Registration Dossier

Toxicological information

Eye irritation

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Administrative data

Endpoint:
eye irritation: in vivo
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-07-29 to 2017-02-06
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 405 (Acute Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.2400 (Acute Eye Irritation)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
Name: 1-Propanaminium, 3-amino-N-(carboxymethyl)-N,N-dimethyl-, N-C18(unsaturated) acyl derivs., hydroxides, inner salts in water,
2-propanol and 1,2-propanediol
Batch No.: DEG4297614
Aggregate State at RT: liquid
Colour: light brown
Storage Conditions: 2 – 8 °C, protected from light
Stability: stable
Active Components: 45.7% incl by-products
Expiry Date: August 2017
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals / tissue source

Species:
rabbit
Strain:
New Zealand White
Details on test animals or tissues and environmental conditions:
Test System
Species/strain: healthy New Zealand White Rabbits, Crl: KBL (NZW)
Source: Charles River Deutschland, 97633 Sulzfeld, Germany
Sex: male
Body weight at the beginning of the study: > 2 kg
Age at the beginning of the study: approximately 24 weeks old
Number of animals: 1
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes. 
This study was performed in an AAALAC-accredited laboratory. According to German animal protection law, the study type has been reviewed and accepted by local authorities. Furthermore, the study has been subjected to Ethical Review Process and was authorised by the Bavarian animal welfare administration.

Housing and Feeding Conditions
- Semi barrier in an air-conditioned room
- Temperature: 18 +/- 3 °C (recommendations of TVT, GV-SOLAS)
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: at least 10 x / hour
- Free access autoclaved hay and to Altromin 2123 maintenance diet for rabbits, rich in crude fibre
- Free access to tap water (drinking water, municipal residue control, microbiological controls at regular intervals)
- Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich
- Housed in ABS-plastic or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Test system

Vehicle:
unchanged (no vehicle)
Controls:
not required
Amount / concentration applied:
A dose of 0.1 mL of the test item was applied to the test site.
Duration of treatment / exposure:
The test item was applied at a single dose in the conjunctival sac of one eye of each test animal after pulling the lower lid away from the eyeball.
The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control.
The treated eye was rinsed with physiological saline 0.9% NaCl 24 hours after the application.

Observation period (in vivo):
The animal was observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period was extended up to 21 days after dosing.
Number of animals or in vitro replicates:
The in vivo test was performed initially using one animal.
The results of the initial test indicated the test item to be corrosive or a severe irritant to the eye using the procedure described. Therefore, no additional animals were treated due to animal welfare reasons.
Details on study design:
Preparation of the Animals
Approximately 24 hours before the test and immediately prior to the application both eyes of the animal were examined. A health inspection was performed to ensure the good state of health of the animal.
Approximately 24 hours before the application the eyes were also examined with the aid of a fluorescein solution (Fluoreszein SE Thilo, Alcon Pharma, lot no. H 503, expiry date: 04/2017). The eyes were rinsed with physiological saline 0.9% NaCl (B. Braun Melsungen, lot no. 1406797, expiry date: 05/2017) after the examination.
The animal did not show eye irritation, ocular defects, or pre-existing corneal injury.

Application
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no.: 5481, expiry date: 01/2018) was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approximately 5 minutes prior to the application of the test item, 1-2 drops of an ocular anaesthetic (proparacaine hydrochloride ophthalmic 0.5% solution, Ursapharm, lot no.: 286171, expiry date: 07/2017) were administered in both the treated and the control eye of the animal.
The test item was applied at a single dose in the conjunctival sac of one eye of the test animal after pulling the lower lid away from the eyeball. The lids were then gently held together for about 1 second in order to prevent loss of the material. The untreated contralateral eye served as control. 
The treated eye was rinsed with physiological saline 0.9% NaCl (see 10.5) 24 hours after the application.
To prevent pain and distress after the application of the test item the animal was treated with the following doses of buprenorphine and meloxicam (Boehringer Ingelheim, lot no.: H20812A-14, expiry date: 07/2018):
Time Analgesic Route and Dose of Administration Frequency on Medication Day
Post-Application
8 h (d 0) – d 6 Buprenorphine subcutaneous: 0.01 mg/kg bw twice
Meloxicam subcutaneous: 0.5 mg/kg bw twice
d 7, d 8, Meloxicam subcutaneous: 0.5 mg/kg bw once
d 15 and d 16
d 9 – d 14, no analgesic medication
d 16 – d 21
h = hour(s); d = day

Clinical Observation
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according to the Grading System for Ocular Lesion.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as daily during the prolonged observation period the treated eye was examined with the aid of a fluorescein solution. The eye was rinsed with physiological saline 0.9% NaCl after the examination.

Body Weight
The animals were weighed prior to the administration and at the end of the observation period.

Evaluation of Results
The animal’s reactions were recorded according to the scoring system described at each time of observation.
For the calculation only the 24, 48 and 72-hour readings were used.
Nature, severity and duration of clinical observations were described.
The body weight changes were summarised in a tabular form.

Results and discussion

In vivo

Results
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
>= 1 <=
Max. score:
1
Reversibility:
not fully reversible within: 21 days
Irritant / corrosive response data:
After the application into the eye of one male NZW rabbit the test item produced irritant and corrosive ocular effects.
Neither mortality nor significant clinical signs of toxicity but local effects were observed.
At the end of the prolonged observation period corneal lesions (with a diameter of approximately 0.2 cm) were found.
No systemic but local changes (slight hypersecretion, hypersecretion with moistening of the lids and surrounding hair, hypersecretion with wetting of the eyelids and hair around the eyes and clear flow secretion) were observed.

Other effects:
The body weight development of the animal was within the expected range.

Any other information on results incl. tables

Result: Eye Irritation Scores in the observation period of up to 72 hours

Observation

Eye Irritation Scores Post-Application after

Average Score (24,48,72 hours)

1 hour

24 hours

48 hours

72 hours

T

C

T

C

T

C

T

C

Redness

2

0

2

0

2

0

2

0

2.00

Chemosis

2

0

1

0

1

0

1

0

1.00

Iris

1

0

1

0

0

0

1

0

0.67

Cornea

1

0

1

0

2

0

1

0

1.00

T = test item; C = control

 

Result: Eye Irritation Scores in the observation period of day 4-12

Observation

Eye Irritation Scores Post-Application after

 

4d

5d

6d

7d

8d

9d

10d

11d

12d

 

T

C

T

C

T

C

T

C

T

C

T

C

T

C

T

C

T

C

Redness

2

0

2

0

1

0

0

0

0

0

0

0

0

0

0

0

1

0

Chemosis

1

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

1

0

Iris

1

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

0

0

Cornea

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

T = test item; C = control; d = day(s)

 

Result: Eye Irritation Scores in the observation period of day 13-21

Observation

Eye Irritation Scores Post-Application after

 

13d

14d

15d

16d

17d

18d

19d

20d

21d

 

T

C

T

C

T

C

T

C

T

C

T

C

T

C

T

C

T

C

Redness

1

0

1

0

1

0

0

0

0

0

0

0

0

0

0

0

1

0

Chemosis

1

0

0

0

1

0

0

0

0

0

0

0

0

0

0

0

0

0

Iris

0

0

0

0

1

0

0

0

0

0

0

0

0

0

1

0

0

0

Cornea

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

1

0

T = test item; C = control; d = day(s)

 

Result: Fluorescein Report

Timepoint

Findings at Fluorescein Examination

1 h – 48 h

-

72 h – 5 d

cornea opacification (ø approx. 0.5 cm)

6 d

cornea opacification (ø approx. 0.3 cm)

7 d – 8 d

cornea opacification (ø approx. 0.2 cm)

9 d – 10 d

slight cornea opacification (ø approx. 0.2 cm)

11 d

slight cornea opacification (ø approx. 0.5 cm)

12 d

cornea opacification (ø approx. 0.5 cm)

13 d

cornea opacification (ø approx. 0.3 cm)

14 d – 18 d

cornea opacification (ø approx. 0.2 cm)

19 d – 20 d

cornea opacification (ø approx. 0.25 cm)

21 d

cornea opacification (ø approx. 0.2 cm)

nsf = no specific findings; h = hour/s; d = days; ø = diameter

Applicant's summary and conclusion

Interpretation of results:
Category 1 (irreversible effects on the eye) based on GHS criteria
Conclusions:
The eye irritation property of the registration substance was investigated according to the Guideline OECD 405. One rabbit was treated with 0.1ml of the test item that corresponded to 45% of the registration substance in water. Based on the result obtained the registration substance should be classified as Cat 1 according to the GHS criteria.
Executive summary:

The eye irritation property of the registration substance was investigated according to the Guideline OECD 405. One rabbit was treated with 0.1ml of the test item that corresponded to 45% of the registration substance in water. Cornea opacity occured that was persistent during the observation time of 21 days. Based on the result obtained  the registration substance should be classified as Cat 1 according to the GHS criteria.