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Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report date:
2012

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)
GLP compliance:
yes

Test material

Constituent 1
Chemical structure
Reference substance name:
2-(methoxymethyl)benzene-1,4-diamine
EC Number:
679-526-3
Cas Number:
337906-36-2
Molecular formula:
C8H12N2O
IUPAC Name:
2-(methoxymethyl)benzene-1,4-diamine
Constituent 2
Reference substance name:
-
EC Number:
474-270-7
EC Name:
-
IUPAC Name:
474-270-7
Constituent 3
Reference substance name:
1,4- diamino-2-methoxymethyl-benzene
IUPAC Name:
1,4- diamino-2-methoxymethyl-benzene
Test material form:
solid: crystalline
Details on test material:
- Name of test material: 2-Methoxymethyl-p-phenylenediamine, MBB (Code # A003825)
- Substance type: pure substance
- Physical state: solid

- Purity test date: provide if available
- Lot/batch No.: 804025
- Stability under test conditions: The substance is considered to be stable for more than 5 yrs, if stored dry and protected from light at room temperature.
- Stability in solutions: Stability in solution: The stability of the test item (MBB) in 2 different solvents r¡as monitored over a total time period of
three days using HPLC/DAD, During the test procedure all test solutions were stored at ambient temperature in the absence of light. The indicated values were standardized on separate initial weights.
Stability in DMSO (10 % solution, wlv) Recovery at t = 0: 99.2 % t = 6h: 100.3 %; t = 2d: 99.8 %; t = 3d. 100.2 %.
Stability, in Water (10 % solution, wlv) Recovery at t = 0: 98.7 % t = 6h: 99.4 %; t= 2d: 99.7 %; t = 3d: 100 %.
Gonclusion:
The test itern of MB4 lot 20080201 is assumed to be stable in Water and DMSO during the course of storage of 3d on the conditions applied.

Test animals / tissue source

Species:
other: chicken eye
Strain:
not specified

Test system

Vehicle:
unchanged (no vehicle)
Amount / concentration applied:
TEST MATERIAL
- Amount(s) applied: 30 mg
- Concentration: 100%

POSITIVE CONTROL: Sodium hydroxyde
- Concentration: neat
- Amount applied: 30 mg
- Batch: Sigma Aldrich, batch # S55833-438
- Expiry date: 30 nov 2013

NEGATIVE CONTROL: physiological Saline
- Concentration: 0.9%
- Amount applied: 30 µL
- Batch: Eurovet, Bladel, the Netherlands
- Expiry date: February 2013
Duration of treatment / exposure:
10 secondes
Observation period (in vivo):
Approx. at 0, 30, 75, 120, 180 and 240 min after treatment. Fluorescein retention was only scored at approx. 30 min after treatment. All examinations were carried out with the slit-lamp microscope.
Details on study design:
DETAILS OF TEST SYSTEM: The isolated chicken eye (ICE)
- Source: Heads of animals were obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, the Netherlands.
- Age at study initiation: 7 wk old
- Weight at study initiation: Body weight range approx. 1.5-2.5 kg
- Sex: Male or female chicken
- Collection and transportation of chicken heads: The heads were removed immediately after sedation of the animals by electric shock and incision of the neck for exsanguination. The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline and were maintained at ambient temperature.

EXPERIMENTAL PROCEDURE: The experimental procedure is as follows:

- Determination of eye for damage: Eye (to corneal surface) was treated with small drop of Fluorescein sodium BP 2% w/v and subsequently rinsed off with isotonic saline. Next, the head with the fluorescein-treated cornea was examined with a slit-lamp microscope (Slit-lamp BP 900, Haag-Streit AG, Liebefeld-Bern, Switzerland), to ensure that the cornea was not damaged.

- Positioning of eye in superfusion apparatus: Undamaged eye was further dissected from the head (without damaging the eye or cornea), without cutting off the optical nerve too short. The enucleated eye was placed in a stainless steel clamp with the cornea positioned vertically and transferred to a chamber of the superfusion apparatus (TNO, Zeist, the Netherlands). The clamp holding the eye was positioned in such a way that the entire cornea was supplied with isotonic saline from a bent, stainless steel tube, at a rate of approx. 0.10 - 0.15 mL/min (peristaltic pump, Watson Marlow 205CA, Rotterdam, the Netherlands). The chambers of the superfusion apparatus as well as the saline were temperature controlled at 32 ± 1.5 °C (water pump, Thermomix 1441, B. Braun Melsungen AG, Melsungen, Germany). After placing in the superfusion apparatus, the eyes were examined again with the slit-lamp microscope to ensure that they were not damaged.

- Measurement of corneal thickness prior to treatment: Corneal thickness was measured by using Depth Measuring Attachment no. I for the Haag-Streit slit-lamp microscope.

-Streit slit-lamp microscope. Corneal thickness of the cornea was expressed in instrument units. An accurate measurement was taken at the corneal apex of each eye. After 45-60 min equilibration period, corneal thickness was measured to determine the zero reference value for corneal swelling calculations.

- Criteria for eye selection: Eyes with a corneal thickness deviating more than 10% of the average corneal thickness of the eyes, or eyes that showed opacity (score higher than 0.5), or were unacceptably stained with fluorescein (score higher than 0.5) indicating the cornea to be permeable, or eyes that showed any other signs of damage, were rejected as test eyes and replaced.

- Number of eyes: 3 for each concentration of the test item, 3 for the positive control NaOH and 1 for the negative control (physiological saline)

- Application of test material: At time t=0 The clamp holding the test eye was placed on paper tissues outside the chamber with the cornea facing upwards and test substance was applied to corneal surface. Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein retention. 30 µL or 30 mg of product (test item or negative or posiitve control) were applied.

- Histological evaluation: After final examination, all treated eyes (test itme, negative and positive control) were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde. The corneas were embedded in paraffin wax, sectioned at 5 µM and stained with PAS (Periodic Acid-Schiff). Histopathological examination was performed by light microscopy.

REMOVAL OF TEST SUBSTANCE
- Washing: The washing was performed with 20 mL saline
- Time after start of exposure: 10 sec
After rinsing, each eye i the holder returned to its chamber.

SCORING SYSTEM: The scoring system is as follows:

1. Corneal swelling: Corneal swelling, expressed as a percentage, was calculated according to the following formula:
Corneal swelling = ((Corneal thickness at time t - Corneal thickness at time t = 0)/ Corneal thickness at time t = 0) × 100

2. Corneal opacity: Opacity degree of density (area most dense taken for scoring)
No opacity…………………………………………………………………………………………………...........................................0
Very faint opacity (= very slight)…………………………………………………………….......................................................0.5
Scattered or diffuse areas, details of iris clearly visible (slight).………………..……………………………………………..1
Easily discernible translucent area, details of iris slightly obscured (= moderate).……….……....…………………......2
Severe corneal opacity, no specific details of iris visible, size of pupil barely iscernible (= sevre)..………................3
Complete corneal opacity, iris invisible (=very severe)..…………………………………………………….………………....4
- The mean corneal opacity value for all test eyes was calculated for the observation time points of 30, 75, 120, 180, and 240 min.
- NOTE. In case of score 4, the thickness assessment will not be possible. Intermediate scores can also be assigned.

3. Fluorescein retention
No fluorescein retention………………………………………………………………………………………………………………..0
Very minor single cell staining (very slight).……………………………………………………………………..………………0.5
Single cell staining scattered throughout the treated area of the cornea (= slight)………………............……..............1
Focal or confluent dense single cell staining (=moderate)..……………………………………………………………………2
Confluent large areas of the cornea retaining fluorescein (=severe)………………………………….……………………..3
Intermediate scores can also be assigned.
- The mean fluorescein retention value for all test eyes was calculated for the observation time point of 30 min only. If desired or in case of test substances that have adhered to the cornea, fluorescein retention can be determined at t=240 min or whenever the test compound is removed.

- Morphological effects: These include "pitting" of corneal epithelial cells, "loosening" of epithelium, "roughening" of the corneal surface and "sticking" of the test substance to the cornea. These findings can vary in severity and may occur simultaneously. The classification of these findings is subjective to the interpretation of the investigator.

- Microscopic effects: Corneal lesions are determined by microscopic examination. The effects include but are not limited to erosion, necrosis and vacuolation of the epithelium, disorder of stromal fibers, pyknotic nuclei in the stroma and necrosis of the endothelium. The classification of these findings is subject to the interpretation of the investigator.

TOOL USED TO ASSESS SCORE: All examinations were performed by using slit lamp microscope (Slit-lamp BP 900, Haag-Streit AG, Liebefeld-Bern, Switzerland).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
other: maximum total score (MMTS) - swelling
Run / experiment:
time point 240 min
Value:
ca. 15
Remarks on result:
other: test substance
Irritation parameter:
other: maximum mean total score (MMTS)
Run / experiment:
other: 30, 75, 120, 180, 240 min
Value:
ca. 2.3
Remarks on result:
other: test substance - distinct loosening of epithelium in two corneas; slight in one cornea
Irritation parameter:
other: fluorescein retention
Run / experiment:
30 min
Value:
ca. 2.7
Remarks on result:
other: test substance
Irritation parameter:
in vitro irritation score
Run / experiment:
irritation index = maximum mean corneal swelling + maximum mean opacity (x20) + mean fluoresein score (x20)
Value:
ca. 115
Remarks on result:
other: test substance
Irritation parameter:
percent corneal swelling
Run / experiment:
30, 75, 120, 180 and 240
Value:
ca. 0
Remarks on result:
other: negative control (saline)
Irritation parameter:
other: maximum mean total score (MMTS)
Run / experiment:
30, 75, 120, 180, 240
Value:
ca. 0
Remarks on result:
other: negative control (saline)
Irritation parameter:
other: maximum mean total score (MMTS) - swelling %
Run / experiment:
180 min
Value:
ca. 56
Remarks on result:
other: positive control (NaOH)
Irritation parameter:
other: maximum mean total score (MMTS) - fluorescein retention
Run / experiment:
30 min
Value:
ca. 0
Remarks on result:
other: negative control (saline)
Irritation parameter:
other: opacity
Run / experiment:
30, 75, 120, 180, 240 min
Value:
ca. 4
Remarks on result:
other: positive control (NaOH)
Irritation parameter:
other: maximum mean total score (MMTS) - fluorescein retention
Run / experiment:
30 min
Value:
ca. 3
Remarks on result:
other: positive control (NaOH)
Irritation parameter:
in vitro irritation score
Run / experiment:
irritation index = maximum mean corneal swelling + maximum mean opacity (x20) + mean fluoresein score (x20)
Value:
ca. 196
Remarks on result:
other: positive control (NaOH)

In vivo

Irritant / corrosive response data:
1,4-diamino-2-methoxymethylbenzene caused slight swelling (15%), moderate to severe opacity (2.3) and moderate or severe fluorescein retention (2.7). in addition, the samples caused slight or distinct loosening of the epithelium.
Other effects:
HISTOPATHOLOGICAL EFFECTS:
- The microscopic examination of the corneas only revealed very slight or slight erosion of the epithelium. No abnormalities of the stroma or endothelium were observed.
- No abnormalities in epithelium, stroma or endothelium were observed in eyes treated with 6.1% of test substance.
- Microscopic examination of the corneas fully confirmed the effects observed by slit-lamp examination.

Any other information on results incl. tables

-Both positive and negative control demonstrated the suitability and sensitivity of the ICE to detect severe eye irritants.

Applicant's summary and conclusion

Interpretation of results:
Category 2A (irritating to eyes)
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
Corneas of isolated chicken eyes were treated with test item at a dose of 30 mg for 10 seconds.
1,4-diamino-2 methoxymethylbenzene caused slight swelling (15%), moderate to severe opacity (2.3) and moderate or severe fluorescein retention (2.7). ln addition, the samples caused slight or distinct loosening of the epithelium.
The calculated lrritation lndex was 115 (see Table of results).
Microscopic examination of the corneas only revealed very slight or slight erosion of the epithelium. No abnormalities of the stroma or endothelium were observed.
According to the classification schemes of the lCE, the following irritation classifications can be assigned:
- Category 2A: "lrritanVcauses eye irritation' (UN-GHS classification)
- Category 2: "lrritating to eyes" (EU-CLP classification).
Executive summary:

Thein-vitr oeye irritation of 1,4-diamino-2-methoxymethyl-benzene (MBB) with isolated chicken eye was determined following the OECD guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants).

The eyes were isolated from either male or female spring chicken heads (ROSS) of 7 wk old obtained from poultry slaughterhouse v.d. Bor, Amersfoortseweg 118, Nijkerkerveen, The Netherlands. The isolated chicken eyes were exposed with 30 mg by a single application of the neat test substance for 10 sec.

Three test eyes/ treatment group were evaluated. NaOH served as a positive control and saline treated eyes served as negative control. Three main parameters, corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells, were measured at approx. 0, 30, 75, 120, 180 and 240 min by using slit microscopy to disclose possible adverse eye effects. In addition, histopathology was performed on the cornea to assess the nature and depth of injury.

Corneas of isolated chicken eyes were treated with test item at a dose of 30 mg for 10 seconds.

1,4-diamino-2 methoxymethylbenzene caused slight swelling (15%), moderate to severe opacity (2.3) and moderate or severe fluorescein retention (2.7). ln addition, the samples caused slight or distinct loosening of the epithelium.

The calculated lrritation lndex was 115. Microscopic examination of the corneas only revealed very slight or slight erosion of the epithelium. No abnormalities of the stroma or endothelium were observed.

Negative control caused no meaningful corneal effects. Positive control caused severe effects after treatment, thus confirmingthe suitability and sensitivity of the ICE to detect severe eye irritants.

1,4-diamino-2-methoxymethyl-benzene was classified as an irritant to the isolated chicken eye (ICE) when applied neat.