Niobium doped titanium dioxide

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

6 February 2019 - 14 February 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Report date:

2019

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2010

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

Version / remarks:

2003

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of study:

mouse local lymph node assay (LLNA)

Test material

Test material information

Constituent 1

Reference substance name:

Niobium doped titanium dioxide

EC Number:

951-670-4

Molecular formula:

Nb:TiO2

IUPAC Name:

Niobium doped titanium dioxide

Test material form:

solid

In vivo test system

Test animals

Species:

mouse

Strain:

CBA

Remarks:

CaOlaHsd

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Envigo (formed partially from Harlan in September 2015), 5800 aN Venray, The Netherlands
- Females nulliparous and non-pregnant: yes
- Microbiological status of animals, when known: SPF
- Age at study initiation: 8-9 weeks
- Weight at study initiation: mean 17.8 g
- Housing: kept in groups of 5 animals in IVC cages, type II L, polysulphone cages on Altromin saw fibre bedding
- Diet: ad libitum, Altromin 1324 maintenance diet for rats and mice
- Water: ad libitum, tap water
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 10
- Air changes (per hr): at least 10 x / hour
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

dimethyl sulphoxide

Concentration:

12.5%, 25% and 50% (w/v), each suspended in DMSO

No. of animals per dose:

5

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: Before the initiation of the prescreen test, a solubility test was performed to define the vehicle and the maximum concentration which is technically applicable to the animals. The maximum technically applicable concentration of the test item in the vehicle was found to be 50% (w/v) in DMSO
- Irritation:
In order to determine the highest tolerated and not excessively irritant test concentration a prescreen test was performed which was conducted under the same conditions as the main study, except there was no assessment of lymph node proliferation. The mice were observed daily for any clinical signs of systemic toxicity or local irritation at the application site. Body weights were recorded pre-test and prior to termination. Both ears were observed for erythema and scored. Ear thickness measurements were performed on day 1 (pre-dose), day 3 (approximately 48 hours after the first dose) day 6. Excessive local irritation was indicated by an erythema score > 3 and/or ear swelling of > 25%.
- Systemic toxicity: Cageside observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge).
- Ear thickness measurements: Immediately before the first application, approximately 48 hours after the first application and shortly before sacrificing the thickness of both ears of the surviving animals was measured.
- Erythema scores:
0 = No erythema
1 = Very slight erythema (barely perceptible)
2 = Well-defined erythema
3 = Moderate to severe erythema
4 = Severe erythema (beef redness) to eschar formation preventing grading of erythema

MAIN STUDY

Topical Application
Immediately before the first application the thickness of both ears of all animals was measured. Each mouse was treated by topical application of 25 µL of the selected solution to the entire dorsal surface of each ear. A second measurement of the ear thickness of all animals was carried out approximately 48 hours after the first application.Topical applications were performed once daily over three consecutive days. The first treatment day is defined as study day 1.

Administration of 3H-Methyl Thymidine
Five days after the first topical application all mice were dosed with 20 µCi 3H-methylthymidine by intravenous injection (tail vein) of 250 µL of 3H-methyl thymidine, diluted with PBS to a working concentration of 80 µCi/mL.

Preparation of Cell Suspension
Approximately 5 hours after the injection of 3H-methyl thymidine all mice were sacrificed by cervical dislocation. Shortly before sacrificing the thickness of the ears of all animals was measured for a third time. The draining auricular lymph nodes were excised, individually pooled for each animal (2 lymph nodes per animal) and collected in phosphate buffered saline (PBS). A single cell suspension of pooled lymph node cells was prepared by gentle mechanical disaggregation through polyamide gauze (200 mesh size). After washing the gauze with PBS the cell suspension was pelleted in
a centrifuge. The supernatant was discarded and the pellets were resuspended with PBS. This washing procedure was repeated. After the final wash each pellet was resuspended in approx. 1 mL 5% TCA at approx. 4 °C for approximately 18 hours for precipitation of macromolecules. Each precipitate was once washed again, resuspended in 1 mL 5% TCA and 7 mL scintillation fluid was added. Then this solution was transferred into scintillation vials and stored at room temperature overnight.

Determination of Incorporated 3H-Methyl Thymidine
The 3H-methyl thymidine - incorporation was measured in a 6-counter and expressed as the number of disintegrations per minute (DPM). Similarly, background 3H-methylthymidine levels were also measured (5% TCA). Determination of radioactivity was performed individually for each animal.

EVALUATION OF RESULTS
A substance is regarded as a 'sensitiser' in the LLNA if at least one concentration of the test item results in a 3-fold or greater increase in 3H-methyl thymidine - incorporation into lymph node cells of the test group animals, relative to that recorded for the lymph nodes of control group animals (Stimulation Index equal to or greater than 3.0). On the basis of the test results, the test substance may be classified into one of the following categories in conformity with the criteria given in Commission Regulation (EU) No 286/2011] as well as in GHS - Globally Harmonised System of Classification and Labelling of Chemicals, seventh revised edition, 2017:
Skin sensitiser
Category 1:
A substance is classified as a skin sensitiser if there is evidence in humans that the substance can lead to sensitisation by skin contact in a substantial number of persons, or if there are positive results from an appropriate animal test.
Sub-category 1A:
Substances showing a high frequency of occurrence in humans and/or a high potency in animals can be presumed to have the potential to produce significant sensitisation in humans. Severity of reaction may also be considered.
EC3 value < 2%
Sub-category 1B:
Substances showing a low to moderate frequency of occurrence in humans and/or a low to moderate potency in animals can be presumed to have the potential to produce sensitisation in humans. Severity of reaction may also be considered.
EC3 value > 2%

Positive control substance(s):

other: 1% phenylenediamine

Statistics:

Grubbs, Nalimov and Dixon

Results and discussion

Positive control results:

The positive-control substance exceeded the stimulation index of 3 confirming the reliability of the test system.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

test group, 12.5%

Results 2

Key result

Parameter:

SI

Value:

1.2

Test group / Remarks:

test group, 25%

Results 3

Key result

Parameter:

SI

Value:

1.4

Test group / Remarks:

test group, 50%

Results 4

Key result

Parameter:

SI

Value:

10.2

Test group / Remarks:

positive control

Results 5

Key result

Parameter:

SI

Value:

1

Test group / Remarks:

negative control

Cellular proliferation data / Observations:

DETAILS ON STIMULATION INDEX CALCULATION
The proliferative response of lymph node cells was expressed as the number of radioactive disintegrations per minute per lymph node (DPM/NODE) and as the ratio of 3H-methyl thymidine - incorporation into lymph node cells of test group animals relative to that recorded for control group animals (STIMULATION INDEX). Before DPM/NODE values were determined, background values were subtracted.

EC3 CALCULATION
The EC3 value (derived by linear interpolation) could not be calculated as the stimulation indices of all concentrations were below 3.

CLINICAL OBSERVATIONS:
All animals survived throughout the test period without showing any clinical signs

BODY WEIGHTS:
All animals showed the expected weight development, which allows for a weight loss of up to 2 g throughout the study.

Any other information on results incl. tables

Table 1: Radioactive Determination of the Test Substance Groups- Main Study

POS	CPM	Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu- lation Index
41	2079.0	Negative Control	100	401	4191.0	4180.0	2090.0
42	581.0			402	1171.0	1160.0	580.0
43	1241.0			403	2499.0	2488.0	1244.0
44	955.0			404	1930.0	1919.0	959.5
45	1498.0			405	3025.0	3014.0	1507.0
MV SD	1270.8 506.2			MV SD	2563.2 1020.4	2552.2 1020.4	1276.1 510.2	1.0
66	1223.0	Test item in DMSO	12.5	1		2^22.(J		10
67	1249.0			2	2516.0	2505.0	1252.5	1.0
68	950.0			3	1925.0	1914.0	957.0	0.7
69	2458.0*			4	4977.0*	n.d.	n.d.	n.d.
70	1471.0			5	3001.0	2990.0	1495.0	1.2
MV	1223.3			MV	2493.8	2482.8	1241.4	1.0
SD	184.9	Test item in DMSO		SD	381.7	381.7	190.9	0.1
71	1557.0		25	6	3150.0	3139.0	1569.5	1.2
72 73	1723.0 427.0**			7 8	3519.0 848.0**	3508.0 n.d.	1754.0 n.d.	1.4 n.d.
74	1120.0			9	2315.0	2304.0	1152.0	0.9
75	1735.0			10	3575.0	3564.0	1782.0	1.4
MV SD	1533.8 249.0			MV SD	3139.8 503.4	3128.8 503.4	1564.4 251.7	1.2 0.2
76	1747.0	Test item in DMSO	50	11	3611.0	3600.0	1800.0	1.4
77 78	1275.0 934.0			12 13	2626.0 1926.0	2615.0 1915.0	1307.5 957.5	1.0 0.8
79	2113.0			14	4371.0	4360.0	2180.0	1.7
80	2512.0			15	5156.0	5145.0	2572.5	2.0
MV	1716.2			MV	3538.0	3527.0	1763.5	1.4
SD	565.4			SD	1161.7	1161.7	580.8	0.5
96	4.0	Background Szinti and TCA			8.0
97	5.0				9.0
98	7.0				14.0
99	6.0				13.0
100 MV	5.0 5.4			MV	11.0 11.0	0.0	0.0	0.0
SD	1.0			SD	2.3

POS = position in counter; CPM = counts per minute; Conc. = concentration
DPM = disintegrations per minute;

* = outlier; failed Grubbs, Nalimov and Dixon; ** = outlier, failed Nalimov; n.d. = not determined
MV = mean value; SD = standard deviation; Szinti = scintillation fluid; TCA = trichloroacetic acid

If not noted individually, the results include both lymph nodes of an animal.

Table 2: Radioactive Determination of the accompanying Positive control group

POS	CPM	Test Item	Conc. [%]	Animal number	DPM	DPM- mean back- ground	DPM/ Node	Stimu- lation Index
41	2079.0	Negative Control DMSO		401	4191.0	4180.0	2090.0
42	581.0			402	1171.0	1160.0	580.0
43	1241.0			403	2499.0	2488.0	1244.0
44	955.0		100	404	1930.0	1919.0	959.5
45	1498.0			405	3025.0	3014.0	1507.0
MV	1270.8			MV	2563.2	2552.2	1276.1	1.0
SD	506.2			SD	1020.4	1020.4	510.2
46	15594.0	Positive Control Phenylene- diamine in DMSO		406	31565.0	31554.0	15777.0	12.4
47 48 49	13068.0 15553.0 12600.0		1	407 408 409	26559.0 31756.0 25519.0	26548.0 31745.0 25508.0	13274.0 15872.5 12754.0	10.4 12.4 10.0
50	8683.0			410	17768.0	17757.0	8878.5	7.0
MV	13099.6			MV	26633.4	26622.4	13311.2	10.4
SD	2529.7			SD	5106.9	5106.9	2553.5	2.0
96	4.0	Background Szinti and TCA			8.0
97	5.0				9.0
98 99 100	7.0 6.0 5.0				14.0 13.0 11.0
MV	5.4			MV	11.0	0.0	0.0	0.0
SD	1.0			SD	2.3

POS = position in counter; CPM = counts per minute; Conc. = concentration;
DPM = disintegrations per minute;

MV = mean value; SD = standard deviation; Szinti = scintillation fluid; TCA = trichloroacetic acid

If not noted individually, the results include both lymph nodes of an animal.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the present Local Lymph Node Assay, the test item tested at concentrations of 12.5 %, 25 %, 50% in a suitable vehicle (DMSO) showed no skin sensitization potential.

Executive summary:

A study according OECD TG 429 was conducted to determine the skin sensitization potential of the test item following dermal exposure to female mice in the Local Lymph Node Assay. The vehicle and maximum dose selection was performed according to the relevant guidelines. Based on the results of a prescreen test the test item was assessed for sensitizing properties at concentrations of 12.5%, 25% and 50% (w/v), each suspended in dimethyl sulphoxide (DMSO). An appropriate positive control (1% phenylenediamine) group, furthermore a negative (vehicle DMSO) control group were employed. 3 test groups (3 different concentrations) and 1 negative control group were tested. All animals survived throughout the test period without showing any clinical signs. All animals showed the expected weight development. Directly prior to the first application, approximately 48 hours after the first application and shortly before excising the lymph nodes the thickness of both ears from all animals was measured. The means of the ear thickness per group showed no relevant difference compared to the negative control. None of the three tested concentrations of the test item reached the stimulation index of 3. The stimulation index at a concentration of 12.5% was 1.0, at a concentration of 25% the SI was 1.2 and at a concentration of 50% the SI was 1.4. The positive control substance exceeded the stimulation index of 3 confirming the reliability of the test system. Under the conditions of the Local Lymph Node Assay, the test item tested at concentrations of 50 %, 25 % and 12.5 % in a suitable vehicle (DMSO) showed no skin sensitization potential.