Reaction mass of isomers disodium 8-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-5-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate, disodium 5-(2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl)-8-(2-{2,5-dimethyl-4-[2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl)naphthalene-2-sulfonate

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014-08-14 to 2014-11-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference 1

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2015

Reference 2

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.5 (Acute Toxicity: Eye Irritation / Corrosion)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Test material

Test material information

1

Reference substance name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

EC Number:

700-946-0

Molecular formula:

C40H35N7Na2O6S2

IUPAC Name:

tetrasodium 5-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-8-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate 8-[(1E)-2-{4-[benzyl(ethyl)amino]-2-methylphenyl}diazen-1-yl]-5-[(1E)-2-{2,5-dimethyl-4-[(1E)-2-(3-sulfonatophenyl)diazen-1-yl]phenyl}diazen-1-yl]naphthalene-2-sulfonate

Test material form:

solid: particulate/powder

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: 19 – 21 weeks old
Number of animals: 3
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. 0724), 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 or Noryl rabbit cages, floor 4200 cm2
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

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 16 - 22 hours before the application the eyes were also examined with the aid of a fluorescein solution
(Fluoreszein SE Thilo, Alcon Pharma, lot no. lot no. H 207, expiry date: 09/2014).
The eyes were rinsed with physiological saline 0.9% NaCl (Serumwerk Bernburg, lot no. B9144, expiry date: 09/2016) after the examination.
None of the animals showed eye irritation, ocular defects, or pre-existing corneal injury.

Test system

Vehicle:

unchanged (no vehicle)

Controls:

not required

Amount / concentration applied:

A dose of 0.1 g of the test item was applied to the test site.

Duration of treatment / exposure:

The treated eye was rinsed with physiological saline 0.9% NaCl 1 hour 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 4 days for animal no. 1, 9 days for animal no. 2 and 6 days for animal no. 3 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, two additional animals were treated in the same manner.

Details on study design:

Application
One hour before the application of the test item, 0.01 mg/kg of buprenorphine (Reckitt Benckiser, lot no. 5416, expiry date: 09/2016) 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. 282598, expiry date: 04/2016) 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 rinsed with physiological saline 0.9% NaCl 1 hour after the application.

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 prolonged observation period the treated eyes were examined with the aid of a fluorescein solution. The eyes were rinsed with physiological saline 0.9% NaCl after the examination.

Evaluation of Results
Individual reactions for each animal were recorded according to the scoring system described in 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.
With few exceptions, toxicology and pathology data were captured, using the validated departmental computerized system E-Workbook (version 9.4.0, ID Business Solutions Ltd).

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 4 days post-application

Results 2

Irritation parameter:

cornea opacity score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 9 days post-application

Results 3

Irritation parameter:

cornea opacity score

Basis:

animal #3

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 6 days post-application

Results 4

Irritation parameter:

iris score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 4 days post-application

Results 5

Irritation parameter:

iris score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 9 days post-application

Results 6

Irritation parameter:

iris score

Basis:

animal #3

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 6 days post-application

Results 7

Irritation parameter:

chemosis score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 4 days post-application

Results 8

Irritation parameter:

chemosis score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

0.33

Reversibility:

fully reversible within: 9 days post-application

Results 9

Irritation parameter:

chemosis score

Basis:

animal #3

Remarks:

male

Time point:

24/48/72 h

Score:

0

Reversibility:

fully reversible within: 6 days post-application

Results 10

Irritation parameter:

conjunctivae score

Basis:

animal #1

Remarks:

male

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 4 days post-application

Results 11

Irritation parameter:

conjunctivae score

Basis:

animal #2

Remarks:

male

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 9 days post-application

Results 12

Irritation parameter:

conjunctivae score

Basis:

animal #3

Remarks:

male

Time point:

24/48/72 h

Score:

1

Reversibility:

fully reversible within: 6 days post-application

Irritant / corrosive response data:

After the application into the eyes of three male NZW rabbits the test item produced slightly irritant but no corrosive ocular effects in all animals, which were fully reversible within 4 days in animal no. 1, within 9 days in animal no. 2 and within 6 days in animal no. 3 post application.
Neither mortalities nor significant clinical signs of toxicity were observed.
Upon fluorescein examinations at the end of the observation period of 72 hours and at the end of the prolonged observation period no corneal lesions were found in any animal. Conjunctival redness, chemosis, discharge were observed in all animals.

Other effects:

The body weight development of all animals was within the expected range.

Applicant's summary and conclusion

Interpretation of results:

other: not eye irritan according to the CLP Regulation (EC) No 1272/2008. 

Conclusions:

Under the conditions of the present study, a single ocular application of the test item to rabbits at a dose of 0.1 g produced slightly irritant effects, which were fully reversible within up to 9 days. Neither mortalities nor significant clinical signs of toxicity were observed.
In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI of Commission Directive 2001/59/EC, the test item has no obligatory labelling requirement for eye irritation.
According to Annex I of Regulation (EC) 1272/2008, the test item has no obligatory labelling requirement for eye irritation.
According to GHS (Globally Harmonized Classification System) the test item has no obligatory labelling requirement for eye irritation.

Executive summary:

Summary Results

On the basis of the test results given below and in conformity with the criteria given in Annex VI to Commission Directive 2001/59/ECsthe substance should be assigned the following risk phrase: no phrase.

On the basis of the test results given below and in conformity with the criteria given in Annex I of Regulation (EC) 1272/2008, the substance should be: not classified

On the basis of the test results given below and in conformity with the criteria given in GHS (Globally Harmonized System of Classification and Labelling of Chemicals), the substance should be: not classified

Species/strain: New Zealand White Rabbits Crl: KBL (NZW)

Number of animals: 3

Amount of substance: 0.1 g per test site

First time of effects: animals no. 1 and no. 3: 1 hour post-application redness grade 1, chemosis grade 1 and discharge grade 2

animal no. 2: 1 hour post-application redness grade 1, chemosis grade 2 and discharge grade 2

Last time of effects: animal no. 1: 72 hours post-application redness grade 1

animal no. 2: 8 days post-application redness grade 1

animal no. 3: 5 days post-application redness grade 1

Reversibility of the observed effects: animal no. 1: the changes were fully reversible within 4 days post-application

animal no. 2: the changes were fully reversible within 9 days post-application

animal no. 3: the changes were fully reversible within 6 days post-application

Method: OECD 405

EC 440/2008, Method B.4

OPPTS 870.2400

The calculated mean scores did not exceed the limit values according to Directive 2001/59/EC, to Regulation (EC) 1272/2008 and to GHS.

 

Conclusion

Under the conditions of the present study, a single ocular application of the test item to rabbits at a dose of 0.1 g produced slightly irritant effects, which were fully reversible within up to 9 days. Neither mortalities nor significant clinical signs of toxicity were observed.

In conformity with the EC criteria for classification and labelling requirements for dangerous substances and preparations according to Annex VI of Commission Directive 2001/59/EC, the test item has no obligatory labelling requirement for eye irritation.

According to Annex I of Regulation (EC) 1272/2008, the test item has no obligatory labelling requirement for eye irritation.

According to GHS (Globally Harmonized Classification System) the test item  has no obligatory labelling requirement for eye irritation.