Registration Dossier

Administrative data

Description of key information

In two LLNA skin sensitisation studies, performed with a structural analogue of 2,2'-[(3 -methylphenyl)imino]ethanol, Accelerator, according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 100%. No in vitro testing was performed, as adequate in vivo studies conducted before 2017 were available.

Key value for chemical safety assessment

Skin sensitisation

Link to relevant study records

Referenceopen allclose all

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
February 27th - March 18th, 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
issued 19 May 2011
Type of study:
mouse local lymph node assay (LLNA)
Species:
mouse
Strain:
other: CBA/J
Sex:
female
Details on test animals and environmental conditions:
- Source: Janvier, Le Genest-Saint-Isle, France.
- Age at study initiation: Young adult females (approx. 9 weeks old)
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (20-23 g).
- Housing: Animals were group housed in labeled Makrolon cages.
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 – 24
- Humidity (%): 40 - 70
- Air changes (per hr): approx 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 06 March 2013 - 18 March 2013
Vehicle:
dimethylformamide
Remarks:
or unchanged (100%)
Concentration:
25, 50, 100%
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
Three test substance concentrations were tested: 25, 50 and 100 % concentration (% w/w). Two young adult animals per concentration were selected (in the range of 8 to14 weeks old). Each animal was treated with one concentration on three consecutive days. Ear thickness measurements were conducted using a digital thickness gauge prior to dosing on Days 1 and 3, and on Day 6. Animals were sacrificed after the final observation.

MAIN STUDY
ANIMAL ASSIGNMENT AND TREATMENT
- Name of test method: Local Lymph Node Assay
- Criteria used to consider a positive response: DPM values are presented for each animal and for each dose group. A Stimulation Index (SI) is calculated for each group. The SI is the ratio of the DPM/group compared to DPM/vehicle control group. If the results indicate a SI ≥ 3, the test substance may be regarded as a skin sensitizer.

ANIMAL ASSIGNMENT
Three groups of five animals were treated with one test substance concentration per group. One group of five animals was treated with vehicle.

TREATMENT PREPARATION AND ADMINISTRATION:
The test substance formulations (w/w) were prepared within 4 hours prior to each treatment. No adjustment was made for specific gravity of the vehicle. Homogeneity was obtained to visually acceptable levels. Correction of the purity/composition of the test substance is not applicable, since the test method requires a logical concentration range rather than specific dose levels to be applied.

Rationale for vehicle: The vehicle was selected based on trial formulations performed at WIL Research Europe and on test substance data supplied by the sponsor.

Induction - Days 1, 2 and 3; Excision of nodes - Day 6; Tissue processing for radioacitivity - Day 6; Radioactivity measurements - Day 7; Performed according to test guidelines.

Observations:
Mortality/Viability: Twice daily.
Body weights: On Day 1 (pre-dose) and Day 6 (prior to necropsy).
Clinical signs: Once daily on Days 1-6 (on Days 1-3 between 3 and 4 hours after dosing).
Irritation: Once daily on Days 1-6 (on Days 1 - 3 immediately after dosing). Furthermore, a description of all other (local) effects was recorded according to guidelines.

Necropsy: No necropsy was conducted on the animals that were found dead or sacrificed for humane reasons.
Positive control substance(s):
hexyl cinnamic aldehyde (CAS No 101-86-0)
Statistics:
Not performed.
Positive control results:
The six-month reliability check with Alpha-hexylcinnamicaldehyde indicates that the Local Lymph Node Assay as performed at WIL Research Europe is an appropriate model for testing for contact hypersensitivity.
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
25% test substance solution
Remarks on result:
other: 25%
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
50% test substance solution
Remarks on result:
other: 50%
Key result
Parameter:
SI
Value:
10.8
Test group / Remarks:
100% test substance
Remarks on result:
other: 100%
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 2008, 2925 and 6725 DPM respectively. The mean DPM/animal value for the vehicle control group was 624 DPM.

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined at 25 and 50%. Both animals at 100% showed very slight erythema on Day 5 only.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Greasy test substance remnants were present on the dorsal surface of the ears of both animals at 100% (Days 2 and 3), which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

No irritation of the ears was observed in any of the animals examined.

Greasy test substance remnants were present on the dorsal surface of the ears of all animals at 50 and 100% (Days 2 and/or 3), which did not hamper scoring of the skin reactions.

The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Interpretation of results:
other: Category 1 (skin sensitising) based on GHS and CLP criteria
Conclusions:
In an LLNA skin sensitisation study, performed according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 100%. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%.
Executive summary:

An LLNA skin sensitisation assay was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 25, 50 or 100%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in any of the animals examined. The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 25, 50 and 100% were 3.2, 4.7 and 10.8 respectively.

These results show that the test substance elicits an SI≥3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. Based on these data, the substance is classified for skin sensitisation.

Endpoint:
skin sensitisation: in vitro
Data waiving:
study scientifically not necessary / other information available
Justification for data waiving:
an in vitro skin sensitisation study does not need to be conducted because adequate data from an in vivo skin sensitisation study are available
Reason / purpose:
data waiving: supporting information
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2013-01-23 - 2013-01-29
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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: 19.2 - 19.8 g
- Housing: single
- Diet: ad libitum (STANRAB (P) SQC; SDS Special Diets Services. 67122 Altrip, Germany)
- Water: ad libitum (Tap water)
- Acclimation period: at least 5 days prior treatment

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 %
No. of animals per dose:
5
Details on study design:
RANGE FINDING TESTS:
- Compound solubility: 100 %
- Irritation: Local Irritation was not observed at both concentrations tested (50, 100 %)
- Lymph node proliferation response: Not determined

MAIN STUDY
TREATMENT PREPARATION AND ADMINISTRATION:
Test Item 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.

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 AOO. The application volume 25 μL/ear/day was spread over the entire dorsal surface (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 (5mg/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 micro plate in triplicate. After fixation and denaturation of the lymph node cells with FixDenat (included inCell 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 inCell Proliferation Elisa Kit) was then added and allowed to produce chromogens. 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, serial number 112 748 2217). Furthermore, the lymph node cell count was determined for each animal. For this, the volume of the cell suspensions mentioned in section 9.3.3 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, serial number 112 748 2217).

Interpretation of Raw Data
The proliferative response of the lymph node cells is expressed as the mean SI. The SI is derived by dividing the mean BrdU labeling index/mouse within each test substance group as well as for the positive control group by the mean BrdU labeling index for the vehicle group. A test item is regarded as a sensitizer in the LLNA if the following criteria are fulfilled:

- First, that exposure to at least one concentration of the test item resulted in an incorporation of BrdU at least 1.6-fold or greater than that recorded in control mice, as indicated by the Stimulation Index.
- Second, that the data are compatible with a conventional dose response, although allowance must be made (especially at high topical concentrations) for either local toxicity or immunological suppression.

Furthermore, an index was calculated for the lymph node weight and -cell count as well as for the ear weight by dividing the mean values of the test item treated groups by the mean of the vehicle control group. For BALB/c mice, a cutoff value for the lymph node cell count index of 1.55 was reported for a positive response (see Ref. 8).Furthermore, 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.

Observations
In addition to the sensitizing reactions the following observations and data were recorded during the test and observation period: Mortality / Viability At least once daily from experimental start to necropsy. Clinical signs (local / systemic) Clinical signs (local irritation at the application site or systemic toxicity) were recorded at least once daily. Especially the treatment sites were observed carefully. Body weights Pre-test and main experiment: prior to the first application and prior to sacrifice. Ear thickness In the pre-test prior to the first application of the test item (day 0), on day 2 and before sacrifice (day 5) the ear thickness was determined using a micrometer (S0247 Kroeplin, 36381 Schlüchtern, Germany). Ear weights In the pre-test and main experiment after sacrifice; biopsy punches were taken from each ear. Lymph node weights After excision, the lymph nodes were pooled per animal and weighed immediately using an analytical balance. Lymph node cell count The lymph node cell count was determined in aliquots taken from the prepared single cell suspensions of the lymph nodes using a cell counter.
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 percent level (p < 0.05). However, both biological and statistical significance were considered together.
Positive control results:
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.
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
25% test substance solution
Remarks on result:
other: 25%
Key result
Parameter:
SI
Value:
3.4
Test group / Remarks:
50% test substance solution
Remarks on result:
other: 50%
Key result
Parameter:
SI
Value:
4.1
Test group / Remarks:
100% test substance
Remarks on result:
other: 100%

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

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 (for details see Annex 2). The positive control group showed very slight erythema during the course of the study in addition to scaling prior sacrifice.

Body Weights

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.

Lymph Node Weights and Cell Counts

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.

Ear Weights

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:
other: Category 1 (skin sensitising) based on GHS and CLP criteria
Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a structural analogue
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substance Accelerator (Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)) is a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol. Accelerator contains ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol as the main constituent. The only difference between 2,2'-[(4-methylphenyl)imino]bisethanol and the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol is the position of the methyl group in the phenyl ring (para vs meta with regard to the imino group). This is not expected to have influnce on toxicological behavior of the substances. Next to 2,2'-[(4-methylphenyl)imino]bisethanol Accelerator contains ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, which contains the same structural moiety of 2,2'-[(4-methylphenyl)imino]bisethanol, but with one alcohol moiety transformed into the ester bond with ethylene glycol. The presence of the ester moiety is not expected to influence skin sensitizing properties of the substances. Furthermore, a structural analogue of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol, differing only by the position of the methyl group in the phenyl ring (meta vs. para) is also present as an impurity up to 7% concentration in the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source chemical is the Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)), containing ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol, ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol and ca. 2-4% of 2-[{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}(4-methylphenyl)amino]ethanol. The purity of the substance is ca. 90-100%.

The target chemical is 2,2'-[(3-methylphenyl)imino]bisethanol with the purity of 90-10%. The substance contains up to 7% 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol as an impurity.

3. ANALOGUE APPROACH JUSTIFICATION
The substance Accelerator (Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)) is a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol. Accelerator contains ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol as the main constituent. The only difference between 2,2'-[(4-methylphenyl)imino]bisethanol and the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol is the position of the methyl group in the phenyl ring (para vs meta with regard to the imino group). This is not expected to have influnce on toxicological behavior of the substances. Next to 2,2'-[(4-methylphenyl)imino]bisethanol Accelerator contains ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, which contains the same structural moiety of 2,2'-[(4-methylphenyl)imino]bisethanol, but with one alcohol moiety transformed into the ester bond with ethylene glycol. The presence of the ester moiety is not expected to influence skin sensitizing properties of the substances. It is probable that the skin sensitizing properties will be mostly governed by basic properties of the amine moiety which is present in both substances. Furthermore, a structural analogue of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol, differing only by the position of the methyl group in the phenyl ring (meta vs. para) is also present as an impurity up to 7% concentration in the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol.

Skin sensitizing properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software. For both substances, the same alerts for protein binding (Schiff Base formation and Micael Acceptor alert) were identified, suggesting that their skin sensitizing properties will be similar.

Furthermore, the data matrix constructed based on the available physico-chemical and (eco)toxicological properties of both substances indicates that the substances have comparable properties across the complete range of endpoints.

Based on this, read-across from Accelerator to 2,2'-[(3-methylphenyl)imino]bisethanol is considered to be justified.

4. DATA MATRIX


Accelerator PT25 2,2'-[(3-methylphenyl)imino]bisethanol

Molecular weight ~217 195.26
State of the substance at ambient conditions Clear slightly yellowish to brown viscous liquid Colourless solidified melt
Melting/freezing point [ºC] Not determined (glass transition temp. of -28ºC) 66.9
Boiling point [ºC] - (decomposition at >275ºC) - (reaction and/or decomposition at > 225°C)
Relative density at 20 ºC 1.11 1.115
Vapour pressure at 25 ºC [Pa] 0.0025 0.000223 (calculated)
Surface tension [mN/m] 65.2 at 1 g/L, not surface active 65.4 at 1 g/L, not surface active
Water solubility at 20 ºC [mg/L] 21800 9330
Partition coefficient n-octanol/water [log Pow] 2.17 1.9
Flash point [ºC] 176 193
Flammability Not flammable, not pyrophoric Not lammable, not pyrophoric
Explosive properties Not explosive Not explosive
Auto-ignition temperature 395 395
Oxidising properties Not oxidizing Not oxidizing
Viscosity (dynamic, mPas) 2797 5474
Ready biodegradability Not readily biodegradable Not readily biodegradable
Hydrolysis as function of pH Half life for hydrolysis >1 y at 25 ºC, hydrolytically stable Read-across
Adsorption/desorption [log Koc] 2.33 (weight-averaged of 4 main components) Read-across
Acute toxicity to daphnia, EC50 [mg/L] 48 107
Growth inhibition algae, EC50, NOEC [mg/L] >100; 100 >100; 100
Acute toxicity to fish, LC50 [mg/L] >100 >102
Acute oral, LD50 [mg/kg bw] 619 300-2000
Skin irritation/corrosion Skin irritant cat 2. Read-across
Eye irritation Corrosive, cat 1. Read-across
Skin sensitisation Sensitiser Read-across
In vitro gene mutation in bacteria (Ames test) Mutagenic Non-mutagenic
In vitro cytogenicity in mammalian cells (CA) Not clastogenic, does not disturb mitotic processes Not clastogenic, does not disturb mitotic processes
In vivo genotox (Comet) Not mutagenic No data
28-day repeated dose toxicity NOAEL 100 mg/kg bw LOAEL 50 mg/kg bw/day



Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 442B (Skin Sensitization: Local Lymph Node Assay: BrdU-ELISA)
Deviations:
no
Qualifier:
according to
Guideline:
other: Commission Regulation (EC) No 440/2008
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Key result
Parameter:
SI
Value:
2.8
Test group / Remarks:
25% test substance solution
Remarks on result:
other: 25%
Key result
Parameter:
SI
Value:
3.4
Test group / Remarks:
50% test substance solution
Remarks on result:
other: 50%
Key result
Parameter:
SI
Value:
4.1
Test group / Remarks:
100% test substance
Remarks on result:
other: 100%

Viability / Mortality

No deaths occurred during the study period.

Clinical Signs

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 (for details see Annex 2). The positive control group showed very slight erythema during the course of the study in addition to scaling prior sacrifice.

Body Weights

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.

Lymph Node Weights and Cell Counts

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.

Ear Weights

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:
other: Category 1 (skin sensitising) based on GHS and CLP criteria
Conclusions:
In a GLP-compliant OECD Guideline OECD 442B study, conducted with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, the Stimulation Indices were 2.8, 3.4 and 4.1 for 25%, 50% and 100% solution of the test substance, respectively. Based on this Accelerator is considered to be sensitizing to skin. This result can be read across to 2,2'-[(3-methylphenyl)imino]bisethanol.
Executive summary:

In a GLP-compliant OECD Guideline OECD 442B study with CDA mice (5 females/group), conducted with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, the Stimulation Indices were 2.8, 3.4 and 4.1 for 25%, 50% and 100% solution of the test substance in acetone/olive oil (4:1 v/v), respectively. The positive control hexyl cinnami aldehyde showed adequate response. No deaths occurred during the study, and 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.

Based on this Accelerator is considered to be sensitizing to skin. This result can be read across to 2,2'-[(3-methylphenyl)imino]bisethanol.

Endpoint:
skin sensitisation: in vivo (LLNA)
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: read-across from a structural analogue
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The substance Accelerator (Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)) is a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol. Accelerator contains ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol as the main constituent. The only difference between 2,2'-[(4-methylphenyl)imino]bisethanol and the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol is the position of the methyl group in the phenyl ring (para vs meta with regard to the imino group). This is not expected to have influence on toxicological behavior of the substances. Next to 2,2'-[(4-methylphenyl)imino]bisethanol Accelerator contains ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, which contains the same structural moiety of 2,2'-[(4-methylphenyl)imino]bisethanol, but with one alcohol moiety transformed into the ester bond with ethylene glycol. The presence of the ester moiety is not expected to influence skin sensitizing properties of the substances. Furthermore, a structural analogue of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol, differing only by the position of the methyl group in the phenyl ring (meta vs. para) is also present as an impurity up to 7% concentration in the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source chemical is the Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)), containing ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol, ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol and ca. 2-4% of 2-[{2-[2-(2-hydroxyethoxy)ethoxy]ethyl}(4-methylphenyl)amino]ethanol. The purity of the substance is ca. 90-100%.

The target chemical is 2,2'-[(3-methylphenyl)imino]bisethanol with the purity of 90-10%. The substance contains up to 7% 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol as an impurity.

3. ANALOGUE APPROACH JUSTIFICATION
The substance Accelerator (Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol)) is a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol. Accelerator contains ca. 50-60% of 2,2'-[(4-methylphenyl)imino]bisethanol as the main constituent. The only difference between 2,2'-[(4-methylphenyl)imino]bisethanol and the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol is the position of the methyl group in the phenyl ring (para vs meta with regard to the imino group). This is not expected to have influnce on toxicological behavior of the substances. Next to 2,2'-[(4-methylphenyl)imino]bisethanol Accelerator contains ca. 35-45% of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, which contains the same structural moiety of 2,2'-[(4-methylphenyl)imino]bisethanol, but with one alcohol moiety transformed into the ester bond with ethylene glycol. The presence of the ester moiety is not expected to influence skin sensitizing properties of the substances. It is probable that the skin sensitizing properties will be mostly governed by basic properties of the amine moiety which is present in both substances. Furthermore, a structural analogue of 2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethanol, 2-{[2-(2-hydroxyethoxy)ethyl](3-methylphenyl)amino}ethanol, differing only by the position of the methyl group in the phenyl ring (meta vs. para) is also present as an impurity up to 7% concentration in the target chemical 2,2'-[(3-methylphenyl)imino]bisethanol.

Skin sensitizing properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software. For both substances, the same alerts for protein binding (Schiff Base formation and Micael Acceptor alert) were identified, suggesting that their skin sensitizing properties will be similar.

Furthermore, the data matrix constructed based on the available physico-chemical and (eco)toxicological properties of both substances indicates that the substances have comparable properties across the complete range of endpoints.

Based on this, read-across from Accelerator to 2,2'-[(3-methylphenyl)imino]bisethanol is considered to be justified.

4. DATA MATRIX


Accelerator PT25 2,2'-[(3-methylphenyl)imino]bisethanol

Molecular weight ~217 195.26
State of the substance at ambient conditions Clear slightly yellowish to brown viscous liquid Colourless solidified melt
Melting/freezing point [ºC] Not determined (glass transition temp. of -28ºC) 66.9
Boiling point [ºC] - (decomposition at >275ºC) - (reaction and/or decomposition at > 225°C)
Relative density at 20 ºC 1.11 1.115
Vapour pressure at 25 ºC [Pa] 0.0025 0.000223 (calculated)
Surface tension [mN/m] 65.2 at 1 g/L, not surface active 65.4 at 1 g/L, not surface active
Water solubility at 20 ºC [mg/L] 21800 9330
Partition coefficient n-octanol/water [log Pow] 2.17 1.9
Flash point [ºC] 176 193
Flammability Not flammable, not pyrophoric Not flammable, not pyrophoric
Explosive properties Not explosive Not explosive
Auto-ignition temperature 395 395
Oxidising properties Not oxidizing Not oxidizing
Viscosity (dynamic, mPas) 2797 5474
Ready biodegradability Not readily biodegradable Not readily biodegradable
Hydrolysis as function of pH Half life for hydrolysis >1 y at 25 ºC, hydrolytically stable Read-across
Adsorption/desorption [log Koc] 2.33 (weight-averaged of 4 main components) Read-across
Acute toxicity to daphnia, EC50 [mg/L] 48 107
Growth inhibition algae, EC50, NOEC [mg/L] >100; 100 >100; 100
Acute toxicity to fish, LC50 [mg/L] >100 >102
Acute oral, LD50 [mg/kg bw] 619 300-2000
Skin irritation/corrosion Skin irritant cat 2. Read-across
Eye irritation Corrosive, cat 1. Read-across
Skin sensitisation Sensitiser Read-across
In vitro gene mutation in bacteria (Ames test) Mutagenic Non-mutagenic
In vitro cytogenicity in mammalian cells (CA) Not clastogenic, does not disturb mitotic processes Not clastogenic, does not disturb mitotic processes
In vivo genotox (Comet) Not mutagenic No data
28-day repeated dose toxicity NOAEL 100 mg/kg bw LOAEL 50 mg/kg bw/day



Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 429 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.42 (Skin Sensitisation: Local Lymph Node Assay)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2600 (Skin Sensitisation)
Deviations:
no
Key result
Parameter:
SI
Value:
3.2
Test group / Remarks:
25% test substance solution
Remarks on result:
other: 25%
Key result
Parameter:
SI
Value:
4.7
Test group / Remarks:
50% test substance solution
Remarks on result:
other: 50%
Key result
Parameter:
SI
Value:
10.8
Test group / Remarks:
100% test substance
Remarks on result:
other: 100%
Cellular proliferation data / Observations:
Mean DPM/animal values for the experimental groups treated with test substance concentrations 25, 50 and 100% were 2008, 2925 and 6725 DPM respectively. The mean DPM/animal value for the vehicle control group was 624 DPM.

Results Pre-screen test:

No irritation and no signs of systemic toxicity were observed in any of the animals examined at 25 and 50%. Both animals at 100% showed very slight erythema on Day 5 only.

Variations in ear thickness during the observation period were less than 25% from Day 1 pre-dose values. Greasy test substance remnants were present on the dorsal surface of the ears of both animals at 100% (Days 2 and 3), which did not hamper scoring of the skin reactions. Based on these results, the highest test substance concentration selected for the main study was a 100% concentration.

Other results - main study:

No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period.

No irritation of the ears was observed in any of the animals examined.

Greasy test substance remnants were present on the dorsal surface of the ears of all animals at 50 and 100% (Days 2 and/or 3), which did not hamper scoring of the skin reactions.

The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals.

 

Interpretation of results:
other: Category 1 (skin sensitising) based on GHS and CLP criteria
Conclusions:
In an LLNA skin sensitisation study with a structural analogue of 2,2-[(3-methylphenyl)imino]bisethanol, Accelerator, was performed according to OECD/EC test guidelines, the substance was considered to be a skin sensitiser, as the SI appeared to be ≥ 3 when tested up to 100%. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. This result can be read across to 2,2-[(3-methylphenyl)imino]bisethanol.
Executive summary:

An LLNA skin sensitisation assay with a structural analogue of 2,2-[(3-methylphenyl)imino]bisethanol, Accelerator, was performed according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 25, 50 or 100%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in any of the animals examined. The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 25, 50 and 100% were 3.2, 4.7 and 10.8 respectively.

These results show that the test substance elicits an SI≥3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. Based on these data, the substance is classified for skin sensitisation. This result can be read across to 2,2-[(3-methylphenyl)imino]bisethanol.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (sensitising)
Additional information:

No skin sensitization studies with 2,2'-[(3 -methylphenyl)imino]bisethanol are available; however, adequate in vivo studies are available with its structural analogue Accelerator (Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-ethanol). Based on structural similarities of the substances and their similar physico-chemical and toxicological properties, read-across from Accelerator to 2,2'-[(3-methylphenyl)imino]bisethanol is considered to be justified.

An LLNA skin sensitisation assay was performed with Accelerator according to OECD/EC guidelines and GLP principles. The test substance was applied at concentrations of 25, 50 or 100%. No mortality occurred and no clinical signs of systemic toxicity were observed in the animals of the main study. Body weights and body weight gain of experimental animals remained in the same range as controls over the study period. No irritation of the ears was observed in any of the animals examined. The lymph nodes were larger in size in animals at 50 and 100%. The largest auricular lymph nodes were found in the higher dose group. No macroscopic abnormalities of the surrounding area were noted in any of the animals. The SI values calculated for the substance concentrations 25, 50 and 100% were 3.2, 4.7 and 10.8 respectively. These results show that the test substance elicits an SI≥3. The EC3 value (the estimated test substance concentration that will give a SI =3) was established to be between 0 and 25%. Based on this Accelerator is considered to be sensitizing to skin. This result can be read across to 2,2'-[(3 -methylphenyl)imino]bisethanol.

In another GLP-compliant OECD Guideline OECD 442B study with CDA mice(5 females/group), conducted with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, the Stimulation Indices were 2.8, 3.4 and 4.1 for 25%, 50% and 100% solution of the test substance in acetone/olive oil (4:1 v/v), respectively. The positive control hexyl cinnami aldehyde showed adequate response. No deaths occurred during the study, and 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. Based on this Accelerator is considered to be sensitizing to skin. This result can be read across to 2,2'-[(3-methylphenyl)imino]bisethanol.


Respiratory sensitisation

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available data, the substance is classified for skin sensitisation according to Regulation (EC) No 1272/2008. As the SI values calculated for the test substance concentration 25% were 3.2 and 2.8 in the two available LLNA studies, it is considered appropriate to assign Category 1B.