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 oral and dermal LD50 value in Wistar rats was established to exceed 2000 mg/kg body weight. No mortality occurred for either route. Upon a 4h inhalation exposure, rats died shortly after exposure to 5 mg/L dust. Rats survived exposure to 1 or 0.52 mg/L and showed no macroscopic findings upon necropsy.

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

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other: GLP compliant guideline study, available as unpublished report, no restrictions, fully adequate for assessment.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 423 (Acute Oral toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EU Method B.1 tris (Acute Oral Toxicity - Acute Toxic Class Method)

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1100 (Acute Oral Toxicity)

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Test type:

acute toxic class method

Limit test:

yes

Species:

rat

Strain:

Wistar

Sex:

female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charies River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approx. 10 weeks old
- Weight at study initiation: Females (animal 1-3) 2000 mg/kg 195 ± 6g; Females (animal 4-6) 2000 mg/kg 224 ± 15g
- Fasting period before study: Food was withheld overnight prior to dosing. Food was given approximately 2-4 hours after the second dosage.
- Housing: Group housing of 3 animals per cage in labelled Macrolon cages (MIV type; height 18 cm.) containing sterilised sawdust as bedding material (Woody-Clean type 3/4, Tecnilab-BMI BV, Someren, The Netheriands) and paper as cage-enrichment (Enviro-dri, Tecnilab-BMI BV, Someren, The Netherlands).
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20.7 - 22.3
- Humidity (%): 40 - 67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

other: 20% Ethylacetate in propylene glycol (Merck, Darmstadt, Germany) (specific gravity 1.009)

Details on oral exposure:

VEHICLE:
- Justification for choice of vehicle: The vehicle was selected based on trial formulations performed at NOTOX and on test substance data supplied by the sponsor.

MAXIMUM DOSE VOLUME APPLIED:
- Two administrations of formulations of the test substance at 10 mL/kg bw.

DOSAGE PREPARATION:
- The formulations (w/w) were prepared within 4 hours prior to dosing. Homogeneity was accomplished to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.

Doses:

2000 mg/kg body weight.
The dose level of 2000 mg/kg bw was reached by administration of two times 1000 mg/kg b.w. The first on t=0 and the second on t=3 hours.

No. of animals per sex per dose:

3/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality/Viability: Twice daily; Bodyweights: Days 1 (pre-administration), 8 and 15; Clinical signs: At periodic intervals on the day of dosing (day 1) and once daily thereafter, until day 15.
- Necropsy of survivors performed: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.

Statistics:

No statistical analysis was performed (The method used is not intended to allow the calculation of a precise LD50 value).

Sex:

female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: Hunched posture, piloerection, red feces and red staining of several body parts were noted among animals between days 1 and 3. Breathing rales were recorded in 1 animal at the last observation time point on day 1.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories B.V., The Netherlands
- Age at study initiation: 8 to 9 weeks
- Weight at study initiation: Males: 256.8 to 285.6 g, Females: 170.5 to 219.3 g
- Fasting period before study: no
- Housing: groups of 5 of the same sex
- Diet: ad libitum
- Water: e.g. ad libitum
- Acclimation period: 5 - 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C,
- Humidity (%): 30 - 70%
- Photoperiod (hrs dark / hrs light): hour fluorescent light / 12 hour dark cycle.

IN-LIFE DATES: From: Jan 31, 2013 To: March 27, 2013

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: flow-past, nose-only exposure system
- Method of holding animals in test chamber: restraint tubes
- Source and rate of air: 1.0 L/min
- System of generating particulates/aerosols: A dust aerosol was generated from the test item using a CR3020 rotating brush aerosol generator
connected to an AirVac TD110M. The aerosol generated was then discharged into the exposure chamber through a 63Ni charge neutralizer.
- Method of particle size determination: Mercer 7 stage cascade Impactor
- Temperature, humidity, pressure in air chamber: The temperature and relative humidity of the test atmosphere was measured continuously during
exposure using a calibrated device. The results were recorded manually and are reported in 30 minute intervals from the start of exposure.

TEST ATMOSPHERE
- Brief description of analytical method used: Gravimetric determination of aerosol concentration was performed twice (group 1) or six times
(groups 2 and 3) during exposure. The samples were collected on a Millipore®durapore filter, Type HVLP loaded in a 47 mm in-line stainless steel filter sampling device. The filters were weighed before and immediately after sampling using a calibrated balance. The test aerosol concentration was calculated from the amount of test item present on the filter and the sample volume.
- Samples taken from breathing zone: yes

TEST ATMOSPHERE : see table

CLASS METHOD (if applicable)
- Rationale for the selection of the starting concentration: Limit dose

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

0.5, 1 and 5 mg/L

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 weighing: Weighing on test days 1 (before exposure), 2, 4, 8 and 15 (before necropsy).
- Necropsy of survivors performed: yes
- Other examinations performed: clinical signs, body weight,organ weights, histopathology, other: Observations for viability were recorded once before exposure on the day of exposure (test day 1), three times during exposure, immediately and 1 h after exposure on test day 1 and twice daily during the observation period. Each animal was examined three times during exposure, immediately and 1 h after exposure on test day 1 and once daily during the observation period. Observations were detailed and carefully recorded using explicitly defined scales as appropriate. Only grossly abnormal signs were detectable during exposure as the animals were restrained in the exposure tubes.

Statistics:

No statistical analysis was performed.

Sex:

male/female

Dose descriptor:

LC0

Effect level:

1 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Sex:

male/female

Dose descriptor:

LC100

Effect level:

5 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Mortality:

All animals exposed to 5.0 mg/l air died within two hours after exposure start. All animals exposed to 0.52 or 1.0 mg/L air survived the scheduled observation period.

Clinical signs:

other: In one male and two females exposed to 5.0 mg/L air, clinical signs such as increased activity, tachypnea and reduced body temperature were recorded before their death during exposure; the remaining seven animals exposed to 5.0 mg/L air died during exposu

Body weight:

Between test days 1 and 2, slight body weight loss was noted in all animals exposed to
0.52 mg/L or 1.0 mg/L air. This effect persisted in two females exposed to 0.5 mg/L air as well
as in two females exposed to 1.0 mg/L air and in up to day 4 of treatment. From test day 4
onwards, these females showed normal body weight gain. In the remaining animals exposed to
0.52 or 1.0 mg/L air, normal body weight development was noted from day 2 of treatment
onwards.

Gross pathology:

After their spontaneous death during exposure, all animals exposed to 5.0 mg/L air showed reddish discolored and incompletely collapsed lungs at necropsy. Although a relation to the treatment with test item cannot be fully excluded (the substance is an orange pigment) - this finding is considered to be most likely
secondary to the spontaneous death of the animals. In animals exposed to 0.52 or 1.0 mg/L air there were no macroscopic findings at scheduled necropsy.

Interpretation of results:

Category 4 based on GHS criteria

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

discriminating conc.

Value:

1 mg/m³ air

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

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

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.1200 (Acute Dermal Toxicity)

Qualifier:

according to guideline

Guideline:

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

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
- Source: Charies River Deutschland, Sulzfeld, Germany.
- Age at study initiation: approx. 8 weeks old
- Weight at study initiation: mean: males 263 ± 6 g, females 188 ± 4 g
- Housing: Individually housed in labeled Macrolon cages (MIll type, height 18 cm.) containing sterilized sawdust as bedding material (Woody-Clean type 3/4; Tecnilab-BMI BV, Someren, The Netherlands) and paper as cage-enrichment (Enviro-dri, Tecnilab-BMI BV, Someren, The Netherlands).
- Diet: Free access to standard pelleted laboratory animal diet (from Altromin (code VRF 1), Lage, Germany).
- Water: Free access to tap water.
- Acclimation period: at least 5 days before start of treatment under laboratory conditions. During the acclimatization period the animals were group housed in Macrolon cages (MIV type).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): actual range 20.1-22.8
- Humidity (%): actual range 36-67
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Type of coverage:

occlusive

Vehicle:

other: 20% Ethylacetate in propylene glycol (Merck, Darmstadt, Germany) (specific gravity 1.009)

Details on dermal exposure:

TEST SITE
- Area of exposure: approx. 25 cm² for males and 18 cm² for females.
- % coverage: approx. 10% of the total body surface
- Type of wrap if used: The formulation was held in contact with the skin with a dressing, consisting of a surgical gauze patch (Surgy ID)(Laboratoires Stella s.a., Liege, Belgium), successively covered with aluminum foil and Coban elastic bandage (3M, St. Paul, Minnesota, U.SA.). A piece of Micropore tape (3M, St. Paul, Minnesota, U.SA.) was additionally used for fixation of the bandages in females only.

REMOVAL OF TEST SUBSTANCE
- Washing: tap water
- Time after start of exposure: 24 hours

VEHICLE
- Amount(s) applied (volume or weight with unit): 20 mL/kg body weight.

Duration of exposure:

24 hours

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5/sex/dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Mortality/Viability: twice daily; Body weights: Days 1 (pre-administration), 8 and 15; Clinical signs: At periodic intervals on the day of dosing (day 1) and once daily thereafter, until day 15.
- Necropsy of survivors performed: At the end of the observation period, all animals were sacrificed by oxygen/carbon dioxide procedure and subjected to necropsy. Descriptions of all internal macroscopic abnormalities were recorded.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Based on:

test mat.

Mortality:

No mortality occurred.

Clinical signs:

other: No signs of systemic toxicity were noted in the animals. Red staining of skin on several body parts, scales and/or scabs were seen on the treated skin of the animals during the observation period.

Gross pathology:

No abnormalities were found at macroscopic post mortem examination of the animals.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

discriminating dose

Value:

2 000 mg/kg bw

Additional information

Acute toxicity oral:

In a GLP compliant acute oral toxicity study, according to OECD guideline 423, the test substance was administered by oral gavage to two subsequent groups of three female Wistar rats at 2000 mg/kg body weight (Notox 2006). Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality occurred. Hunched posture, piloerection, red feces and red staining of several body parts were noted among animals between days 1 and 3. Breathing rales were recorded in 1 animal at the last observation time point on day 1. The body weight gain shown by the animals over the study period was considered to be normal. No abnormalities were found at macroscopic post mortem examination of the animals. The oral LD50 value of the test substance in Wistar rats was established to exceed 2000 mg/kg body weight.

Acute toxicity dermal:

In a GLP compliant study, according to OECD guideline 402, the acute dermal toxicity of the test substance, following administration to five Wistar rats of each sex by a single dermal application at 2000 mg/kg body weight for 24 hours, was assessed (Notox, 2006). Animals were subjected to daily observations and weekly determination of body weight. Macroscopic examination was performed after terminal sacrifice (day 15). No mortality occurred. No signs of systemic toxicity were noted in the animals. Red staining of skin on several body parts, scales and/or scabs were seen on the treated skin of the animals during the observation period. The body weight gain during the observation period was within the range expected for rats used in this type of study. No abnormalities were found at macroscopic post mortem examination of the animals. The dermal LD50 value of the test substance in Wistar rats was established to exceed 2000 mg/kg body weight.

Acute Inhalation:

Three groups of five male and five female albino rats [RccHanTM:WIST(SPF)] were exposed by nose-only, flow-past inhalation for four hours to the test item at gravimetrically determined mean concentrations of 5.0 mg/L air, 0.52 mg/L air and 1.0 mg/L air. The procedure followed OECD testing guideline 403 and GLP. All animals were observed for clinical signs and mortality during the inhalation exposure and the subsequent 14-day

observation period or until they were found dead. Body weights were recorded prior to exposure on test day 1, and during the observation period on test days 2, 4, 8 and 15 before necropsy. On test day 15 all animals were sacrificed and necropsied. Animals which died were necropsied as soon as they were found dead.

All animals exposed to 5.0 mg/l air died within two hours after exposure start. All animals exposed to 0.52 or 1.0 mg/L air survived the scheduled observation period. In one male and two females exposed to 5.0 mg/L air, clinical signs such as increased activity, tachypnea and reduced body temperature were recorded before their death during exposure; the remaining seven animals exposed to 5.0 mg/L air died during exposure without recording of clinical signs. In animals exposed to 0.52 or 1.0 mg/L air, clinical signs were mostly limited to the day of treatment and consisted of slightly decreased activity, slightly ruffled fur and tachypnea immediately and one hour after exposure. Animals exposed to 1.0 mg/L air additionally showed labored breathing. In single animals exposed to 0.52 mg/L air, slightly ruffled fur persisted until test days 2 or 3; in single animals exposed to 1.0 mg/L air, slightly ruffled fur and tachypnea persisted until test day 2. All animals exposed to 0.52 or 1.0 mg/L air did not show any clinical

signs from test day 4 until the end of the observation period.

Between test days 1 and 2, slight body weight loss was noted in all animals exposed to 0.52 mg/L or 1.0 mg/L air. This effect persisted in two females exposed to 0.52 mg/L air as well as in two females exposed to 1.0 mg/L air and in up to day 4 of treatment. From test day 4 onwards, these females showed normal body weight gain. In the remaining animals exposed to 0.52 or 1.0 mg/L air, normal body weight development was noted from day 2 of treatment onwards.

After their spontaneous death during exposure, all animals exposed to 5.0 mg/L air showed reddish discolored and incompletely collapsed lungs at necropsy. Although a relation to the treatment with test item cannot be fully excluded, this finding is considered to be most likely secondary to the spontaneous death of the animals. In animals exposed to 0.52 or 1.0 mg/L air there were no macroscopic findings at scheduled necropsy.

The LD50 for acute inhalation in rats is therefore higher than 1 mg/L, but lower than 5 mg/L.

Justification for classification or non-classification

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 or dermal toxicity under Regulation (EC) No. 1272/2008, as amended for the thirteenth time in Regulation (EC) No. 2018/1480. Since the LD50 for acute inhalation toxicity is lower than 5 mg/L, but higher than 1 mg/L, the substance needs to be classified for acute inhalation toxicity in GHS Category 4.