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:
2012-07-18 to 2012-10-26
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

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

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
Details on test material:
Name: (Tetrapropylensuccinimido)-caproic acid
Product (common name/code): Hostacor 4221
Batch No.: 12-YS032
Chemical Name: (Tetrapropylensuccinimido)-capronic acid
Purity (qualitative and
quantitative): 100%
Physical State at RT: liquid
Active Components (%): 98%
Colour: brown
Expiry Date: 17.04.2014
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: female
Body weight at the beginning of the study: > 2 kg
Age at the beginning of the study: approximately 14 - 21 weeks old
Number of animals: 2
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.

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 to autoclaved hay and to Altromin 2123 maintenance diet for rabbits (lot no. 1530), 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 at BSL BIOSERVICE
- Housed in ABS - plastic 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 treated eye was not rinsed 24 hours after the application.
Observation period (in vivo):
The animals were observed for 72 hours after dosing. To determine the reversibility of the observed effects, the observation period
was extended up to 18 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 did not indicate the test item to be corrosive or a severe irritant to the eye using the procedure described.
In order to confirm the response, an additional animal was treated in the same way.
Details on study design:
Preparation of the Animals
Approximately 24 hours before the test and immediately prior to the application both eyes of each animal were examined.
A health inspection was performed to ensure the good state of health of the animals.
Approximately 24 hours before the application the eyes were also examined with the aid of a fluorescein solution
(Fluoreszein SE Thilo, Laboratories Alcon, lot no. H 002, expiry date: 08/2012 (animal no.1) / H 201, expiry date: 12/2013 (animal no.2)).
The eyes were rinsed with physiological saline 0.9% NaCl (AlleMan Pharma, lot no. 111214, expiry date: 11/2014) after the examination.
None of the animals showed 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.: 5230, expiry date: 02/2016)
was administered subcutaneously in order to achieve a therapeutic level of systemic analgesia. Approximately 5 minutes prior to the
application of the test item, 2-3 drops of an ocular anaesthetic (proparacaine hydrochloride ophtalmic 0.5% solution, Ursapharm
Arzneimittel, lot no.: 273851, expiry date: 05/2013) were administered in both the treated and the control eye of each animal.
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 not rinsed 24 hours after the application.
Animals showing pain or distress after the application of the test item were treated with the following doses of buprenorphine:
Animal No. Dose Administration
1 1 ampulla (0.3 mg) of buprenorphine via 600 mL drinking water
2 1 ampulla (0.3 mg) of buprenorphine via 600 mL drinking water
Based on an average daily water intake from 15 to 50 mL/kg body weight, buprenorphine was administered at a dose range from 0.0075 to 0.025 mg/kg body weight, diluted in water.

Clinical Observation
The eyes were examined for signs of irritation throughout the observation period. The eye irritation was scored and recorded according
to the grades in the table below.
For the calculation only the 24, 48 and 72-hour readings were used.
72 hours post-application as well as at the end of the observation period the treated eyes were examined with the aid of a fluorescein solution
(Fluoreszein SE Thilo, Laboratories Alcon, lot no 420361, expiry date: 12/2013). The eyes were rinsed with physiological saline 0.9% NaCl
after the examination.

Evaluation of Results
Individual reactions of each animal were recorded at each time of observation.
Nature, severity and duration of all lesions observed were described.
For the calculation only the 24, 48 and 72-hour readings were used.
On the basis of the test results, the test substance was classified in any of the following classes in conformity with the criteria
given in Annex VI to Commission Directive 2001/59/EC:
R36 Irritating to eyes
- Substances and preparations which, when applied to the eye of the animal, cause significant ocular lesions which occur within 72 hours
after exposure and which persists for at least 24 hours.
Ocular lesions are significant if the mean scores of the eye irritation test cited in Annex B.5 to Commission Regulation (EC)
No. 440/2008, L 142 have any of the following values:
- corneal opacity ≥ 2 but < 3,
- iris lesion ≥ 1 but ≤ 1.5,
- redness of the conjunctiva ≥ 2.5,
- oedema of the conjunctiva (chemosis) ≥ 2,
or, in case where Annex B.5 test have been completed using three animals if the lesions, on two or more animals,
are equivalent to any of the above values except that for iris lesion the value should be ≥ 1 but < 2 and for redness of
the conjunctiva the value should be ≥ 2.5.
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the
respective mean values.
- Substances or preparations which cause significant ocular lesions, based on practical experience in humans.
- Organic peroxides except where evidence to the contrary is available.
R41 Risk of serious damage to eyes
Substances and preparations which, when applied to the eye of the animal cause severe ocular lesions which occur within 72 hours
after exposure and which persist for at least 24 hours.
Ocular lesions are severe if the means of the scores of the eye irritation test cited in Annex B.5 to Commission Regulation (EC)
No. 440/2008, L 142 have any of the values:
- cornea opacity ≥ 3,
- iris lesion > 1.5.
The same shall be the case where the test has been completed using three animals if these lesions, on two or more animals,
have any of the values:
- cornea opacity ≥ 3,
- iris lesion = 2.
In both cases all scores at each of the reading times (24, 48 and 72 hours) for an effect should be used in calculating the respective mean values.
Ocular lesions are also severe when they are still present at the end of the time of observation.
Ocular lesions are also severe if the substance or preparation causes irreversible colouration of the eyes.
-Substances and preparations which cause severe ocular lesions, based on practical experience in humans.
Note:
When a substance or preparation is classified as corrosive and assigned R34 or R35, the risk of severe damage to eyes is considered implicit
and R41 is not included in the label.
On the basis of the test results, the following risk phrases shall be assigned in conformity with the criteria
given in Annex I of Regulation (EC) 1272/2008:
Eye irritant Category 1:
Irreversible effects on the eyes / serious damage to the eyes
An eye irritant category 1 is a test material that produces:
- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or that have not fully reversed
within 21 days; and/or
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 3; and/or
(ii) iritis > 1.5
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material.
Eye irritant Category 2:
Reversible effects on the eyes/Irritating to the eyes
An eye irritant category 2 is a test material that produces:
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 1; and/or
(ii) iritis ≥ 1; and/or
(iii) conjunctival redness ≥ 2; and/or
(iv) conjunctival oedema (chemosis) ≥ 2
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material, and which fully
reverse within an observation period of 21 days.
On the basis of the test results, the following risk phrases may be assigned in conformity with the criteria given in GHS -
Globally Harmonized System of Classification and Labelling of Chemicals, third revised edition, July 2009:
Category 1: An eye irritant Category 1 (irreversible effects on the eye) is a test material that produces:
- at least in one animal effects on the cornea, iris or conjunctiva that are not expected to reverse or that have not fully reversed
within 21 days; and/or
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 3; and/or
(ii) iritis > 1.5
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material.
Category 2A: An eye irritant Category 2A (irritating to eyes) is a test material that produces:
- at least in 2 of 3 animals, a positive response of
(i) corneal opacity ≥ 1; and/or
(ii) iritis ≥ 1; and/or
(iii) conjunctival redness ≥ 2; and/or
(iv) conjunctival oedema (chemosis) > 2
calculated as the mean scores following the grading at 24, 48 and 72 hours after instillation of the test material, and which fully
reverse within 21 days.
Category 2B: Within this category an eye irritant is considered mildly irritating to eyes (Category 2B), when the effects listed above
are fully reversible within 7 days of observation.

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
1.8
Max. score:
2
Reversibility:
fully reversible within: 18 days
Irritation parameter:
conjunctivae score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
2.33
Max. score:
3
Reversibility:
fully reversible within: 18 days
Irritation parameter:
chemosis score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
2.5
Max. score:
4
Reversibility:
fully reversible within: 18 days
Irritation parameter:
iris score
Basis:
mean
Time point:
other: 24, 48 and 72 hours
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 18 days
Irritant / corrosive response data:
After the application into the eyes of three female NZW rabbits the test item produced irritant but not corrosive effects in all animals.
Neither mortalities nor significant clinical signs of toxicity were observed.
The eyes were not rinsed after the application.
Upon fluorescein examinations at the end of the observation period of 72 hours corneal lesions (approximately 50% of the area) were
found in one animal.
At the end of the prolonged observation period no corneal lesions were found in any animal.
Conjunctival redness, chemosis, discharge, corneal effects and iris lesion were observed in all animals.
Other effects:
There were no significant body weight changes during the observation period.

Any other information on results incl. tables

Table  Eye Irritation Scores – Animal No. 1

Animal No. 1

Single Data

 

Time Post-Application

Average Score
(24, 48 and 72 hours)

1 hour

24 hours

48 hours

72 hours

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

3/0

3/0

2.67

Conjunctival Chemosis

3/0

2/0

2/0

2/0

2

Iris

1/0

1/0

1/0

1/0

1

Cornea

1/0

2/0

2/0

1/0

1.67

T =testitem,     C = control


 

Animal No. 1

Single Data - Prolonged Observation Period

 

Time Post-Application

4 days

5 days

6 days

7 days

8 days

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

3/0

n.a.

2/0

1/0

0/0

Conjunctival Chemosis

2/0

n.a.

1/0

1/0

1/0

Iris

1/0

n.a.

1/0

1/0

1/0

Cornea

1/0

n.a.

1/0

1/0

0/0

 

9 days

10 days

11 days

12 days

13 days

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

0/0

1/0

1/0

0/0

0/0

Conjunctival Chemosis

1/0

1/0

1/0

1/0

1/0

Iris

0/0

0/0

0/0

0/0

0/0

Cornea

0/0

0/0

0/0

0/0

0/0

 

14 days

15 days

16 days

17 days

18 days

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

0/0

0/0

0/0

0/0

0/0

Conjunctival Chemosis

1/0

1/0

1/0

1/0

0/0

Iris

0/0

0/0

0/0

0/0

0/0

Cornea

0/0

0/0

0/0

0/0

0/0

                                    T =testitem,     C = control       n.a. = no data available


 

Table:  Eye Irritation Scores – Animal No. 2

Animal No. 2

Single Data

 

Time Post-Application

Average Score
(24, 48 and 72 hours)

1 hour

24 hours

48 hours

72 hours

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

2/0

2/0

2

Conjunctival Chemosis

4/0

4/0

3/0

2/0

3

Iris

2/0

1/0

1/0

1/0

1

Cornea

2/0

2/0

2/0

2/0

2

T =testitem,     C = control

Animal No. 2

Single Data - Prolonged Observation Period

 

Time Post-Application

4 d

5 d

6 d

7 d

8 d

9 d

T/C

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

2/0

2/0

2/0

1/0

1/0

1/0

Conjunctival Chemosis

1/0

1/0

1/0

1/0

0/0

0/0

Iris

1/0

1/0

0/0

0/0

0/0

0/0

Cornea

1/0

1/0

1/0

0/0

0/0

0/0

 

10 d

11 d

12 d

13d

14 d

15 d

T/C

T/C

T/C

T/C

T/C

T/C

Conjunctival Redness

1/0

1/0

1/0

1/0

1/0

0/0

Conjunctival Chemosis

0/0

0/0

0/0

0/0

0/0

0/0

Iris

0/0

0/0

0/0

0/0

0/0

0/0

Cornea

0/0

0/0

0/0

0/0

0/0

0/0

T =testitem,     C = control


 

Table:  Clinical Signs of Toxicity

Animal No. 1

Time after
Test Item Application

Systemic Findings

Local Findings
(apart from findings in Table 3)

1 hour

nsf

discharge grade 3

24 hours

nsf

discharge grade 2

48 hours

nsf

discharge grade 1

72 hours

nsf

discharge grade 1

day 4

nsf

discharge grade 1

day 5

n.a.

n.a.

day 6

nsf

discharge grade 1

day 7

nsf

nsf

day 8

nsf

nsf

day 9

nsf

nsf

day 10

nsf

discharge grade 1

day 11

nsf

nsf

day 12

nsf

nsf

day 13

nsf

nsf

day 14

nsf

discharge grade 1

day 15

nsf

discharge grade 1

day 16

nsf

nsf

day 17

nsf

nsf

day 18

nsf

nsf

nsf: no specific findings;n.a. = no data available


 

Table 5:  Clinical Signs of Toxicity (Cont.)

Animal No. 2

Time after
Test Item Application

Systemic Findings

Local Findings
(apart from findings in Table 4)

1 hour

nsf

discharge grade 3

24 hours

nsf

discharge grade 2

48 hours

nsf

discharge grade 2

72 hours

nsf

discharge grade 1

day 4

nsf

discharge grade 1

day 5

nsf

nsf

day 6

nsf

nsf

day 7

nsf

nsf

day 8

nsf

nsf

day 9

nsf

nsf

day 10

nsf

nsf

day 11

nsf

nsf

day 12

nsf

nsf

day 13

nsf

nsf

day 14

nsf

nsf

day 15

nsf

nsf

nsf: no specific findings

Table:  Absolute Body Weights in kg

 

Animal No. 1

Animal No. 2

Start of Study
(weight in kg)

3.0

3.9

72 Hours Post-Application
(weight in kg)

3.1

3.9

End of Observation Period
(weight in kg)

3.4

4.2

Applicant's summary and conclusion

Interpretation of results:
irritating
Remarks:
Migrated information Criteria used for interpretation of results: EU
Conclusions:
The registration substance was investigated for its eye irritation potential according to OECD 405. The substance should be classified as Xi, R36 according to DSD 67/548/EEC.
Executive summary:

The registration substance was investigated for its eye irritation potential according to OECD 405. The substance should be classified as Xi, R36 according to DSD 67/548/EEC.