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

Endpoint summary

Description of key information

The substance was found to be not irritating to the skin and eye.

Key value for chemical safety assessment

Skin irritation / corrosion

Link to relevant study records

Reference

Endpoint:

skin irritation: in vivo

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.

Qualifier:

according to guideline

Guideline:

OECD Guideline 404 (Acute Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EU Method B.4 (Acute Toxicity: Dermal Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2500 (Acute Dermal Irritation)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charies River Deutschland, Kisslegg, Germany
- Age at study initiation: 7 - 14 weeks
- Weight at study initiation: 1458 - 2552 g
- Housing: Individually in labelled cages with perforated floors (Scanbur, Denmark, dimensions 56x44x37.5 cm).
- Diet: Standard laboratory rabbit diet (Charies River Breeding and Maintenance Diet for Rabbits, Altromin, Lage, Germany) approx. 100 g. per day. Hay (TecniLab-BMl BV, Someren, The Netherlands) was provided at least three times a week.
- Water: Free access to tap water.
- Acclimation period: Acclimatisation period was at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.0- 21.8
- Humidity (%): 35-65
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

semiocclusive

Preparation of test site:

clipped

Vehicle:

other: watery ethanol (50% v/v) (water; Milli-U, Millipore Corporation, Bedford, USA; ethanol, Merck, Darmstadt, Germany)

Controls:

no

Amount / concentration applied:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): 0.5g of the test substance. The test substance was moistened with 1 mL of the vehicle


VEHICLE
- Amount(s) applied (volume or weight with unit): 1 mL
- Concentration (if solution): watery ethanol (50% v/v) (water; Milli-U, Millipore Corporation, Bedford, USA; ethanol, Merck, Darmstadt, Germany)

Duration of treatment / exposure:

4 hours

Observation period:

72 hours

Number of animals:

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 skin irritation observed in the first animal.

TREATMENT
Approximately 24 hours before treatment, the dorsal fur was clipped with electric clippers, exposing an area of approximately 150 square centimeters (10x15 cm²). To facilitate scoring, treated skin areas were re-clipped at least 3 hours before the observations. Each animal was treated by dermal application of 0.5g of the test substance. The test substance was moistened with 1 mL of the vehicle and applied to the skin of one flank, using a metalline patch of 2x3 cm. The patch was mounted on Micropore tape, which was wrapped around the abdomen and secured with Coban elastic bandage. Four hours after the application, the dressing was removed and the skin cleaned of residual test substance using tap water, watery ethanol (50% v/v) (water; Milli-U, Millipore Corporation, Milli-U, Millipore Corporation, Bedford, USA; acetone, Labscan, Dublin, Ireland). For one animal the skin was also cleaned on day 2 with watery acetone (50% v/v) (tap water; acetone, Labscan, Dublin, Ireland). The treated skins were still stained by the test substance, but erythema could be assessed from 48 hours after application of the test substance onwards. After the final observation, the animals were sacrificed by intra-venous injection of pentobarbital (Euthesate®; Sanofi Sante B.V., Maassluis, The Netherlands).

OBSERVATIONS
Mortality/Viability: Twice daily; Toxicity: At least once daily; Body Weight: Day of treatment (prior to application) and at termination; Irritation: The skin reactions were assessed at approximately 1, 24,48 and 72 hours after the removal of the dressings and test substance. The irritation scores and a description of all other (local) effects were recorded. Adjacent areas of the untreated skin of each animal served as controls.

Irritation parameter:

erythema score

Basis:

mean

Time point:

other: 48 - 72 hours

Score:

0

Max. score:

4

Remarks on result:

other: Orange staining of the skin by the test substance, therfore at the 1 and 24 hours time point no scoring possible.

Irritation parameter:

edema score

Basis:

mean

Time point:

other: 1 - 72 hours

Score:

0

Max. score:

4

Irritant / corrosive response data:

No skin irritation was caused by 4 hours exposure to the test substance. The observed mean values (24 - 72 h or 48 - 72 h) of individual animals for edema and erythema were 0 without exception.

Other effects:

Colouration / Remnants: Orange staining of the treated skin by the test substance was observed throughout the observation period. This hampered the skin irritation assessment on day 1 and 2 only.
Toxicity / Mortality: No symptoms of systemic toxicity were observed in the animals during the test period and no mortality occurred.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Eye irritation

Link to relevant study records

Reference

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

Qualifier:

according to guideline

Guideline:

OECD Guideline 405 (Acute Eye Irritation / Corrosion)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.2400 (Acute Eye Irritation)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

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

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.

Irritation parameter:

cornea opacity score

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

iris score

Basis:

mean

Time point:

other: 1, 24, 48 and 72 hours

Score:

0

Max. score:

2

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 1 hours

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 24 hours

Score:

1.33

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 48 hours

Score:

0.33

Max. score:

3

Reversibility:

fully reversible within: 72 hours

Irritation parameter:

other: Conjunctivae: Redness

Basis:

mean

Time point:

other: 72 hours

Score:

0

Max. score:

3

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 1 hour

Score:

0.33

Max. score:

4

Reversibility:

fully reversible within: 24 hours

Irritation parameter:

chemosis score

Basis:

mean

Time point:

other: 24, 48 and 72 hours

Score:

0

Max. score:

4

Irritation parameter:

other: Conjunctivae: Discharge

Basis:

mean

Time point:

other: 1 hour

Score:

1

Max. score:

3

Reversibility:

fully reversible within: 48 hours

Irritation parameter:

other: Conjunctivae: Discharge

Basis:

mean

Time point:

other: 24 hours

Score:

0.66

Max. score:

3

Reversibility:

fully reversible within: 48 hours

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.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (not irritating)

Respiratory irritation

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Skin irritation:

In a GLP-compliant primary skin irritation/corrosion study, performed according to OECD guideline 404, the test substance was tested on the skin of Albino New Zealand rabbits (Notox 2006). Three rabbits were exposed to 0.5g applied onto the clipped skin for 4 hours using a semi-occlusive dressing. Skin reactions were assessed 1, 24, 48 and 72 hours after exposure. No skin irritation was caused by 4 hours exposure. The observed mean values (24 - 72 h or 48 - 72 h) of individual animals for edema and erythema were 0 without exception. Orange staining of the treated skin by the test substance was observed throughout the observation period. This hampered the skin irritation assessment on day 1 and 2 only. Based on these results the test substance did not induce significant or irreversible damage to the rabbit skin and is considered not to be a skin irritant.

Eye irritation:

In a GLP-compliant acute eye irritation/corrosion study, tested according to OECD guideline 405, single samples of approximately 34 mg of test substance (a volume of approximately 0.1 mL) were instilled into one eye of each of three Albino New Zealand White rabbits (Notox 2006). Observations were made 1, 24, 48 and 72 hours after instillation. Instillation of the test substance 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. 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. 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. Based on these results the test substance did not induce significant or irreversible damage to the rabbit eyes and is considered not to be an eye irritant.

Justification for classification or non-classification

Based on the results of the skin and eye irritation study, the test material does not need to be classified according to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008 as amended for the thirteenth time in Regulation (EC) No. 2018/1480.