Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Skin sensitisation

Administrative data

Endpoint:

skin sensitisation: in vivo (LLNA)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 Feb - 28 Mar 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

The study was conducted within the scope of global registration outside the European Union and outside REACH.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted in 2010

Deviations:

yes

Remarks:

no data on cellular proliferation provided (BrdU incorporation)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2600 (Skin Sensitisation)

GLP compliance:

yes

Type of study:

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

Test material

Test material information

Constituent 1

Reference substance name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

EC Number:

265-723-8

EC Name:

Bis[2-[(2-aminoethyl)amino]ethanolato][2-[(2-aminoethyl)amino]ethanolato-O](propan-2-olato)titanate

Cas Number:

65380-84-9

Molecular formula:

not applicable (UVCB substance)

IUPAC Name:

2-methyl-4,4-bis(propan-2-yloxy)-3,5-dioxa-8-aza-4-titanadecan-10-amine; 7,7-bis(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7,7-bis({2-[(2-aminoethyl)amino]ethoxy})-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; 7-{2-[(2-aminoethyl)amino]ethoxy}-7-(propan-2-yloxy)-6,8-dioxa-3,11-diaza-7-titanatridecane-1,13-diamine; tetrakis(propan-2-yloxy)titanium

In vivo test system

Test animals

Species:

mouse

Strain:

CBA:J

Sex:

female

Details on test animals and environmental conditions:

TEST ANIMALS
- Source: Jackson Laboratories (Bar Harbor, Maine, USA)
- Females nulliparous and non-pregnant: yes
- Weight at study initiation: 18.2 – 25.3 g
- Housing: individually in suspended wire-bottom cages
- Diet: PMI Rodent Chow Diet No. 5001, ad libitum
- Water: ad libitum
- Acclimation period: at least five days
- Indication of any skin lesions: no

ENVIRONMENTAL CONDITIONS
- Temperature and humidity were continuously monitored, no further detaisl were provided
- Photoperiod (hrs dark / hrs light): 12/12

Study design: in vivo (LLNA)

Vehicle:

other: Dimethylacetamide:Acetone:Ethanol 4:3:3; DaAE

Concentration:

Preliminary dermal irritation screen: 5, 10 and 25% (v/v)
Main study: 5, 10 and 25% (v/v)

No. of animals per dose:

2 (preliminary dermal irritation screen), 5 (main study)

Details on study design:

PRE-SCREEN TESTS:
- Compound solubility: The test item at a concentration of 50% was found to be soluble in dimethylacetamide:acetone:ethanol 4:3:3 (DaAE), dimethyl sulfoxide (DMSO) and N,N-dimethylformamide (DMF). DaAE was chosen as vehicle for the study.
- Systemic toxicity: The animals of the low and mid dose group did not show clinical signs on Day 1 up to Day 6. The animals treated with concentrations of 25% showed localized hair loss around the ears on Day 3 to 6.
- Ear thickness as a measure for dermal irritation: No increase in ear thickness of 25% or more at test item concentrations of 5, 10 and 25% (v/v) was noted on Day 1, 3 and 6 and thus, the test substance was not considered irritating at these concentrations.
- Erythema scores: Test item concentrations of 10% (v/v) revealed a mean erythema score of 0.3 on Day 3. 25% (v/v) of the test item resulted in mean erythema scores of 0.8 (Days 3 and 6), 1.0 (Day 4) and 1.5 (Day 5).

MAIN STUDY
Based on the pre-screen test results, test substance concentrations of 5, 10 and 25% (v/v) were chosen for the main study.

ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local lymph node assay BrdU ELISA
- Criteria used to consider a positive response: The test substance is considered to have a positive response if treatment results in a 1.6-fold or greater increase in the mean LNC proliferation (BrdU ELISA OD values) relative to that obtained for the vehicle control. Therefore, the test substance was considered as sensitizer if a SI value of 1.6 or more was achieved.

TREATMENT PREPARATION AND ADMINISTRATION:
The test item at concentrations of 5, 10 and 25% (v/v) in DaAE was topically applied to the dorsum of each ear once daily for three consecutive days. The test substance was spread over the entire dorsal surface of each ear using a micropipette to deliver 25 µL/ear. Both ears were observed daily for erythema and scored. On Days 1, 3 and 6 ear thickness measurements were performed. On Day 5 of the main test, approximately 96 h following the initial dose and 24 h prior to euthanasia, 500 µL of a 10 mg/mL concentrated BrdU solution in Dulbecco’s phosphate-buffered saline (DPBS) was intraperitoneally injected into the mice. On Day 6, each mouse was euthanized using CO2 asphyxiation, and the jugular vein was opened for complete exsanguination. Gross observations of the auricular lymph nodes were made and the lymph nodes were collected. The auricular lymph nodes were combined for each animal and single-cell suspensions were prepared in RPMI-10 medium for individual animals. BrdU incorporation was detected with a BrdU-specific Enzyme-linked Immunosorbent Assay (ELISA) kit. An aliquot of 100 µL of the lymph node cell (LNC) suspension preparation 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 bind. Excess unbound anti-BrdU antibody solution was then removed by washing and the substrate solution was added and allowed to generate chromogen. The absorbance of each well was measured using a MicroQuant plate reader (Bio-Tek Instruments) at 370 nm with a reference wavelength of 492 nm.

CLINICAL SIGNS
Animals were observed once daily throughout the study for clinical signs, for local irritation at the application site and for mortality.

BODY WEIGHT
The body weight of each mouse was recorded on Day 1 immediately prior to dosing, and on Day 6 prior to euthanasia.

DETAILS ON STIMULATION INDEX CALCULATION
Calculation of the Stimulation Index (SI) per animal:
SI = Mean OD test article / Mean OD vehicle

Calculation of the EC1.6:
EC1.6 = c + (1.6-d) / (b-d) x (a-c)

a: test article concentration at SI value “b”
b: SI value nearest to but greater than 1.6
c: test article concentration at SI value “d”
d: SI value nearest to but less than 1.6

When no treatment produces an SI value less than 1.6, the concentration at which SI = 1.6 (EC1.6) was estimated from the lowest positive (SI ≥ 1.6) test article concentration and the vehicle (i.e. 0% test article) according to the following formular:

EC1.6 = c + (1.6-d) / (b-d) x (a-c)

a: test article concentration at SI value “b”
b: SI value nearest to but greater than 1.6
c: 0% test article (vehicle only) i.e. concentration at SI value “d”
d: SI value of vehicle control = 1.0

Positive control substance(s):

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

Statistics:

For each dose group, the individual animal SI values along with the mean group SI and standard deviation were calculated, and ANOVA followed by the Student-Newman-Keuls test was performed to statistically compare each test article dose group to the vehicle control group. Although specified in the test guidelines, these calculations and results were not incorporated into the interpretation of the data. An SI value of 1.6 or more is the sole determinant for a positive sensitization response.

Results and discussion

Positive control results:

A 25% solution of HCA (v/v) in dimethylacetamide : acetone : ethanol (4:3:3) yielded a SI value of 3.4 ± 0.7. The 25% HCA positive control produced no more than very slight erythema. Ear thickness on Day 6 increased greater than 25% and enlargement of the lymph nodes were observed, which is consistent with historical results reported in the literature.

In vivo (LLNA)

Results

Results 1

Key result

Parameter:

SI

Value:

1.5

Variability:

± 0.6

Test group / Remarks:

5% (v/v)

Results 2

Key result

Parameter:

SI

Value:

1.6

Variability:

± 0.6

Test group / Remarks:

10% (v/v)

Results 3

Key result

Parameter:

SI

Value:

1.3

Variability:

± 0.6

Test group / Remarks:

25% (v/v)

Results 4

Parameter:

SI

Value:

1

Variability:

± 0.5

Test group / Remarks:

Vehicle control

Results 5

Parameter:

SI

Value:

3.4

Variability:

± 0.7

Test group / Remarks:

Positive Control

Results 6

Key result

Parameter:

other: EC1.6

Remarks:

(%)

Value:

10

Cellular proliferation data / Observations:

CLINICAL OBSERVATIONS:
No mortality was not observed in the treatment groups or in the control groups. Localized hair loss around the ears was noted at a test item concentration of 25% in 2/5 animals on Day 1, in 4/5 animals on Day 2 and in 5/5 animals on Days 3 to 6.

ERYTHEMA AND EAR THICKNESS: Erythema was observed at test substance concentrations of 10 and 25% (refer to Table 1). A greater than 25% increase in ear thickness on Day 6 was not measured in animals treated with the test substance (refer to Table 2).

BODY WEIGHTS:
Body weight losses were noted but were not considered significant (less than 2 grams).

Any other information on results incl. tables

Table 1: Ear thickness (Mean over 5 animals)

	Mean Ear Thickness (mm)			Change (%)
Treatment	Pre- dosing (Day 1)	48 Hr (Day 3)	End In-Life (Day 6)	Day 3 – Day 1	Day 6 – Day 1
DaAE (Vehicle Control)	0.19	0.19	0.19	0.00%	0.00%
25% HCA (Positive Control)	0.18	0.23	0.25	27.8%a	38.9%a
5% (v/v)	0.19	0.2	0.19	5.30%	0.00%
10% (v/v)	0.18	0.19	0.19	5.60%	5.60%
25% (v/v)	0.19	0.23	0.2	21.10%	5.30%
DaAE: Dimethylacetamide:Acetone:Ethanol 4:3:3, solvent control HCA: hexyl cinnamic aldehyde, positive control  a = an increase in ear thickness of 25% or more indicates a more-than-moderate dermal irritation response

Table 2: Erythema score

Treatment	Animal I.D	Day 1		Day 2		Day 3		Day 4		Day 5		Day 6
		Left Ear	Right Ear	Left Ear	Right Ear	Left Ear	Right Ear	Left Ear	Right Ear	Left Ear	Right Ear	Left Ear	Right Ear
DaAE (Vehicle Control)	19	0	0	0	0	0	0	0	0	0	0	0	0
	20	0	0	0	0	0	0	0	0	0	0	0	0
	21	0	0	0	0	0	0	0	0	0	0	0	0
	22	0	0	0	0	0	0	0	0	0	0	0	0
	23	0	0	0	0	0	0	0	0	0	0	0	0
	Mean	0		0		0		0		0		0
25% HCA (Positive Control)	24	0	0	1	0	2	1	1	1	1	1	0	1
	25	0	0	0	0	1	1	1	1	1	1	0	1
	26	0	0	0	1	1	1	1	1	1	1	1	1
	27	0	0	0	0	1	1	1	1	1	1	0	0
	28	0	0	1	0	1	1	1	1	1	1	0	1
	Mean	0		0.3		1.1		1		1		0.5
5% (v/v)	29	0	0	0	0	0	0	0	0	0	0	0	0
	30	0	0	0	0	0	0	0	0	0	0	0	0
	31	0	0	0	0	0	0	0	0	0	0	0	0
	32	0	0	0	0	0	0	0	0	0	0	0	0
	33	0	0	0	0	0	0	0	0	0	0	0	0
	Mean	0		0		0		0		0		0
10% (v/v)	34	0	0	0	0	0	0	0	0	0	0	0	0
	35	0	0	0	0	0	0	1	0	0	0	0	0
	36	0	0	0	0	1	1	1	0	0	0	0	0
	37	0	0	0	0	0	0	1	0	1	1	0	1
	38	0	0	0	0	1	1	1	1	0	1	0	1
	Mean	0		0		0.4		0.5		0.3		0.2
25% (v/v)	39	0	0	0	0	0	0	a	a	1	a	1	a
	40	0	0	0	0	a	0	a	1	a	1	0	0
	41	0	0	0	0	1	a	1	a	2	a	2	a
	42	0	0	0	0	a	0	0	1	1	1	0	0
	43	0	0	0	0	a	1	a	a	1	2	1	1
	Mean	0		0		0.3		0.8		1.3		0.6
DaAE: Dimethylacetamide:Acetone:Ethanol 4:3:3, solvent control; HCA: hexyl cinnamic aldehyde, positive control 0 = No erythema; 1 = Very slight erythema (barely perceptible); 2 = Well-defined erythema; a = due to the extent of test article residue (opaque white) on the ears, erythema could not be evaluated

Table 3: Results Stimulation Index

Treatment	Animal ID	Optical Density			Mean OD	SI
		OD1	OD 2	OD 3
DaAE vehicle	19	0.04	0.037	0.047	0.041	0.3
	20	0.108	0.094	0.112	0.105	0.8
	21	0.201	0.212	0.17	0.194	1.5
	22	0.112	0.111	0.15	0.124	1
	23	0.215	0.142	0.19	0.182	1.4
	Mean				0.129	1
	StDev				0.062	0.5
25% HCA pos. Control	24	0.433	0.539	0.451	0.474	3.7
	25	0.506	0.521	0.478	0.502	3.9
	26	0.473	0.528	0.609	0.537	4.1
	27	0.397	0.345	0.354	0.365	2.8
	28	0.339	0.375	0.266	0.327	2.5
	Mean				0.441	3.4a
	StDev				0.09	0.7
5% (v/v)	29	0.227	0.223	0.167	0.205	1.6
	30	0.176	0.067	0.118	0.12	0.9
	31	0.163	0.242	0.111	0.172	1.3
	32	0.272	0.387	0.34	0.333	2.6
	33	0.158	0.203	0.123	0.161	1.2
	Mean				0.198	1.5
	StDev				0.081	0.6
10% (v/v)	34	0.183	0.277	0.191	0.217	1.7
	35	0.391	0.308	0.332	0.343	2.7
	36	0.127	0.195	0.165	0.162	1.3
	37	0.194	0.134	0.169	0.165	1.3
	38	0.128	0.161	0.112	0.133	1
	Mean				0.204	1.6a
	StDev				0.083	0.6
25% (v/v)	39	0.144	0.164	0.141	0.149	1.2
	40	0.167	0.202	0.192	0.187	1.4
	41	0.101	0.15	0.117	0.122	0.9
	42	0.065	0.106	0.106	0.092	0.7
	43	0.292	0.234	0.331	0.285	2.2
	Mean				0.167	1.3
	StDev				0.075	0.6
a = SI of 1.6 or more indicates a sensitizing response DaAE: Dimethylacetamide:Acetone:Ethanol 4:3:3, solvent control HCA: hexyl cinnamic aldehyde, positive control

Applicant's summary and conclusion

Interpretation of results:

other: Skin Sens. 1 (H317) according to Regulation (EC) No. 1272/2008

Conclusions:

CLP: Skin Sens. 1 (H317)