4,4'-bis(chloromethyl)-1,1'-biphenyl

Eye irritation

Administrative data

Endpoint:

eye irritation: in vitro / ex vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 March 2016 to 07 March 2016

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 438 (Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test material

Test material information

1

Reference substance name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

EC Number:

216-784-4

EC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Cas Number:

1667-10-3

Molecular formula:

C14H12Cl2

IUPAC Name:

4,4'-bis(chloromethyl)-1,1'-biphenyl

Test material form:

solid: particulate/powder

Details on test material:

- Pysical description: White crystalline powder
- Storage conditions: Controlled Room Temperature (15-25oC), protected from humidity, under inert gas.

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source: Provided by sponsor
- Batch/Lot Number: 150701
- Expiration date of the lot/batch: 14 July 2017

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25 ˚C), protected from humidity, under inert gas

Test animals / tissue source

Species:

chicken

Strain:

other: ROSS 308

Details on test animals or tissues and environmental conditions:

SOURCE OF COLLECTED EYES
- Number of animals: Three test item treated eyes, three positive control treated eyes and one negative control eye were examined during the study.
- Characteristics of donor animals: Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption.
- Storage, temperature and transport conditions of ocular tissue (e.g. transport time, transport media and temperature, and other conditions): Heads were collected by a slaughter house technician and heads transported to CiToxLAB Hungary Ltd. at ambient temperature at the earliest convenience.
After collection, the heads were inspected for appropriate quality and wrapped with tissue paper moistened with saline, then placed in a plastic box which was closed (4-5 heads per box). The heads were received at CiToxLAB Hungary Ltd. and processed within approximately 2 hours of collection.
- indication of any existing defects or lesions in ocular tissue samples: One small drop of 2 % (w/v) fluorescein solution was applied onto the cornea surface for a few seconds and subsequently rinsed off with 20 mL physiological saline. Then the fluorescein-treated cornea was examined with a hand-held slit lamp or slit lamp microscope, with the eye in the head, to ensure that the cornea was not damaged.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

yes, concurrent positive control

yes, concurrent negative control

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 30 mg of the powdered test item.

VEHICLE
The test item was applied as supplied. No formulation was required; although the test item was powdered.

Duration of treatment / exposure:

10 seconds from the end of the application to the cornea surface.

Duration of post- treatment incubation (in vitro):

The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

Number of animals or in vitro replicates:

3 corneas were exposed to the test material. Three positive control treated eyes and one negative control eye were examined during the study.

Details on study design:

SELECTION AND PREPARATION OF ISOLATED EYES
After removing the head from the plastic box, it was put on soft paper. The eyelids were carefully cut away with scissors, avoiding damaging the cornea. Fluorescein was used to ensure that the cornea was not damaged. If the cornea was in good condition, the eyeball was carefully removed from the orbit.
Preparation of eyes
The eye ball was carefully removed from the orbit by holding the nictitating membrane with a surgical forceps, while cutting the eye muscles with bent scissors. Care was taken to remove the eyeball from the orbit without cutting off the optical nerve too short. The procedure avoided pressure on the eye while removing the eyeball from the orbit, in order to prevent distortion of the cornea and subsequent corneal opacity. Once removed from the orbit, the eye was placed onto damp paper and the nictitating membrane was cut away with other connective tissue. The prepared eyes were kept on the wet papers in a closed box so that the appropriate humidity was maintained.
The prepared eye was placed in a steel clamp with the cornea positioned vertically with the eye in the correct relative position (same position as in the chicken head). Again avoid too much pressure on the eye by the clamp. Because of the relatively firm sclera of the chicken eyeball, only slight pressure was needed to fix the eye properly. The clamp with the eyeball was 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 physiological saline solution dripping from a stainless steel tube, at a rate of approximately 3-4 drops/minute or 0.1 to 0.15 mL/minutes. The door of the chamber was closed except for manipulations and examinations, to maintain temperature and humidity.
The appropriate number of eyes was selected and after being placed in the superfusion apparatus. There they were examined again with the slit lamp microscope to ensure that they were in good condition. The focus was adjusted to see clearly the physiological saline which was flowing on the cornea surface. Eyes with a high baseline fluorescein staining (i.e., > 0.5) or corneal opacity score (i.e., > 0.5) were rejected. The cornea thickness was measured, any eye with cornea thickness deviating more than 10 % from the mean value for all eyes, or eyes that showed any other signs of damage, were rejected and replaced.

EQUILIBRATION AND BASELINE RECORDINGS
If the selected eyes were appropriate for the test, acclimatization started and it was conducted for approximately 45 to 60 minutes. The chambers of the superfusion apparatus were at controlled temperature (32±1.5 °C) during the acclimatization and treatment periods.
The baseline assessments
At the end of the acclimatization period, a zero reference measurement was recorded for cornea thickness and opacity to serve as a baseline (t=0) for each individual eye. The cornea thickness of the eyes should not change by more than 5 % within the -45 min and the zero time. No changes in thickness (0.0 %) were observed in the eyes. Following the equilibration period, the fluorescein retention was measured. Baseline values were required to evaluate any potential test item related effect after treatment. All eyes were considered to be suitable for the assay.

NUMBER OF REPLICATES
Three

NEGATIVE CONTROL USED
30 µL physiological saline (0.9 % (w/v) NaCl).

SOLVENT CONTROL USED (if applicable)
Not applicable. The test item was applied as supplied. No formulation was required; although the test item was powdered.

POSITIVE CONTROL USED
30 mg of powdered Imidazole.

APPLICATION DOSE AND EXPOSURE TIME
After the zero reference measurements, the eye in its retainer was taken out of the chamber and placed on a layer of tissue with the cornea facing upwards. The eye was held in horizontal position, while the test item was applied onto the centre of the cornea. For treatment, 30 mg of the powdered test item was applied onto the entire surface of the cornea attempting to cover the cornea surface uniformly with the test item, taking care not to damage or touch the cornea.
After an exposure period of 10 seconds from the end of the application the cornea surface was rinsed thoroughly.

OBSERVATION PERIOD
The control eyes and test eyes were evaluated pre-treatment and at approximately 30, 75, 120, 180 and 240 minutes after the post-treatment rinse. Minor variations within approximately ±5 minutes were considered acceptable.

REMOVAL OF TEST SUBSTANCE
- Volume and washing procedure after exposure period:
10 seconds from the end of the application the cornea surface was rinsed thoroughly with 20 mL physiological saline solution at ambient temperature, taking care not to damage the cornea but attempting to remove all residual test item if possible.
Additional gentle rinsing with 20 mL saline was performed at each time point when the control material remaining on the cornea was observed.

METHODS FOR MEASURED ENDPOINTS:
- Corneal opacity: Corneal thickness and corneal opacity were measured at all time points. Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse. Haag-Streit Bern 900 slit-lamp microscope was used for the measurements.
- Damage to epithelium based on fluorescein retention: Fluorescein retention was measured on two occasions, at baseline (t=0) and approximately 30 minutes after the post-treatment rinse
- Swelling: measured with optical pachymeter on a slit-lamp microscope; slit-width setting: Corneal thickness was measured at all time points using a Haag-Streit Bern 900 slit-lamp microscope

SCORING SYSTEM:
- Mean corneal swelling (%)
Corneal swelling was calculated according to the following formulae:

CS at time t =( (CT at time t-CT at t=0) / (CT at t = 0)) x 100
Mean CS at time t = (FECS(at time t) + SECS(at time t) + TECS(at time t)) / 3

Where:
CS = cornea swelling
CT = cornea thickness
FECS(at time t) = first eye cornea swelling at a given time-point
SECS(at time t) = second eye cornea swelling at a given time-point
TECS(at time t) = third eye cornea swelling at a given time-point
Small negative numbers for swelling (0 to - 5 %) following application are evaluated as class I. Large negative numbers (>12 % below control) are probably due to erosion and indicate a severe effect (scored as class IV). Cases of values of -5 % to -12 % are evaluated on a case by case basis but in the absence of other findings do not indicate a severe effect (class II).

- Mean maximum opacity score
Cornea opacity was calculated according to the following formulae:

ΔCO at time t = CO at time t – CO at t = 0

Mean ΔCOmax = (FECO(30 min to 240 min) + SECO(30 min to 240 min) + TECO(30 min to 240 min)) / 3

Where:
CO at time t = cornea opacity at (30, 75, 120, 180 and 240) minutes after the post-treatment rinse
CO at t=0 = baseline cornea opacity
ΔCO at time t = difference between cornea opacity at t time and cornea opacity baseline
FECO = first eye cornea opacity
SECO = second eye cornea opacity
TECO= third eye cornea opacity
max(30min to 240min) = maximum opacity of the individual eye at 30 to 240 minutes minus baseline cornea opacity of the individual eye

- Mean fluorescein retention score at 30 minutes post-treatment
Fluorescein retention was calculated according to the following formulae:

ΔFR at time t = FR at time t –FR at t = 0
Mean FRΔ = (FEFR(30 min) + SEFR(30 min) + TEFR(30 min)) / 3

Where:
FR at time t = fluorescein retention at 30 minutes after the post-treatment rinse
FR at t=0 = baseline fluorescein retention
ΔFR at time t = difference between fluorescein retention at t time and fluorescein retention baseline
FEFR = first eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
SEFR = second eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention
TEFR = third eye fluorescein retention at 30 minutes after the post-treatment rinse minus baseline fluorescein retention

DECISION CRITERIA: The conclusion on eye irritancy was based on the OECD guideline quantitative assessments.

Results and discussion

In vitro

Results

Results 1

Irritation parameter:

cornea opacity score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Remarks:

It is concluded that further information is required for classification.

Results 2

Irritation parameter:

fluorescein retention score

Run / experiment:

Mean

Value:

0

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Remarks:

It is concluded that further information is required for classification.

Results 3

Irritation parameter:

percent corneal swelling

Remarks:

at up to 240 min

Run / experiment:

Mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: -1.6 % It is concluded that further information is required for classification.

Remarks:

Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

Results 4

Irritation parameter:

other: Overall ICE Class

Run / experiment:

Mean

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 3xI

Other effects / acceptance of results:

OTHER EFFECTS:
Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Based on these in vitro eye irritation assays in isolated chicken eyes, the test item is not classified as a severe irritant and not classified as non-irritant. It is concluded that further information is required for classification.

ACCEPTANCE OF RESULTS:
- Acceptance criteria met for negative control: The negative control, physiological saline, was classified as non-irritating.
- Acceptance criteria met for positive control: The positive control, Imidazole, was classified as severely irritating; UN GHS Classification: Category 1.

Any other information on results incl. tables

Validity of the test

The results from all eyes used met the quality control standards. The negative control and positive control results were within the historical data range in experiment. This experiment was considered to be valid.

Test item

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	-0.5 %	I
Mean maximum corneal swelling at up to 240 min	-1.6 %	I
Mean maximum corneal opacity	0.00	I
Mean fluorescein retention	0.0	I
Other Observations	Test item was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	3xI

The mean maximum corneal swelling up to 240 min, the mean maximum corneal opacity and the mean fluorescein retention ICE classes are used for classification.

 

Positive control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	8.1 %	II
Mean maximum corneal swelling at up to 240 min	25.8 %	III
Mean maximum corneal opacity	4.00	IV
Mean fluorescein retention	3.00	IV
Other Observations	Imidazole was stuck on all cornea surfaces after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.
Overall ICE Class	1xIII; 2xIV

The positive control (Imidazole) was classified as severely irritating, UN GHS Classification: Category 1.

 

Negative control

Observation	Value	ICE Class
Mean maximum corneal swelling at up to 75 min	0.0 %	I
Mean maximum corneal swelling at up to 240 min	0.0 %	I
Mean maximum corneal opacity	0.0 %	I
Mean fluorescein retention	0.0 %	I
Other Observations	None
Overall ICE Class	3 x I

The negative control Physiological saline was classified as non-irritating, UN GHS Classification: Non-classified.

 

Historical Control data (on 24 September 2015): Physiological saline

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-3.2 %	3.4 %
Maximum corneal swelling at up to 240 min	-4.8 %	3.4 %
Maximum corneal opacity change	0.00	0.50
Fluorescein retention	0.00	0.50
Number of studies	200

 

Historical Control data (on 24 September 2015): Imidazole

Observation	Min. Value	Max. Value
Maximum corneal swelling at up to 75 min	-6.6 %	25.0 %
Maximum corneal swelling at up to 240 min	-15.9 %	35.9 %
Maximum corneal opacity change	3.50	4.00
Fluorescein retention	2.00	3.00
Number of studies	96

Applicant's summary and conclusion

Interpretation of results:

study cannot be used for classification

Conclusions:

Under the conditions of the study, the test material is not considered an irritant. Further information is required for classification.

Executive summary:

The eye irritancy potential of the test material was investigated in vitro in a study conducted in accordance with the standardised guidelines OECD 438 and EU Method B.48 under GLP conditions.

30 mg powdered test material was applied onto the centre of the cornea in such a way that the entire surface of the cornea was covered. After 10 seconds, the surface was rinsed with physiological saline. Positive control eyes were treated with 30 mg powdered Imidazole. The negative control eye was treated with 30 μL of physiological saline (0.9 % (w/v) NaCl solution). In the study, three test material treated eyes, three positive control treated eyes and one negative control treated eye were examined. The results from all eyes used in the study met the quality control standards. The negative control and positive control results were within the historical control data range in experiment. Thus, the experiment was considered to be valid. No significant corneal swelling and no corneal opacity change were observed during the four hour observation period. No fluorescein retention change was noted on all three eyes. However, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse. This fact might indicate morphological changes in an in vivo system (although during in vivo conditions the eyelids will probably clear the surface, but abrasion may occur).

Under the conditions of the study, the test material is not considered an irritant. However, It is concluded that further information is required for classification.