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Administrative data

Description of key information

There are no studies available for 2,2'-(phenylimino)diethanol, but for the structural analoge diethanol-para-toluidine [Reaction mass of 2,2'[(4 -methylphenyl)imino]bisethanol and Ethanol 2 -[[2 -(2(hydroxyethoxy)ethyl](4 -methylphenyl)amino]-].

In a GLP compliant study according to OECD guideline 442B the possible skin sensitisation potential of the test substance was studied (Bioassay, 2013). Test item concentrations of 25, 50 and 100 % (w/w; weight per weight) were used. Doses are based on a previously performed pretest in the same animal strain in which 6 mice in total were used.

In the pre-test the highest concentration that could be technically used was 100 % (w/w). Signs of systemic toxicity were not observed in the pre-test. At the tested concentrations of 50 and 100 % the animals showed no relevant signs of local irritation as confirmed by ear weight measurements and ear swelling measurements. Very slight erythema and test item residues (colorless) were noted in all animals; in addition incrustations were observed in one animal. Therefore, the highest concentration tested in the main study was 100 % (w/w).

In the main study the animals did not show any signs of systemic toxicity. In the high dose group (100 %) very slight erythema was noted during the course of the study; while scaling was observed in the 50 % dose group prior sacrifice only (for details see Annex 2). According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25 % was considered to be indicative for excessive local skin irritation. This threshold was not exceeded in any test item treated group and no statistical significant increase of all dose groups compared to the vehicle control was observed. A test item is regarded as a sensitizer in the LLNA if exposure to one or more test item concentration results in a 1.6-fold or greater increase in incorporation of BrdU compared with concurrent controls, as indicated by the Stimulation Index (S.I.). The estimated test item concentration required to produce a S.I. of 1.6 is referred to as the EC1.6 value. In this study, Stimulation Indices (S.I.) of 2.8, 3.4 and 4.1 were determined with the test item at concentrations of 25, 50 and 100 % (w/w) in AOO, respectively. An EC1.6 value could not be calculated, because the S.I. values in every dose group exceeded the value of 1.6. A clear dose response was observed. A statistically significant and biologically relevant increase in BrdU labeling was observed for the low (25 %), middle (50 %) and high (100 %) dose group. This was also observed for these dose groups in lymph node weights and lymph node cell count. Furthermore, the cutoff value for a positive response regarding the lymph node cell count index of 1.55 reported for BALB/c mice was exceeded in the low, middle and high dose group (index of 1.9, 2.3 and 2.3). As expected, a statistical and biological relevant increase in BrdU labelling, lymph node weight and lymph node cell count was determined in the positive control. Ear weight measurement revealed statistical significance without reaching biological relevance.

The test substance was thus a skin sensitizer under the test conditions of this study (BASF SE, 2013).

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
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP compliant, guideline study, available as unpublished report, no restrictions, fully adequate for assessment
Justification for type of information:
see chapter 13 read across justification
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
GLP compliance:
yes (incl. certificate)
Remarks:
Bioassay Labor für biologische Analytik GmbH, INF 515, 69120 Heidelberg, Germany
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
CBA
Sex:
female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 8 weeks
- Weight at study initiation: 18.2 - 21.0 g
- Housing: single in Markrolon cages, type II; H 15005-29 bedding; Ssniff.Spezialitäten GmbH (Experimental AnimalDiets Inc., 59494 Soest, Germany)
- Diet: STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany
- Water: Tap water, ad libitum
- Acclimation period: at least 5 days prior to the start of dosing

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 30 - 70
- Air changes (per hr): approx. 10
- Photoperiod (hrs dark / hrs light): 12/12
Vehicle:
acetone/olive oil (4:1 v/v)
Concentration:
25, 50, 100% (w/w)
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: A solubility experiment was performed according to the recommendations given by OECD 442B. The highest test item concentration, which could be technically used, was 100%.
- Irritation: To determine the highest non-irritant test concentration that does not induce signs of systemic toxicity at the same time, a pre-test was performed in six animals (2 animals per test item concentration and vehicle, each). Six mice were treated by topical application to the dorsal surface of each ear with test item concentrations of 50 and 100% (w/w) and with the vehicle once daily each on three consecutive days. At the tested concentrations of 50 and 100% the animals showed no relevant signs of local irritation as confirmed by ear weight measurements and ear swelling measurements. Very slight erythema and test item residues (colorless) were noted in all animals; in addition incrustations were observed in one animal prior sacrifice only. Therefore, the highest concentration tested in the main study was 100%

MAIN STUDY
- Topical application: Each test group of mice was treated by topical (epidermal) application to the dorsal surface of each ear with test item concentrations of 25, 50, and 100% (w/w) in the vehicle. The application volume 25 μL/ear/day was spread over the entire dorsal surface (Ø ~8 mm) of each ear once daily for three consecutive days. The control test group of mice was treated with an equivalent volume of the relevant vehicle alone. The positive control test group of mice was treated with 25% α-hexyl cinnamaldehyde dissolved in acetone: olive oil (4:1 v/v).
- Administration of BrdU: BrdU was purchased from Roche Diagnostics GmbH. For injection it was dissolved in DPBS (10 mg/mL) before administration and stored in a refrigerator until use. Four days after the first topical application (day 5) 0.5 mL of BrdU solution (5 mg/per mouse/injection) were intraperitoneally injected.
- Determination of Incorporated BrdU: Approximately 24 hours after treatment with BrdU the mice were sacrificed and the draining lymph nodes were rapidly excised and pooled for each animal (2 nodes per animal). Single cell suspensions of pooled lymph node cells were prepared by gentle mechanical disaggregation through a 70 μm nylon mesh. The lymph node cells were resuspended in 6 mL DPBS. After cell count with a cell counter (Casy Cell Counter, Innovatis), cell suspensions of 100,000 cells / mL were adjusted. The incorporation of BrdU into lymph node cells was determined using a commercial cell proliferation assay kit as follows (Roche Applied Science. Mannheim): 100 μL of the lymph node cell suspension (100,000 cells /mL) were added to the wells of a flat-bottom microplate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included in Cell Proliferation Elisa Kit), 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 TMB (included in Cell Proliferation Elisa Kit) was then added and allowed to produce chromogen. After stopping the substrate reaction the absorbance was measured at 450 nm with a reference wavelength of 690 nm (Absorbance-Reader SUNRISE,Tecan).
- Determination of Lymph Node Weight and Cell Count: After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance (XS 204, Mettler Toledo). Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned above was adjusted to an equal final volume and vortexed. Subsequently, individual cell counts were determined using a cell counter (Casy Cell Counter, Innovatis). The values obtained were taken down manually.
- Determination of Ear Weights: After the lymph nodes were excised, both ears of mice were punched at the apical area using a biopsy punch (Stiefel, Ø 8 mm corresponding to 0.5 cm2). For each animal both punches were immediately weighed (pooled per animal) using an analytical balance (XS 204, Mettler Toledo).

TREATMENT PREPARATION
The test item preparations were produced on a weight per weight (w/w) basis. The test item was emulsified in the vehicle AOO. The positive control preparation was produced on a weight per weight (w/w) basis shortly before application by stirring with a magnetic stirrer. After stirring with a magnetic stirrer the positive control was soluble in the vehicle AOO.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
The mean values and standard deviations were calculated in the body weight tables, for the ear weights, the lymph node weights and lymph node cell count, and for the BrdU values. For the calculations excel (Version 2010) was applied. No EC1.6 value could be calculated, because all S.I.s obtained were above the threshold of 1.6. A statistical analysis was conducted on the BrdU values, the ear weights, the lymph node weights and the lymph node cell count to assess whether the difference was statistically significant between test item groups and negative control group. For all statistical calculations Statistica (Version 10) was used. A Mann-Whitney-U test for non-parametric comparison was applied as statistical test. Statistical significance was set at the five per cent level (p < 0.05). However, both biological and statistical significance were considered together.
Positive control results:
A stimulation indice of 2.9 was determined for 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v) in the current experiment. Stimulation Indices of 2.6, 5.0, 5.4, 1.9 and 3.4 were determined in 5 previous consecutive positive control experiments, with 25% alfa-hexyl cinnamaldehyde in acetone: olive oil (4:1 v/v).
Parameter:
SI
Remarks on result:
other: see Remark
Remarks:
In this study Stimulation Indices (S.I.) of 2.8, 3.4 and 4.1 were determined with the test item at concentrations of 25, 50 and 100% (w/w) in AOO, respectively. An EC1.6 value could not be calculated, because the S.I. values in every dose group exceeded the value of 1.6. A clear dose response was observed.

No deaths occurred during the study period. The animals did not show any signs of systemic toxicity. Very slight erythema was observed in the 100% dose group during the course of the study, while scaling was noted in the 50% dose group prior sacrifice only. The positive control group showed very slight erythema during the course of the study in addition to scaling prior sacrifice. The body weight of the animals recorded prior to the first application and prior to sacrifice were within the range commonly recorded for animals of this strain and age. 

The measured lymph node weights and –cell counts of all animals treated were recorded after sacrifice. A statistically significant increase in lymph node weights and - cell counts was observed in the 25%, 50% and 100% dose group and in the positive control in comparison to the vehicle control group. 

The measured ear weights of all animals treated were recorded after sacrifice. A statistically significant increase in ear weights was not observed in the test item groups. In the positive control ear weight measurement reached statistical significance without biological relevance in comparison to the vehicle control group. According to OECD guideline 442B, an increase in ear weight exceeding the threshold value of 25% was considered to be indicative for excessive local skin irritation. This threshold was neither exceeded in any test item treated group nor in the positive control.

Interpretation of results:
sensitising
Remarks:
Migrated information
Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)

Justification for classification or non-classification

Based on the results of the skin sensitization testing, the test item was classified and labelled as skin sens. cat 1 (H317, may cause an allergic skin reaction) according to Regulation (EC) No 1272/2008 (CLP).