29H,31H-phthalocyanine

Endpoint summary

Description of key information

- Oral: LD50 > 2000 mg/kg bw

- Inhalation: LC50 > 2.046 mg/L air

- Dermal: LD50 > 5000 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:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

no individual data reported, only summaries

GLP compliance:

no

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Heliogengrün 9360

Species:

rat

Strain:

not specified

Sex:

not specified

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Doses:

10000 mg/kg bw

Control animals:

no

Details on study design:

14 day observation period

Sex:

not specified

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed.

Mortality:

none

Gross pathology:

no adverse findings at necropsy

Interpretation of results:

GHS criteria not met

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:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 423 (Acute Oral toxicity - Acute Toxic Class Method)

Deviations:

yes

Remarks:

Individual findings not reported; only group averages

GLP compliance:

no

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Heliogengrün 9140

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: no data
- Age at study initiation: no data
- Weight at study initiation: mean 147g
- Fasting period before study: 16 hours
- Housing: five rats per cage
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least three days, at maximum 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-23°C
- Humidity (%): 54-56%
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

other: 20% Lutrol in 0.5% aqueous carboxymethylcellulose

Details on oral exposure:

Application volume 31.6 mL/kg bw

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

10

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations daily, weighing weekly
- Necropsy of survivors performed: yes

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed

Mortality:

none

Clinical signs:

other: none

Gross pathology:

No adverse findings were observed at necropsy

Other findings:

Rats had soft stool.

Interpretation of results:

GHS criteria not met

Reference 3

Endpoint:

acute toxicity: oral

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 401 (Acute Oral Toxicity)

GLP compliance:

yes (incl. QA statement)

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Synonyms: Copper tri/tetrachlorophthalocyanine pigment
- Substance type: blue powder
- Analytical purity: >85%
- Lot/batch No.: M200058
- Stability under test conditions: guaranteed for 4 hours
- Storage condition of test material: darkness at approx. 20 °C in a fume cupboard
- Other: different CAS No. are existing: 29719-96-8, 68987-63-3, 16040-69-0, 27614-71-7

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann, Borchen, Germany
- Age at study initiation: 6-10 weeks
- Average weight at study initiation: males 189 g; females 178 g
- Fasting period before study: from ca. 16 h before to 3 - 4 h after treatment
- Housing: in fully air-conditioned rooms in macrolon cages (type 4) on soft wood granulate in groups of 5 animals
- Diet: ssniff (R) R/M-H (V 1534), ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +- 3 °C
- Humidity: 50 +- 20 %
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: gavage

Vehicle:

other: sesame oil

Details on oral exposure:

The animals received the compound as a 20 % suspension in sesame oil, the administration volume being 10 ml/kg bw.

Doses:

2000 mg/kg bw

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

The prepared test substance was administered by gavage to fasted animals at the stated dosage. The observation period following treatment lasted for 14 days. Symptoms were recorded twice every day (in the morning and in the afternoon), on weekends and public holidays only once. During this time the animals were weighed weekly. At the end of the observation period, the animals were killed by CO2 asphyxilation, dissected and examined for macroscopically visible changes.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Remarks on result:

other: no mortality occurred

Mortality:

No deaths occured during the whole study.

Clinical signs:

other: Bluish discoloured feces were observed after the administration of the test material. From day 4 until the end of the study no findings were observed.

Gross pathology:

No macroscopically visible changes were seen.

Table 1: Individual body weights of rats

		bodyweight (g) at day
animal no.	sex	1	8	15
1	f	176	196	220
2	f	176	199	207
3	f	178	207	216
4	f	180	200	207
5	f	178	216	203
1	m	187	263	294
2	m	189	261	298
3	m	191	259	295
4	m	182	228	268
5	m	197	258	286

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the limit test (accrding to OECD guideline 401) for acute toxicity after oral application, the LD50 for the test material is > 2000 mg/kg body weight for male and female rats.

Reference 4

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Remarks:

pre-GLP

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 100 %

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Firma Gassner, SPF breeding, Ottobrunn, Germany
- Weight at study initiation: males ca. 163 g; females ca. 145 g
- Diet: Altromin-R (Altrogge, Lage/L.), ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: water solution containing CMC

Details on oral exposure:

Concentration of the test material in vehicle:
- 30 % (6400 and 3200 mg/kg bw),
- 16 % (1600 mg/kg bw),
- 2 % (200 mg/kg bw)

Amount of test material applied per gavage:
- 21.3 ml/kg bw for 6400 mg/kg bw;
- 10.6 ml/kg bw for 3200 mg/kg bw.
- 10 ml/kg bw for 1600 mg/kg bw;
- 10 ml/kg bw for 200 mg/kg bw.

Doses:

200, 1600, 3200 and 6400 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: Animals were observed and examined for clinical signs of toxicity during the first hour following application, after 4 and 5 hours and further on day 1, 2, 5, 6, 7, 8, 9, 12, 13 and 14 after dosing.
- The body weights of the individual animals were gathered prior to application of the test material.
- Necropsy of survivors performed: Deceased animals and those sacrificed at the end of the observation period (on day 14 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 6 400 mg/kg bw

Mortality:

All animals survived, no mortality was observed.

Clinical signs:

other: Green feces were observed in all animals treated 24 hours after application of the test material. Dyspnea was observed in animals of the 1600 mg/kg bw group immediately after treatment, but was reversible.

Gross pathology:

No abnormality was found in the organs.

Interpretation of results:

GHS criteria not met

Reference 5

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- lot No.: 92.10.9
- purity: 99.04 %

Species:

rat

Strain:

Crj: CD(SD)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Source: Charles River Japan, Inc.
- Age at study initiation: 5 weeks
- Weight at study initation: 153 - 160g and 121 - 135g (male/female)
- Housing: 5 animals per cage
- Diet: laboratory animal solid feed (MF: Oriental Yeast Co., Ltd.).
- Water (ad libitum): tap water (filtered + ultraviolet radiation)
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 - 25
- Humidity (%): 40 - 70
- Air changes (per hr): approx. 12
- Photoperiod (hrs dark / hrs light): 12h each

Route of administration:

oral: gavage

Vehicle:

olive oil

Details on oral exposure:

VEHICLE
- Amount of vehicle (if gavage): 0.5 ml / 100g body weight
- Justification for choice of vehicle: The test substance is insoluble in water and suspendible in olive oil.

CLASS METHOD:
- Rationale for the selection of the starting dose: Limit dose as the test substance was expected to be non toxic.

Doses:

2000 mg /kg bw

No. of animals per sex per dose:

5

Control animals:

not specified

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: observations daily, weighing immediately before administration then 3, 7 and 14 days after administration
- Necropsy of survivors performed: yes

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 2 000 mg/kg bw

Remarks on result:

other: No mortality was observed.

Mortality:

No deaths occured in either sex during the observation period.

Clinical signs:

other: A greenish stool, that matched the color ofthe test substance, was observed in all rats of both sexes 1 day after administration. No other ahnormalities were found.

Gross pathology:

No abnormal findings were recorded.

Interpretation of results:

GHS criteria not met

Reference 6

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Remarks:

pre-GLP

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Analytical purity: ca. 98 %

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Average weight at study initiation: males: 180 g; females 160 g
- Fasting period before study: 15 to 20 hours prior to application
- Diet: Herilan MRH-Haltung (H. Eggersmann KG), ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: suspension containing 0.5 % CMC and 1-2 drops Cremophor EL

Details on oral exposure:

- Concentration of the test material in vehicle: 50 %
- Amount of test material applied per gavage: 10 ml/kg bw

Doses:

5000 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: Animals were observed and examined for clinical signs of toxicity during the first hour following application at 15 min, 30 min and 60 min, after 2, 4 and 5 hours and further on day 1, 2, 3, 6, 7, 8, 9, 10, 13 and 14 after dosing.
- The body weights of the individual animals were gathered prior to application of the test material and on day 3, 7 and 13 after dosing.
- Necropsy of survivors performed: Deceased animals and those sacrificed at the end of the observation period (on day 14 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

discriminating dose

Effect level:

> 5 000 mg/kg bw

Mortality:

All animals survived, no mortality was observed.

Clinical signs:

other: Dyspnea, apathy, green feces as well as a poor general state were observed.

Gross pathology:

Autopsy revealed no relevant findings.

Table 1: Mean body weight (g) of rats after oral application of the test substance

	Males	Females
Dose level [mg/kg bw]	5000	5000
Day 2-4	213	187
Day 7	237	198
Day 13	266	173

Interpretation of results:

GHS criteria not met

Reference 7

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Name of test material (as cited in study report): Heliogenblau MFA
- Analytical purity: ca. 100 %

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Average weight at study initiation: males: 220 g; females 180 g
- Fasting period before study: 15 to 20 hours prior to application
- Diet: Herilan MRH-Haltung (H. Eggersmann KG), ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

0.5% in water

Details on oral exposure:

- Concentration of the test material in vehicle: 50 %
- Amount of test material applied per gavage: 10 ml/kg bw

Doses:

5000 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: Animals were observed and examined for clinical signs of toxicity during the first hour following application at 15 min, 30 min and 60 min, after 2, 4 and 5 hours and further on day 1, 4, 5, 6, 7, 8, 11, 12, 13 and 14 after dosing.
- The body weights of the individual animals were gathered prior to application of the test material and on day 4, 7 and 13 after dosing.
- Necropsy of survivors performed: Deceased animals and those sacrificed at the end of the observation period (on day 14 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed.

Mortality:

All animals survived, no mortality was observed.

Clinical signs:

other: No findings were seen.

Gross pathology:

Autopsy revealed no relevant findings.

Table 1: Mean body weight (g) of rats after oral application of phthalocyanine

	Males	Females
Dose level [mg/kg bw]	5000	5000
Day 4	265	210
Day 7	279	213
Day 13	310	225

Interpretation of results:

GHS criteria not met

Reference 8

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Average weight at study initiation: males: 210 g; females 180 g
- Fasting period before study: 15 to 20 hours prior to application
- Diet: Herilan MRH-Haltung (H. Eggersmann KG), ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

other: suspension containing 0.5 % CMC

Details on oral exposure:

- Concentration of the test material in vehicle: 50 %
- Amount of test material applied per gavage: 10 ml/kg bw

Doses:

5000 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: Animals were observed and examined for clinical signs of toxicity during the first hour following application at 15 min, 30 min and 60 min, after 2, 4 and 5 hours and further on day 1, 2, 5, 6, 7, 8, 9, 12 and 13 after dosing.
- The body weights of the individual animals were gathered prior to application of the test material and on day 2, 7 and 13 after dosing.
- Necropsy of survivors performed: Deceased animals and those sacrificed at the end of the observation period (on day 14 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Based on:

test mat.

Remarks on result:

other: No mortality was observed.

Mortality:

All animals survived, no mortality was observed.

Clinical signs:

other: No findings were seen.

Gross pathology:

Autopsy revealed no relevant findings.

Table 1: Mean body weight (g) of rats after oral application of Heliogenblau MFA

	Males	Females
Dose level [mg/kg bw]	5000	5000
Day 2	233	194
Day 7	258	204
Day 13	280	212

Interpretation of results:

GHS criteria not met

Reference 9

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Limited reporting details

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

less reporting details on conditions and test material. Only 8 days observation

GLP compliance:

no

Test type:

acute toxic class method

Limit test:

no

Specific details on test material used for the study:

- Purity: 100%

Species:

rat

Strain:

other: CFE (RAC/SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Healthy young random bred rats of the CFE (RAC, SPF) strain purchased from the breeder were used for these experiments. The mean initial body weight was between 126 and 138 g. Before starting, the animals were acclimatized for at least 5 days in our laboratories to a constant room temperature of 22+/-1°C, relative humidity of 55+/-5% and 14 hours light/day. They were housed in groups of 5 in macrolon cages (size 3). A standard diet of Nafag and drinking water were given "ad libitum".

Route of administration:

oral: gavage

Vehicle:

other: 0.5% Carboxymethylcellulose in water

Details on oral exposure:

- Amount of vehicle (if gavage): 20 and 30 ml/kg bw

Doses:

5000 and 10000 mg/kg bw

No. of animals per sex per dose:

5 (at 5000 mg/kg bw)
2 and 3 (at 10000 mg/kg bw)

Control animals:

no

Details on study design:

Symptoms and mortality after administration were recorded during an observation period of 8 days.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 10 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

none

Other findings:

Blue staining of skin, feces, urine

Interpretation of results:

GHS criteria not met

Reference 10

Endpoint:

acute toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Deviations:

yes

Remarks:

; the study was conducted with mice (rat is the standard species, recommended in OECD guideline 401).

GLP compliance:

not specified

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Analytical purity: no data given
- Lot/batch No.: 3507

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: HAM/ICR
- Weight at study initiation: 15 - 22 g
- Fasting period before study: overnight before treatment

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

Concentration of the test material in vehicle:
- 40 % w/v suspension

- Amount of test material applied per gavage:
- 40 ml/kg bw for 16000 mg/kg bw

The control animals were treated with vehicle alone.

Doses:

16000 mg/kg bw

No. of animals per sex per dose:

5 animals per sex per dose.

Control animals:

yes

Details on study design:

During the observation period of 14 days, a record was kept of all signs of toxicity. All mice were killed terminally and examined macroscopically in attempt to identify any target organ.

Key result

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 16 000 mg/kg bw

Based on:

test mat.

Remarks on result:

not determinable due to absence of adverse toxic effects

Mortality:

There were no mortalities.

Clinical signs:

other: Signs of reaction to treatment were observed shortly after dosing in all treated mice and were pilo-erection and abnormal body carriage, (hunched posture) and abnormal gait (waddling). These findings were accompanied by lethargy (4 animals), ptosis (1 ani

Gross pathology:

Terminal autopsy revealed no findings.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the test for acute toxicity after oral application, the LD50 for the test material is > 16000 mg/kg body weight for male and female mice.

Reference 11

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: see 'Remark'

Remarks:

Meets generally accepted scientific standards, well documented study with the following restriction: In some cases in the highest dose group the application volume per animal slightly exceeded the volume of 2 ml/100 g bw, which is recommended in OECD guideline 401 as maximum application volume for aqueous solutions.

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 401 (Acute Oral Toxicity)

Principles of method if other than guideline:

BASF-Test:
In principle, the methods described in the OECD Guideline 401 were used. Young adult laboratory rats were purchased from breeder. Several groups of 5 rats per sex and dose were treated simultaneously by gavage with preparations of the test substance in suitable vehicle. The concentrations of these preparations were used to achieve comparable volumes per kg body weight. Group-wise documentation of clinical signs was performed over the 14 day study period. The LD50 value was estimated on the basis of the observed mortalities.

GLP compliance:

no

Remarks:

GLP was not compulsory at the time the study was conducted

Test type:

standard acute method

Limit test:

no

Specific details on test material used for the study:

- Analytical purity: 100 %

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Firma Gassner, Ottobrunn; SPF breeding
- Average weight at study initiation: males: ca. 170 g; females: ca. 145 g
- Diet: Altromin-R (Altrogge, Lage/L., Germany), ad libitum
- Water: ad libitum

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

Concentration of the test material in vehicle:
- 30 % (6400 and 3200 mg/kg bw)
- 16 % (1600 mg/kg bw),
- 2 % (200 mg/kg bw)

Amount of test material applied per gavage:
- 21.3 ml/kg bw for 6400 mg/kg bw;
- 10.6 ml/kg bw for 3200 mg/kg bw;
- 10.0 ml/kg bw for 1600 mg/kg bw;
- 10.0 ml/kg bw for 200 mg/kg bw

Doses:

200, 1600, 3200, 6400 mg/kg bw

No. of animals per sex per dose:

5 animals per sex per dose

Control animals:

no

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations: Animals were observed and examined for clinical signs of toxicity during the first hour following application, after 4 hours and further on day 1, 4, 5, 6, 7, 8, 11, 12, and 13 after dosing.
- The body weights of the individual animals were gathered prior to application of the test material.
- Necropsy of survivors performed: yes. Deceased animals and those sacrificed at the end of the observation period (on day 14 after dosing) were necropsied.

Sex:

male/female

Dose descriptor:

LD50

Effect level:

> 6 400 mg/kg bw

Mortality:

No mortality was observed at any dose group.

Clinical signs:

other: - 6400 - 3200 mg/kg bw: Ca. 4 hours after application the animals were found in a crouched down position with intermittent breath. On the following morning slight apathy,a crouched down position and intermittent breath were observed. The stool of the anim

Gross pathology:

Autopsy revealed no relevant findings.

Interpretation of results:

GHS criteria not met

Conclusions:

Under the conditions of the BASF test (similar to OECD guideline 401) for acute toxicity after oral application, the LD50 for the test material is > 6400 mg/kg body weight for male and female rats.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 2 000 mg/kg bw

Acute toxicity: via inhalation route

Link to relevant study records

Reference

Reference 1

Endpoint:

acute toxicity: inhalation

Type of information:

other: in silico

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Current OECD guidelines for acute inhalation toxicity assessments incorporate a limit test aerosol concentration for poorly soluble particles that are either known or expected to be virtually non-toxic (OECD, 2009). Depending upon regulatory requirements, the limit test concentration for aerosols can be as high as 5 mg/L (or the maximum attainable concentration) if the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is used.
During the acute dust exposure study (GLP, OECD 403), rats exposed to 5 mg/L of the test substance died of suffocation after agglutinated pigment blocked the airways, whereas all rats survived the 4h dust exposure at the low dose of 1 mg/L.
It is widely recognized that external exposure concentrations of airborne particulates are not directly equivalent to the amounts that are inhaled, deposited, and retained in airways of laboratory animals and humans due to major differences in airway anatomy and physiology. In line with this, with regard to dose adjustment, the ECHA R.8 document on “Characterisation of dose[concentration]-response for human health” (Version 2.1, 2012) suggests to account for differences in respiratory rates of the experimental animal (at rest) and the human as well as for the increased respiratory rate of a worker, when inhalation DNELs are derived.
Without factoring in these species differences, results from inhalation toxicity studies are not directly translatable to human health risk. Accordingly, the results from the acute inhalation study on rats alone are regarded as insufficient to derive a potential human health hazard posed by the test substance.
Therefore, additional computational approaches that incorporate species-specific anatomy, physiology, and the physics of aerosol transport and deposition were utilized to assess whether the results from the acute inhalation study is relevant for humans.
Focusing upon acute toxicity testing guidelines, the MPPD model was applied to provide a generic (not chemical-specific) respiratory dosimetry assessment of rats exposed in nose-only inhalation chambers for 4-hr to aerosols with a respirable particle size of 2.75 µm mass median aerosol diameter (MMAD) and a geometric standard deviation (GSD) of 1.0 at a limit test concentration of 5 mg/L – similar to the settings of the in vivo study and analog to the particle properties of the test item used. Particular emphasis was placed upon particle load and retention in individual airways to assess potential vulnerabilities for physical obstruction of bronchial and pulmonary airways in the rat due to high rates of local aerosol deposition. In addition, human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non-toxic materials.
As a result, the MPPD simulations indicate that the results from the high dose group of the acute dust exposure study (GLP, OECD 403) in rats are not relevant for resting or light exercising humans, since deviating internal exposure patterns of rat and humans were observed.
The major difference between both species is that 50% of the material is deposited in the head-region in humans (only 30% in rats) and can be blown or spit out. Moreover, at tracheobronchial and bronchial level, the rat receives ~5-10-fold higher dose/cm2 airway surface (when cumulative deposited mass is normalized to respective airway surface areas) than the human under resting nasal breathing ventilation conditions. The pulmonary region of humans receives a greater fraction of inhaled aerosol than the rat, though when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose.
As to the applicability of limit test concentrations for aerosols with presumed low toxicity in guideline acute inhalation toxicity studies with rats, the potential for airway obstruction which could lead to decreases in airflows and pulmonary function was assessed using a geometric analysis of MPPD simulations. Given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols during a 4-hr nose-only exposure to aerosols at 5 mg/L atmospheric concentrations, the size (diameter) of retained masses in each airway segment were compared with their corresponding airway diameter. In this analysis, which assumed that the total retained mass in each airway at the end of the 4-hr exposure formed one spherical particle, a significant a percentage of pulmonary airways and to a lesser extent bronchiolar airways may be at risk of occlusion and impairment of gas-exchange. Such a potential for physical impact on pulmonary function under limit test exposure conditions for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans.
Finally, in comparison to the rat, more material is deposited in the head regions in human. Second, the wider airways in human hamper fast agglutination of the test material and therefore obstruction of the airways. At last, a 4h-exposure of worker to such high concentrations is rather unlikely even in case of an accident. The suffocation at 5 mg/L as observed in rats is therefore not regarded as relevant for humans and a classification as acute cat. 4 H332 not justified.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

The Multiple Path Particle Dosimetry (MPPD) model was used to predict aerosol deposition patterns representative of a guideline limit test acute inhalation toxicity study in the Sprague Dawley rat. Specifically, a 4-hr nose-only inhalation exposure to generic aerosol particles having a mass median aerosol diameter (MMAD) of 2.75 μm and a geometric standard deviation (GSD) of 1.0 (i.e., monodisperse distribution), a unit density of 1 g/cm3, and an airborne concentration of 5000 mg/m3 (5 mg/L) was simulated. Human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non toxic materials.

GLP compliance:

no

Test type:

other: MPPD model

Limit test:

yes

Species:

rat

Strain:

Sprague-Dawley

Sex:

male

Details on test animals or test system and environmental conditions:

not applicable

Route of administration:

inhalation: dust

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.75 µm

Geometric standard deviation (GSD):

1

Duration of exposure:

4 h

Concentrations:

5000 mg/m3 (5 mg/L)

No. of animals per sex per dose:

not applicable

Control animals:

other: not applicable

Remarks on result:

other: not applicable for test type

Mortality:

not applicable

Clinical signs:

other: not applicable

Body weight:

not applicable

Gross pathology:

not applicable

Other findings:

Rat Simulations

Aerosol Deposition in the Rat:
Slightly over 50% of respirable 2.75 μm particles deposited in the head (~32%; nose through larynx/throat) and conducting airways in the chest (~20-22%; tracheobronchial, TB) regions in both the Symmetric and Asymmetric rat models (Figure 1). With the enhanced variability in airway branching and surface areas associated with respiratory bronchioles that branch from upper conducting airways, not just the deep lung, slightly greater pulmonary deposition occurred in the Asymmetric (~4.7%) vs. the Symmetric (~3.7%) rat model.
Physiology profiles for each breathing mode are summarized in Table 1.
In the Symmetric Sprague Dawley rat model, tracheobronchial airways cover the first 13 generations with the generation 1 representing the trachea. Eight additional generations represented the pulmonary or gas-exchange region for a total of 21 generations in the Symmetric Sprague Dawley rat model (Table 2). MPPD provides multiple choices for dose metrics that can be used to compare across exposure scenarios, aerosol characteristics, and species. Of these options, deposition fraction, deposited mass (μg/breath), deposited mass rate (μg/min), total deposited over a single 4-hr exposure (μg) and total deposited mass/airway surface area (μg/cm2) for each airway generation for the Symmetric Sprague Dawley rat model are summarized in Table 2. Most of the deposited aerosol mass was predicted to occur in the tracheobronchial vs. pulmonary region. While significant amounts of deposited aerosol mass were predicted to deposit in the first three generations of pulmonary airways, when corrected for airway surface areas, the pulmonary region received very little deposited dose in 4 hr (μg/cm2) in the pulmonary airways of the symmetric lung model.
For the Asymmetric Sprague Dawley rat model, tracheobronchial airways cover the first 28 generations (generation 1 representing the trachea) with pulmonary airways branching off or tracheobronchial airways from generation 8—28 with an additional 8 generations terminating each complete tracheobronchial airway for a total of 36 generations (Table 3). This more accurate representation of the monopodial pulmonary airway branching system of the rat provides insights into the heterogeneities in local deposited aerosol doses in the lung than with the symmetric model. The same deposited dose metrics summarized in Table 2 for the symmetric model are shown in Table 3 for the entire lung (all airways in each generation) of the asymmetric mode. Several-fold differences between lobes were predicted. Such variability and differences in airway deposition between lobes in the asymmetric model can uncover local airway deposited dose differences that are masked using the symmetric model assumption. Airway generation-specific deposited mass as well as deposited mass per airway surface area are shown graphically in Figure 4. A larger fraction of pulmonary airways was exposed to inhaled aerosols without penetrating as deep into the tracheobronchial region in the asymmetric (monopodial) lung model than would be predicted with the simpler, symmetric (bipodial) lung airway model. However, when normalized to airway surface area, the deposited doses (μg/cm2) in the pulmonary regions were similar between the two models.


Theoretical Distribution of Aerosol Deposition Within the Head Region of the Rat:
The MPPD model uses a simple empirical model to determine aerosol deposition in the head region (nose through larynx) of rats without differentiating local regions within the head that are common target sites for deposition and potential toxicity in inhalation studies due to their unique airway anatomy and breathing patterns. However, a recently published computational fluid particle dynamics (CFPD) model provided site-specific deposition patterns in the upper conducting airways of the Sprague Dawley rat with comparable aerosol exposures that can inform the results from the MPPD simulations as both the CFPD and MPPD models utilized the same source of CT-derived airway geometry data (Figure 5; Corley et al., 2021).
In the CFPD rat model, regional deposition fractions in the nasal vestibule (dry squamous epithelium); wet squamous, respiratory, transitional, and olfactory epithelium; and the nasopharynx and larynx regions were determined for a single breath of exposure to 2.72 μm MMAD aerosols at 4.03 mg/L air concentration (Figure 5). The main differences between the simulations were with the simplified one-dimensional airway geometry used in the empirical MPPD head model vs. the actual three-dimensional airway geometry used in the CFPD model and the respective breathing profiles used in each simulation (CFPD: 217 ml minute volume at 100 bpm; MPPD: 353 ml minute volume, 166 bpm). In addition, the use of an inhalability correction in MPPD which reduced the inhalable fraction to 0.79, but not with the CFPD simulation (no inhalability adjustment) was largely responsible for differences in total inhaled fractions deposited in the head regions between the two models (CFPD: 0.59 vs. MPPD: 0.32).
Regardless, estimated fractional deposition of inhalable aerosols within each head region of the CFPD model could still be used to normalize the total fraction deposited from the MMPD head simulations to corresponding theoretical regions within the Symmetric and Asymmetric Sprague Dawley rat models (Table 4). Thus, most inhalable aerosols from the MPPD simulations are predicted to deposit in the anterior most portions of the nose (nasal vestibule and wet squamous epithelium) followed by the most anterior portions of respiratory and transitional epithelium of the nasoturbinates and maxilloturbinates, and larynx. Of these tissues, the anterior portions of respiratory and transitional epithelium of the nasoturbinates and maxilloturbinates and the larynx are common target sites for inhaled materials that produce local inflammation, irritation, or cytotoxicity in the head region. Since the Symmetric and Asymmetric Sprague Dawley rat models utilized the same empirical head model, results for the two models were essentially the same.


Tracheobronchial and Pulmonary Airway Clearance in the Rat:
While cumulative deposited dose (total mass or mass per airway or regional surface areas) may be appropriate dose-metrics for short-term acute exposures, they are not appropriate for repeated or long-term exposures as particle clearance mechanisms can play a significant role in respiratory tissue exposures (Brown et al., 2005; Corley et al, 2021). The MPPD model includes equations for mucociliary clearance in the tracheobronchial airways and macrophage clearance for the pulmonary regions of the rat and human. No clearance model is currently available in MPPD for the head region of any species.
The effect of mucociliary clearance in the tracheobronchial region is the most dramatic as the only 22-27% of the cumulative deposited mass (87—93 mg) in the tracheobronchial region of the whole lung of the Symmetric and Asymmetric Sprague Dawley rat simulations were retained at the end of exposure (Table 5). Following the 4-hr exposure, the retained mass was rapidly cleared within the first 12 hr from the start of the exposures with <0.1% remaining after 7 days.
For the pulmonary region, the slow clearance rate by macrophages results in minimal clearance of the cumulative deposited dose both during the exposure and for up to 7 days postexposure leading a higher retained mass (9.94 —13.6 mg) in each model (Table 5). Even with the slow clearance, the retained dose per pulmonary airway surface area remains low, but persistent. Given the pulmonary lung burden of 16-20 mg and over 100 mg when the tracheobronchial region is included at the end of the 4-hr exposure, it is possible that particle overload could occur that reduces clearance rates at this aerosol concentration, potentially confounding results and ability to extrapolate to the human (Bevan et al., 2018).


Potential for Obstructive Disruption of Airway Function in the Rat:
While MPPD is not designed to specifically address the question of whether deposited aerosols can be of sufficient magnitude to alter local airflows and thus, airway function, the significant amounts of aerosol that deposit and are retained in individual bronchiolar and pulmonary regions of the Symmetric and Asymmetric Sprague Dawley rat simulations suggest that the potential for disruption in airflows and lung function are possible. To assess this possibility, the total mass of aerosol particles that were retained in each tracheobronchial and pulmonary airway at the end of the 4-hr exposure were assumed to coalesce into a single spherical mass whose dimensions (diameter) were then compared with each airway diameter. To be clear, MPPD has no equations describing the influence of particles on airflows included in the current mode versions. Thus, while significant deposition and accumulation certainly occurs in discrete regions within each airway over a 4-hr exposure especially in pulmonary regions with slower clearance rates, this comparative calculation from MPPD results represents a worst-case scenario.
Given this scenario, the ratios of the diameters of the total retained mass in each airway to the diameters of corresponding airways in the Symmetric and Asymmetric Sprague Dawley rat simulations were determined at the end of the 4-hr exposure. As the ratios for each airway generation (Symmetric model) or airway within each generation (Asymmetric model) approaches unity, the potential for airflow obstruction increases. The results of this analysis for each generation of the Symmetric Sprague Dawley rat model are all below 1.0 but several generations have ratios >0.5 indicating some airways may be especially vulnerable to altered airflows.
The Asymmetric Sprague Dawley rat model provides a significantly more detailed assessment as nearly 40,000 airway segments are specifically defined according to realistic measurements from high resolution CT scans of silicone lung casts where pulmonary airways begin branching off of bronchiolar airways as high as generation 8, not just the terminal bronchiolar region as assumed in the Symmetric Sprague Dawley rat model in MPPD (Table 3), leading to a greater number of small diameter airways being exposed to higher airway concentrations of aerosol than occurs in the deep lung. In this model, ratios of retained aerosol mass diameters to their corresponding airway diameters of the tracheobronchial and pulmonary regions show a significant number of airways with ratios >0.5 and in some cases, ratios significantly >1.0. For the tracheobronchial region, most airways (4925 or 59.1%) have ratios between 0.25 and 0.5. For ratios >0.5, 23% or 1,915 airways are between 0.5 and 1.0 with 6.2% of airways (517 airways) with ratios >1.0. For the pulmonary region, the number and percentages of airways are increased, with over 42% having ratios >0.5 which is consistent with their smaller airway diameters and the high numbers of airways that branch off from upper bronchiolar airways.
While this computational analysis of model simulations is worst-case, it does reveal that the total mass deposited and retained by many airways at the end of 4 hr of exposure can be significant and may potentially lead to obstructive losses in function that is not reflective of chemical-specific toxicity. The higher up the airway generations this occurs, the greater the effect on associated downstream pulmonary gas-exchange regions. There is experimental evidence that phenomenon occurs based upon histopathologic observations from rats exposed in acute 4-hr studies to 5 mg/L aerosol concentrations of materials with high hydrophobicity and greater likelihood to clump up rather than disperse on airway surfaces when deposited (Hofmann et al., 2018). The result from this hypothetical computational analysis from a biological and aerosol physics based mechanistic model like MPPD supports these experimental observations.


Species Comparisons:
For 2.75 μm-sized aerosols, only 79.2% of atmospheric aerosols are inhalable by the rat while 100% is respirable for the human. For nasal breathing under resting ventilation conditions, approximately 32% of respirable 2.75 μm aerosols deposit in the head of the rat vs. 50% in the human. For the tracheobronchial region, nearly twice the fractional deposition occurred in the rat (20-22%) vs. only 8% in the human. Overall, there is a generally greater fractional pulmonary deposition of 2.75 μm aerosols in the human (20-33%) than in the rat (3.7- 4.7%). There is also a shift toward significantly less deposition in the head and greater pulmonary deposition and deeper penetration into pulmonary airways following oral breathing in humans.
When cumulative deposited mass is normalized to respective airway surface areas, the rat receives ~5-10-fold higher dose/cm2 airway surface in the tracheobronchial regions than the human under resting nasal breathing ventilation conditions, This margin is reduced between species in the first 2-4 generations of the conducting airways for human nasal breathing under light exercise conditions. When the cumulative deposited mass per cm2 airway surface in the rat is compared with human oral breathing the differences between species is similar under resting conditions but light or even heavy exercise results in deposited mass per cm2 airway surface that are near or even exceeding the equivalent surface area normalized dose in the first 2-5 generations of bronchial airways.
Both species have very low cumulative deposited mass/cm2 airway surface in the pulmonary regions but what mass is deposited is very slowly cleared with over 87% retained over 7 days in the rat (asymmetric model) and over 97% retained in the human (all breathing and activity conditions). Mucociliary clearance is faster in the tracheobronchial airways of the rat than in the human with the time to clear at least 95% of retained mass in tracheobronchial airways is ~12 hr for rats vs. ~48-72 hr for humans depending upon breathing mode and activity pattern.
These comparisons between species are limited to the same exposure conditions for a generic aerosol to highlight the impact of species-specific anatomy and physiology on aerosol deposition. If toxicity in the respiratory airways of the rat was an outcome of this exposure scenario, MPPD can also be used to calculate the human equivalent exposure concentrations (HEC) for a variety of scenarios that result in the same target region delivered or, preferably, retained doses from the simulations of the rat exposure under bioassay conditions.

Table 2. Aerosol deposition by generation (all airways in each generation) in the Symmetric Sprague Dawley rat model (EPA v1.01) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration.

 

Generation	Structure	No. Airways	Surface Area (cm2)	Deposited Fraction	Deposited Mass (µg/Breath)	Deposited Mass Rate (µg/min)	Total Deposited Mass, 4-hr (µg)	Total Deposited Mass, 4- hr/Surface Area (µg/cm2)
1	Trachea	1	0.50	2.60E-04	0.0028	0.46	109.9	220.9
2	Main Bronchi	2	0.55	1.82E-02	0.1933	32.08	7,699.2	13,957.9
3	Bronchi(ole)	4	0.76	1.59E-02	0.1686	27.99	6,717.6	8,868.1
4	Bronchi(ole)	8	0.81	1.57E-02	0.1664	27.62	6,628.8	8,167.6
5	Bronchi(ole)	16	0.82	2.39E-02	0.2544	42.24	10,137.6	12,299.9
6	Bronchi(ole)	32	0.87	2.62E-02	0.2790	46.31	11,114.4	12,756.1
7	Bronchi(ole)	64	0.98	2.60E-02	0.2760	45.81	10,994.4	11,227.9
8	Bronchi(ole)	128	1.18	2.72E-02	0.2894	48.04	11,529.6	9,795.8
9	Bronchi(ole)	256	1.70	2.38E-02	0.2533	42.05	10,092.0	5,950.5
10	Bronchi(ole)	512	2.65	1.74E-02	0.1849	30.70	7,368.0	2,777.2
11	Bronchi(ole)	1024	4.28	1.11E-02	0.1177	19.54	4,689.6	1,095.4
12	Bronchi(ole)	2048	7.74	7.60E-03	0.0808	13.40	3,216.0	415.7
13	Bronchi(ole)	4096	12.59	6.76E-03	0.0718	11.93	2,863.2	227.4
14	Prox.AlvRegion	8,192	35.72	9.81E-03	0.1043	17.31	4,154.4	116.3
15	Prox.AlvRegion	16,384	60.06	1.88E-02	0.2002	33.24	7,977.6	132.8
16	DistalAlvRegion	32,768	90.96	8.26E-03	0.0878	14.58	3,499.2	38.5
17	DistalAlvRegion	65,536	169.70	0	0	0	0	0
18	DistalAlvRegion	131,072	330.80	0	0	0	0	0
19	DistalAlvRegion	262,144	670.10	0	0	0	0	0
20	DistalAlvRegion	524,288	1,132.00	0	0	0	0	0
21	DistalAlvRegion	1,048,576	2,478.00	0	0	0	0	0

 

 

Table 3. Aerosol deposition by generation in entire lung (all airways in each generation) in the Asymmetric Sprague Dawley rat model (EPA v1.01) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration.

 

Generation	Structure	TB Surface Area (cm2)	Pulmonary Surface Area (cm2)	Combined Surface Area (cm2)	Deposition Fraction	Deposited Mass (µg/Breath)	Deposited Mass Rate (µg/min)	Total Deposited Mass 4-hr (µg)	Total Dep Mass 4-hr (µg/cm2)
1	TB	0.37	0	0.37	2.23E-04	2.37E-03	0.39	94.4	252.73
2	TB	0.39	0	0.39	2.70E-02	0.2868	47.61	11426.1	29163.12
3	TB	0.58	0	0.58	2.14E-02	0.227	37.68	9043.7	15512.32
4	TB	0.57	0	0.57	1.22E-02	0.1296	21.51	5163.3	9009.36
5	TB	0.76	0	0.76	1.07E-02	0.1141	18.94	4545.7	5976.52
6	TB	0.76	0	0.76	9.57E-03	0.1017	16.88	4051.7	5365.11
7	TB	0.97	0	0.97	1.17E-02	0.1238	20.55	4932.2	5069.06
8	TB+Pulm	1.02	0.00	1.02	1.28E-02	0.1358	22.54	5410.3	5293.81
9	TB+Pulm	1.39	0.02	1.41	1.27E-02	0.1353	22.46	5390.4	3828.38
10	TB+Pulm	1.87	0.15	2.01	1.43E-02	0.1519	25.22	6051.7	3006.31
11	TB+Pulm	1.99	0.55	2.54	1.36E-02	0.1449	24.05	5772.8	2277.24
12	TB+Pulm	2.36	1.69	4.06	1.25E-02	0.1328	22.04	5290.8	1304.75
13	TB+Pulm	2.38	4.00	6.38	1.25E-02	0.133	22.08	5298.7	830.26
14	TB+Pulm	2.25	8.73	10.98	1.10E-02	0.1173	19.47	4673.2	425.61
15	TB+Pulm	2.14	18.09	20.23	1.07E-02	0.1135	18.84	4521.8	223.52
16	TB+Pulm	1.94	35.77	37.71	1.04E-02	0.1108	18.39	4414.3	117.06
17	TB+Pulm	1.60	68.51	70.11	9.25E-03	0.0984	16.33	3920.3	55.92
18	TB+Pulm	1.40	113.30	114.70	8.22E-03	0.0873	14.49	3478.0	30.32
19	TB+Pulm	1.13	168.80	169.90	7.34E-03	0.078	12.95	3107.5	18.29
20	TB+Pulm	0.81	201.20	202.00	6.08E-03	0.0646	10.72	2573.7	12.74
21	TB+Pulm	0.59	234.10	234.60	4.68E-03	0.0497	8.25	1980.0	8.44
22	TB+Pulm	0.32	240.10	240.40	3.81E-03	0.0405	6.72	1613.5	6.71
23	TB+Pulm	0.18	217.60	217.80	3.03E-03	0.0322	5.35	1282.8	5.89
24	TB+Pulm	0.08	204.10	204.20	2.39E-03	0.0254	4.22	1011.9	4.96
25	TB+Pulm	0.06	177.60	177.70	1.66E-03	0.0177	2.94	705.2	3.97
26	TB+Pulm	0.05	160.70	160.70	1.11E-03	0.0118	1.96	470.1	2.93
27	TB+Pulm	0.02	129.20	129.20	6.38E-04	6.78E-03	1.13	270.1	2.09
28	TB+Pulm	0.01	99.50	99.51	3.13E-04	3.33E-03	0.55	132.7	1.33
29	Pulmonary	0	65.94	65.94	1.47E-04	1.56E-03	0.26	62.2	0.94
30	Pulmonary	0	36.32	36.32	7.46E-05	7.93E-04	0.13	31.6	0.87
31	Pulmonary	0	20.11	20.11	3.77E-05	4.00E-04	0.07	15.9	0.79
32	Pulmonary	0	10.06	10.06	1.42E-05	1.51E-04	0.03	6.0	0.60
33	Pulmonary	0	5.59	5.59	3.65E-08	3.88E-07	0.00	0.0	0.00
34	Pulmonary	0	3.91	3.91	0	0	0	0.0	0
35	Pulmonary	0	1.12	1.12	0	0	0	0.0	0
36	Pulmonary	0	0.56	0.56	0	0	0	0.0	0

 

 

Table 4. Theoretical distributions of the fractions of deposited mass estimated for the head region of the rat MPPD simulations based upon published computational fluid particle dynamics (CFPD) simulations with comparable aerosols (Corley et al., 2001).

 

 

From Table 2, Corley et al. (2021)			Normalized Deposition in MPPD Head Region (EPA v1.01) Symmetric SD Rat				Normalized Deposition in MPPD Head Region (EPA v1.01) Asymmetric SD Rat
Airway Region	Surf. Area (cm2)	Dep. Fract.	Est. Dep. Fract.	Est. Dep Mass Rate (µg/Breath)	Est. Dep Mass Rate (µg/min)	Est. Total Dep Mass 4-hr (µg)	Est. Dep. Fract.	Est. Dep Mass Rate (µg/Breath)	Est. Dep Mass Rate (µg/min)	Est. Total Dep Mass 4-hr (µg)
Vestibule, Dry Squamous	0.45	0.5310	0.2900	3.08	511.6	122,786.0	0.2915	3.10	514.3	123,439.0
Wet Squamous	0.63	0.0483	0.0264	0.28	46.5	11,170.6	0.0265	0.28	46.8	11,230.0
Respiratory	5.69	0.0010	0.0005	0.01	0.9	227.0	0.0005	0.01	1.0	228.2
Transitional	2.16	0.0016	0.0009	0.01	1.6	372.4	0.0009	0.01	1.6	374.4
Olfactory	6.75	0.0002	0.0001	0.00	0.2	46.7	0.0001	0.00	0.2	46.9
Pharynx	1.32	0.0002	0.0001	0.00	0.2	44.0	0.0001	0.00	0.2	44.3
Larynx	0.38	0.0032	0.0017	0.02	3.1	737.2	0.0018	0.02	3.1	741.2
Total	17.39	0.5855	0.3197	3.3982	564.1	135,384.0	0.3214	3.4163	567.1	136,104.0

 

 

Table 5. Cumulative mass deposited and retained in the tracheobronchial and pulmonary regions of the Symmetric and Asymmetric Sprague Dawley rat models (EPA v1.01) at the end of a 4-hr exposure to 2.75 μm MMAD particles at 5 mg/L air concentration.

 

Region	SD Rat Model	Cumulative Mass Deposited in 4- hr (mg)	Retained Mass at 4-hr (mg)	Fraction Retained at 4- hr	Retained Mass at 7 days (mg)	Fraction Retained at 7 days
TB	Symmetric	93.2	20.3	0.22	0.0559	0.0006
Alveolar	Symmetric	15.6	15.6	1.00	9.94	0.9819
TB	Asymmetric	87	23.3	0.27	0.07	0.0008
Alveolar	Asymmetric	20	20	1.00	13.60	0.87

 

Interpretation of results:

study cannot be used for classification

Conclusions:

This analysis showed that a significant percentage of pulmonary airways and to a lesser extent, bronchiolar airways, may be at risk of occlusion and impairment of lung function and gas exchange in the rat. Such a potential for physical impact on lung function for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans based upon guideline acute toxicity studies in the rat at limit test concentrations.

Executive summary:

Rats are obligate nose breathers and with over 30% of inhalable aerosol depositing in the head region, anterior nasal tissues and laryngeal regions are likely to receive a significant amount of deposition based upon a computational fluid-particle dynamics (CFPD)-informed analysis of MPPD simulations. Given the smaller airways and higher breathing frequencies and minute volumes per unit body weight in rats vs. humans, greater amounts of aerosol mass are deposited in the tracheobronchial region in rats, while in humans, a greater mass is deposited in the pulmonary region. However, when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose. Mucociliary clearance rates are also slower in humans than in the rat and the time to clear tracheobronchial airways varies between ~48 and 72 hr depending upon breathing mode and activity level vs. ~12 hr in the rat. Both species have slow macrophage clearance rates in the pulmonary region with neither species clearing much material (<87-97% of deposited mass) by 7 days. However, given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols in individual airways, the potential for airway obstruction and resulting decreases in airflows and pulmonary function was assessed in individual airways using a geometric analysis of MPPD simulations. This analysis showed that a significant percentage of pulmonary airways and to a lesser extent, bronchiolar airways, may be at risk of occlusion and impairment of lung function and gas exchange in the rat.

Reference 2

Endpoint:

acute toxicity: inhalation

Type of information:

other: in silico

Adequacy of study:

weight of evidence

Study period:

2022

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Justification for type of information:

Current OECD guidelines for acute inhalation toxicity assessments incorporate a limit test aerosol concentration for poorly soluble particles that are either known or expected to be virtually non-toxic (OECD, 2009). Depending upon regulatory requirements, the limit test concentration for aerosols can be as high as 5 mg/L (or the maximum attainable concentration) if the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is used.
During the acute dust exposure study (GLP, OECD 403), rats exposed to 5 mg/L of the test substance died of suffocation after agglutinated pigment blocked the airways, whereas all rats survived the 4h dust exposure at the low dose of 1 mg/L.
It is widely recognized that external exposure concentrations of airborne particulates are not directly equivalent to the amounts that are inhaled, deposited, and retained in airways of laboratory animals and humans due to major differences in airway anatomy and physiology. In line with this, with regard to dose adjustment, the ECHA R.8 document on “Characterisation of dose[concentration]-response for human health” (Version 2.1, 2012) suggests to account for differences in respiratory rates of the experimental animal (at rest) and the human as well as for the increased respiratory rate of a worker, when inhalation DNELs are derived.
Without factoring in these species differences, results from inhalation toxicity studies are not directly translatable to human health risk. Accordingly, the results from the acute inhalation study on rats alone are regarded as insufficient to derive a potential human health hazard posed by the test substance.
Therefore, additional computational approaches that incorporate species-specific anatomy, physiology, and the physics of aerosol transport and deposition were utilized to assess whether the results from the acute inhalation study is relevant for humans.
Focusing upon acute toxicity testing guidelines, the MPPD model was applied to provide a generic (not chemical-specific) respiratory dosimetry assessment of rats exposed in nose-only inhalation chambers for 4-hr to aerosols with a respirable particle size of 2.75 µm mass median aerosol diameter (MMAD) and a geometric standard deviation (GSD) of 1.0 at a limit test concentration of 5 mg/L – similar to the settings of the in vivo study and analog to the particle properties of the test item used. Particular emphasis was placed upon particle load and retention in individual airways to assess potential vulnerabilities for physical obstruction of bronchial and pulmonary airways in the rat due to high rates of local aerosol deposition. In addition, human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non-toxic materials.
As a result, the MPPD simulations indicate that the results from the high dose group of the acute dust exposure study (GLP, OECD 403) in rats are not relevant for resting or light exercising humans, since deviating internal exposure patterns of rat and humans were observed.
The major difference between both species is that 50% of the material is deposited in the head-region in humans (only 30% in rats) and can be blown or spit out. Moreover, at tracheobronchial and bronchial level, the rat receives ~5-10-fold higher dose/cm2 airway surface (when cumulative deposited mass is normalized to respective airway surface areas) than the human under resting nasal breathing ventilation conditions. The pulmonary region of humans receives a greater fraction of inhaled aerosol than the rat, though when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose.
As to the applicability of limit test concentrations for aerosols with presumed low toxicity in guideline acute inhalation toxicity studies with rats, the potential for airway obstruction which could lead to decreases in airflows and pulmonary function was assessed using a geometric analysis of MPPD simulations. Given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols during a 4-hr nose-only exposure to aerosols at 5 mg/L atmospheric concentrations, the size (diameter) of retained masses in each airway segment were compared with their corresponding airway diameter. In this analysis, which assumed that the total retained mass in each airway at the end of the 4-hr exposure formed one spherical particle, a significant a percentage of pulmonary airways and to a lesser extent bronchiolar airways may be at risk of occlusion and impairment of gas-exchange. Such a potential for physical impact on pulmonary function under limit test exposure conditions for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans.
Finally, in comparison to the rat, more material is deposited in the head regions in human. Second, the wider airways in human hamper fast agglutination of the test material and therefore obstruction of the airways. At last, a 4h-exposure of worker to such high concentrations is rather unlikely even in case of an accident. The suffocation at 5 mg/L as observed in rats is therefore not regarded as relevant for humans and a classification as acute cat. 4 H332 not justified.

Reason / purpose for cross-reference:

reference to same study

Qualifier:

no guideline available

Principles of method if other than guideline:

The Multiple Path Particle Dosimetry (MPPD) model was used to predict aerosol deposition patterns representative of a guideline limit test acute inhalation toxicity study in the Sprague Dawley rat. Specifically, a 4-hr nose-only inhalation exposure to generic aerosol particles having a mass median aerosol diameter (MMAD) of 2.75 μm and a geometric standard deviation (GSD) of 1.0 (i.e., monodisperse distribution), a unit density of 1 g/cm3, and an airborne concentration of 5000 mg/m3 (5 mg/L) was simulated. Human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non toxic materials.

GLP compliance:

no

Test type:

other: MPPD model

Limit test:

yes

Species:

other: human

Sex:

male

Details on test animals or test system and environmental conditions:

not applicable

Route of administration:

inhalation: dust

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

2.75 µm

Geometric standard deviation (GSD):

1

Duration of exposure:

4 h

Concentrations:

5000 mg/m3 (5 mg/L)

No. of animals per sex per dose:

not applicable

Control animals:

other: not applicable

Remarks on result:

other: not applicable for test type

Mortality:

not applicable

Clinical signs:

other: not applicable

Body weight:

not applicable

Gross pathology:

not applicable

Other findings:

Human Simulations

Aerosol Dosimetry in the Human:
Following nasal breathing under resting conditions, 50.5% of the inhaled fraction of aerosols deposited in the head, while 8.4% deposited in the tracheobronchial and 19.6% in the pulmonary regions (79% total; Figure 9). Under light exercise conditions, the inhaled fraction of aerosols depositing in the head following nasal breathing increased from 50.5 to 80% while deposition correspondingly decreased in tracheobronchial (8.4 to 3.0%) and pulmonary regions (19.6 to 10.5%).
This is in contrast with oral breathing where under resting conditions only 3.1% deposited in the head, 13.2% deposited in the tracheobronchial and 30.8% deposited in the pulmonary regions (47.1% total; Figure 9). Under light exercise conditions, deposition in the head also increased for oral breathing (3.1 to 6.4%) with a corresponding decrease in deposition in tracheobronchial (13.2 to 8.6%) but little change in pulmonary regions (30.8 to 30.4%).
For oral breathing with normal nasal augmentation that commonly occurs under heavy exercise conditions, deposition of inhaled aerosols was 48% in the head, 6.6% in the tracheobronchial and 17.6% in pulmonary regions. Physiology profiles for each breathing mode are summarized in Table 1.
Similar in structural organization to the Symmetric Sprague Dawley rat model, the Symmetric Yeh and Schum human model consists of 17 generations of tracheobronchial airways (trachea is generation 1) with 7 generations of pulmonary airways appended to each terminal bronchiole airway for a total of 24 generations. Several potential dose-metrics for acute exposures, including deposition fractions, deposited mass, deposition rate and total deposited mass, number of particles and mass/cm2 airway surface by generation for each breathing modality in the Symmetric Yeh and Schum human model are summarized in Tables 6 to 10.
Deposition fractions and cumulative deposited mass per generation over 4-hr exposures were significantly greater in pulmonary than tracheobronchial regions for each breathing modality with a general shift towards deeper pulmonary airway generations during exercise and with oral vs. nasal breathing. However, with the large airway surfaces in the pulmonary region, the cumulative deposited mass in 4 hr per airway surface area remained significantly lower in pulmonary regions with the greatest cumulative surface area normalized dose occurring in the first few generations of tracheobronchial airways (Figure 12).

Theoretical Distribution of Aerosol Deposition Within the Head Region of the Human:
As with the rat, the theoretical distributions of airway deposition across cell type or anatomic region (Figure 5) in the empirical head region of the Symmetric Yeh and Schum MPPD simulations for resting oral and nasal breathing were calculated using published CFPD simulations and are summarized in Table 4b (Corley et al., 2021). Based upon these calculations, the respiratory epithelium and the larynx are predicted to receive the greatest deposited fractions and cumulative deposited masses of aerosols following nasal breathing vs. the mouth and larynx following oral breathing. While each of these regions have high rates of aerosol clearance (ICRP, 2015), depending upon the aerosol toxicity mode of action, the nasal respiratory epithelium (nasal breathing) and larynx (both nasal and oral breathing) could represent tissues of potential concern just based upon local deposited dose in the head region of MPPD following exposures to such high concentrations of aerosols of this size (5 mg/L, 2.75 μm) unless cell-specific sensitivity factors across this region are known and suggest otherwise.

Tracheobronchial and Pulmonary Airway Clearance in the Human:
Mucociliary clearance rates are typically slower in humans than in rats. The deposited masses retained in tracheobronchial airways following each breathing mode ranged from 53 to 81% of the cumulative amounts deposited during the 4 hr exposure with over 95% cleared by 72 hr and 97% by 7 days (Table 5). For the pulmonary airways, very little deposited mass was cleared during the 4- hr exposure with less than 5% cleared over 7 days for any breathing mode (Table 5).

Species Comparisons:
For 2.75 μm-sized aerosols, only 79.2% of atmospheric aerosols are inhalable by the rat while 100% is respirable for the human. For nasal breathing under resting ventilation conditions, approximately 32% of respirable 2.75 μm aerosols deposit in the head of the rat vs. 50% in the human. For the tracheobronchial region, nearly twice the fractional deposition occurred in the rat (20-22%) vs. only 8% in the human. Overall, there is a generally greater fractional pulmonary deposition of 2.75 μm aerosols in the human (20-33%) than in the rat (3.7- 4.7%). There is also a shift toward significantly less deposition in the head and greater pulmonary deposition and deeper penetration into pulmonary airways following oral breathing in humans.
When cumulative deposited mass is normalized to respective airway surface areas, the rat receives ~5-10-fold higher dose/cm2 airway surface in the tracheobronchial regions than the human under resting nasal breathing ventilation conditions, This margin is reduced between species in the first 2-4 generations of the conducting airways for human nasal breathing under light exercise conditions. When the cumulative deposited mass per cm2 airway surface in the rat is compared with human oral breathing the differences between species is similar under resting conditions but light or even heavy exercise results in deposited mass per cm2 airway surface that are near or even exceeding the equivalent surface area normalized dose in the first 2-5 generations of bronchial airways.
Both species have very low cumulative deposited mass/cm2 airway surface in the pulmonary regions but what mass is deposited is very slowly cleared with over 87% retained over 7 days in the rat (asymmetric model) and over 97% retained in the human (all breathing and activity conditions). Mucociliary clearance is faster in the tracheobronchial airways of the rat than in the human with the time to clear at least 95% of retained mass in tracheobronchial airways is ~12 hr for rats vs. ~48-72 hr for humans depending upon breathing mode and activity pattern.
These comparisons between species are limited to the same exposure conditions for a generic aerosol to highlight the impact of species-specific anatomy and physiology on aerosol deposition. If toxicity in the respiratory airways of the rat was an outcome of this exposure scenario, MPPD can also be used to calculate the human equivalent exposure concentrations (HEC) for a variety of scenarios that result in the same target region delivered or, preferably, retained doses from the simulations of the rat exposure under bioassay conditions.

Table 4. Theoretical distributions of the fractions of deposited mass estimated for the head region of the human MPPD simulations based upon published computational fluid particle dynamics (CFPD) simulations with comparable aerosols (Corley et al., 2001).

 

From Tables 4 and 5, Corley et al. (2021)			Normalized Deposition in MPPD Head Region (v3.041)
Nose Breathing			Nose Breathing (Resting)
Airway Region	Surf. Area (cm2)	Dep. Fract. (a)	Est. Dep. Fract.	Est. Dep Mass Rate (µg/Breath)	Est. Dep Mass Rate (µg/min)	Est. Total Dep Mass 4-hr (µg)
Vestibule	33.0	0.00034	0.0269	83.9	1,006.9	241,656.6
Respiratory	181.4	0.00359	0.2849	890.3	10,683.0	2,563,930.8
Olfactory	21.3	0.00033	0.0258	80.6	966.7	232,013.1
Pharynx	29.1	0.00048	0.0381	119.1	1,429.5	343,068.4
Larynx	33.0	0.00164	0.1297	405.3	4,863.9	1,167,331.1
Total	297.732	0.00637	0.5054	1,579.2	18,950.0	4,548,000.0
Oral Breathing			Oral Breathing
Mouth	77.8	0.00597	0.0226	70.58	846.9	203,263.1
Oropharynx	44.0	0.00070	0.0026	8.23	98.7	23,693.1
Larynx	33.9	0.00155	0.0059	18.36	220.3	52,883.8
Total	155.6	0.00822	0.0311	97.17	1,166.0	279,840.0

a) Interpolated from deposition fractions in airway regions for 2.75 μm aerosols from 1 and 3 μm aerosol simulations (Tables 4 and 5, Corley et al. (2021)).

 

 

Table 5. Cumulative mass deposited and retained in the tracheobronchial and pulmonary regions of the Symmetric Yeh and Schum human models (v3.041) for each breathing mode at the end of a 4-hr exposure to 2.75 μm MMAD particles at 5 mg/L air concentration.

 

Region	Breathing Mode	Cumulative Mass Deposited in 4- hr (mg)	Retained Mass at 4-hr (mg)	Fraction Retained at 4- hr	Retained Mass at 7 days (mg)	Fraction Retained at 7 days
TB	Nasal, Resting	754	613	0.81	11.3	0.015
TB	Nasal, Lt. Exercise	887	591	0.67	20.7	0.023
TB	Oral, Resting	1,186	963	0.81	17.8	0.015
TB	Oral, Lt. Exercise	2,569	1,710	0.67	60.0	0.023
TB	Oronasal, Heavy Exercise	3,928	2,090	0.53	71.3	0.018
Alveolar	Nasal, Resting	1,765	1,760	1.00	1,690	0.957
Alveolar	Nasal, Lt. Exercise	3,144	3,140	1.00	3,010	0.957
Alveolar	Oral, Resting	2,771	2,770	1.00	2,660	0.960
Alveolar	Oral, Lt. Exercise	9,115	9,110	1.00	8,720	0.957
Alveolar	Oronasal, Heavy Exercise	10,540	10,500	1.00	10,100	0.958

 

 

Table 6. Aerosol deposition by generation (all airways in each generation) in the Symmetric Yeh and Schum human model (v3.041) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration for nasal breathing under resting conditions.

 

Generation	Airway	Surface Area (cm2)	Dep Fraction	Dep Mass Rate (µg/Breath)	Dep Mass Rate (µg/min)	Dep Mass Rate (µg/min/cm2)	Total Dep Mass 4-hr (µg)	Total Dep Particles (no.)	Total Dep Mass 4- hr (µg/cm2)
1	TB	4.46E+01	0.0000	0.08	1.01	0.02	242.5	2.23E+07	5.44
2	TB	3.02E+01	0.0026	8.25	99.00	3.28	23,760.0	2.18E+09	787.02
3	TB	1.79E+01	0.0023	7.17	86.04	4.82	20,649.6	1.90E+09	1,156.19
4	TB	1.42E+01	0.0015	4.71	56.52	3.99	13,564.8	1.25E+09	957.29
5	TB	2.30E+01	0.0014	4.50	54.00	2.35	12,960.0	1.19E+09	563.48
6	TB	4.12E+01	0.0015	4.77	57.24	1.39	13,737.6	1.26E+09	333.68
7	TB	5.50E+01	0.0016	5.09	61.08	1.11	14,659.2	1.35E+09	266.58
8	TB	1.02E+02	0.0023	7.08	84.96	0.83	20,390.4	1.87E+09	200.10
9	TB	1.59E+02	0.0032	10.00	120.00	0.76	28,800.0	2.64E+09	181.59
10	TB	1.95E+02	0.0038	11.80	141.60	0.73	33,984.0	3.12E+09	174.46
11	TB	2.50E+02	0.0045	14.10	169.20	0.68	40,608.0	3.73E+09	162.30
12	TB	3.16E+02	0.0055	17.10	205.20	0.65	49,248.0	4.52E+09	155.75
13	TB	3.85E+02	0.0063	19.70	236.40	0.61	56,736.0	5.21E+09	147.33
14	TB	4.60E+02	0.0085	26.60	319.20	0.69	76,608.0	7.04E+09	166.54
15	TB	5.63E+02	0.0099	31.00	372.00	0.66	89,280.0	8.20E+09	158.66
16	TB	7.33E+02	0.0123	38.40	460.80	0.63	110,592.0	1.02E+10	150.88
17	TB	1.05E+03	0.0165	51.60	619.20	0.59	148,608.0	1.36E+10	141.26
18	Pulmonary	4.56E+03	0.0249	77.90	934.80	0.21	224,352.0	2.06E+10	49.25
19	Pulmonary	1.13E+04	0.0318	99.50	1194.00	0.11	286,560.0	2.63E+10	25.45
20	Pulmonary	3.54E+04	0.0436	136.00	1632.00	0.05	391,680.0	3.60E+10	11.06
21	Pulmonary	5.71E+04	0.0522	163.00	1956.00	0.03	469,440.0	4.31E+10	8.22
22	Pulmonary	1.00E+05	0.0416	130.00	1560.00	0.02	374,400.0	3.44E+10	3.73
23	Pulmonary	1.79E+05	0.0021	6.47	77.64	0.00	18,633.6	1.71E+09	0.10
24	Pulmonary	3.30E+05	0.0000	0.00	0.00	0.00	0.0	0.00E+00	0.00

 

 

Table 7. Aerosol deposition by generation (all airways in each generation) in the Symmetric Yeh and Schum human model (v3.041) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration for nasal breathing under light exercise conditions.

 

Generation	Airway	Surface Area (cm2)	Dep Fraction	Dep Mass Rate (µg/Breath)	Dep Mass Rate (µg/min)	Dep Mass Rate (µg/min/cm2)	Total Dep Mass 4- hr (µg)	Total Dep Particles (no.)	Total Dep Mass 4- hr (µg/cm2)
1	TB	4.46E+01	2.13E-06	0.01	0.27	0.01	63.8	5.86E+06	1.43
2	TB	3.02E+01	2.41E-03	15.10	302.00	10.00	72,480.0	6.66E+09	2,400.79
3	TB	1.79E+01	2.13E-03	13.30	266.00	14.89	63,840.0	5.86E+09	3,574.47
4	TB	1.42E+01	1.40E-03	8.75	175.00	12.35	42,000.0	3.86E+09	2,964.01
5	TB	2.30E+01	1.29E-03	8.09	161.80	7.03	38,832.0	3.57E+09	1,688.35
6	TB	4.12E+01	1.09E-03	6.82	136.40	3.31	32,736.0	3.01E+09	795.14
7	TB	5.50E+01	8.99E-04	5.62	112.40	2.04	26,976.0	2.48E+09	490.56
8	TB	1.02E+02	1.07E-03	6.66	133.20	1.31	31,968.0	2.94E+09	313.72
9	TB	1.59E+02	1.22E-03	7.60	152.00	0.96	36,480.0	3.35E+09	230.01
10	TB	1.95E+02	1.12E-03	6.98	139.60	0.72	33,504.0	3.08E+09	171.99
11	TB	2.50E+02	1.36E-03	8.52	170.40	0.68	40,896.0	3.76E+09	163.45
12	TB	3.16E+02	1.55E-03	9.71	194.20	0.61	46,608.0	4.28E+09	147.40
13	TB	3.85E+02	1.74E-03	10.90	218.00	0.57	52,320.0	4.80E+09	135.86
14	TB	4.60E+02	2.24E-03	14.00	280.00	0.61	67,200.0	6.17E+09	146.09
15	TB	5.63E+02	2.57E-03	16.10	322.00	0.57	77,280.0	7.10E+09	137.34
16	TB	7.33E+02	3.21E-03	20.10	402.00	0.55	96,480.0	8.86E+09	131.62
17	TB	1.05E+03	4.26E-03	26.60	532.00	0.51	127,680.0	1.17E+10	121.37
18	Pulmonary	4.56E+03	6.55E-03	40.90	818.00	0.18	196,320.0	1.80E+10	43.10
19	Pulmonary	1.13E+04	8.88E-03	55.50	1110.00	0.10	266,400.0	2.45E+10	23.66
20	Pulmonary	3.54E+04	1.35E-02	84.60	1692.00	0.05	406,080.0	3.73E+10	11.47
21	Pulmonary	5.71E+04	2.01E-02	126.00	2520.00	0.04	604,800.0	5.55E+10	10.59
22	Pulmonary	1.00E+05	2.77E-02	173.00	3460.00	0.03	830,400.0	7.63E+10	8.28
23	Pulmonary	1.79E+05	2.61E-02	163.00	3260.00	0.02	782,400.0	7.19E+10	4.37
24	Pulmonary	3.30E+05	1.93E-03	12.00	240.00	0.00	57,600.0	5.29E+09	0.17

 

 

Table 8. Aerosol deposition by generation (all airways in each generation) in the Symmetric Yeh and Schum human model (v3.041) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration for oral breathing under resting conditions.

 

Generation	Airway	Surface Area (cm2)	Dep Fraction	Dep Mass Rate (µg/Breath)	Dep Mass Rate (µg/min)	Dep Mass Rate (µg/min/cm2)	Total Dep Mass 4- hr (µg)	Total Dep Particles (no.)	Total Dep Mass 4- hr (µg/cm2)
1	TB	4.46E+01	4.24E-05	0.13	1.5888	0.04	381.312	3.50E+07	8.5
2	TB	3.02E+01	4.15E-03	13.00	156	5.17	37440	3.44E+09	1240.1
3	TB	1.79E+01	3.61E-03	11.30	135.6	7.59	32544	2.99E+09	1822.2
4	TB	1.42E+01	2.37E-03	7.41	88.92	6.28	21340.8	1.96E+09	1506.1
5	TB	2.30E+01	2.26E-03	7.07	84.84	3.69	20361.6	1.87E+09	885.3
6	TB	4.12E+01	2.40E-03	7.49	89.88	2.18	21571.2	1.98E+09	524.0
7	TB	5.50E+01	2.56E-03	7.99	95.88	1.74	23011.2	2.11E+09	418.5
8	TB	1.02E+02	3.56E-03	11.10	133.2	1.31	31968	2.94E+09	313.7
9	TB	1.59E+02	5.04E-03	15.70	188.4	1.19	45216	4.15E+09	285.1
10	TB	1.95E+02	5.93E-03	18.50	222	1.14	53280	4.89E+09	273.5
11	TB	2.50E+02	7.10E-03	22.20	266.4	1.06	63936	5.87E+09	255.5
12	TB	3.16E+02	8.62E-03	26.90	322.8	1.02	77472	7.11E+09	245.0
13	TB	3.85E+02	9.93E-03	31.00	372	0.97	89280	8.20E+09	231.8
14	TB	4.60E+02	1.34E-02	41.80	501.6	1.09	120384	1.11E+10	261.7
15	TB	5.63E+02	1.56E-02	48.70	584.4	1.04	140256	1.29E+10	249.3
16	TB	7.33E+02	1.93E-02	60.40	724.8	0.99	173952	1.60E+10	237.3
17	TB	1.05E+03	2.60E-02	81.10	973.2	0.93	233568	2.14E+10	222.0
18	Pulmonary	4.56E+03	3.92E-02	122.00	1464	0.32	351360	3.23E+10	77.1
19	Pulmonary	1.13E+04	5.00E-02	156.00	1872	0.17	449280	4.13E+10	39.9
20	Pulmonary	3.54E+04	6.84E-02	214.00	2568	0.07	616320	5.66E+10	17.4
21	Pulmonary	5.71E+04	8.20E-02	256.00	3072	0.05	737280	6.77E+10	12.9
22	Pulmonary	1.00E+05	6.54E-02	204.00	2448	0.02	587520	5.40E+10	5.9
23	Pulmonary	1.79E+05	3.26E-03	10.20	122.4	0.00	29376	2.70E+09	0.2
24	Pulmonary	3.30E+05	0.00E+00	0.00	0	0.00	0	0.00E+00	0.0

 

 

Table 9. Aerosol deposition by generation (all airways in each generation) in the Symmetric Yeh and Schum human model (v3.041) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration for oral breathing under light exercise conditions.

 

Generation	Airway	Surface Area (cm2)	Dep Fraction	Dep Mass Rate (µg/Breath)	Dep Mass Rate (µg/min)	Dep Mass Rate (µg/min/cm2)	Total Dep Mass 4-hr (µg)	Total Dep Particles (no.)	Total Dep Mass 4- hr (µg/cm2)
1	TB	4.46E+01	6.18E-06	0.04	0.77	0.02	185.3	1.70E+07	4.2
2	TB	3.02E+01	6.98E-03	43.6	872.0	28.88	209280.0	1.92E+10	6932.1
3	TB	1.79E+01	6.18E-03	38.6	772.0	43.23	185280.0	1.70E+10	10374.0
4	TB	1.42E+01	4.06E-03	25.4	508.0	35.85	121920.0	1.12E+10	8604.1
5	TB	2.30E+01	3.75E-03	23.4	468.0	20.35	112320.0	1.03E+10	4883.5
6	TB	4.12E+01	3.16E-03	19.8	396.0	9.62	95040.0	8.73E+09	2308.5
7	TB	5.50E+01	2.61E-03	16.3	326.0	5.93	78240.0	7.19E+09	1422.8
8	TB	1.02E+02	3.09E-03	19.3	386.0	3.79	92640.0	8.51E+09	909.1
9	TB	1.59E+02	3.52E-03	22.0	440.0	2.77	105600.0	9.70E+09	665.8
10	TB	1.95E+02	3.24E-03	20.2	404.0	2.07	96960.0	8.90E+09	497.7
11	TB	2.50E+02	3.95E-03	24.7	494.0	1.97	118560.0	1.09E+10	473.9
12	TB	3.16E+02	4.50E-03	28.1	562.0	1.78	134880.0	1.24E+10	426.6
13	TB	3.85E+02	5.03E-03	31.4	628.0	1.63	150720.0	1.38E+10	391.4
14	TB	4.60E+02	6.48E-03	40.5	810.0	1.76	194400.0	1.79E+10	422.6
15	TB	5.63E+02	7.45E-03	46.5	930.0	1.65	223200.0	2.05E+10	396.7
16	TB	7.33E+02	9.30E-03	58.2	1,164.0	1.59	279360.0	2.57E+10	381.1
17	TB	1.05E+03	1.23E-02	77.1	1,542.0	1.47	370080.0	3.40E+10	351.8
18	Pulmonary	4.56E+03	1.90E-02	119.0	2,380.0	0.52	571200.0	5.25E+10	125.4
19	Pulmonary	1.13E+04	2.57E-02	161.0	3,220.0	0.29	772800.0	7.10E+10	68.6
20	Pulmonary	3.54E+04	3.92E-02	245.0	4,900.0	0.14	1176000.0	1.08E+11	33.2
21	Pulmonary	5.71E+04	5.82E-02	364.0	7,280.0	0.13	1747200.0	1.60E+11	30.6
22	Pulmonary	1.00E+05	8.03E-02	502.0	10,040.0	0.10	2409600.0	2.21E+11	24.0
23	Pulmonary	1.79E+05	7.57E-02	473.0	9,460.0	0.05	2270400.0	2.08E+11	12.7
24	Pulmonary	3.30E+05	5.59E-03	34.9	698.0	0.00	167520.0	1.54E+10	0.5

 

 

Table 10. Aerosol deposition by generation (all airways in each generation) in the Symmetric Yeh and Schum human model (v3.041) following a 4-hr exposure to 2.75 µm MMAD particles at 5 mg/L air concentration for oronasal (normal augmenter) breathing under heavy exercise conditions.

 

Generation	Airway	Surface Area (cm2)	Dep Fraction	Dep Mass Rate (µg/Breath)	Dep Mass Rate (µg/min)	Dep Mass Rate (µg/min/cm2)	Total Dep Mass 4-hr (µg)	Total Dep Particles (no.)	Total Dep Mass 4- hr (µg/cm2)
1	TB	4.46E+01	2.84E-06	0.03	0.71	0.02	170.4	1.56E+07	3.8
2	TB	3.02E+01	7.81E-03	75.1	1952.60	64.68	468,624.0	4.30E+10	15,522.5
3	TB	1.79E+01	7.01E-03	67.4	1752.40	98.12	420,576.0	3.86E+10	23,548.5
4	TB	1.42E+01	4.92E-03	47.3	1229.80	86.79	295,152.0	2.71E+10	20,829.4
5	TB	2.30E+01	4.85E-03	46.6	1211.60	52.68	290,784.0	2.67E+10	12,642.8
6	TB	4.12E+01	3.91E-03	37.6	977.60	23.75	234,624.0	2.15E+10	5,698.9
7	TB	5.50E+01	2.70E-03	25.9	673.40	12.25	161,616.0	1.48E+10	2,939.0
8	TB	1.02E+02	3.10E-03	29.8	774.80	7.60	185,952.0	1.71E+10	1,824.8
9	TB	1.59E+02	2.97E-03	28.5	741.00	4.67	177,840.0	1.63E+10	1,121.3
10	TB	1.95E+02	2.15E-03	20.6	535.60	2.75	128,544.0	1.18E+10	659.9
11	TB	2.50E+02	2.62E-03	25.2	655.20	2.62	157,248.0	1.44E+10	628.5
12	TB	3.16E+02	2.78E-03	26.7	694.20	2.20	166,608.0	1.53E+10	526.9
13	TB	3.85E+02	2.93E-03	28.2	733.20	1.90	175,968.0	1.62E+10	456.9
14	TB	4.60E+02	3.53E-03	33.9	881.40	1.92	211,536.0	1.94E+10	459.9
15	TB	5.63E+02	3.84E-03	36.9	959.40	1.70	230,256.0	2.11E+10	409.2
16	TB	7.33E+02	4.66E-03	44.8	1164.80	1.59	279,552.0	2.57E+10	381.4
17	TB	1.05E+03	5.71E-03	54.9	1427.40	1.36	342,576.0	3.15E+10	325.6
18	Pulmonary	4.56E+03	8.33E-03	80.1	2082.60	0.46	499,824.0	4.59E+10	109.7
19	Pulmonary	1.13E+04	1.12E-02	108.0	2808.00	0.25	673,920.0	6.19E+10	59.9
20	Pulmonary	3.54E+04	1.73E-02	167.0	4342.00	0.12	1,042,080.0	9.57E+10	29.4
21	Pulmonary	5.71E+04	2.69E-02	258.0	6708.00	0.12	1,609,920.0	1.48E+11	28.2
22	Pulmonary	1.00E+05	4.09E-02	393.0	10218.00	0.10	2,452,320.0	2.25E+11	24.4
23	Pulmonary	1.79E+05	5.05E-02	486.0	12636.00	0.07	3,032,640.0	2.78E+11	17.0
24	Pulmonary	3.30E+05	2.05E-02	197.0	5122.00	0.02	1,229,280.0	1.13E+11	3.7

 

Interpretation of results:

study cannot be used for classification

Conclusions:

This analysis showed that a significant percentage of pulmonary airways and to a lesser extent, bronchiolar airways, may be at risk of occlusion and impairment of lung function and gas exchange in the rat. Such a potential for physical impact on lung function for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans based upon guideline acute toxicity studies in the rat at limit test concentrations.

Executive summary:

In general, greater deposition of aerosols occurs in the head region of humans following nasal than with oral breathing. However, oral breathing and increased activity (increased bpm and minute volume) can lead to deeper penetration of aerosols within the pulmonary region. Given the smaller airways and higher breathing frequencies and minute volumes per unit body weight in rats vs. humans, greater amounts of aerosol mass are deposited in the tracheobronchial region in rats, while in humans, a greater mass is deposited in the pulmonary region. However, when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose. Mucociliary clearance rates are also slower in humans than in the rat and the time to clear tracheobronchial airways varies between ~48 and 72 hr depending upon breathing mode and activity level vs. ~12 hr in the rat. Both species have slow macrophage clearance rates in the pulmonary region with neither species clearing much material (<87-97% of deposited mass) by 7 days.

Reference 3

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

July - Sept 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Current OECD guidelines for acute inhalation toxicity assessments incorporate a limit test aerosol concentration for poorly soluble particles that are either known or expected to be virtually non-toxic (OECD, 2009). Depending upon regulatory requirements, the limit test concentration for aerosols can be as high as 5 mg/L (or the maximum attainable concentration) if the United Nations (UN) Globally Harmonized System of Classification and Labelling of Chemicals (GHS) is used.
During the acute dust exposure study (GLP, OECD 403), rats exposed to 5 mg/L of the test substance died of suffocation after agglutinated pigment blocked the airways, whereas all rats survived the 4h dust exposure at the low dose of 1 mg/L.
It is widely recognized that external exposure concentrations of airborne particulates are not directly equivalent to the amounts that are inhaled, deposited, and retained in airways of laboratory animals and humans due to major differences in airway anatomy and physiology. In line with this, with regard to dose adjustment, the ECHA R.8 document on “Characterisation of dose[concentration]-response for human health” (Version 2.1, 2012) suggests to account for differences in respiratory rates of the experimental animal (at rest) and the human as well as for the increased respiratory rate of a worker, when inhalation DNELs are derived.
Without factoring in these species differences, results from inhalation toxicity studies are not directly translatable to human health risk. Accordingly, the results from the acute inhalation study on rats alone are regarded as insufficient to derive a potential human health hazard posed by the test substance.
Therefore, additional computational approaches that incorporate species-specific anatomy, physiology, and the physics of aerosol transport and deposition were utilized to assess whether the results from the acute inhalation study is relevant for humans.
Focusing upon acute toxicity testing guidelines, the MPPD model was applied to provide a generic (not chemical-specific) respiratory dosimetry assessment of rats exposed in nose-only inhalation chambers for 4-hr to aerosols with a respirable particle size of 2.75 µm mass median aerosol diameter (MMAD) and a geometric standard deviation (GSD) of 1.0 at a limit test concentration of 5 mg/L – similar to the settings of the in vivo study and analog to the particle properties of the test item used. Particular emphasis was placed upon particle load and retention in individual airways to assess potential vulnerabilities for physical obstruction of bronchial and pulmonary airways in the rat due to high rates of local aerosol deposition. In addition, human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non-toxic materials.
As a result, the MPPD simulations indicate that the results from the high dose group of the acute dust exposure study (GLP, OECD 403) in rats are not relevant for resting or light exercising humans, since deviating internal exposure patterns of rat and humans were observed.
The major difference between both species is that 50% of the material is deposited in the head-region in humans (only 30% in rats) and can be blown or spit out. Moreover, at tracheobronchial and bronchial level, the rat receives ~5-10-fold higher dose/cm2 airway surface (when cumulative deposited mass is normalized to respective airway surface areas) than the human under resting nasal breathing ventilation conditions. The pulmonary region of humans receives a greater fraction of inhaled aerosol than the rat, though when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose.
As to the applicability of limit test concentrations for aerosols with presumed low toxicity in guideline acute inhalation toxicity studies with rats, the potential for airway obstruction which could lead to decreases in airflows and pulmonary function was assessed using a geometric analysis of MPPD simulations. Given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols during a 4-hr nose-only exposure to aerosols at 5 mg/L atmospheric concentrations, the size (diameter) of retained masses in each airway segment were compared with their corresponding airway diameter. In this analysis, which assumed that the total retained mass in each airway at the end of the 4-hr exposure formed one spherical particle, a significant a percentage of pulmonary airways and to a lesser extent bronchiolar airways may be at risk of occlusion and impairment of gas-exchange. Such a potential for physical impact on pulmonary function under limit test exposure conditions for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans.
Finally, in comparison to the rat, more material is deposited in the head regions in human. Second, the wider airways in human hamper fast agglutination of the test material and therefore obstruction of the airways. At last, a 4h-exposure of worker to such high concentrations is rather unlikely even in case of an accident. The suffocation at 5 mg/L as observed in rats is therefore not regarded as relevant for humans and a classification as acute cat. 4 H332 not justified.

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

Specific details on test material used for the study:

purity > 99%
Expiry date: 22 Feb 2031
Storage: Room temperature
physical appearance: solid/blue

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: on study day 0: male animals 54 – 64 days, female animals 54 – 80 days
- Weight at study initiation: animals of comparable weight (+/- 20% of the mean body weight)
- Fasting period before study: no
- Housing: single houing or up to 5 animals
- Diet: Kliba laboratory diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20–24 °C
- Humidity: 45-65 %
- Air changes: 15 changes per hour
- Photoperiod: 12 hours light / 12 hours dark

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

> 1.9 - < 3 µm

Geometric standard deviation (GSD):

> 2.6 - < 3.2

Remark on MMAD/GSD:

Two samples were analyzed.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: For each test group the dusts were produced inside the inhalation system with a brush dust generator and compressed air and passed into the inhalation system. The concentrations were adjusted by varying the apertural width rotation of the dosing wheel of the dust generator.
- Exposure chamber volume: 34 L
- Method of holding animals in test chamber: restraining tubes
- Source and rate of air: central air conditioning system,1.5 m³/h
- Method of conditioning air: Central air conditioning system provided cold air of about 15°C. This cold air passed through an activated charcoal filter, adjusted to room temperature of 20 to 24°C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The test item was stirred in its container before a sample for dust generation was taken. The test item was desagglomerated in a mixer (mixing for 10 seconds) before introduction into the dust generator via the dosing wheel (Gericke/BASF).
- Method of particle size determination: Stack Sampler Mark III (Andersen). Before sampling, impactor stages were assembled with preweighed glass fiber collecting discs, and equipped with a backup particle filter. The impactor was connected to a vacuum pump, and for each test group samples were taken from the breathing zone of the animals. Sampling occurred 30 minutes (or later) after the beginning of the exposure.
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric, filtration equipment with probe, internal diameter: 7 mm, Sampling velocity 1.25 m/s, 4 samples at about hourly intervals, Sampling Volume 15L (low dose) and 6L (high dose)
- Samples taken from breathing zone: yes



VEHICLE
none apart from air


TEST ATMOSPHERE:
- Particle size distribution: Based on cascade impactor measurements. The calculation of particle size distribution was carried out by means of mathematical methods for evaluating particle measurements.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

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 observations and weighing: Individual body weight was determined once during the acclimatization period, at the start of the exposure period (Day 0) and at least on Days 1, 3 and 7, and weekly thereafter, or before the sacrifice of the animals at the end of the observation period.
- Clinical observations were recorded for each animal before exposure, separately several times during exposure (usually hourly) and after exposure. At least once daily on the preexposure day and during the post exposure observation period.
- Necropsy of survivors performed: yes

Pathology: At the end of the observation period the surviving animals were sacrificed with CO2-inhalation in a chamber with increasing concentration over time and were subjected to gross pathological examination as well as the animal which died before. To clarify the gross pathological findings, selected organs of individual animals were examined histopathologically.

Statistics:

LC50 values were calculated for males, females and both sexes combined using a binomial test.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 1.084 - < 5.212 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: High dose animals suffocated because the particles blocked the airways.

Mortality:

5.212 mg/L: All of the male and female animals died. Lethality was observed either during exposure, after exposure on study day 0 or on study day 1 or study day 2.
1.084 mg/L: All animals survived.

Clinical signs:

irregular respiration

Body weight:

1.084 mg/L: The mean body weights of the animals decreased on the first post-exposure observation day but increased thereafter.
5.212 mg/L: The mean body weights of the surviving animals decreased during the first post-exposure observation days. No further body weight data were available for the animals because all animals died.

Gross pathology:

1.084 mg/L: No gross pathological findings were noted during the necropsy of the animals at the termination of the post-exposure observation period.
5.212 mg/L: During necropsy of the dead five male and five female animals, many black foci were seen in the lung lobes with sunken surface. Blue discoloration of content of the stomach was seen in four males and three females and blue depositions in the trachea were present in four males and two females.

Other findings:

low dose (1 mg/L)
Clinical signs of toxicity in animals exposed to 1.084 mg/L comprised accelerated respiration, intermittent respiration, abdominal respiration, respiration sounds, feces substance like discolored, piloerection, substance-discolored fur and substance-contaminated fur. Findings were observed in the males from hour 1 of exposure through study day 11. No findings were detected in the male animals during the post-exposure observation period from study day 12 onwards. Findings were observed in the females from hour 1 of exposure until the end of the post-exposure observation period.

high dose (5 mg/L)
Clinical signs of toxicity in animals exposed to 5.212 mg/L comprised accelerated respiration, depressed respiration, abdominal respiration, no feces, activity: attention reduced, piloerection, substance-discolored fur and substance-contaminated fur. Findings were observed from hour 1 of exposure until the death of the animals. The mean body weights of the surviving animals decreased during the first post-exposure observation days. For further evaluation, histopathological examinations of the respiratory tract (nasal cavity, larynx, pharynx, trachea, and lung) from three animals were performed. The lung showed large amount of blue pigment within the bronchi, bronchioles and terminal bronchioles, leading to obstruction of the airways in two animals with one of them also showing emphysema. The trachea of all examined animals was dilated and contained blue pigment. The larynx at level I - II showed obstruction by blue pigment and large amounts of blue pigment at level III in 2 animals. The third animal presented with large amounts of blue pigment at larynx level I-II and small amounts at the third level. The nasal cavity at level I - IV contained small to moderate amounts of blue pigment.
The histopathological findings in the lung, the trachea, and the larynx of animal Nos. 793, 798 and 800 indicate an airway obstruction caused by the inhaled blue pigment as cause of death.

Table 1: Clinical signs and duration in the low concentration group

Test group 1 (1.084 mg/L)	Male animals	Female animals
Feces, discolored feces,substance like	d1	d1
Fur, discolored, substance like	d2 – d11	d2 – d14
Fur, piloerection	d0 – d1	d0 – d1
Fur, substance-contaminated	d0 – d1	d0 – d1
Respiration, abdominal	-	d0 – d1
Respiration, accelerated	h1 – d4	h1 – d4
Respiration, intermittent	d5 – d6	d5 – d6
Respiration, sounds	d2 – d4	d2 – d4

No findings were detected in the male animals during the post-exposure observation period from study day 12 onwards.

Table 2: Clinical signs and duration, high concentration group

Test group 2 (5.212 mg/L)	Male animals	Female animals
Lethality (number of animals)	5 / 5	5 / 5
Activity/behavior, attention reduced	d0	d0 – d1
Feces, no feces	-	d1
Fur, discolored, substance like	d0	d0 – d1
Fur, piloerection	d0	d0 – d1
Fur, substance-contaminated	d0	d0
Respiration, abdominal	d0	d0 – d1
Respiration, accelerated	h1 – h2	h1 – h2
Respiration, depressed	h3 – h4	h3 – h4

Table 3: Necropsy findings in animals that died during the study period

Findings	high concentration
Number of animals	5 males + 5 females
Lung: many black foci in all lobes (Æ4 mm), surface sunken	1 male + 3 females
Lung: many black foci in all lobes (Æ8 mm), surface sunken	4 males + 2 females
Stomach: blue discoloration of the content	4 males + 2 females
Trachea: blue deposition	4 males + 2 females

Table 4: Necropsy findings of animals at termination of the post exposure period

Findings	low concentration
Number of animals	5 males + 5 females
Organs without particular findings	5 males + 5 females

Table 5: Histopathology findings

	Animal No.:
	793	798	800
Lung
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment	x	-	-
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment, which obstructs the lumen	-	x	-
Bronchi, bronchioles and terminal bronchioles contain large amount of blue pigment, which obstructs the lumen, emphysema	-	-	x
Trachea
Dilation, contains blue pigment	x	x	x
Larynx
Level I Obstructed by blue pigment	x	-	x
Level I Contains large amounts of blue pigment	-	x	-
Level II Obstructed by blue pigment	x	-	x
Level II Contains large amounts of blue pigment, edema	-	x	-
Level III Contains large amounts of blue pigment	x	-	x
Level III Contains small amounts of blue pigment	-	x	-
Nasalcavity
Level IContains small amounts of blue pigment	x	x	x
Level II Contains small amounts of blue pigment	x	-	x
Level II Contains moderate amounts of blue pigment		x
Level III Contains small amounts of blue pigment	x	x	x
Level IV Contains small amounts of blue pigment	x	x	x

Interpretation of results:

Category 4 based on GHS criteria

Reference 4

Endpoint:

acute toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

January - March 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 403 (Acute Inhalation Toxicity)

Deviations:

yes

Remarks:

Low relative humidity in exposure chamber during 4h exposure

GLP compliance:

yes (incl. QA statement)

Test type:

acute toxic class method

Limit test:

yes

Specific details on test material used for the study:

Pigment Blue 16
Lot. 130004P040
Solid / blue
Expiry date: 14 Feb 2023
Storage conditions:Room temperature

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH,
- Age at study initiation: on study day 0: (male animals 58 - 62 days, female animals 73 - 77 days)
- Weight at study initiation: animals of comparable weight (+/- 20% of the mean body weight)
- Fasting period before study: no
- Housing: single houing or up to 5 animals
- Diet: Kliba laboratory diet, ad libitum
- Water: tap water, ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20–24 °C
- Humidity: 30-65 %
- Air changes: 15 changes per hour
- Photoperiod: 12 hours light / 12 hours dark

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 2.58 - <= 2.67 µm

Geometric standard deviation (GSD):

>= 3.12 - <= 3.49

Remark on MMAD/GSD:

Two samples were analyzed.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: For each test group the dusts were produced inside the inhalation system with a dust generator and compressed air and passed into the inhalation system. The concentrations were adjusted by varying the apertural width rotation of the dosing wheel of the dust generator.
- Exposure chamber volume: 34 L
- Method of holding animals in test chamber: restraining tubes
- Source and rate of air: central air conditioning system,1.5 m³/h
- Method of conditioning air: Central air conditioning system provided cold air of about 15°C. This cold air passed through an activated charcoal filter, adjusted to room temperature of 20 to 24°C and passed through a second particle filter (H13 (HEPA) Camfil Farr, Germany). The so generated conditioned air was used to generate inhalation atmospheres.
- System of generating particulates/aerosols: The test item was stirred in its container before a sample for dust generation was taken. The test item was desagglomerated in a mixer (mixing for 10 seconds) before introduction into the dust generator via the dosing wheel (Gericke/BASF).
- Method of particle size determination: Stack Sampler Mark III (Andersen). Before sampling, impactor stages were assembled with preweighed glass fiber collecting discs, and equipped with a backup particle filter. The impactor was connected to a vacuum pump, and for each test group samples were taken from the breathing zone of the animals. Sampling occurred 30 minutes (or later) after the beginning of the exposure.
- Treatment of exhaust air: The exhaust air was filtered and conducted into the exhaust air of the building.

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric, filtration equipment with probe, internal diameter: 7 mm, Sampling Volume 15L, 4 samples at about hourly intervals
- Samples taken from breathing zone: yes

VEHICLE
none apart from air


TEST ATMOSPHERE:
- Particle size distribution: Based on cascade impactor measurements. The calculation of particle size distribution was carried out by means of mathematical methods for evaluating particle measurements.

Analytical verification of test atmosphere concentrations:

yes

Duration of exposure:

4 h

Concentrations:

7.3 mg/L (nominal)
The actual measured concentration was 2.046 mg/L

No. of animals per sex per dose:

5 rats

Control animals:

yes

Details on study design:

- Duration of observation period following administration: 14 days
- Frequency of observations and weighing: Individual body weight was determined once during the acclimatization period, at the start of the exposure period (Day 0) and at least on Days 1, 3 and 7, and weekly thereafter, or before the sacrifice of the animals at the end of the observation period.
- Clinical observations were recorded for each animal before exposure, separately several times during exposure (usually hourly) and after exposure. At least once daily on the preexposure day and during the post exposure observation period.
- Necropsy of survivors performed: yes

Pathology: At the end of the observation period the surviving animals were sacrificed with CO2-inhalation in a chamber with increasing concentration over time and were subjected to gross pathological examination as well as the animal which died before. To clarify the gross pathological findings, selected organs of individual animals were examined histopathologically.


Selection of target concentration:
The target concentration of 2 mg/L was tested. The concentration of 2 mg/L was chosen following the recommendation of OECD guidance document No. 39 (OECD guidance document on inhalation toxicity studies. Series on testing and assessment No. 39 (second edtion). 6 July 2018)
1) Paragraph 8 “Testing in GHS category 5 is generally discouraged and should only be considered when there is a strong likelihood that results of such a test would have direct relevance for protecting human health (see APPENDIX II of the OECD guidance document No. 39).”
2) Paragraph 70 “Testing aerosols, for acute toxicity, the primary goal should be to achieve a respirable particle size (MMAD equal to or less than 2 micrometer). This is possible with most test chemicals at a
concentration of 2 mg/L.”
The measured MMAD was 2.58 - 2.67 µm.

Statistics:

LC50 values were calculated for males, females and both sexes combined using a binomial test.

Sex:

male/female

Dose descriptor:

LC50

Effect level:

> 2.046 mg/L air (analytical)

Based on:

test mat.

Exp. duration:

4 h

Remarks on result:

other: 1 of 10 animals died. Relative humidity 7.2%

Remarks:

Death was observed in the 2nd hour of exposure.

Mortality:

No lethality was observed in the five females. One of five male animals died on study day 0 during exposure

Clinical signs:

irregular respiration

Body weight:

The mean body weights of the animals decreased on the first post exposure observation days but increased thereafter.

Gross pathology:

Clinical signs of toxicity in animals comprised abdominal and intermittent respiration,
respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur
substance like. Findings were observed from hour 1 of exposure until the end of the 14-day
post-exposure observation period. The mean body weights of the surviving animals decreased
on the first post-exposure observation day but increased thereafter. This is a typical finding for
this test design. Gross necropsy of the male animal that died on study day 0 showed blue
discoloration of the fur on the entire body. During necropsy of the surviving animals at the
termination of the post-exposure observation period the following gross pathological findings
were noted: many blue foci in all lung lobes, grey/blue discoloration of the mediastinal lymph
nodes and blue discoloration of the fur of the tail and/or head region.

Other findings:

Clinical signs of toxicity in animals comprised abdominal and intermittent respiration, respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur substance like. Findings were observed from hour 1 of exposure until the end of the 14-day post-exposure observation period.

To further evaluate the macroscopic findings, histopathological examination was carried out of the respiration tract from the male animal No. 721 which died on study day 0. Minimal amount of green-blue crystals revealing the presence of the test substance were seen in the lumen of the larynx, pharynx and nasal cavity. Furthermore, in the larynx, minimal grey granular material in the lumen and a slight edema in the region of the ventral pouch were noted. All these findings are regarded as treatment-related, however they are not considered sufficiently severe to explain the death of this animal.

Sample	MMAD (µm)	Geometrical standard deviation
1	2.67	3.49
2	2.58	3.12

Test group 1 (2.046 mg/L)	Male animals	Female animals
Lethality (number of animals)	1 / 5	-
Fur, discolored, substance like	d1 – d14	d1 – d14
Fur, substance-contaminated	d0	d0
Respiration, abdominal	d0 – d2	d0 – d2
Respiration, intermittent	h1 – h4	h1 – h4
Respiration, sounds	d0 – d1	d0 – d1
Piloerection	d1 – d2	d1 – d2
Posture, hunched posture	d0	d0

 

Histopathological findings in the animal no. 721 that died on study day 0

Animal No.	Organ	Finding	Modifier
721	Tracheobronchial and mediastinal lymph nodes	No finding	-
	Larynx:	Edema, slight Crystals, minimal multifocal Granular, material, slight	ventral pouch green-blue, luminal   grey, luminal
	Pharynx	Crystals, minimal multifocal	green-blue
	Trachea	No finding	-
	Lungs	No finding	-
	Nasal cavity	Crystals, minimal multifocal

Interpretation of results:

Category 5 based on GHS criteria

Executive summary:

To determine the acute inhalation toxicity (single 4-hour exposure, nose only) of 29H,31H-phthalocyanine as a dust, a study was performed in male and female Wistar rats according to OECD-Guideline method 403. The test was run with an actual measured concentration of 2.064 mg/L (analytical concentration). Cascade impactor measurements resulted in particle size distributions with mass median aerodynamic diameters (MMADs) of 2.67 and 2.58 µm, which are well within the respirable range.

One of the five males was died at 2.064 mg/L on study day 0 during exposure.

The death was determined in the 2nd hour of the exposure. N

o mortality was observed in female animals. Clinical signs of toxicity in animals comprised abdominal and intermittent respiration, respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur substance like. Findings were observed from hour 1 of exposure until the end of the 14-day post-exposure observation period. The mean body weights of the surviving animals decreased on the first post-exposure observation day but increased thereafter.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LC50

Value:

> 2.046 mg/L air

Physical form:

inhalation: dust

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:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

non-GLP

Qualifier:

according to guideline

Guideline:

OECD Guideline 402 (Acute Dermal Toxicity)

GLP compliance:

no

Test type:

standard acute method

Limit test:

yes

Specific details on test material used for the study:

- Analytical purity: 100 %

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Breeding Farm VELAZ
- Age at study initiation: 7-9 weeks
- Weight at study initiation: 236-278 g
- Housing: individually in plastic breeding cage Velaz T4
- Diet: DOS 2b - standard pelleted diet, ad libitum
- Water: drinking tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30-70 %
- Photoperiod: 12 hrs dark / 12 hrs light

Type of coverage:

occlusive

Vehicle:

water

Details on dermal exposure:

The amount of the test substance for each animal was weighed (acc. to its body weight) and moistened with water for injection immediately before application.
Approx. 24 h before application of the test substance, the fur on the back of the animals was shaved at area about 6 cm x 6 cm.
Aliquot part of the test material was applied on the depilated area of skin. The application site was covered by mull, plastic foil and held in contact by plaster (strapping). After 24 h the occlusive dressing and the remains of the test substance were removed.

Duration of exposure:

24 h

Doses:

5000 mg/kg bw

No. of animals per sex per dose:

5 males

Control animals:

no

Details on study design:

Body weight:
Animals were weighed before application, at 8th day and before euthanasia of animals. Average body weight in a group was calculated from individual body weights.

Clinical examination:
After application of the test material the animals were observed individually. Observations included changes of health conditions. Observations were performed daily during the 14 days observation period.

Pathological examination:
All test animal survivors to the end of the study were sacrificed on the 15th day and gross necropsy was carried out. Normally the nutritious state, body surface, body foramina, thoracic, abdominal and cranial cavity were evaluated.

Sex:

male

Dose descriptor:

LD50

Effect level:

> 5 000 mg/kg bw

Mortality:

All animals survived the 14 day observation period and the day of sacrifice.

Clinical signs:

other: No clinical signs of toxicity were observed during the study period.

Gross pathology:

No individual records of macroscopic changes were found.

Interpretation of results:

GHS criteria not met

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

LD50

Value:

> 5 000 mg/kg bw

Additional information

All substances in this category share a similar structure with a molecular weight > 500 g/mol. Based on the high molecular weight and insolubility, the substances in this category will not be able to penetrate biological membranes. Thus, no systemic exposure is expected. This is confirmed by the acute toxicity studies for four substances in this category: CAS 147-14-8, CAS 1328-53-6, CAS 574-93-6, CAS 27614-71-7 and CAS 68512-13-0. The acute toxicity data can be used for read-across to all substances in this category.

 

CAS No. 147-14-8:

Oral

There are valid data available for the assessment of the acute oral toxicity of Copper phthalocyanine. Five male and five female Sprague-Dawley rats were treated with 200, 1600, 3200, 6400 mg/kg bw under standardized conditions; the test method is comparable to OECD TG 401. The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was > 6400 mg/kg bw for male and female rats. No mortality was observed at any dose group and autopsy revealed no relevant findings. Clinical signs were reported for all groups. In the 6400 - 3200 mg/kg bw groups, ca. 4 hours after application the animals were found in a crouched down position with intermittent breath. On the following morning slight apathy, a crouched down position and intermittent breath were observed. The stool of the animals was bluish discoloured until day 4. On the following days the animals were without any findings. In the 1600 - 200 mg/kg bw groups, immediately after application, irregular breath and masticatory movement were observed, after 4 hours the animals were found in a crouched down position. On the following days until day 3, the stool of the animals was bluish discoloured but later on the animals were without any findings (BASF 1971, K2).

In another supporting study, conducted with five male and five female mice per group, the animals were treated with 0 or 16000 mg/kg bw under standardized conditions and were observed for 14 d, necropsy was performed with all animals. The LD50 was > 16000 mg/kg bw for male and female mice. No mortality was observed and autopsy revealed no relevant findings. The following clinical signs were reported shortly after dosing in all treated mice: pilo-erection and abnormal body carriage (hunched posture) and abnormal gait (waddling). These findings were accompanied by lethargy (4 animals), ptosis (1 animal), diarrhoea, coloured blue (1 animal). Recovery of all treated mice as judged by external appearance and behaviour, was apparently complete within 4 days of dosing (Huntingdon Research Centre 1981, K2).

A supporting study provided a LD50 of > 10000 mg/kg bw in rats (BASF 1972, K2).

 

Dermal

There are valid data available for the assessment of the acute dermal toxicity of Copper phthalocyanine. Five male Wistar rats were treated with 5000 mg/kg bw under standardized conditions; the test method was acc. to OECD TG 402 (limit test). The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was > 5000 mg/kg bw for male rats. No mortality was observed, autopsy revealed no relevant findings and no clinical signs of toxicity were reported. Body weight gain was within the normal range (Synthesia 2009, K2).

 

 

CAS No. 1328-53-6:

Oral

There are valid data available for the assessment of the acute oral toxicity of polychloro copper phthalocyanine. Five male and five female Sprague-Dawley rats were treated with doses of 200, 1600, 3200 and 6400 mg/kg bw of polychloro copper phthalocyanine under standardized conditions; the test method is comparable to OECD TG 401 (BASF 1971, K2). The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was > 6400 mg/kg bw for male and female rats. All animals survived, no mortality was observed, and no abnormalities were found in the organs. Green feces were observed in all animals treated 24 hours after application of the test material. Dyspnea was observed in animals of the 1600 mg/kg bw group immediately after treatment, but was reversible.

In a supporting limit test,comparable to OECD TG 401, five Sprague-Dawley rats per sex were administered to a single dose of 5000 mg/kg bw polychloro copper phthalocyanine (BASF 1980, K2). The animals were observed for 14 d, necropsy was performed even with the survivors. Here, the LD50 was > 5000 mg/kg bw. All animals survived, no mortality was observed, and no abnormalities were found in the organs. Dyspnea, apathy, green feces as well as a poor general state were observed. A slight decrease of the average body weight in the female group was seen on day 13.

Another study in rat provided a LD50 range of > 2000 to > 10000 mg/kg bw polychloro copper phthalocyanine in rats, in all three cases no mortalities occured (MHLW 2001, K1).

 

CAS No. 574-93-6:

Oral

There are valid data available for the assessment of the acute oral toxicity of Heliogen Blue MFA. In a limit test (comparable to OECD TG 401), five Sprague-Dawley rats per sex were administered 5000 mg/kg bw Heliogen Blue (BASF 1979, K2). The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was > 5000 mg/kg bw. All animals survived, no mortality was observed. No clinical signs of toxicity were seen. Autopsy revealed no abnormal findings.

In a supporting study using a test method also comparable to OECD TG 401, five Sprague-Dawley rats per sex were administered 5000, 6400, 8000 and 10000 mg/kg bw Heliogen Blue MFA, respectively (BASF 1980, K2). The animals were observed for 14 d, necropsy was performed even with the survivors. The LD50 was > 5000 mg/kg bw. All animals survived, no mortality was observed. No clinical signs of toxicity were seen. Autopsy revealed no abnormal findings.

 

Inhalation

To determine the acute inhalation toxicity (single 4-hour exposure, nose only) of 29H,31H-phthalocyanine as a dust, a study was performed in male and female Wistar rats according to OECD TG 403 (BASF 2021, K1). The test was run with an actual measured concentration of 2.064 mg/L. Cascade impactor measurements resulted in particle size distributions with mass median aerodynamic diameters (MMADs) of 2.67 and 2.58 µm, which are well within the respirable range. One of the five males was died at 2.064 mg/L on study day 0 during exposure. The death was determined in the 2nd hour of the exposure. No mortality was observed in female animals. Clinical signs of toxicity in animals comprised abdominal and intermittent respiration, respiration sounds, hunched posture, piloerection, substance-contaminated and discolored fur substance like. Findings were observed from hour 1 of exposure until the end of the 14-day post-exposure observation period. The mean body weights of the surviving animals decreased on the first post-exposure observation day but increased thereafter.

 

CAS No. 27614-71-7:

Oral

There are valid data available for the assessment of the acute oral toxicity of Hostaperm Blau BT-729-D. In a limit test five Sprrague-Dawley rats of both sexes were administered to a single dose of 2000 mg/kg bw (acc. OECD TG 401, Aventis Pharma 2000, K1). The animals were observed for 14 d, necropsy was performed with all animals. The LD50 was > 2000 mg/kg bw. All animals survived, no mortality was observed, and no abnormalities were found in the organs. Development of body weight was not impaired, except one female, which suffered a loss of body weight between day 8 and day 15. Bluish discoloured feces were observed after the administration of the test material. From day 4 until the end of the study no findings were observed.

 

Inhalation

In a procedure following OECD TG 403 and GLP, five male and five female albino rats per group were exposed to the test substance by nose-only inhalation for four hours at gravimetrically determined mean concentrations of 1.1 and 5.2 mg/L air (BASF 2021, K1). All animals exposed to 5.2 mg/L air died within two days after exposure start. All animals exposed to 1.1 mg/L air survived the scheduled observation period. Clinical signs of toxicity in animals exposed to 1.1 mg/L comprised accelerated respiration, intermittent respiration, abdominal respiration, respiration sounds, faeces substance like discolored, piloerection, substance-discolored fur and substance-contaminated fur. Findings were observed in the males from hour 1 of exposure through study day 11. No findings were detected in the male animals during the post exposure observation period from study day 12 onwards. Findings were observed in the females from hour 1 of exposure until the end of the post-exposure observation period. No gross pathological findings were noted during the necropsy of the animals at the termination of the post-exposure observation period. Clinical signs of toxicity in animals exposed to 5.2 mg/L comprised accelerated respiration, depressed respiration, abdominal respiration, no feces, activity: attention reduced, piloerection, substancediscolored fur and substance-contaminated fur. Findings were observed from hour 1 of exposure until the death of the animals. The mean body weights of the surviving animals decreased during the first post-exposure observation days. The LD50 was estimated to be > 1.084 < 5.212 mg/L in rats. 

 

Two additional in silico studies have been performed by Greek Creek Toxicokinetics Consulting (2022, K2), which used the Multiple Path Particle Dosimetry (MPPD) model was to predict aerosol deposition patterns representative of a guideline limit test acute inhalation toxicity study in the Sprague Dawley rat. Specifically, a 4-hr nose-only inhalation exposure to generic aerosol particles having a mass median aerosol diameter (MMAD) of 2.75 µm and a geometric standard deviation (GSD) of 1.0 (i.e., monodisperse distribution), a unit density of 1 g/cm3, and an airborne concentration of 5000 mg/m3 (5 mg/L) was simulated. Human simulations for a variety of hypothetical activity and breathing conditions for the same exposure were also conducted to highlight cross-species differences, although it is unlikely that such high aerosol exposures would be considered tolerable, even for non-toxic materials. Rats are obligate nose breathers and with over 30% of inhalable aerosol depositing in the head region, anterior nasal tissues and laryngeal regions are likely to receive a significant amount of deposition based upon a computational fluid-particle dynamics (CFPD)-informed analysis of MPPD simulations. In general, greater deposition of aerosols occurs in the head region of humans following nasal than with oral breathing. However, oral breathing and increased activity (increased bpm and minute volume) can lead to deeper penetration of aerosols within the pulmonary region. Given the smaller airways and higher breathing frequencies and minute volumes per unit body weight in rats vs. humans, greater amounts of aerosol mass are deposited in the tracheobronchial region in rats, while in humans, a greater mass is deposited in the pulmonary region. However, when normalized to airway surface areas, both species receive a relatively low overall pulmonary regional dose. Mucociliary clearance rates are also slower in humans than in the rat and the time to clear tracheobronchial airways varies between ~48 and 72 hr depending upon breathing mode and activity level vs. ~12 hr in the rat. Both species have slow macrophage clearance rates in the pulmonary region with neither species clearing much material (<87%-97% of deposited mass) by 7 days. However, given the smaller airways in the rat and the potential for significant amounts of deposited and retained mass of aerosols in individual airways, the potential for airway obstruction and resulting decreases in airflows and pulmonary function was assessed in individual airways using a geometric analysis of MPPD simulations. This analysis showed that a significant percentage of pulmonary airways and to a lesser extent, bronchiolar airways, may be at risk of occlusion and impairment of lung function and gas exchange in the rat. Such a potential for physical impact on lung function for certain materials that are presumed to be of low toxicity raises questions for assessing acute toxicity risks for humans based upon guideline acute toxicity studies in the rat at limit test concentrations.

 

CAS 68512-13-0

Oral

In a limit test (comparable to OECD TG 423), ten Sprague-Dawley rats per sex were administered 5000 mg/kg bw test substance (BASF 1978, K2). The LD50 was > 5000 mg/kg bw. All animals survived, no mortality was observed. No clinical signs of toxicity were seen. Autopsy revealed no abnormal findings.

 

In another limit test (comparable to OECD TG 423), rats per sex were administered 10000 mg/kg bw test substance (BASF 1975, K2). The LD50 was > 10000 mg/kg bw. 

Justification for classification or non-classification

Based on the data for acute oral, acute dermal and acute inhalation toxicity, classification for acute toxicity is not warranted in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. (EC) 1272/2008.