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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:
2009-02-19 - 2010-01-28
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP-Guideline Study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

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
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material: bis(2.6-diisopropylphenyl)carbodiimide
- Substance type: organic technical product
- Appearance: solid, crystalline waxy
- Lot/batch no.: 08100014
- Storage: room temperature
- Expiration date: 2009-05-11
-Other: Confirmation of the identity of the test item was performed. The test compound is not corrosive to the skin.

Test animals / tissue source

Species:
rabbit
Strain:
other: Crl:KBL(NZW)BR
Details on test animals or tissues and environmental conditions:
For reasons of animal welfare a sequential testing strategy was followed in accordance with the current version of the EEC Directive No. 440/2008 and the OECD Guideline No. 405, irrespective of the requirements of other guidelines for testing for eye irritation/corrosion in rabbits.
This testing strategy comprised a stepwise approach including the evaluation of existing human and/or animal data showing effects on the eye or the skin, the performance of a SAR evaluation for eye and skin corrosion/irritation, measurement of pH value, the evaluation of data on systemic toxicity via the oral route, the performance of a validated in vitro test for skin corrosion (Human 3D Epidermal Skin Model) and in vivo testing for skin irritation/corrosion in rabbits before in vivo testing for eye irritation/corrosion in rabbits.

TEST ANIMALS: Healthy adult albino rabbits free of clinical signs. The health of the animals was routinely examined for the main specific pathogens by the breeder. Before the study the rabbits had been vaccinated against pasteurellosis and all animals were medicated against coccidiosis. No treatment with antibiotics was performed prior to receipt of the animals, or during the acclimatisation phase or study period. In some cases the animals had already been used in earlier studies. This has no influence to the results of this study. Females were nulliparous and not pregnant.
- Age at study initiation: young adult animals
- Weight at study initiation: 3.5 kg-3.6 kg
- Housing: individually in cage units metal/noryl by EBECO. Excrement trays below the cages contained low dust wood granulate bedding (J. Rettenmaier & Sons, Rosenberg, Germany). The wood granulate was changed at least twice weekly. The animals were regularly transferred to clean cages.
- Diet (e.g. ad libitum): standard diet "Ssniff K-Z" 4mm (manufacturer: Ssniff Spezialdiäten GmbH, Soest, Germany), approximately 100 g per animal per day (To satisfy the needs of roughage, hay was offered additionally (hay, irradiated, delivered by Harlan Nederland, 5961 NM Horst, Netherlands or Harlan Winkelmann GmbH, Borchen, Germany, respectively hay pellets, manufacturer: Ssniff Spezialdiäten GmbH, 59494 Soest, Germany). The nutritive composition and the contaminant content of the standard diet were checked and analysed routinely in random samples. No unwanted ingredients were detected.)
- Water (e.g. ad libitum): tap water ad libitum from polycarbonate bottles (The tap water was of drinking water quality (according to the Drinking Water Decree in the current version) and analysed routinely. The wood granulate was randomly checked for contaminants at regular intervals.)
- Acclimation period: at least 5 days
- Other: The animals were identified by labels on the cages stating at least study number, test compound, animal number and by tattooed number assigned by the breeder.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ±3°C
- Humidity (%): 50 ±25%
- Photoperiod (hrs dark / hrs light): light/ dark cycle with a 12 hours rhythm.
- Other: The humidity and air temperature were continuously recorded. Occasionally deviations from the standards occurred, e.g. during cleaning of the animal room or effects of the weather. They did not have any apparent influence on the outcome of the study. The animal room was provided with sound from a radio program.

The animal room was cleaned at least once a week and disinfected at least once a month. It was ensured that the diet was not contaminated, and that there was no contact of the cleaning or disinfecting solution with the test animals.

Test system

Vehicle:
unchanged (no vehicle)
Remarks:
0.1 g of powder for each animal was placed into the conjunctival sac
Controls:
other: the untreated eye served as the control
Amount / concentration applied:
The solid test compound was heated in a drier to approximately 60°C (duration: approx. 2h). When the test substance was liquid, approx. 1 mL was taken and was given in a mortar. When the substance was crystallised again (duration approx. 1 min.), it was pulverised. From this powder 0.1 g was weighed for each animal.
Duration of treatment / exposure:
1 hour (eye was rinsed approximately 1 hour after instillation.)
Observation period (in vivo):
approximately at 1, 24, 48 and 72 hours post application.
If no irritation indices were observed after 72 h, the study was finished.
If eye irritations were observed, animals were monitored usually on day 7, 14 and 21 after application until the changes had completely subsided, however for not more than 21 days after application.
Number of animals or in vitro replicates:
3
Details on study design:
On the day before the test, both eyes of each animal were examined including fluorescein examination. Only animals with healthy intact eyes were used.
0.1 g of the pulverized test substance was placed into the conjunctival sac of one eye of the first animal after having gently pulled the lower lid away from the eyeball. The lids were gently held together for about one second in order to prevent loss of the test compound. The other eye, which remained untreated, served as control.
Because of the fact that one hour after treatment a severe irritation was not observed, a further two rabbits were treated as described.

REMOVAL OF TEST SUBSTANCE
- Washing (if done): The eye was rinsed approximately 1 hour following instillation.
- Time after start of exposure: 1 hour

SCORING SYSTEM:
The degree of ocular lesions was recorded as specified by DRAIZE and any serious lesions or toxic effects other than ocular lesions were also recorded and fully described.
As general criteria the body weight of each animal was recorded at the beginning of the study. If clinical findings other than eye irritations occur they were recorded daily.

TOOL USED TO ASSESS SCORE: fluorescein

Results and discussion

In vivo

Resultsopen allclose all
Irritation parameter:
cornea opacity score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
cornea opacity score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #1
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #2
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
iris score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
24 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
48 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
24 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
48 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
chemosis score
Basis:
animal #3
Time point:
24/48/72 h
Score:
0
Max. score:
0
Reversibility:
other: not applicable
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
24 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
48 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #1
Time point:
72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
24 h
Score:
2
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
48 h
Score:
1
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #2
Time point:
72 h
Score:
0
Max. score:
2
Reversibility:
fully reversible within: 3 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
24 h
Score:
1
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
48 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritation parameter:
conjunctivae score
Basis:
animal #3
Time point:
72 h
Score:
0
Max. score:
1
Reversibility:
fully reversible within: 2 days
Irritant / corrosive response data:
Only redness of the conjunctivae was noted (up to 48 hours after instillation). No effects on iris or cornea were observed.
Other effects:
No other effects reported

Any other information on results incl. tables

Table1: Irritant Effects on the Eye 
Animal 1,Body Weight3.6kg
Observation 1h 24 h 48 h 72 h day7 day14 day21
Degree of cornea opacity 0 0 0 0 - - -
Area of cornea opacity 0 0 0 0 - - -
Iris 0 0 0 0 - - -
Redness conjunctivae 2 2 1 0 - - -
Chemosis conjunctivae 0 1 0 0 - - -
Animal 2,Body Weight3.5kg            
Observation 1h 24 h 48 h 72 h day7 day14 day21
Degree of cornea opacity 0 0 0 0 - - -
Area of cornea opacity 0 0 0 0 - - -
Iris 0 0 0 0 - - -
Redness conjunctivae 2 2 1 0 - - -
Chemosis conjunctivae 0 1 0 0 - - -
Animal 3,Body Weight3.5kg            
Observation 1h 24 h 48 h 72h day7 day14 day21
Degree of cornea opacity 0 0 0 0 - - -
Area of cornea opacity 0 0 0 0 - - -
Iris 0 0 0 0 - - -
Redness conjunctivae 2 1 0 0 - - -
Chemosis conjunctivae 0 0 0 0 - - -
- :no further examination
Animal 1, 2 and 3, 1 h p.a.:   test compound adhered to cornea and conjunctiva 

Table 2: Summary of Irritant Effects (Score)  
          Mean Reversible
Animal   24h 48h 72h scores Response (days)
  Corneal opacity 0 0 0 0.0 na
1 Iritis 0 0 0 0.0 na
Redness conjunctivae 2 1 0 1.0 3
  Chemosis conjunctivae 1 0 0 0.3 2
  Corneal opacity 0 0 0 0.0 na
2 Iritis 0 0 0 0.0 na
Redness conjunctivae 2 1 0 1.0 3
  Chemosis conjunctivae 1 0 0 0.3 2
  Corneal opacity 0 0 0 0.0 na
Iritis 0 0 0 0.0 na
3 Redness conjunctivae 1 0 0 0.3 2
  Chemosis conjunctivae 0 0 0 0.0 na
response:
corneal opacity : mean scores < 2 = -,  > 2 < 3 = +, > 3 = ++
iritis : mean scores < 1 = -,  > 1 < 2  = +, =2=++
conjunctival redness : mean scores < 2,5 = -, > 2.5  = +,
conjunctival oedema : mean scores < 2 = -, > 2  = +,
na : not applicable 

Applicant's summary and conclusion

Interpretation of results:
not irritating
Remarks:
Migrated information Criteria used for interpretation of results: other: Eu-GHS
Conclusions:
The study was performed according to the OECD TG405 without deviations and according to the good laboratory practice principles, it is considered to be of the highest quality (reliability Klimisch 1). The criteria of validity of the test system are fulfilled. The test material did induce only slight reversible irritation on rabbit eyes. The test material was considered to be not irritating under the conditions of the test.
Executive summary:

The test substance bis(2.6-diisopropylphenyl)carbodiimide was investigated for its potential to cause irritation in the rabbit eye according to OECD TG405 (Gmelin, 2010). 0.1 g of pure substance were instilled into the conjunctival sac, the untreated eye served as control. Under the conditions of this experiment bis(2.6-diisopropylphenyl)carbodiimide was found to cause no irritation when applied to the rabbit eye mucosa. No corrosion effect was observed at each of the measuring intervals. No discoloration of the cornea and conjunctivae which could be related to effects of the test material was observed. Additionally no corrosive or acute toxicological signs were observed in the animals during the test period. According to classification criteria in the European regulation (EC) No. 1272/2008, amending regulation EC no. 1907/2006 (REACh) the test substance is not irritating to eyes and should be unclassified. There were no systemic intolerance reactions.