4-amino-m-cresol

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 29 June 2016 to 25 September 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying i) Chemicals Inducing Serious Eye Damage and ii) Chemicals Not Requiring Classification for Eye Irritation or Serious Eye Damage)

Version / remarks:

Adopted : 26 July 2013

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

Constituent 1

Reference substance name:

4-amino-m-cresol

EC Number:

220-621-2

EC Name:

4-amino-m-cresol

Cas Number:

2835-99-6

Molecular formula:

C7H9NO

IUPAC Name:

4-amino-m-cresol

Test material form:

solid: particulate/powder

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: supplied by Grocter & Gamble, Batch No. DRDGAMC110703W
- Expiration date of the lot/batch: 30 June 2018
- Purity test date: 13 May 2016

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: stored at ambient temperature (15-25 deg celsius)
- Stability under test conditions: not specified
- Solubility and stability of the test substance in the solvent/vehicle: the test item was used pure
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: not specified

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test item was used neat
- Final dilution of a dissolved solid, stock liquid or gel: no dilution was used
- Final preparation of a solid:not specified

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS, spring chickens

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Bor poultry slaughterhouse (Netherlands)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 1.5 - 2.5 kg

ENVIRONMENTAL CONDITIONS
The heads were placed in small plastic boxes on a bedding of paper tissues moistened with isotonic saline. Next, they were transported to the testing facility. During transportation, the heads were kept at ambient temperature.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30mg of test item ; 30 µL of positive control
- Concentration (if solution): the positive control was used 5% (v/v) aqueous diluted

VEHICLE
The test item was used pure

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

POSITIVE CONTROL
- Amount(s) applied (volume or weight with unit): 30 mL
- Concentration (if solution): 5% (v/v) aqueous

Duration of treatment / exposure:

10 seconds

Observation period (in vivo):

240 minutes

Duration of post- treatment incubation (in vitro):

240 minutes

Number of animals or in vitro replicates:

Number of animals or in vitro replicates
1 for Negative Control
3 for Positive Control
3 for Test group

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
Within 2 hours after kill, eyes were carefully dissected and placed in a superfusion apparatus using t
he following procedure: First the eye lids were carefully removed without damaging the cornea and a
small drop of Fluorescein sodium 2.0% w/v (Minims, England) was applied to the corneal surface for
a few seconds and subsequently rinsed off with isotonic saline at ambient temperature. Next, the h
ead with the fluorescein treated cornea was examined with a slit lamp microscope to ensure that the
cornea was not damaged. If undamaged (e.g., fluorescein retention and corneal opacity scores of ≤
0.5), the eye was further dissected from the head without damaging the eye or cornea. Care was take
n to remove the eye ball from the orbit 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. 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 target rate
of 0.10 0.15 mL/min (peristaltic pump set at speed 5.00). The chambers of the superfusion apparat
us as well as the saline were temperature controlled at approximately 32 deg Celsius (water pump se
t at 36.4 deg Celsius). After placing in the superfusion apparatus, the eyes were examined again wi
th the slit lamp microscope to ensure that they were not damaged. Corneal thickness was measured
using the Depth Measuring Attachment No. I for the Haag Streit slit lamp microscope, set at 0.095
mm. Corneal thickness was expressed in instrument units. An accurate measurement was taken at
the corneal apex of each eye. Eyes with a corneal thickness deviating more than 10% of the average
corneal thickness of the eyes, eyes showing 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.

EQUILIBRATION AND BASELINE RECORDINGS
Each eye provided its own baseline values for corneal swelling, corneal opacity and fluorescein re
tention. For that purpose, after an equilibration period of 45-60 minutes, the corneal thickness of the
eyes was measured again to determine the zero reference value for corneal swelling calculations.

NUMBER OF REPLICATES
1 for Negative Control ; 3 for Positive Control ; 3 for Test group

NEGATIVE CONTROL USED
Physiological Saline NaCl

POSITIVE CONTROL USED
Benzalkonium Chloride (BAC)

APPLICATION DOSE AND EXPOSURE TIME
30 μL of negative control or 30 mg of postive control and test item were applied for 10 seconds

OBSERVATION PERIOD
0, 30, 75, 120, 180 and 240 minutes

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period: The eye was rinsed 10 seconds after treatment with test item or control. They were rinsed with 20 mL saline solution.
- Indicate any deviation from test procedure in the Guideline : no deviation

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: slit lamp microscope
- Damage to epithelium based on fluorescein retention: Slit lamp microscope
- Swelling: measured with optical pachymeter on a slit-lamp microscope
- Macroscopic morphological damage to the surface: No
- Others (e.g, histopathology): Histopathological examination

SCORING SYSTEM:
Corneal swelling, expressed as a percentage, is calculated according to the following formula:
“Corneal thickness at time t minus corneal thickness at time t = 0, divided by corneal thickness at time
t = 0 and multiplied by 100”.
The mean percentage of swelling for the three test eyes will be calcula¬ted for each of the observa
tion time points of 30, 75, 120, 180, and 240 minutes. The maximum mean percentage (can be at any
of the time points) will be used for classification into one of four categories

- Mean maximum opacity score
Opacity degree of density (area most dense taken for scoring)
0 = no opacity
0.5 = very faint opacity (= very slight)
1 = scattered or diffuse areas, details of iris clearly visible (= slight)
2 = easily discernible translucent area, details of iris slightly obscured (= moderate)
3 = severe corneal opacity, no specific details of iris visible, size of pupil barely discernible (= severe)
4 = complete corneal opacity, iris invisible (= very severe)
The mean corneal opacity value for all test eyes is calculated for the observation time points of 30, 75
, 120, 180, and 240 minutes. The maximum mean opacity score (can be at any of the time points) will
be used for classification into one of four categories

- Mean fluorescein retention score at 30 minutes post-treatment
0 = no fluorescein retention
0.5 = very minor single cell staining (= very slight)
1 = single cell staining scattered throughout the treated area of the cornea (= slight)
2 = focal or confluent dense single cell staining (= moderate)
3 = confluent large areas of the cornea retaining fluorescein (= severe)

DECISION CRITERIA: please specify if the decision criteria as indicated in the TG was used.

On the basis of the severity of the observed findings for corneal swelling, corneal opacity and fluoresc
ein retention, the effects are divided into four categories, viz. I = no effect; II = slight effect; III = m
oderate effect; IV = severe effect.
Interpretation of corneal swelling, corneal opacity, and fluorescein retention and categorisation into
the four categories is done according the following methodology:
Corneal swelling:
Mean corneal swelling (%) Category
0 5 I
>5 12 II
>12 - 18 (>75 min. after treatment) II
(≤75 min. after treatment) III
>18 26 III
>26 - 32 (>75 min. after treatment) III
(≤75 min. after treatment) IV
>32 IV
Corneal opacity:
Max. mean opacity score Category
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 4.0 IV
Fluorescein retention:
mean fluorescein retention score Category
at 30 min after treatment:
0.0 0.5 I
0.6 1.5 II
1.6 2.5 III
2.6 3.0 IV

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

percent corneal swelling

Run / experiment:

Test item

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 2

Irritation parameter:

cornea opacity score

Run / experiment:

Test item

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 3

Irritation parameter:

fluorescein retention score

Run / experiment:

Test item

Value:

0

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Results 4

Irritation parameter:

in vitro irritation score

Run / experiment:

Test item

Value:

1

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

no indication of irritation

Other effects / acceptance of results:

OTHER EFFECTS:
- Visible damage on test system: No

DEMONSTRATION OF TECHNICAL PROFICIENCY: Benzalkonium Chloride (BAC) was sued as positive control and provided informations about suitability of the test system

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: Yes
- Acceptance criteria met for positive control: Yes
- Range of historical values if different from the ones specified in the test guideline: Not specified

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met

Conclusions:

Under experimental conditions of the study, the registered substance 4-amino-m-cresol did not cause corneal effects other than very slight corneal swelling (mean swelling 1%). Microscopic examination of the corneas did not reveal any abnormalities. The 4-amino-m-Cresol was not classified as irritating for the eye according to CLP regulation.

Executive summary:

This GLP-compliant study was performed to assess the potential irritation/corrosion property of the registered substance 4 -amino-m-Cresol in a Isolated Chicken Eye test (ICE test) according to OECD guideline 438 method.

4-Amino-M-Cresol (A074) was evaluated neat for eye irritation potential in the Isolated Chicken Eye (ICE) test. In addition, the test included a negative control (saline) and a positive control (BAC). Chicken eyes were obtained from slaughter animals used for human consumption. The isolated chicken eyes were exposed to a single application of 30 mg for 10 seconds followed by a 20 mL saline rinse. Three main parameters were measured to disclose possible adverse eye effects: corneal thickness (expressed as corneal swelling), corneal opacity and fluorescein retention of damaged epithelial cells. In addition, histopathology of the corneas was performed.

4-Amino-M-Cresol (A074) did not cause corneal effects other than very slight corneal swelling (mean swelling 1%).  Microscopic examination of the corneas did not reveal any abnormalities.

Under experimental conditions of the study, the registered substance 4-amino-m-cresol did not cause corneal effects other than very slight corneal swelling (mean swelling 1%).  Microscopic examination of the corneas did not reveal any abnormalities. The 4-amino-m-Cresol was not classified as irritating for the eye according to CLP regulation.