Registration Dossier

Administrative data

Endpoint:
eye irritation: in vitro / ex vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
03 November 2017 - 09 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report Date:
2018

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
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:
OECD Guidelines for Guidelines for Testing of Chemicals No.438 (09 October 2017) “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”
Deviations:
no
Qualifier:
according to
Guideline:
EU method B.48 (Isolated chicken eye test method for identifying occular corrosives and severe irritants)
Version / remarks:
Commission Regulation (EU) 2017/735 of 14 February 2017 amending, for the purpose of its adaptation to technical progress, the Annex to Regulation (EC) No 440/2008 method B.48 laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH) “Isolated Chicken Eye Test Method for Identifying Ocular Corrosives and Severe Irritants”
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Details on test material:
Chemical name: α,α’-Dichloro-p-xylene
CAS number: 623-25-6
Batch/Lot Number: EWP6O
Description: White-Pale yellow green, Solid
Purity*: >98.0% (GC)
Expiry date: 17 August 2018 / 30 October 2019
Storage conditions: Controlled room temperature (15-25 °C, ≤ 70 RH%), protected from humidity (tight closed container), protected from light
Safety precautions: Enhanced safety precautions were applied considering the supplied safety data sheet to assure personnel health and safety. Fatal if inhaled!
Specific details on test material used for the study:
The test item of a suitable chemical purity was supplied by the Sponsor. All precautions required in the handling and disposal of the test item were outlined by the Sponsor. These documents are part of the raw data. Test item was identified on the basis of the information provided by the Sponsor by the Pharmacy of Citoxlab Hungary Ltd.

Test item solubility
The solubility of the test item in physiological saline was tested prior to the experiment (30 mg test material in 1 mL physiological saline). The test item did not dissolve in physiological saline.

Formulation
The test item was applied in its original form, although it was powdered.

Test animals / tissue source

Species:
chicken
Strain:
other:
Remarks:
COBB 500
Details on test animals or tissues and environmental conditions:
Chicken heads were collected after slaughter in a commercial abattoir from chickens (approximately 7 weeks old) which are used for human consumption. 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 2 hours of collection in each experiment.

Eyes selection
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. 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 fluoresceintreated 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. 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

Eyes examination and acclimatization time
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.

Test system

Vehicle:
unchanged (no vehicle)
Controls:
yes, concurrent positive control
yes, concurrent negative control
Amount / concentration applied:
The test item was applied in its original form, although it was powdered.
Duration of treatment / exposure:
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. Slight corneal thickness changes (0.0 - 1.6 %) were observed in the eyes, this is considered normal when maintaining enucleated 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.

Treatment
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 material was applied onto the centre of the cornea. In each experiment, 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. In each experiment negative control eye was treated with 30 μL of physiological saline; positive control eyes were treated with 30 mg powdered imidazole. One eye was treated with physiological saline, three eyes with the test item and another three eyes with powdered imidazole in each experiment.
Observation period (in vivo):
Observation and assessment of corneal effects 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.
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 BP 900® slit-lamp microscope was used for the measurements.

Morphological effects
Morphological effects 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 according to the interpretation of the investigator.
Duration of post- treatment incubation (in vitro):
Test item removal
The time of application was noted, then after an exposure period of 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 material if possible. Additional gentle rinsing with 20 mL saline was performed after treatment and at each time point when the test item or positive control material remaining on the cornea was observed in each experiment.
Number of animals or in vitro replicates:
The appropriate number of eyes was selected and after being placed in the superfusion apparatus, 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. 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.
Details on study design:
The Enucleated Eye Test with isolated eyes of chickens is a well validated and accepted in vitro test system. It has been recognised as a valuable alternative to the Draize eye irritation test, because it represents a test system nearest to the in vivo test, without the need to use live animals. It can also be used as a screening tool for corrosivity/severe irritancy to avoid unacceptable effects in vivo. In the Isolated Chicken Eye Test (ICET) the test compound is applied in one single dose onto the cornea of isolated eyes. Chicken heads are obtained from a veterinary-inspected, commercial slaughter-house, processing chickens for human consumption. This method can provide detailed information about the effects of test items on the cornea, and can be used to identify chemicals not requiring classification for eye irritation, or for serious eye damage and chemicals inducing serious eye damage as defined by the UN GHS (UN GHS non-classified or UN GHS Category 1). The test is described in OECD No. 438 and is approved by international regulatory agencies as a replacement for the identification of non-irritant, corrosives/severe irritants in the in vivo Rabbit Eye Assay (OECD No. 405).

Results and discussion

In vitro

Resultsopen allclose all
Irritation parameter:
morphological effects
Value:
>= 1.1 - <= 1.6
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
cornea opacity score
Value:
>= 0 - <= 0
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Irritation parameter:
fluorescein retention score
Value:
>= 0 - <= 0.17
Vehicle controls validity:
not applicable
Negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
no indication of irritation
Other effects / acceptance of results:
The test item α,α’-Dichloro-p-xylene showed no significant corneal effect in the first experiment. As the test item was solid, the negative results were confirmed by a second experiment according to the recommendations of the OECD No. 438 guideline. The second experiment confirmed the negative results. Therefore, based on these in vitro eye irritation tests in isolated chicken eyes with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.

MORPHOLOGICAL EFFECTS RESULTS
The positive control material was stuck on all cornea surfaces after the post-treatment rinse, the cornea surfaces were not cleared at 240 minutes after the post-treatment rinse in each experiment.
No other morphological effect was observed in the study.

Any other information on results incl. tables

TEST ITEM

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.1%

I

Mean maximum corneal swelling at up to 240 min

1.6%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.17

I

Other Observations

None

Overall ICE Class

3xI

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

1.1%

I

Mean maximum corneal swelling at up to 240 min

1.1%

I

Mean maximum corneal opacity

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

POSITIVE CONTROL

Experiment I

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

12.2%

III

Mean maximum corneal swelling at up to 240 min

28.2%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces at 30 minutes 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

 

Experiment II

Observation

Value

ICE Class

Mean maximum corneal swelling at up to 75 min

13.4%

III

Mean maximum corneal swelling at up to 240 min

27.3%

III

Mean maximum corneal opacity

4.00

IV

Mean fluorescein retention

3.00

IV

Other Observations

Imidazole was stuck on all cornea surfaces at 30 minutes 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

 

NEGATIVE CONTROL

Experiment I

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

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

Experiment II

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

0.00

I

Mean fluorescein retention

0.00

I

Other Observations

None

Overall ICE Class

3xI

 

TABLES OF INDIVIDUAL DATA

Table of individual data α,α’-Dichloro-p-xylene (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

11

61

61

0.0%

61

0.0%

61

0.0%

0.0%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

12

60

61

1.7%

61

0.0%

62

1.6%

1.6%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0.5

0.5

13

61

61

0.0%

61

0.0%

62

1.6%

1.6%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

Mean values:

0.0%

 

1.1%

1.1%

 

1.6%

 

1.6%

 

1.6%

1.6%

 

 

 

 

 

 

0.00

 

 

0.17

 

Table of individual data α,α’-Dichloro-p-xylene (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

11

63

63

0.0%

63

0.0%

3.

0.0%

0.0%

64

1.6%

64

1.6%

64

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

12

63

63

0.0%

63

0.0%

63

0.0%

0.0%

63

0.0%

63

0.0%

63

0.0%

0.0%

0

0

0

0

0

0

0.0

0.5

0.5

0.0

13

61

61

0.0%

61

0.0%

61

0.0%

0.0%

62

1.6%

62

1.6%

62

1.6%

1.6%

0

0

0

0

0

0

0.0

0

0

0.0

Mean values:

0.0%

 

0.0%

0.0%

 

1.1%

 

1.1%

 

1.1%

1.1%

 

 

 

 

 

 

0.00

 

 

0.00

 

Table of individual data (Imidazole) (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

14

60

60

0.0%

64

6.7%

67

11.7%

11.7%

70

16.7%

74

23.3%

78

30.0%

30.0%

0

4

4

4

4

4

4.0

0

3

3.0

15

61

61

0.0%

65

6.6%

68

11.5%

11.5%

70

14.8%

73

19.7%

77

26.2%

26.%

0

4

4

4

4

4

4.0

0

3

3.0

16

60

60

0.0%

65

8.3%

68

13.3%

13.3%

71

18.3%

74

23.3%

77

28.3%

28.3%

0

4

4

4

4

4

4.0

0

3

3.0

Mean values:

7.2%

 

12.2%

12.2%

 

16.9%

 

22.1%

 

28.2%

28.2%

 

 

 

 

 

 

4.00

 

 

3.00

Note: Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Table of individual data (Imidazole) (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

14

62

62

0.0%

66

6.5%

70

12.9%

12.9%

73

17.7%

76

22.6%

79

27.4%

27.4%

0

4

4

4

4

4

4.0

0

3

3.0

15

63

63

0.0%

68

7.9%

71

12.7%

12.7%

74

17.5%

77

22.2%

79

25.4%

25.4%

0

4

4

4

4

4

4.0

0

3

3.0

16

62

62

0.0%

68

9.7%

71

14.5%

14.5%

73

17.7%

77

24.2%

80

29.0%

29.0%

0

4

4

4

4

4

4.0

0

3

3.0

Mean values:

8.0%

 

13.4%

13.4%

 

17.6%

 

23.0%

 

27.3%

27.3

 

 

 

 

 

 

4.00

 

 

3.00

Note: Imidazole was stuck on all cornea surfaces at 30 minutes after the post-treatment rinse. The cornea surfaces (3/3) were not cleared at 240 minutes after the post-treatment rinse.

 

Table of individual data (Physiological saline) (Experiment I)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

17

62

62

0.0%

62

0.0%

62

0.0%

0.0%

62

0.0%

62

0.0%

62

0.0%

0.0%

0

0

0

0

0

0

0.00

0

0

0.00

 

Table of individual data (Physiological saline) (Experiment II)

Chamber number ↓

Corneal thickness (instrument units)

Corneal opacity score

Fluorescein retention

Relative observation time (min) →

-45

0

Change

30

Change at 30

75

Change at 75

Max change up to 75

120

Change at 120

180

Change at 180

240

Change at 240

Max change up to 240

0

30

75

120

180

240

Max ∆ Opac

0

30

∆ Flu ret

17

63

63

0.0%

63

0.0%

63

0.0%

0.0%

63

0.0%

63

0.0%

63

0.0%

0.0%

0

0

0

0

0

0

0.00

0

0

0.00

 

Applicant's summary and conclusion

Interpretation of results:
GHS criteria not met
Conclusions:
Based on these in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.
Executive summary:

SUMMARY

An in vitro eye irritation study of the test item was performed in isolated chicken’s eyes. The irritation effects of the test item were evaluated according to the OECD No. 438 (09 October 2017).

In each experiment after the zero reference measurements, the eye was held in horizontal position and 30 mg powdered test item 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 item treated eyes, three positive control treated eyes and one negative control treated eye were examined in each experiment. 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 each experiment. Thus, the study was considered to be valid.

Experiment I: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No significant fluorescein retention change (severity 0.5) was noted on one test item treated eye and no fluorescein retention change was noted on two test item treated eyes. No other corneal effect was observed.

Experiment II: No significant corneal swelling (mean ≤5%) was observed during the four-hour observation period on test item treated eyes. No corneal opacity change was noted on test item treated eyes. No fluorescein retention change was noted on test item treated eyes. No other corneal effect was observed.

Based on these in vitro eye irritation in the isolated chicken eyes tests with α,α’-Dichloro-p-xylene, the test item was non-irritant, UN GHS Classification: No Category.