2,2'-(m-tolylimino)diethanol

Endpoint summary

Description of key information

Based on the results of three in vitro skin irritation/corrosion studies with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, conducted according to OECD guidelines and GLP principles, 2,2'-[(3-methylphenyl)imino]bisethanol is considered to be irritating to skin.

Based on the results of two in vitro and one in vivo studies with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, conducted according to OECD guidelines and GLP principles,2,2'-[(3-methylphenyl)imino]bisethanol is considered to be causing serious damage to eyes.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Reference 1

Endpoint:

skin corrosion: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January 24th - February 22nd, 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.40 (In Vitro Skin Corrosion: Transcutaneous Electrical Resistance Test (TER))

Version / remarks:

Official Journal of the European Union No. L142, 31 May 2008.

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 431 (In Vitro Skin Corrosion: Human Skin Model Test)

Version / remarks:

adopted 13 April 2004

Deviations:

no

GLP compliance:

yes

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.3 – 37.0
- Humidity (%): 72 – 88

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Volume applied: 50 μl

NEGATIVE CONTOL:
- Volume applied: 25 µl milliQ.

POSITIVE CONTROL
- Volume applied: 50 µl
- Concentration: 8N KOH

Details on study design:

TEST SITE
- EpiDerm Skin Model (EPI-200, Lot no.:16850 kits A and B). The model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multilayered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm tissues (surface 0.6 cm²) were cultured on polycarbonate membranes of 10 mm cell culture inserts. The model was obtained from MatTek Corporation, Ashland MA, U.S.A.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 3 minutes or 1 hour

POST INCUBATION PERIOD
- 3 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

BACKGROUND:
- Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction in the Skin irritation test using EpiskinTM as a skin model (project 501366). Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal 1-hour procedure, one freeze-killed tissue treated with test substance and one freeze-killed non treated tissue must be used for the cytotoxicity evaluation with MTT.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

14%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

8%

Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction in the Skin irritation test using EpiskinTM as a skin model (project 501366). Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal 1-hour procedure, one freeze-killed tissue treated with test substance and one freeze-killed non-treated tissue was used for the cytotoxicity evaluation with MTT. In the present experiment no non-specific reduction was measured.

Interpretation of results:

other: Not corrosive in the in vitro skin corrosion test

Conclusions:

An in vitro skin corrosion test was conducted according to OECD guideline 431 and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) according to OECD guideline 431 and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.6 cm² cultured skin (25mg, in presence of 25 μl Milli-Q water). After 3 minutes or 1 hour, the substance was removed and cells were cultured for 3 hours in the presence of MTT. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% and 8% after resp. 3 minutes or 1 hour exposure whereas the test substance showed cell viability of 85% and 56% resp. Since the mean relative tissue viability after exposure to the test substance was above 50% or 15% after resp. 3 minutes or 1 hour exposure, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test.

Reference 2

Endpoint:

skin corrosion: in vitro / ex vivo

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 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 irritating properties of the substances. It is probable that the skin irritating 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.

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 irritating properties of the substances. It is probable that the skin irritating 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 irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software. For both substances, skin irritating properties were predicted, which is consistent with the results obtained for Accelerator.

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 H 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 (main component)
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 for cross-reference:

read-across source

Species:

human

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

3-minute exposure

Value:

85

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

14%

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

1-hour exposure

Value:

56

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

8%

An analogue substance Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction in the Skin irritation test using EpiskinTM as a skin model (project 501366). Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal 1-hour procedure, one freeze-killed tissue treated with test substance and one freeze-killed non-treated tissue was used for the cytotoxicity evaluation with MTT. In the present experiment no non-specific reduction was measured.

Interpretation of results:

other: Not corrosive in the in vitro skin corrosion test

Conclusions:

An in vitro skin corrosion test with a structural analogue of ethanol, 2,2'-[(3-methylphenyl)imino]bis-, Accelerator, was conducted according to OECD guideline 431 and GLP principles. It is concluded that this test is valid and that the test substance is not corrosive in the in vitro skin corrosion test. This result can be read across to ethanol, 2,2'-[(3-methylphenyl)imino]bis-.

Executive summary:

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) according to OECD guideline 431 and GLP principles conducted with a structural analogue of ethanol, 2,2'-[(3-methylphenyl)imino]bis-, Accelerator, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.6 cm² cultured skin (25 mg, in presence of 25 μl Milli-Q water). After 3 minutes or 1 hour, the substance was removed and cells were cultured for 3 hours in the presence of MTT. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% and 8% after resp. 3 minutes or 1 hour exposure whereas the test substance showed cell viability of 85% and 56% resp. Since the mean relative tissue viability after exposure to the test substance was above 50% or 15% after resp. 3 minutes or 1 hour exposure, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test. This result can be read across to ethanol, 2,2'-[(3-methylphenyl)imino]bis-.

Reference 3

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2012-11-09 - 2013-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 640/2012

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiDerm™ 200 kit

Details on test animals or test system and environmental conditions:

TEST SYSTEM:
- Source: MatTek Corp., Ashland MA, USA

Three dimensional human epidermis model
The EpiDerm™ model consists of normal, human-derived epidermal keratinocytes which have been cultured to form a multi layered, highly differentiated model of the human epidermis. It consists of organized basal, spinous and granular layers, and a multi-layered stratum corneum containing intercellular lamellar lipid layers arranged in patterns analogous to those found in vivo. The EpiDerm™ tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs®, 10 mm ∅) and commercially available as kits (EpiDerm™ 200), containing 24 tissues on shipping agarose.
Based on the results of ECVAM (European Center for Validation of Alternative Methods) funded validation studies, it was concluded by the ECVAM Scientific Advisory Committee that the EpiDerm™ human epidermis model is suitable to be used for distinguishing between corrosive and non-corrosive chemicals (ECVAM: ESAC statement on the application of the Epiderm™ human skin model for skin corrosivity testing of 14-15 Mar 2000) as well as between irritant and non-irritant chemicals (ECVAM: ESAC statement on the scientific validity of in-vitro tests for skin irritation testing of 5 Nov 2008).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 37

Vehicle:

unchanged (no vehicle)

Controls:

other: Concurrent treatment with 30 µL PBS (negative control, NC) or 30 µL of 5% SDS (positive control, PC)

Amount / concentration applied:

- Amount(s) applied (volume or weight with unit): 30 µL

Duration of treatment / exposure:

The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator.

Observation period:

The tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period.

Details on study design:

EXPERIMENTAL PROCEDURE
Pre-Test MTT reduction:
The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurred and visible residues of the test substance remained on the tissues after washing, subsequent testing of killed controls (one freeze-killed control tissue (KC)) was considered. Killed controls are treated with, each, the test article and the negative control, in the same way as described in section “Experimental procedure” (3.6), additionally.

Main Test
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 0.9 mL assay medium and preconditioned in the incubator at 37°C. After 1 hour the preincubation medium was replaced with fresh medium and preconditioning continued for 18 ± 3 hours. Three tissues were treated with the test substance, the PC and NC, respectively. Thirty microliter (30 μL) of the undiluted test substance was applied using a pipette. Control tissues were concurrently applied with 30 μL of sterile PBS (negative control, NC) or with 30 μL of 5% SDS (positive control, PC). A nylon mesh was placed carefully onto the tissue surface afterwards. The tissues were kept under the laminar flow hood at room temperature for 25 minutes overall and for 35 minutes in the incubator. The tissues were washed with sterile PBS to remove residual test material 1 hour after start of application. Rinsed tissues were blotted on sterile absorbent paper and transferred into new 6-well plates, pre-filled with 0.9 mL fresh medium. When all tissues were rinsed, the surface of each tissue was carefully dried with a sterile cotton swab. Subsequently, the tissues were incubated in the incubator at 37°C for 24 ± 2 hours. After 24 ± 2 hours the tissues were transferred into new 6-well plates pre-filled with 0.9 mL of fresh medium and placed into the incubator for additional 18 ± 2 hours post-incubation period. After the postincubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 6 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION
Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of three tissues treated in the same way is calculated.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
Assay acceptance criterion for the negative control (NC)
The absolute OD570 of the negative control tissues in the MTT test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Assay acceptance criterion for the positive control (PC)
5% SDS is used as PC and reflects the sensitivity of the tissues used in the test conditions. A viability of ≤ 20% is acceptable.

Assay acceptance criterion for tissue variability
For every treatment, 3 tissues are treated in parallel. The intertissue variability is considered to be acceptable if the SD of %-viability is ≤ 20.

EVALUATION OF RESULTS
Irritant potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile PBS. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%.

Irritation / corrosion parameter:

% tissue viability

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% (mean OD570: 2.407)

Positive controls validity:

valid

Remarks:

4% (mean OD570: 0.097)

Other effects / acceptance of results:

Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

Interpretation of results:

other: Not classified based on GHS and CLP criteria

Conclusions:

Based on the observed results and applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

Executive summary:

The substance was tested in a reliable in vitro skin irritation test with reconstructed human epidermis, performed according to OECD guidelines and according to GLP principles. Based on the observed results and applying the evaluation criteria, it was concluded, that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

Reference 4

Endpoint:

skin irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

November 13th - December 24th, 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 24 August 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 22 July 2010

Deviations:

no

GLP compliance:

yes

Species:

human

Details on test animals or test system and environmental conditions:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 36.9 - 37.8
- Humidity (%): 69 - 95

Vehicle:

unchanged (no vehicle)

Amount / concentration applied:

TEST MATERIAL
- Amounts applied: The liquid test substance was applied undiluted (25 μl) directly on top of the tissue. No correction was made for the purity/composition of the test compound.

NEGATIVE CONTOL:
- Amount applied: 25 µl Phosphate buffered saline.

POSITIVE CONTROL
- Amount applied: 25 µl
- Concentration: 5% (aq) Sodium dodecyl sulphate in PBS.

Duration of treatment / exposure:

15 minutes

Details on study design:

TEST SITE
- EPISKIN Small ModelTM (EPISKIN-SMTM, 0.38 cm2, Batch no.: 12-EKIN-032). This model is a three-dimensional human epidermis model, which consists of adult human-derived epidermal keratinocytes which have been seeded on a dermal substitute consisting of a collagen type I matrix coated with type IV collagen. The keratinocytes were cultured for 13 days, which results in a highly differentiated and stratified epidermis model comprising the main basal, supra basal, spinous and granular layers and a functional stratum corneum. The EPISKIN human epidermis was obtained from SkinEthic Laboratories, Lyon, France.

REMOVAL OF TEST SUBSTANCE
- Washing: phosphate buffered saline
- Time after start of exposure: 15 minutes

POST INCUBATION PERIOD
- 43 hours

SCORING SYSTEM:
- Cytotoxicity is expressed as the reduction of mitochondrial dehydrogenase activity measured by formazan production from MTT at the end of the treatment. Cell viability was calculated for each tissue as a percentage of the mean of the negative control tissues.

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-min exposure

Value:

23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

31%

Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Accelerator (PT 25E or PT 25E/2) was 0.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Interpretation of results:

other: Category 2 (irritant) based on GHS and CLP criteria

Conclusions:

An in vitro skin irritation test was conducted according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that the test substance is irritating in the in vitro skin irritation test.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (25 μL). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 23%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test.

Reference 5

Endpoint:

skin irritation: in vitro / ex vivo

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 irritating properties of the substances. It is probable that the skin irritating 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.

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 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 irritating properties of the substances. It is probable that the skin irritating 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 irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software. For both substances, skin irritating properties were predicted, which is consistent with the results obtained for Accelerator.

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 [ºC] 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 H 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 (main constituent)
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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No 640/2012

Deviations:

no

Vehicle:

unchanged (no vehicle)

Irritation / corrosion parameter:

% tissue viability

Value:

99

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% (mean OD570: 2.407)

Positive controls validity:

valid

Remarks:

4% (mean OD570: 0.097)

Other effects / acceptance of results:

Based on the observed results and applying the evaluation criteria, it was concluded, that the read-across source test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen.

Interpretation of results:

other: Not classified based on GHS and CLP criteria

Conclusions:

In a study with a structural analogue Accelerator, based on the observed results and applying the evaluation criteria, it was concluded that the test substance does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen. This result can be read-across to 2,2'-[(3-methylphenyl)iminodiethanol.

Executive summary:

A structural analogue of 2,2'-[(3-methylphenyl)iminodiethanol, Accelerator, was tested in a reliable in vitro skin irritation test with reconstructed human epidermis, performed according to OECD guidelines and according to GLP principles. Based on the observed results and applying the evaluation criteria, it was concluded that Accelerator does not show a skin irritation potential in the EpiDerm (TM) skin irritation test under the test conditions chosen. This result can be read across to 2,2'-[(3-methylphenyl)iminodiethanol.

Reference 6

Endpoint:

skin irritation: in vitro / ex vivo

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 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 irritating properties of the substances. It is probable that the skin irritating 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.

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 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 irritating properties of the substances. It is probable that the skin irritating 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 irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software. For both substances, skin irritating properties were predicted, which is consistent with the results obtained for Accelerator.

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 [ºC] 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 H 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 (main component)
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 Not clastogenic, does not disturb mitotic processes (CA) 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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

EU Method B.46 (In Vitro Skin Irritation: Reconstructed Human Epidermis Model Test)

Version / remarks:

adopted 24 August 2009

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 439 (In Vitro Skin Irritation: Reconstructed Human Epidermis Test Method)

Version / remarks:

adopted 22 July 2010

Deviations:

no

Duration of treatment / exposure:

15 minutes

Irritation / corrosion parameter:

% tissue viability

Run / experiment:

15-min exposure

Value:

23

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100%

Positive controls validity:

valid

Remarks:

31%

The source substance Accelerator (PT 25E or PT 25E/2) was checked for possible direct MTT reduction by adding the test substance to MTT medium. Because a colour change was observed it was concluded that Accelerator (PT 25E or PT 25E/2) did interact with MTT. In addition to the normal procedure, three killed tissues treated with test substance and three killed non treated tissues were used for the cytotoxicity evaluation with MTT. The non-specific reduction of MTT by Accelerator (PT 25E or PT 25E/2) was 0.6% of the negative control tissues. The net OD of the treated killed tissues was subtracted from the ODs of the test substance treated viable tissues.

Interpretation of results:

other: Category 2 (irritant) based on GHS and CLP criteria

Conclusions:

An in vitro skin irritation test was conducted with a structural analogue of 2,2'-[(3-methylphenyl)imino]diethanol, Accelerator, according to OECD 439 guideline and GLP principles. It is concluded that this test is valid and that Accelerator is irritating in the in vitro skin irritation test. This result can be read across to 2,2'-[(3-methylphenyl)imino]diethanol.

Executive summary:

In an in vitro skin irritation test using a human skin model ( EPISKIN Standard Model) according to OECD 439 guideline and GLP principles conducted with a structural analogue of 2,2'-[(3-methylphenyl)imino]diethanol, Accelerator, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 23%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that Accelerator is irritating in the in vitro skin irritation test. This result can be read-across to 2,2'-[(3-methylphenyl)imino]diethanol.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irritating)

Eye irritation

Link to relevant study records

Reference

Reference 1

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

in vitro

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-11-13 - 2013-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not applicable

GLP compliance:

yes (incl. QA statement)

Species:

other: EpiOcular™ OCL-200 kit

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
Source: MatTek Corp., Ashland MA, USA

Three dimensional human cornea model
The EpiOcular™ model (OCL-200) is a three-dimensional non-keratinized tissue construct composed of normal human derived epidermal keratinozytes used to model the human corneal epithelium. The EpiOcularTM tissues (surface 0.6 cm²) are cultured on specially prepared cell culture inserts (MILLICELLs, 10 mm ∅) and are commercially available as kits (EpiOcular™ 200), containing 24 tissues on shipping agarose.

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 50 µL

Duration of treatment / exposure:

30 min

Details on study design:

EXPERIMENTAL PROCEDURE
Pre-Test
To assess the ability of the test material to directly reduce MTT a pretest was performed as described below. The test substance was added to 0.9 mL of the MTT solution. The mixture was incubated in the dark at about 37 °C for 55 to 65 minutes. A negative control (de-ionized water) was tested concurrently. If the MTT solution color or, in case of water-insoluble test substances the border to the water-phase, turned blue / purple, the test substance was presumed to directly reduce MTT. The direct reduction of MTT by a test substance interferes with the color density produced by metabolic capacity of the tissue and would falsify the test results. In case that direct MTT reduction occurred, two freeze-killed control tissues were treated with, each, the test article and the negative control.

Main Test
Two tissues were treated with the test substance, the PC and NC, respectively. In addition two killed tissues were used for the test substance and NC, respectively, in order to detect direct MTT reduction. There are two separate protocols for liquids and solids, differing in exposure time and postincubation period. Due to the physical condition of the test substance the protocol for liquids was applied.

Pre-incubation of the tissues
On the day of arrival in the laboratory, the tissues were transferred to sterile 6-well plates with 1 mL assay medium and preconditioned in the incubator at standard culture conditions for 16 – 24 hours (pre-incubation).

Pretreatment of the tissues
After the pre-incubation the tissues were pre-treated with 20 μL of PBS in order to wet the tissue surface. The tissues were incubated at standard culture conditions for 30 minutes.

Application of the test substance
Using a pipette, fifty microliter (50 μL) of the undiluted liquid test substance was applied covering the whole tissue surface. Control tissues were concurrently applied with 50 μL of sterile de-ionized water (NC) or with 50 μL of methyl acetate (PC) or test substance (killed tissue control, KC). After application, the tissues were placed into the incubator until the total exposure time of 30 minutes was completed.

Removal of the test substance and postincubation period
To remove the test substance, the tissues were washed with sterile PBS. For this purpose the tissues were immersed and swiveled three times in each of three beakers filled with PBS. Washed tissues were immediately immersed into 12-well plates, pre-filled with 5 mL/well prewarmed medium (post-soak immersion) in order to remove residual test substance. After 12 minutes of post-soak immersion, each tissue was dried on absorbent paper and transferred to fresh 6-well plates filled with 1 mL/well pre-warmed medium. Subsequently, the tissues were incubated at standard culture conditions for 2 hours (postincubation period).

MTT incubation
After the post-incubation period, the assay medium was replaced by 0.3 mL MTT solution and the tissues were incubated in the incubator for 3 hours. After incubation, the tissues were washed with PBS to stop the MTT-incubation. The formazan that was metabolically produced by the tissues was extracted by incubation of the tissues in isopropanol at room temperature overnight or for at least 2 hours on a plate shaker. The optical density at a wavelength of 570 nm (OD570) of the extracts was determined spectrophotometrically. Blank values were established of 4 microtiter wells filled with isopropanol for each microtiter plate.

DATA EVALUATION
Principle
The OD570 values determined for the various tissues are measures of their viability. The quotient of the OD570 of tissues treated with the test material and the mean OD570 values of the NC (percent of control) is used for evaluating whether or not a test material is an irritant.

Calculation of individual and mean optical densities
The individual tissue OD570 is calculated by subtracting the mean blank value of the respective microtiter plate from the respective individual tissue OD570 value. The mean OD570 for a test group of two tissues treated in the same way is calculated.

Application of measurements using killed control tissues
In case of direct reduction of MTT by the test substance, the OD570 values measured in the freeze-killed control tissues (KC) will be used to correct the mean OD570 of the test substance treated tissues (mean OD570 KC corrected). Since killed tissue might still have a residual enzyme activity that is able to produce some formazan net OD570 KC is calculated by subtracting the mean OD570 KC of the NC from the mean OD570 KC of the test substance. In case the mean net OD570 KC is greater than 0.1 it is subtracted from the respective mean OD570 to result in the mean OD570 KC corrected. The mean OD570 KC corrected represents the formazan production linked to the tissue viability and therefore indicates the cytotoxic potency of the test substance.

Tissue viability
The quantification of tissue viability is presented as the quotient of the mean OD570 (or mean OD570 KC corrected, if applicable) divided by the respective OD570 NC value in percent.

ACCEPTANCE CRITERIA
Assay acceptance criterion for the NC
The absolute OD570 of the NC-tissues in the MTT-test is an indicator of tissue viability obtained in the testing laboratory after the shipping and storing procedure and under specific conditions of the assay. Tissue viability is acceptable if the mean OD570 of the NC is ≥ 1.0. The mean OD570 of the NC should not exceed 2.5.

Acceptance criteria for the PC
Methyl acetate used as PC usually leads to a tissue viability of approx. 25%. A viability of < 50% is acceptable.

Assay acceptance criterion for tissue variability
Two tissues were treated under the same conditions. A variability between the tissues is considered to be acceptable if the difference of the viability is ≤ 20%.

Acceptance criteria for the KC
The OD570 of the killed control tissues treated as negative control should be ≤ 0.35. The value for direct MTT-reduction of a test substance should be ≤ 30% of the NC.

EVALUATION OF RESULTS
The irritation potential of the test materials is predicted from the mean relative tissue viabilities compared to the negative control tissues concurrently treated with sterile water. A chemical is considered as "irritant", if the mean relative tissue viability with a test material is less than or equal to 50%. At present no prediction is performed if the mean relative tissue viability with a test material is > 50 ≤ 60% as the cut off value is currently being evaluated to lie in this range.

Irritation parameter:

other: Mean tissue viability (%)

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% (OD570: 1.856)

Positive controls validity:

valid

Remarks:

29% (OD570: 0.534)

Remarks on result:

other: OD570: 0.187

Other effects / acceptance of results:

Based on the observed results and applying the evaluation criteria cited in chapter 3.8 it was concluded, that Diethanol-para-toluidine shows an eye irritation potential in the EpiOcular™ eye irritation test under the test conditions chosen.

Interpretation of results:

study cannot be used for classification

Conclusions:

In a GLP-compliant EpiOcular test, the test substance Accelerator produced a positive response (mean tissue viability 10%). As EpiOcular test does not allow distinguishing between Category 1 and Category 2 eye irritants, the results of the study cannot be used for classification purposes.

Executive summary:

In a GLP-compliant EpiOcular test, the test substance Accelerator produced a positive response. The mean tissue viability, measured as a mean of two tissues, was 10%. The acceptability criteria were met, and the positive and negative controls produced satisfactory response, confirming the sensitivity of the assay. As EpiOcular test does not allow distinguishing between Category 1 and Category 2 eye irritants, the results of the study cannot be used for classification purposes.

Reference 2

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2012-12-10 - 2013-01-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 1152/2010

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Species:

other: Isolated Bovine Cornea

Details on test animals or tissues and environmental conditions:

TEST SYSTEM
The test system (target tissue) is the isolated bovine cornea. Bovine eyes are obtained as a by-product of freshly slaughtered cattle (age of the animals: minimum 12 months, maximum 60 months).

SOURCE
Schlachthof Bensheim, Am Schlachthof 7-9, 64625 Bensheim, Germany

Vehicle:

unchanged (no vehicle)

Controls:

yes

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 750 µL

Duration of treatment / exposure:

10 min (exposure time for liquids and surfactants)

Observation period (in vivo):

Determination of Permeability: 90 ± 5 min

Number of animals or in vitro replicates:

3 corneas per treatment group were used.

Details on study design:

EXPERIMENTAL PROCEDURE
Preparation of the bovine corneas and measurement of initial corneal opacity
Corneas free of defects (opacity, scratches, pigmentation etc.) were dissected with a 2 to 3 mm rim of sclera. Isolated corneas were mounted in specially designed corneal holders that consisted of anterior and posterior chambers. Both chambers were filled to excess with prewarmed Eagles’s MEM (without phenol red) and then equilibrated in a vertical position at about 32 °C for at least 1 hour. After the equilibration period the medium in both chambers was replaced with fresh prewarmed medium and initial corneal opacity readings were taken for each cornea with an opacitometer. Any corneas that show macroscopic tissue damage or an opacity value < 512 opacity units were discarded. Corneas with opacity values close to the median value of all corneas were selected as negative control (NC). The remaining corneas were then distributed into treatment and positive control groups. Each corneal holder was uniquely identified with a number on the chambers.

Application of the test substance and washing
Each treatment group (test substance, NC and PC) consisted of 3 corneas. Before application the medium in the anterior chamber was removed using a syringe. 750 μL of the undiluted liquid test substance was applied directly to the epithelial surface of the cornea using a pipette (open chamber method). Control tissues were concurrently applied into the anterior chamber with 750 μL of de-ionized water (negative control, NC) or with 750 μL of 1% (w/v) solution of sodium hydroxide in deionized water (positive control, PC) using a pipette. The corneas were incubated in a horizontal position at about 32 °C for approximately 10 minutes (liquids and surfactants). The NC and PC were then removed from the anterior chamber using a syringe and the epithelium was washed at least 3 times with Eagle’s MEM (containing phenol red) and once with Eagle’s MEM (without phenol red). Both chambers were then refilled with fresh Eagle’s MEM (without phenol red). Because the test substance could not be removed using a syringe, the epithelium was rinsed with the open chamber method.

Post-exposure incubation for liquid test substances and surfactants
The corneas were incubated for further 2 hours at about 32 °C. After the incubation period the medium was removed and both chambers were then refilled with fresh Eagle’s MEM.

Measurement of final corneal opacity
Before measurement, each cornea was observed visually and observations were recorded. Final corneal opacity readings were taken for each cornea with an opacitometer.

Determination of permeability
For determination of permeability the medium in the anterior chamber was replaced by 1 mL sodium fluorescein solution (4 mg/mL for liquid test substances and surfactants) and incubated for 90 ± 5 min in a horizontal position at about 32 °C. The amount of sodium fluorescein that permeated through the corneas into the posterior chamber was measured spectrophotometrically. Three aliquots per cornea were transferred to a 96-well microtiter plate and the optical density (OD490) was determined.

DATA EVALUATION
Calculation of the corneal opacity value
First, the opacity was calculated using the opacitometer specific algorithm, with a and b device specific and with Io being the illuminance (lux) through the empty corneal holder with windows and liquid and I being the illuminance (lux) through the holder with the cornea (more details see specific SOP).

• opacity value = a * Io/I + b

Then the opacity change per cornea was calculated by subtracting the initial from the final opacity.

• opacity change per cornea = final opacity - initial opacity

Subsequently, the corrected opacity change was calculated by subtracting the mean opacity change of the negative control.

• corrected opacity change = opacity change - mean opacity change of NC

Finally, the mean opacity value for each test substance could be determined as the mean of all corrected opacity changes per treatment group.

• mean opacity value = mean of all corrected opacity changes per group

Calculation of permeability value
First, the OD490 value was calculated by subtracting the mean blank OD490 (blank = Eagle´s MEM w/o phenol red) from the OD490 of each cornea.

• OD490 value = OD490 - mean blank OD490

If the OD490 value of the treated cornea was above 1.5, the OD490 of a 1:5 dilution was used to calculate the OD490 value:

• OD490 value = 5 * (OD490 of a 1:5 dilution - mean blank OD490)

Subsequently, the corrected OD490 value was calculated by subtracting the mean OD490 value of the negative control.

• corrected OD490 value = OD490 value - mean OD490 value of NC

Finally, the mean OD490 value for each test substance could be determined as the mean of all corrected OD490 values per treatment group.

• mean OD490 value = mean of all corrected OD490 values per group

Calculation of the In Vitro Irritancy Score (IVIS)
The IVIS could be calculated per treated cornea and finally the mean IVIS per treatment group ± standard deviation was determined:
• IVIS per cornea = corrected opacity change + 15 * corrected permeability OD change
• IVIS per treatment group = mean opacity value + 15 * mean permeability OD value

ACCEPTANCE CRITERIA
A study is considered acceptable if the positive control gives an IVIS that falls within two standard deviations of the current historical mean.
The negative control responses should result in opacity and permeability values that are less than the established upper limits.
Since the IVIS per treatment group is determined from the mean of three single corneas, the variability between the corneas treated per test substance should be acceptably low. If no clear prediction is possible, e.g. different predictions are obtained for single corneas, the test will be repeated.

EVALUATION OF RESULTS
The following rules of assessment apply:
IVIS > 55 risk of serious damage to the eye
IVIS =< 55 no risk of serious damage to the eye

Irritation parameter:

in vitro irritation score

Run / experiment:

10-min exposure

Value:

46.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Other effects / acceptance of results:

Based on the observed results and applying the evaluation criteria (see EVALUATION OF RESULTS) it was concluded, that the test substance does not cause serious eye damage in the Bovine Corneal Opacity and Permeability Test (BCOP Test) under the test conditions chosen.

Interpretation of results:

study cannot be used for classification

Conclusions:

In the GLP-compliant OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test) the test substance Accelerator did not cause serious damage to eyes. Based on the obtained in vitro irritancy score (IVIS 46.9) which is below the cut-off for the classification as Category 1 (IVIS of 55), the study cannot be used for classification purposes.

Executive summary:

In the GLP-compliant OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test) the test substance Accelerator did not cause serious damage to eyes. The in vitro irritance score (IVIS) was 46.9, based on the average result from 3 corneas. Positive and negative controls were valid, confirming the sensitivity of the system. Based on the obtained in vitro irritancy score (IVIS 46.9) which is below the cut-off for the classification as Category 1 (IVIS of 55), the study cannot be used for classification purposes.

Reference 3

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 9th-30th, 1995

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

GLP compliance:

yes (incl. QA statement)

Remarks:

issued 31JAN1994

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: David Percival Ltd., Moston, Sandbach, Cheshire, UK
- Age at study initiation: 12 - 16 weeks old
- Weight at study initiation: 3.1 kg
- Housing: individually in a suspended metal cage
- Diet: ad libitum (STANRAB SQC Rabbit diet, Special Diets Services, Witham, Essex, UK)
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 17-21
- Humidity (%): 54 - 74
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12/12

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

0.1 ml

Duration of treatment / exposure:

Single instillation

Observation period (in vivo):

21 days

Number of animals or in vitro replicates:

1

Details on study design:

REMOVAL OF TEST SUBSTANCE
- Washing (if done): no

SCORING SYSTEM: According to the OECD guideline.

TOOL USED TO ASSESS SCORE: ophthalmoscope

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: At 21 days, circumcorneal vascularisation invading up to 2 mm onto the cornea was noted (irreversible effect)

Irritation parameter:

other: area of opacity

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Ectropion was noted at day 21 (irreversible effect)

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

other: discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Areas of diffuse corneal opacity were noted in the treated eye one hour after treatment and at the 24 and 48-hour observations with areas of translucent corneal opacity noted at the 72-hour and 7-day observations. Diffuse corneal opacity was also noted at the 14-day observation.

Iridial inflammation was noted in the treated eye one hour after treatment and at the 24, 48, 72-hour and 7-day observations with minimal conjunctival irritation noted at the 14-day observation.

Other effects:

Ectropion was noted in the treated eye at the 7, 14 and 21-day observations with circumcorneal vascularisation invading o to 2 mm onto the cornea also noted at these times. These reactions were considered to be irreversible.

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on GHS and CLP criteria

Conclusions:

An in vivo eye irritation test was performed according to OECD guideline and GLP principles. Based on irreversibel effects to the eye, BISOMER PTE is classified cat. 1 for eye irritancy.

Executive summary:

An in vivo eye irritation test was performed according to the OECD guideline and GLP principles. One rabbit was treated and observed for 21 days. The average scores for observations at 24, 48 and 72 hours were 1.3 (cornea score), 1 (iris score) and 2 (chemosis score). At the end of the observation period of 21 days, ectropion was noted in the treated eye with circumcorneal vascularisation invading up to 2 mm onto the cornea. Since these reactions were considered to be irreversible, BISOMER PTE is classified cat. 1 for eye irritancy (corrosive).

Reference 4

Endpoint:

eye irritation: in vitro / ex vivo

Remarks:

in vitro

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 eye irritating properties of the substances. It is likely that the eye irritating properties will be mostly governed by basic nature of the imine 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.

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 eye irritating properties of the substances. It is probable that the eye irritating 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.

Eye irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software, but the results were inconclusive in both cases.

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 [ºC] 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 (main constituent)
Acute toxicity to daphnia, EC50 [mg/L] 48 107 mg/L
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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

other: MatTek Corporation, Ashland, MA 01721, USA: EpiOcularTM human cell construct: Procedure details, Version 3.1a of February 10, 2010

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Harbell J.W. et al. (2009): COLIPA Program on Optimization of Existing In Vitro Eye Irritation Assays for Entry into Formal Validation: Technology Transfer and Intra/Inter Laboratory Evaluation of EpiOcular Assay for Chemicals.

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

not applicable

Irritation parameter:

other: Mean tissue viability (%)

Value:

10

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Remarks:

100% (OD570: 1.856)

Positive controls validity:

valid

Remarks:

29% (OD570: 0.534)

Remarks on result:

other: OD570: 0.187

Interpretation of results:

study cannot be used for classification

Conclusions:

In a GLP-compliant EpiOcular test conducted with a structural analogue of 2,2'-[(3 -methylphenyl)imino]bisethanol, Accelerator, the test substance Accelerator produced a positive response. This result can be read across to 2,2'-[(3 -methylphenyl)imino]bisethanol. As EpiOcular test does not allow distinguishing between Category 1 and Category 2 eye irritants, the results of the study cannot be used for classification purposes.

Executive summary:

In a GLP-compliant EpiOcular test conducted with a structural analogue of 2,2'-[(3 -methylphenyl)imino]bisethanol, Accelerator, the test substance Accelerator produced a positive response. The mean tissue viability, measured as a mean of two tissues, was 10%. The acceptability criteria were met, and the positive and negative controls produced satisfactory response, confirming the sensitivity of the assay. This result can be read across to

2,2'-[(3 -methylphenyl)imino]bisethanol. As EpiOcular test does not allow distinguishing between Category 1 and Category 2 eye irritants, the results of the study cannot be used for classification purposes.

Reference 5

Endpoint:

eye irritation: in vitro / ex vivo

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 eye irritating properties of the substances. It is likely that the eye irritating properties will be mostly governed by basic nature of the imine 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.

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 eye irritating properties of the substances. It is probable that the eye irritating 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.

Eye irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software, but the results were inconclusive in both cases.

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 (main constituent)
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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Commission Regulation (EC) No. 1152/2010

Deviations:

no

Irritation parameter:

in vitro irritation score

Run / experiment:

10-min exposure

Value:

46.9

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Interpretation of results:

study cannot be used for classification

Conclusions:

In the GLP-compliant OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test) with a structural analogue of 2,2'-[(3 -methylphenyl)imino]diethanol, Accelerator, the test substance Accelerator did not cause serious damage to eyes. This result can be read across to 2,2'-[(3 -methylphenyl)imino]diethanol. Based on the obtained in vitro irritancy score (IVIS 46.9) which is below the cut-off for the classification as Category 1 (IVIS of 55), the study cannot be used for classification purposes.

Executive summary:

In the GLP-compliant OECD Guideline 437 (Bovine Corneal Opacity and Permeability Test) with a structural analogue of 2,2'-[(3 -methylphenyl)imino]diethanol, Accelerator, the test substance Accelerator did not cause serious damage to eyes. The in vitro irritance score (IVIS) was 46.9, based on the average result from 3 corneas. Positive and negative controls were valid, confirming the sensitivity of the system. This result can be read across to 2,2'-[(3 -methylphenyl)imino]diethanol. Based on the obtained in vitro irritancy score (IVIS 46.9) which is below the cut-off for the classification as Category 1 (IVIS of 55), the study cannot be used for classification purposes.

Reference 6

Endpoint:

eye irritation: in vivo

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 eye irritating properties of the substances. It is likely that the eye irritating properties will be mostly governed by basic nature of the imine 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.

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 eye irritating properties of the substances. It is probable that the eye irritating 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.

Eye irritating properties of both substances were predicted based on their structural features using Toxtree v.2.6.13 software, but the results were inconclusive in both cases.

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 (main constituent)
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 for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Irritation parameter:

cornea opacity score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.3

Max. score:

4

Reversibility:

not reversible

Remarks on result:

other: At 21 days, circumcorneal vascularisation invading up to 2 mm onto the cornea was noted (irreversible effect)

Irritation parameter:

other: area of opacity

Basis:

animal #1

Time point:

24/48/72 h

Score:

4

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

iris score

Basis:

animal #1

Time point:

24/48/72 h

Score:

1

Max. score:

2

Reversibility:

fully reversible within: 14 days

Irritation parameter:

conjunctivae score

Remarks:

(redness)

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

3

Reversibility:

not reversible

Remarks on result:

other: Ectropion was noted at day 21 (irreversible effect)

Irritation parameter:

chemosis score

Basis:

animal #1

Time point:

24/48/72 h

Score:

2

Max. score:

4

Reversibility:

fully reversible within: 21 days

Irritation parameter:

other: discharge

Basis:

animal #1

Time point:

24/48/72 h

Score:

1.67

Max. score:

3

Reversibility:

fully reversible within: 7 days

Irritant / corrosive response data:

Areas of diffuse corneal opacity were noted in the treated eye one hour after treatment and at the 24 and 48-hour observations with areas of translucent corneal opacity noted at the 72-hour and 7-day observations. Diffuse corneal opacity was also noted at the 14-day observation.

Iridial inflammation was noted in the treated eye one hour after treatment and at the 24, 48, 72-hour and 7-day observations with minimal conjunctival irritation noted at the 14-day observation.

Other effects:

Ectropion was noted in the treated eye at the 7, 14 and 21-day observations with circumcorneal vascularisation invading o to 2 mm onto the cornea also noted at these times. These reactions were considered to be irreversible.

Interpretation of results:

other: Category 1 (irreversible effects on the eye) based on GHS and CLP criteria

Conclusions:

An in vivo eye irritation test with a structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, was performed according to OECD guideline and GLP principles. Based on irreversibel effects to the eye, the test substance Accelerator is classified cat. 1 for eye irritancy. This result can be read across to 2,2'-[(3-methylphenyl)imino]bisethanol.

Executive summary:

An in vivo eye irritation test was performed with the structural analogue of 2,2'-[(3-methylphenyl)imino]bisethanol, Accelerator, according to the OECD guideline and GLP principles. One rabbit was treated and observed for 21 days. The average scores for observations at 24, 48 and 72 hours were 1.3 (cornea score), 1 (iris score) and 2 (chemosis score). At the end of the observation period of 21 days, ectropion was noted in the treated eye with circumcorneal vascularisation invading up to 2 mm onto the cornea. Since these reactions were considered to be irreversible, the test substance Accelerator is classified cat. 1 for eye irritancy (corrosive). This result can be read across to 2,2'-[(3-methylphenyl)imino]bisethanol.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (irreversible damage)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

No skin and eye irritation/corrosion studies with 2,2'-[(3-methylphenyl)imino]bisethanol were available; however, reliable in vitro and in vivo studies were available for 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.

In an in vitro skin irritation test using a human skin model (EPISKIN Standard Model) according to OECD 439 guideline and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.38 cm² cultured skin (25 μl). After 15 minutes, the substance was removed and cells were cultured for 43 hours. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 31% whereas the test substance showed cell viability of 23%. Since the mean relative tissue viability after exposure to the test substance was below 50%, it can be concluded that the test substance is irritating in the in vitro skin irritation test. This result can be read across to 2,2'-[(3 -methylphenyl)imino]bisethanol.

In an in vitro skin corrosion test using a human skin model (EpiDerm Skin Model) according to OECD guideline 431 and GLP principles, the influence of Accelerator (PT 25E or PT 25E/2) on the viability of human skin was tested. The test substance was applied directly to 0.6 cm² cultured skin (25 mg, in presence of 25 μl Milli-Q water). After 3 minutes or 1 hour, the substance was removed and cells were cultured for 3 hours in the presence of MTT. The viability of the cells was tested by reduction of MTT. Survival of unexposed skin was set at 100%, the positive control had a mean cell viability of 14% and 8% after resp. 3 minutes or 1 hour exposure whereas the test substance showed cell viability of 85% and 56% resp. Since the mean relative tissue viability after exposure to the test substance was above 50% or 15% after resp. 3 minutes or 1 hour exposure, it can be concluded that the test substance is not corrosive in the in vitro skin corrosion test. This result can be read across to 2,2'-[(3 -methylphenyl)imino]bisethanol.

In an in vitro eye irritation study (Bovine Corneal Opacity and Permeability test) conducted according to the OECD guideline and GLP principles, the test substance Accelerator did not cause serious damage to eyes. The in vitro irritance score (IVIS) was 46.9, based on the average result from 3 corneas. Positive and negative controls were valid, confirming the sensitivity of the system. This result can be read across to 2,2'-[(3 -methylphenyl)imino]diethanol. Based on the obtained in vitro irritancy score (IVIS 46.9) which is below the cut-off for the classification as Category 1 (IVIS of 55), the study cannot be used for classification purposes.

In a GLP-compliant EpiOcular test conducted with a structural analogue of 2,2'-[(3 -methylphenyl)imino]bisethanol, Accelerator, the test substance Accelerator produced a positive response. The mean tissue viability, measured as a mean of two tissues, was 10%. The acceptability criteria were met, and the positive and negative controls produced satisfactory response, confirming the sensitivity of the assay. This result can be read across to

2,2'-[(3 -methylphenyl)imino]bisethanol. As EpiOcular test does not allow distinguishing between Category 1 and Category 2 eye irritants, the results of the study cannot be used for classification purposes. Therefore in vivo testing was considered necessary.

An in vivo eye irritation test with Accelerator was performed according to the OECD guideline and GLP principles. One rabbit was treated and observed for 21 days. The average scores for observations at 24, 48 and 72 hours were 1.3 (cornea score), 1 (iris score) and 2 (chemosis score). At the end of the observation period of 21 days, ectropion was noted in a treated eye with circumcorneal vascularisation invading up to 2 mm onto the cornea. Since this reaction was considered to be irreversible, Accelerator is classified cat. 1 for eye irritancy (corrosive). This result can be read across to 2,2'-[(3 -methylphenyl)imino]bisethanol.

Justification for classification or non-classification

Based on the available data, the substance is irritating to the skin, but not corrosive and is classified as skin irritant (Skin Irrit. 2, H315) according to CLP Regulation (EC) No. 1272/2008.

Based on the available data, it is concluded that the substance is corrosive to the eye, and is classified as Eye Damage 1, H318 according to CLP Regulation (EC) No. 1272/2008.