0.08

Test group / Remarks:

35 % concentration of test item

Results 3

Key result

Parameter:

SI

Value:

0.91

Variability:

Standard deviation: 0.03

Test group / Remarks:

70 % concentration of test item

Cellular proliferation data / Observations:

CELLULAR PROLIFERATION DATA
The following BrdU-index was determined for the control group and treatment groups (mean values):
- control group: 1.062
- 10 % concentration: 1.008
- 35 % concentration: 0.929
- 70 % concentration: 0.968
Please also refer to the field "Attached background material" below.

DETAILS ON STIMULATION INDEX CALCULATION
Results for each treatment group were expressed as the mean SI. The SI was derived by dividing the mean BrdU labelling index/mouse within each test group by the mean BrdU labelling index for the control group.

EC3 CALCULATION
No stimulation index higher than 1.6 was recorded for any concentration. Therefore, the EC1.6 cannot be determined due to the absence of a SI value higher than 1.6.

CLINICAL OBSERVATIONS / MORTALITY / LOCAL IRRITATION:
No mortality was noted in the test and control animals during the test.
No signs of systemic toxicity were noted in the test animals treated at 10%, 35%, 70% and control animals during the test.

No erythema was observed in animals treated at 10%, 35% and 70%, respectively.
No increase in ear thickness (≥ 25 %) and in ear weight was noted in animals treated at 10%, 35% and 70%, respectively.
Therefore, the test item has to be considered as not excessively irritant at these concentrations.
Please also refer to the field "Attached background material" below.

BODY WEIGHTS
No statistical significant change in body weight was noted for all treated groups versus control group (Student’s test).

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

The substance is not a skin sensitiser.
According to Regulation (EC) No 1272/2008 and subsequent adaptations, the substance does not require classification as skin sensitiser.