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

Endpoint summary

Description of key information

- Acute oral toxicity according to OECD TG 423, rats; LD50  > 2000 mg/kg bw; no mortality occurred and no signs of systemic toxicity were observed up to 2000 mg/kg bw (Ciba-Geigy Ltd 1993a).
- Acute dermal toxicity: LD50 > 2000 mg/kg bw  (OECD 402, GLP), (RCC Ltd (1993a).
- Acute inhalation toxicity: LC50 > 2.2 mg/L (highest attainable concentration, read-across to Pigment Red 254, EC 401-540-3, OECD 403, GLP, Ciba 1991)

Key value for chemical safety assessment

Acute toxicity: via oral route

Link to relevant study records

Reference

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1993-05-13 - 1993-06-29

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 401) performed under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Version / remarks:

adopted on 24th February 1987

Deviations:

no

GLP compliance:

yes

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

other: albino (Tif: RAI f (SPF))

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CIBA-GEIGY Limited Animal Production, 4332 Stein, Switzerland
- Age at study initiation: young adult animals
- Weight at study initiation: 172 to 220 g
- Housing: 5 animals/sex housed in Macrolon cagestype 4, with standardized soft wood bedding
- Diet: Rat diet ad libitum, prior to dosing, the animals were fasted overnight
- Water: ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 55 +/- 10 °C
- Photoperiod: 12 hours dark / 12 hours light

IN-LIFE DATES: From: 1993-06-01 To: 1993-06-22

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % (w/v) carboxymethylcellulose in 0.1 % (w/v) aqueous polysorbate 80

Details on oral exposure:

VEHICLE:
- Concentration in vehicle: 200 mg/mL, based on dose volume
- Amount of vehicle (if gavage): 10 mL/kg body weight
- Justification for choice of vehicle: to make dosing by gavage possible
- Purity: no data

MAXIMUM DOSE VOLUME APPLIED:
- 2000 mg/kg bw (males and females)

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing:
- once daily for 14 days for clinical signs and mortality
- Body weights were recorded immediately before administration and on days 7 and 14,
- Necropsy of survivors performed: yes

Statistics:

No statistical testing was performed.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortalities occurred in this study.

Clinical signs:

other: Piloerection, hunched posture, and dyspnea were seen, being common symptoms in acute tests. The animals recovered within 4 to 5 days.

Gross pathology:

At necropsy, spotted lungs were found in one female. No deviations from normal morphology were found in the remaining animals.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study conducted according to GLP.

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.

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Acute toxicity: via dermal route

Link to relevant study records

Reference

Endpoint:

acute toxicity: dermal

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-11-17 - 1998-12-08

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study (OECD TG 402) performed under GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Version / remarks:

adopted on 24th February 1987

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.3 (Acute Toxicity (Dermal))

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OTS 798.1100 (Acute Dermal Toxicity)

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: Testing Methods for New Chemical Substances according to the Revised Japanese Chemical Substance Law (March 31, 1987)

Deviations:

not specified

GLP compliance:

yes (incl. QA statement)

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
- Source: BRL, Biological Research Laboratories Ltd, Füllinsdorf, Switzerland
- Age at study initiation: Males 10 weeks, Females 11 weeks
- Body weight at study initiation: 283.9 – 292.7 g (males), 200.1 – 213.6 g (females)
- Housing:
- 5 animals/sex/cage in Makrolon type-4 cages, standardized softwood bedding (acclimatization)
- individually in Makrolon type-3 cages (treatment and observation period)
- Diet: Pelleted standard Provimi Kliba 3433 rat/mouse maintenance diet (Provimi Kliba AG, Kaiseraugst, Switzerland), ad libitum
- Water: Community tap-water from Füllinsdorf, ad libitum
- Acclimation period: at least 7 days (only animals without any visible signs of illness were used for the study)

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 40 – 70 %
- Air changes: 10-15 air changes/hour
- Photoperiod: 12-hour fluorescent light/12-hours dark cycle, music during the light period

IN-LIFE DATES:
From: 1993-11-24 To: 1993-12-08

Type of coverage:

semiocclusive

Vehicle:

corn oil

Details on dermal exposure:

TEST SITE
- Area of exposure: Backs of the animals
- Coverage: Approximately 10 % of the total body surface
- On test day 1, the test item was applied evenly on the intact skin with a syringe and covered with a semi-occlusive dressing
- Type of wrap: Dressing was wrapped around the abdomen and fixed with an elastic adhesive bandage.

REMOVAL OF TEST SUBSTANCE
- Washing: The skin was flushed with lukewarm tap water and dried.

TEST MATERIAL
- Amount applied: 500 mg/mL
- Constant volume or concentration used: Yes, 4 mL/kg bw

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 rats

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Mortality/Viability: Four times during test day 1 (according to the raw data the last check was conducted 5 hours after application), and once daily during days 2 - 15
- Frequency of observations (clinical signs): Each animal was examined for changes in behaviour and appearance (with special emphasis on the application area, except for time interval of semi-occlusive dressing) four times during day 1, and once daily during days 2-15. All abnormalities were recorded.
- Frequency of weighing: On test days 1 (pre-administration), 8 and 15
- Necropsy of survivors performed: yes, the animals were examined macroscopically and all abnormalities recorded
- Dose formulation: The test item was placed into a glass beaker on a balance and the vehicle (corn oil) was added. A weight by volume dilution was prepared using a homogenizer. Homogeneity of the test item in the vehicle was maintained during treatment using a magnetic stirrer. The preparation was made shortly prior to dosing.
- Rationale for route of exposure: Dermal administration was used as this is one possible route of human exposure during manufacture, handling and use of the test item.

Statistics:

No statistical analysis was used. The LOGIT-Model could not be applied, since no deaths occurred.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No deaths occurred during the study period.

Clinical signs:

other: No clinical signs of systemic toxicity were observed in any animals during the observation period.

Gross pathology:

The macroscopic examination at study termination revealed no organ abnormalities.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Additional information

Acute toxicity, oral route:

In an acute oral toxicity study performed according to the Acute Toxic Class method (CIBA-GEIGY Ltd, 1993a), doses of 2000 mg/kg bw of the substance (purity > 90%; preparations in 0.5 % (w/v) carboxymethylcellulose in 0.1 % (w/v) aqueous polysorbate 80) were administered to two test groups of three fasted Wistar rats each (first step: 2000 mg/kg bw in 3 females; second step: 2000 mg/kg bw in 3 females; each following step conducted in the absence of mortality in the previous) by gavage in a sequential manner.No mortality occurred, no macroscopic pathological findings were observed, and the mean body weight increased within the normal range. Therefore the acute oral LD50 was calculated to be greater than 2000 mg/kg bw (greater than 5000 mg/kg bw when the results are interpreted based on the Annex 2 of the OECD TG 423).

The study was conducted according to the OECD test guideline 401 and GLP and is therfore assigned as valid without restriction.

Acute toxicity, inhalation route:

No experimental data is available for the test substance, but an a valid acute inhalation study was performed with a similar substance. The read-across substance shares the diketo-pyrrolo-pyrrol (DPP) core with the two phenyl substituents. It differs in the substituents at the phenyl ring. In the read-across substance, this is chlorine, whereas in the target substance, it is a tertiary butyl group. Both pigments have shown absence of systemic toxicity upon oral and dermal dosing and are considered to be too insoluble for transport in biological fluids. The available data on the read-across pigment indicates that the core structure of the DPP pigments does not represent a specific hazard for acute inhalation toxicity. The additional investigations shown below indicate typical local responses for exposure to inert nuisance dust.

In the study with Pigment Red 254 (EC 401-540-3), an acute inhalation toxicity GLP conform test, performed according to OECD Guideline 403, a nose inhalation system was used to expose 5 Wistar rats per sex to an aerosol at a concentration of 2.25 mg/L for 4 hours (Ciba-Geigy Ltd, 1991a, b).

The animals were observed for a post-dosing period of 14 days for mortality, body weight changes, clinical signs of intoxication and pathological findings. No mortality and no macroscopic findings at necropsy were observed in males and females. It was not possible to generate higher concentrations of the test compound. The exposure to the maximum attainable concentration was thus considered a limit test as stated in the OECD test guideline 403.Clinical symptoms were piloerection, hunched posture and dyspnea. From this, the animals recovered within 5 to 9 days. Histopathological examinations of the lungs revealed minimal congestion, minimal emphysema and minimal and multifocal bronchiolar dilatation in all animals. These changes are common response in rats treated by inhalation with a nuisance dust.

 

In a mechanistic follow-up study the acute lung response, especially acute inflammatory/cytotoxic responses in the rat lung following a single administration by inhalation of an aerosol for 4 hours was assessed by examination of various biochemical and cellular parameters in bronchoalveolar lavage fluid obtained 24 hours after exposure.

The investigation was based on a publication by Lindenschmidt et al. (“The comparison of a fibrogenic and two nonfibrogenic dusts by bronchoalveolar lavage.” Toxicology and Applied Pharmacology, 102, 268 – 281 (1990)). As it is a non-standard study, historical control data was not available.

Measured concentrations were 1.1 mg/L for the test item, the negative control titanium dioxide (nuisance dust) and the positive control Sikron F600 ((also known as HSE Standard Quartz, fibrinogenic agent). The MMAD was 2.5, 2.0 and 2.7 µm for titanium dioxide, Sikron F600 and the test item, respectively. At least 90% of the particles were had a diameter of less than 7 µm. Another control group was exposed to air only. There were no signs indicative of a toxic or irritant effect following exposure to the test compound. Red stained feces and staining of the skin/fur were noted in both sexes post exposure to the test item. Exposure exaggerated respiratory movements were evident in a proportion of rats exposed to the test compound from 15 minutes of exposure. This finding was also apparent after inhalation of titanium dioxide (2 h) and Sikron F600 (15 minutes), respectively, but not the air control.

There were no treatment-related macroscopic findings following the 24 hour post exposure observation period. No effects on lungs weights after exposure to 3,6-bis(4-chlorophenyl)-2,5-dihydropyrrolo[3,4-c]pyrrole-1,4-dione were seen.

After laboratory investigations of bronchoalveolar lavage samples, differences to air control samples were evident in animals treated with the test compound, titanium dioxide and Sikron F600. Biochemical examinations showed that β-glucuronidase, N-acetyl-glucosaminidase and lactate dehydrogenase levels in animals exposed to aerosols of the three dust samples were higher than in air control values. Total protein values as well as the total and viable cell counts were also higher than in air controls.

The order of magnitude of effects was similar for both control dusts and the test item, with the responses to the test item being generally higher than those to both dust controls. The experimental design was set up to test equal particle load in regard to number and size, but the density and the surface area of the particles were not taken into account. Therefore, a quantitative interpretation of the results is not possible. This acute response is typical of the lung response to inert and biologically active particles and it is only several weeks after exposure that the cellular response to inert and biologically active particles differs (Lindenschmidt 1990).

 

Acute toxicity, dermal route:

The test article (purity > 90%; preparations in olive oil Ph.Eur.)

was administered to a group of 5 male and 5 female rats by dermal application at a single dose of 2000 mg

test article/kg body weight (RCC Ltd 1993a). No deaths occurred during the study period. No clinical signs of systemic toxicity were observed during the observation period. The test article stained the animals skin red at the application site. There were no test article-related effects on the body weight of the animals during the observation period. The macroscopic examination at study termination revealed no organ abnormalities.

The study was conducted according to the OECD test guideline 402 and GLP and is therfore assigned as valid without restriction.

Justification for classification or non-classification

 

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. As a result the substance is not considered to be classified for acute oral and dermal toxicity under Directive 67/548/EEC, as amended for the

the 31st time in Directive 2009/2/EG.

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for acute oral and dermal toxicity under Regulation (EC) No. 1272/2008, as amended for the third time in Directive EC 618/2012.