Reaction mass of 3,6-Bis(3-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione, 3-(3-Chlorophenyl)-6-(4-chlorophenyl)-2,5-dihydro- pyrrolo[3,4-c]pyrrole-1,4-dione and 3,6-Bis(4-chlorophenyl)-2,5-dihydro-pyrrolo[3,4-c]pyrrole-1,4-dione

Eye irritation

Administrative data

Endpoint:

eye irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Test guideline 4

Qualifier:

according to guideline

Guideline:

other: Japanese Ministry of Agriculture, Forestry and Fisheries (JMAFF), 12 Nousan, Notification No 8147

GLP compliance:

yes

Test material

Test material information

Constituent 1

Test material form:

solid: nanoform, no surface treatment

Details on test material:

- Description: Orange-red powder
- Test substance storage: At room temperature in the dark
- Stability under storage conditions: Stable
- Expiry date: 15 August 2007

Test animals / tissue source

Species:

rabbit

Strain:

New Zealand White

Details on test animals or tissues and environmental conditions:

TEST ANIMALS
- Source: Charies River Deutschland, Kisslegg, Germany
- Age at study initiation: 8-12 weeks
- Weight at study initiation: 1835 - 2286g
- Housing: Individually in labeled cages with perforated floors (Scanbur, Denmark, dimensions 56x44x37.5 cm).
- Diet: Standard laboratory rabbit diet (Charles River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany) approx. 100 g. per day. In addition, hay (TecniLab-BMl BV, Someren, The Netherlands) was provided at least three times weekly.
- Water: Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.8 - 21.9
- Humidity (%): 36-63
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Test system

Vehicle:

unchanged (no vehicle)

Controls:

other: Negative control: untreated eye

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied: Animals were treated by instillation of, on average, 34.4 mg (range 33.6 - 35.2 mg) of the test substance

Duration of treatment / exposure:

72 hours

Observation period (in vivo):

72 hours

Number of animals or in vitro replicates:

3

Details on study design:

STUDY DESIGN:
The study was performed in a stepwise manner and was started by treatment of a single rabbit (sentinel). The two other animals were treated in a similar manner 7 days later, after considering the degree of eye irritation observed in the first animal.

TREATMENT:
Animals were treated by instillation of, on average, 34.4 mg (range 33.6 - 35.2 mg) of the test substance (a volume of approximately 0.1 mL), as the maximum required amount in the conjunctival sac of one of the eyes after gentiy pulling the lower lid away from the eyeball. The lids were then gentiy held together for about one second to prevent loss of the test substance. The other eye remained untreated and served as the reference control.
Immediately after the 24-hour observation, a solution of 2% fluorescein (Merck, Darmstadt, Germany) in water (adjusted to pH 7.0) was instilled into both eyes of each animal to quantitatively determine corneal epithelial damage. Any bright green stained area, indicating epithelial damage, was estimated as a percentage of the total corneal area. Immediately after fluorescein examination on day 2, the treated eye of one animal was rinsed with approx 50 mL tepid tap water, using a velocity of flow that did not affect the eye, to remove residual test substance. For reference control the other eye was also rinsed. After the final observation, the animals were sacrificed by intra-venous injection of pentobarbital (Euthesate; Sanofi Sante B.V., Maassluis, The Netheriands).

OBSERVATIONS:
Mortality/Viability: Twice daily; Toxicity: At least once daily; Body Weight: Day of treatment (prior to instillation) and at termination.

SCORING SYSTEM:
- Irritation: The eyes of each animal were examined approximately 1, 24, 48 and 72 hours after instillation of the test substance. The irritation scores and a description of all other (local) effects were recorded.
- The following irritaiton scores were assessed: corneal irritation, area of cornea involved; iris; conjunctival irritation, chemosis (refers to lids and/or nictitating membranes), discharge.

Results and discussion

In vivo

Results

Results 1

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hours

Score:

0

Max. score:

4

Results 2

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hours

Score:

0

Max. score:

2

Results 3

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 1 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 4

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 24 hours

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 5

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 48 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Results 6

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

3

Results 7

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 1 hour

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Results 8

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Results 9

Irritation parameter:

other: Conjunctivae: Discharge

Basis:

mean

Time point:

other: 1 hour

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 10

Irritation parameter:

other: Conjunctivae: Discharge

Basis:

mean

Time point:

other: 24 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Results 11

Irritation parameter:

other: Conjunctivae: Discharge

Basis:

mean

Time point:

other: 48 and 72 hours

Score:

0

Max. score:

3

Irritant / corrosive response data:

Instillation of approximately 34 mg of the test substance (a volume of approximately 0.1 mL) into one eye of each of three rabbits resulted in irritation of the conjunctivae, which consisted of redness, chemosis and discharge. The irritation had completely resolved within 48 hours in two animals, and 72 hours in one animal. No iridial irritation or corneal opacity was observed, and treatment of the eyes with 2% fluorescein, 24 hours after test substance instillation revealed no corneal epithelial damage. The observed mean values (24 - 72 h) of individual animals were 0.3, 0.3, 1.0 for redness and 0 in each case for chemosis, iritis and cornea opacity.

Other effects:

Colouration / Remnants: Remnants of the test substance were present in the eye on day 1 in two animals, and on days 1 and 2 in one animal. Red staining of the fur on the head and paws, caused by the test substance, was noted during the observation period.
Toxicity / Mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Applicant's summary and conclusion

Interpretation of results:

GHS criteria not met