Pyrrolo[3,4-c]pyrrole-1,4-dione, 3,6-bis(4-chlorophenyl)-2,5-dihydro-

Endpoint summary

Description of key information

Acute toxicity oral: LD50: > 2000 mg/kg bw

Acute toxicity inhalation: LD50 : > 2.25 mg/L

Acute toxicity dermal: LD50: > 2000 mg/kg bw

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance to GLP. However, purity of test article is unknown.

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain of animals as stated in the report: KFM-Han. Wistar (outbred, SPF-Quality)
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 214 - 237 g (males), 172 - 200 g (females)
- Housing: groups of 5 in Makrolon type-3 cages
- Diet: pelleted standard Kliba 343, rat maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/-3
- Humidity (%):40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

The test article was suspended in the vehicle (weight by volume). Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. The preparation was made immediately prior to dosing.

MAXIMUM DOSE VOLUME APPLIED: no data available

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

10 animals (5 females, 5 males)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations on mortality and viability and clinical signs: 4-times during test day 1, daily during days 2 - 15
- Frequency of weighing: body weight was assessed on day 1, 8 and 15.
- Necropsy of survivors performed: yes, all animals were sacrifieced and subjected to gross pathology.

Statistics:

No statistics were performed.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: None of the animals died after treatment.

Mortality:

No mortality occured.

Clinical signs:

other: After administration of 5000 mg/kg bw sedation, dyspnea, hunched body position and ruffled fur were observed in animals of both sexes. Additionally rales were observed in males. All rats recovered after 24 h. In addition to the above symptoms the extremit

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

No other findings

Table 1 Mean body weights in grams (Dose level: 5000 mg/kg bw)

Test day	1 *	8	15
Male	224 +/- 8.5	264 +/- 10	289 +/- 12
Female	187 +/- 11	211 +/- 11	217 +/- 12

* body weights on day 1 were assessed before application of 5000 mg/kg bw.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD TG 401 using the limit test dose of 5000 mg/kg bw, the LD50 oral of the substance was determined to be > 5000 mg/kg bw in both females and males.

Executive summary:

The acute oral toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 401 (version 1981). Suspended in polyethylene glycol, 5000 mg/kg bw of the substance were given to 5 male and 5 female Wistar rats by gavage. The rats were observed for clinical signs and body weight change for 15 days. After sacrifice, the rats were subjected to gross pathology. Besides some clinical effect in the first 24 hours, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LD 50 was > 5000 mg/kg bw for both sexes.

Reference 2

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted in accordance to GLP. However, purity of test article is unknown.

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified. In addition, the zeta-potential of both forms indicated that agglomeration increases with lower pH, which further supports the read-across for studies with oral exposure. The low gastric pH increases agglomeration, which supports the conclusion that both forms are not bioavailable upon oral exposure.

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain of animals as stated in the report: KFM-Han. Wistar (outbred, SPF-Quality)
- Source: Kleintierfarm Madoerin AG, CH-4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 214 - 237 g (males), 172 - 200 g (females)
- Housing: groups of 5 in Makrolon type-3 cages
- Diet: pelleted standard Kliba 343, rat maintenance diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C):22 +/-3
- Humidity (%):40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on oral exposure:

The test article was suspended in the vehicle (weight by volume). Homogeneity of the test article in the vehicle was maintained during treatment using a magnetic stirrer. The preparation was made immediately prior to dosing.

MAXIMUM DOSE VOLUME APPLIED: no data available

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

10 animals (5 females, 5 males)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations on mortality and viability and clinical signs: 4-times during test day 1, daily during days 2 - 15
- Frequency of weighing: body weight was assessed on day 1, 8 and 15.
- Necropsy of survivors performed: yes, all animals were sacrifieced and subjected to gross pathology.

Statistics:

No statistics were performed.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: None of the animals died after treatment.

Mortality:

No mortality occured.

Clinical signs:

other: After administration of 5000 mg/kg bw sedation, dyspnea, hunched body position and ruffled fur were observed in animals of both sexes. Additionally rales were observed in males. All rats recovered after 24 h. In addition to the above symptoms the extremit

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

No other findings

Table 1 Mean body weights in grams (Dose level: 5000 mg/kg bw)

Test day	1 *	8	15
Male	224 +/- 8.5	264 +/- 10	289 +/- 12
Female	187 +/- 11	211 +/- 11	217 +/- 12

* body weights on day 1 were assessed before application of 5000 mg/kg bw.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD TG 401 using the limit test dose of 5000 mg/kg bw, the LD50 oral of the substance was determined to be > 5000 mg/kg bw in both females and males.

Executive summary:

The acute oral toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 401 (version 1981). Suspended in polyethylene glycol, 5000 mg/kg bw of the substance were given to 5 male and 5 female Wistar rats by gavage. The rats were observed for clinical signs and body weight change for 15 days. After sacrifice, the rats were subjected to gross pathology. Besides some clinical effect in the first 24 hours, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LD 50 was > 5000 mg/kg bw for both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

5 000 mg/kg bw

Quality of whole database:

The one available study is reliable with restrictions.

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

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 403 (Acute Inhalation Toxicity)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: (Tif: RAI f (SPF) hybrids of RII/1 x RII/2)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited Animal Production, Stein /Switzerland
- Weight at study initiation: 177 - 227 g
- Fasting period before study: at least 5 d
- Housing: Groups of 5 in Makrolon type-4 cages
- Diet (ad libitum): Rat diet Nafag 890
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus:
The exposure apparatus was developed by Battelle Research Center (Geneve/Switzerland). The internal active volume was less than 1 l and the flow in any individual aerosol delivery chamber was standardised to 2 l/min (velocity 1.25 m/s).

- Method of holding animals in test chamber:
For inhalation period rats were placed in Macrolon animal holders.

- Treatment of exhaust air:
The exhaust air was decontaminated by passage through a Pall HDC absolute filter.

VEHICLE:
The test compound tended to form secondary agglomerates. Therefore, it was mixed with inert silica. A 10% mixture of Sipernat 50S with the test article was used in the animal exposure tests.


TEST ATMOSPHERE:
The aerosol concentration was determined gravimetrically five times during exposure period. The Particle size determination was conducted four times during exposure using an eight-stage cascade impactor. In the same intervall temperature, relative humidity and oxygen content of the inhalation chambers were assessed.


The test substance was administered as an aerosol in a nose-only exposure system that ensures uniform exposure and avoids re-breathing of the aerosol. During exposure, the animals were placed in Macrolon animal holders positioned radially around the exposure chamber, so that only the snouts and nostrils were exposed. The aerosol was generated from the solid test material blended with 10 % Sipernat 50S (Degussa, Germany) by means of a brush-feed micronizing jet mill. A cyclone-type classifier ensures that only particles of the desired diameter leave the jet mill.
The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as described above.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetrically

Duration of exposure:

4 h

Concentrations:

2.25 mg/l; Due to the properties of the test material, it was not possible to generate higher concentrations of the test compound with the equipment used in this study.

No. of animals per sex per dose:

10 (5 males, 5 females)

Control animals:

yes

Details on study design:

The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as treated animals.

- Duration of observation period following administration: 14 days
- Frequency of observations of clinical symptoms and mortality: During and after exposure, therafter daily.
- Frequency of weighing: Body weights were recorded prior to treatment and on day 7 and 14.
- Necropsy of survivors performed: yes, all animals were sacrificed and subjected to gross pathology.

Statistics:

Body weights of treated and untreated animals were compared by analysis of variance.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: None of the animals died.

Mortality:

None of the animals died.

Clinical signs:

other: Piloerection, hunched posture and dyspnea were seen in animals exposed to the test material. They recovered within 5 days.

Body weight:

Males exposed to the test substance showed a significantly higher body weight gain during the first and the second observation week as compared to control animals.

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

- Histopathology:
- In all examined tissue samples, the alveolar lumen contained alveolar macrophages (phagocytic cells) filled with brown
pigment, most likely representing the test article. This change was minimal in males and moderate in females. The pneumocytes
type II in the alveolar epithelium of all animals were activated. This activation was minimal and multifocal in
3 males and one female, moderate and multifocal in 2 males and 4 females. The bronchial lymph node of one male and one female
showed moderate brown pigmentation, regarded to represent the test article. In one male the bronchiolar epithelium was
minimally and focally hyperplastic.
- The minimal congestion, the minimal emphysema and the minimal and multifocal bronchiolar dilatation seen in all animals are a common response in rats treated by inhalation. Therefore, it was considered not to be treatment-related.

Table 1: Mean body weights in grams (Dose level: 2.25 mg/l)

Test day	1*	7	14
Control males	212 +/-4	246 +/- 12	278 +/-19
Treated males	202 +/-6	254 +/- 7	297 +/- 9
Control females	187 +/- 5	199 +/- 4	215 +/- 8
Treated females	186 +/- 6	199 +/- 8	215 +/- 7

*body weights on day 1 were assessed before application of 2.25 mg/l.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD test guideline 403, the substance was not toxic at a concentration of 2.25 mg/l air. Therefore, it is considered to be not acutely toxic via the inhalation route.

Executive summary:

The acute inhalation toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 403. Five male and five female rats were exposed nose only to the maximum technically achieveable concentration of 2.25 mg/L air for four hours. The rats were observed for clinical signs, body weight change and mortality for 14 days. After sacrifice, the rats were subjected to gross pathology. Besides some clinical effect in the first five days, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LC50 was > 2,25 mg/L air for both sexes.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified. In addition, the zeta-potential of both forms indicated that agglomeration increases with lower pH, which further supports the read-across for studies with oral exposure. The low gastric pH increases agglomeration, which supports the conclusion that both forms are not bioavailable upon oral exposure. As the inhalation route is of particular concern for nanoforms, read-across is supported by a 5-day in vivo inhalation study (Hofmann et al., 2016). This short-term inhalation toxicity study compared, among others, a coarse and a fine form of PR254, which are similar to the two forms considered in this analogue approach. The same minor effects were observed at the identical concentration (of 30 mg/m3).

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Version / remarks:

(1981)

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: (Tif: RAI f (SPF) hybrids of RII/1 x RII/2)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited Animal Production, Stein /Switzerland
- Weight at study initiation: 177 - 227 g
- Fasting period before study: at least 5 d
- Housing: Groups of 5 in Makrolon type-4 cages
- Diet (ad libitum): Rat diet Nafag 890
- Water: ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 55 +/- 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION

- Exposure apparatus:
The exposure apparatus was developed by Battelle Research Center (Geneve/Switzerland). The internal active volume was less than 1 l and the flow in any individual aerosol delivery chamber was standardised to 2 l/min (velocity 1.25 m/s).

- Method of holding animals in test chamber:
For inhalation period rats were placed in Macrolon animal holders.

- Treatment of exhaust air:
The exhaust air was decontaminated by passage through a Pall HDC absolute filter.

VEHICLE:
The test compound tended to form secondary agglomerates. Therefore, it was mixed with inert silica. A 10% mixture of Sipernat 50S with the test article was used in the animal exposure tests.


TEST ATMOSPHERE:
The aerosol concentration was determined gravimetrically five times during exposure period. The Particle size determination was conducted four times during exposure using an eight-stage cascade impactor. In the same intervall temperature, relative humidity and oxygen content of the inhalation chambers were assessed.


The test substance was administered as an aerosol in a nose-only exposure system that ensures uniform exposure and avoids re-breathing of the aerosol. During exposure, the animals were placed in Macrolon animal holders positioned radially around the exposure chamber, so that only the snouts and nostrils were exposed. The aerosol was generated from the solid test material blended with 10 % Sipernat 50S (Degussa, Germany) by means of a brush-feed micronizing jet mill. A cyclone-type classifier ensures that only particles of the desired diameter leave the jet mill.
The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as described above.

Analytical verification of test atmosphere concentrations:

yes

Remarks:

gravimetrically

Duration of exposure:

4 h

Concentrations:

2.25 mg/l; Due to the properties of the test material, it was not possible to generate higher concentrations of the test compound with the equipment used in this study.

No. of animals per sex per dose:

10 (5 males, 5 females)

Control animals:

yes

Details on study design:

The control animals were exposed to an inhalation atmosphere of Sipernat 50S at a nominal concentration of 0.48 mg/l under the same conditions as treated animals.

- Duration of observation period following administration: 14 days
- Frequency of observations of clinical symptoms and mortality: During and after exposure, therafter daily.
- Frequency of weighing: Body weights were recorded prior to treatment and on day 7 and 14.
- Necropsy of survivors performed: yes, all animals were sacrificed and subjected to gross pathology.

Statistics:

Body weights of treated and untreated animals were compared by analysis of variance.

Key result

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.25 mg/L air

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: None of the animals died.

Mortality:

None of the animals died.

Clinical signs:

other: Piloerection, hunched posture and dyspnea were seen in animals exposed to the test material. They recovered within 5 days.

Body weight:

Males exposed to the test substance showed a significantly higher body weight gain during the first and the second observation week as compared to control animals.

Gross pathology:

No macroscopic findings were observed at necropsy.

Other findings:

- Histopathology:
- In all examined tissue samples, the alveolar lumen contained alveolar macrophages (phagocytic cells) filled with brown
pigment, most likely representing the test article. This change was minimal in males and moderate in females. The pneumocytes
type II in the alveolar epithelium of all animals were activated. This activation was minimal and multifocal in
3 males and one female, moderate and multifocal in 2 males and 4 females. The bronchial lymph node of one male and one female
showed moderate brown pigmentation, regarded to represent the test article. In one male the bronchiolar epithelium was
minimally and focally hyperplastic.
- The minimal congestion, the minimal emphysema and the minimal and multifocal bronchiolar dilatation seen in all animals are a common response in rats treated by inhalation. Therefore, it was considered not to be treatment-related.

Table 1: Mean body weights in grams (Dose level: 2.25 mg/l)

Test day	1*	7	14
Control males	212 +/-4	246 +/- 12	278 +/-19
Treated males	202 +/-6	254 +/- 7	297 +/- 9
Control females	187 +/- 5	199 +/- 4	215 +/- 8
Treated females	186 +/- 6	199 +/- 8	215 +/- 7

*body weights on day 1 were assessed before application of 2.25 mg/l.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD test guideline 403, the substance was not toxic at a concentration of 2.25 mg/l air. Therefore, it is considered to be not acutely toxic via the inhalation route.

Executive summary:

The acute inhalation toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 403. Five male and five female rats were exposed nose only to the maximum technically achieveable concentration of 2.25 mg/L air for four hours. The rats were observed for clinical signs, body weight change and mortality for 14 days. After sacrifice, the rats were subjected to gross pathology. Besides some clinical effect in the first five days, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LC50 was > 2,25 mg/L air for both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

2 250 mg/m³ air

Quality of whole database:

The one available study is reliable without restrictions.

Acute toxicity: via dermal route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP. However, purity of test article is unknown.

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

Semi-occlusive dressing is recommended by guideline. However, occlusive dressing was used (worst case).

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain as stated in the report: KFM-Han. Wistar (outbred, SPF)
- Source: Kleintierfarm Madoerin AG CH-4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 228 - 256 g (males), 197 - 211 g (females)
- Housing: individually in Makrolon type-2 cages
- Diet (ad libitum): pelleted standar Kliba 343, rat maintenance diet
- Water (ad libitum): tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on dermal exposure:

TEST SITE
- Area of exposure: back of animals
- % coverage: 10 % of total body surface
- Type of wrap: Occlusive dressing fixed with elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: Skin was washed with lukewarm tap water
- Time after start of exposure: 24 h

TEST MATERIAL
- Application Volume/kg bw: 4 ml, corresponding to 2000 mg/kg bw.
- For solids, paste formed: Yes, test article was homogenised in PEG 400.

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

10 animals (5 males, 5 females)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations for mortality and clinical symptoms: 4-times at day one, afterwards daily.
- Frequency of weighing: Weights of animals were assessed on day 1, 8 and 15.
- Necropsy of survivors performed: Yes, all animals were sacrificed and subjected to gross pathology.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: None of the treated animals died.

Mortality:

No animal died.

Clinical signs:

other: After treatment a reddish skin discoloration was observed in all rats during the observation period. No other signs of local or systemic toxicity were observed.

Gross pathology:

No macroscopic findings were observed at necropsy.

Table 1: Mean body weights in grams (Dose level: 2000 mg/kg bw)

Test day	1*	8	15
Male	240 +/- 10	255 +/- 11	275 +/- 14
Female	206 +/- 5.4	211 +/- 8	219 +/- 8.7

body weights on day 1 were assessed before application of 2000 mg/kg bw.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD TG 402 using the limit test dose of 2000 mg/kg bw, the dermal LD50 of the substance was determined to be > 2000 mg/kg bw in both females and males.

Executive summary:

The acute dermal toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 402 (version 1981). Suspended in polyethylene glycol, 2000 mg/kg bw of the substance were occlusively applied to the skin of 5 male and 5 female Wistar rats for 24 hours. The rats were observed for clinical signs, body weight change and mortality for 15 days. After sacrifice, the rats were subjected to gross pathology. Besides some reddish skin discoloration, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LD50 was > 2000 mg/kg bw for both sexes.

Reference 2

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP. However, purity of test article is unknown.

Justification for type of information:

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
The read-across is performed between two forms of the same substance. The identities of the two forms are describe below.

2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The source form is 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrol-1,4-dione (EC-no. 401-540-3), referred to here as PR254. PR254 is an organic mono-constituent substance with a typical purity of > 99.5% (w/w). It does not contain any impurity relevant for classification or labelling of the substance. The target form is the nanoform of the source substance, referred to here as PR254 nanoform. As the source form, it has a typical purity of > 99.5% (w/w) and it does not contain any impurity relevant for classification or labelling of the substance. The PR254 nanoform is spheroidal with a pure polyhedral shape and is not surface-treated.

3. ANALOGUE APPROACH JUSTIFICATION
The two analogue forms have the same structure. Under ambient atmosphere, the specific surface energy of particles increases with decreasing particle size. Therefore, particle aggregate to reach an energy minimum. The driving forces are hydrogen bonds and van der Waals forces (π-π interaction). Substantial energy is required to disperse the PR254 nanoform aggregates to particles that fall under the nanoform definition.
PR254 was been tested extensively addressing information requirements of Annexes VII to IX without identifying any biological target. PR254 nanoform could potentially have biological targets due to the different particle size distribution, which would require processes capable of dispersing the aggregates, e.g. in aqueous milieu. However, both forms have been tested according to OECD Test Guideline 318, demonstrating that PR254 nanoform cannot be dispersed under the condition of the study, i.e. immediately after sonification re-forms aggregates. Also, PR254 aggregates to a large extent, but can be more easily dispersed than the nanoform. The experiments demonstrated that exposure in aqueous milieu will be primarily to aggregates, regardless of the PR254 form.
Therefore, it is concluded that both forms will behave identically in studies, in which they are applied under atmospheric conditions and/or in aqueous milieus, so that for the PR254 nano-form no specific biological targets need to be considered.
As both forms form non-dispersible aggregates in aqueous milieu and under ambient conditions, read-across of toxicological studies from the source to the target form is scientifically justified.

4. DATA MATRIX
The data matrix is included as Annex 1 in the assessment report ‘PR254 bulk nano analogue approach 210111’ attached here below under ‘Attached justification’.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

(1981)

Deviations:

yes

Remarks:

Semi-occlusive dressing is recommended by guideline. However, occlusive dressing was used (worst case).

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
-Strain as stated in the report: KFM-Han. Wistar (outbred, SPF)
- Source: Kleintierfarm Madoerin AG CH-4414 Fuellinsdorf / Switzerland
- Age at study initiation: 9 - 11 weeks
- Weight at study initiation: 228 - 256 g (males), 197 - 211 g (females)
- Housing: individually in Makrolon type-2 cages
- Diet (ad libitum): pelleted standar Kliba 343, rat maintenance diet
- Water (ad libitum): tap water
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

occlusive

Vehicle:

polyethylene glycol

Remarks:

PEG 400

Details on dermal exposure:

TEST SITE
- Area of exposure: back of animals
- % coverage: 10 % of total body surface
- Type of wrap: Occlusive dressing fixed with elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: Skin was washed with lukewarm tap water
- Time after start of exposure: 24 h

TEST MATERIAL
- Application Volume/kg bw: 4 ml, corresponding to 2000 mg/kg bw.
- For solids, paste formed: Yes, test article was homogenised in PEG 400.

Duration of exposure:

24 h

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

10 animals (5 males, 5 females)

Control animals:

no

Details on study design:

- Duration of observation period following administration: 15 days
- Frequency of observations for mortality and clinical symptoms: 4-times at day one, afterwards daily.
- Frequency of weighing: Weights of animals were assessed on day 1, 8 and 15.
- Necropsy of survivors performed: Yes, all animals were sacrificed and subjected to gross pathology.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: None of the treated animals died.

Mortality:

No animal died.

Clinical signs:

other: After treatment a reddish skin discoloration was observed in all rats during the observation period. No other signs of local or systemic toxicity were observed.

Gross pathology:

No macroscopic findings were observed at necropsy.

Table 1: Mean body weights in grams (Dose level: 2000 mg/kg bw)

Test day	1*	8	15
Male	240 +/- 10	255 +/- 11	275 +/- 14
Female	206 +/- 5.4	211 +/- 8	219 +/- 8.7

body weights on day 1 were assessed before application of 2000 mg/kg bw.

Interpretation of results:

GHS criteria not met

Conclusions:

In a GLP-study according to OECD TG 402 using the limit test dose of 2000 mg/kg bw, the dermal LD50 of the substance was determined to be > 2000 mg/kg bw in both females and males.

Executive summary:

The acute dermal toxicity of the substance was inevstigated in a GLP-compliant staudy according to OECD Test Guideline 402 (version 1981). Suspended in polyethylene glycol, 2000 mg/kg bw of the substance were occlusively applied to the skin of 5 male and 5 female Wistar rats for 24 hours. The rats were observed for clinical signs, body weight change and mortality for 15 days. After sacrifice, the rats were subjected to gross pathology. Besides some reddish skin discoloration, no effects of the substance, especially mortality and on body weight, were observed. Therefore, the LD50 was > 2000 mg/kg bw for both sexes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

2 000 mg/kg bw

Quality of whole database:

The one available study is reliable with restrictions.

Justification for classification or non-classification

As the substance is practically non-toxic, it does not need to be classified for acute toxic effects.

The maximum technically achieveable concentration was 2.25 mg/L, which did not induce any mortality or effects on body weight. This is considered to be sufficient evidence to justify no acute inhalation classification, even though the limit concentration acc. to the CLP regulation is 5 mg/L.