2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutyramide]

Endpoint summary

Description of key information

No toxic effects were observed in an oral repeated dose toxicity study in rats (highest dose tested: 523 mg/kg bw/day). The only effect observable after subacute inhalation exposure was minimal deposition of test material in the lung, which was accompanied by minmal, fully reversible macrophage reaction (BALF).

The systemic NOAECs in these studies were 30 mg/m³.

No data on chronic repeated dose toxicity after inhalation or dermal exposure are available.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From JAN 1974 to APR 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD Guideline 451 (Carcinogenicity Studies)

Deviations:

not applicable

Remarks:

The study was performed to test carcinogenicity of the test item. That's the reason why some details usually requested in a repeated dose toxicity study (e.g. data on clinical biochemistry, haematology) are missing.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: S. IVANOVAS GmbH & Co, Med. Versuchstierzucht KG (Kissleg, Germany)
- Age at study initiation: 38 (males) -42 (females) days
- Weight at study initiation: 100 - 109 g
- Housing: in groups of 2 or 3 animals in Macrolon cages (Type III)
- Diet: Altromin 1321 (Altromin, Lage), ad libitum
- Water: tap water, ad libitum
- Acclimation period: no data


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 +/- 0.5
- Humidity (%): 60 +/- 3
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): weekly

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

104 weeks

Frequency of treatment:

daily

Dose / conc.:

1 000 other: ppm in diet

Dose / conc.:

3 000 other: ppm in diet

Dose / conc.:

9 000 other: ppm in diet

No. of animals per sex per dose:

50

Control animals:

yes, plain diet

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice per day


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: weekly


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once, immediately before sacrifice
- Dose groups that were examined: all animals


HAEMATOLOGY: No


CLINICAL CHEMISTRY: No

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
at the end of the exposure period the following investigations were performed:
- audiometry (using a simple sound test)
- inspection of denture
- organ weights of 7-8 organs (heart, liver, lungs, spleen, kidney, thymus, brain, testis)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes (animals of the control and highest dose group; paraffin sections, Haematoxylin-Eosin staining):
heart, lung, liver (additionally: frozen sections with Sudan staining), kidney, spleen, adrenal, thymus, pituitary, brain, gonads, thyroid, prostate/uterus, seminal vesicle/mammary gland, stomach, duodenum, colon, salivary gland, lymph nodes, eye and optic nerve, urinary bladder, bone marrow, neoplastic lesions, bones
- histopathological investigations of animals of the lower dose groups were performed, if they died or were sacrificed in the meantime and revealed macroscopic findings

Statistics:

- variance analysis according to Peto
- Student's t-test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

9 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no substance related toxicity or carcinogenicity was observed; 9000 mg/kg diet (ppm) correspond to 533.1 mg/kg bw/day and 523.0 mg/kg bw/day in male and female rats, respectively (calculated in the study report on basis of food consumption)

Key result

Critical effects observed:

no

 - no effects on behaviour, appearance, faeces, feed and drinking water uptake, eyes, hearing, dentition, mortality, body weight development

- no substance induced macroscopic or histological changes

- no substance related effects on the tumour incidence

- 1000, 3000, 9000 ppm in diet correspond to 58, 174, 533 mg/kg bw/day in male and 59, 180, 523 mg/kg bw/day in female rats, respectively

 - no 3,3'-dichlorobenzidine was detected in the urine samples (limit of detection: 3 µg/10 ml urine; 0.3 ppm)

Conclusions:

Chronic feeding of Sprague Dawley rats with up to 9000 ppm of the test item in diet did not cause any adverse effect. The NOAEL in this study was 9000 ppm in diet (corresponding to 533.1 mg/kg bw/da and 523.0 mg/kg bw/day in male and female rats, respectively).

Executive summary:

Sprague Dawley rats (50 per sex per dose) were exposed to 1000, 3000, 9000 ppm of the test item in diet (corresponding to 58, 174, 533 mg/kg bw/day and 59, 180, 523 mg/kg bw/day in male and female rats, respectively) for 104 weeks. The test item did neither induce toxicity nor tumorigenicity. The NOAEL in this study was 9000 ppm in diet.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 9 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: THERE WERE NO EFFECTS AT ALL

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Reference 3

Endpoint:

chronic toxicity: oral

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

See justification document in chapter 13

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

ca. 9 000 mg/kg diet

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: There wer no adverse effects at all

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

523 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

reliable

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

yes

Remarks:

reduced exposure period, males only

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced exposure period, males only

GLP compliance:

yes (incl. QA statement)

Remarks:

The study was performed in a GLP certified institute according to standard methods and procedures but no GLP-compliance statement is included.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca 240 g

- Housing:
- Diet (e.g. ad libitum): ad lib. except during exposure
- Water (e.g. ad libitum): ad lib. except during exposure
- Acclimation period: 13 d

DETAILS OF FOOD AND WATER QUALITY:
The rats were housed together (up to 5 animals per cage) in Typ 2000P ca. 2065 cm2
(polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used
in this study (the present supplier is documented in the raw data). For enrichment wooden
gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna,
Austria and Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

> 0.3 - <= 0.4 µm

Details on inhalation exposure:

The nose-only exposure technique was preferably selected for this dust inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.
Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of t he final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume.
A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all t est groups were exposed for 6 hours on each workday over a time period suitable to reach 5 exposures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study means and standard deviations of test substance concentrations
Test group Target concentration (mg/m³) Measured concentration (mg/m³) Nominal concentration (mg/m³) Effectiveness of generation (%)
Mean SD
0 - - - - -
1 3 3,3 0,9
2 10 10, 3 0,7
3 30 31,4 4,2

Duration of treatment / exposure:

6h x 5 days + 3 weeks recovery

Frequency of treatment:

daily

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

8 males (main:5; recovery: 3)

Control animals:

yes, concurrent vehicle

Details on study design:

Nine week old male Wistar rats (16 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule: before, during and after exposure period (3/day)

BODY WEIGHT: Yes
- Time schedule for examinations: start & end of exposure period, then 2x weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters: WBC, RBC, HGB, MCV, HCT, MCHC, PLT, diff. count, Retic.

CLINICAL CHEMISTRY: Yes
- Parameters: ALAT, ASAT, ALP, GGT, phosphate, Ca, urea, creatinine, Glucose, Bilirubin, Albumin, Triglycerides, Protein, Globuline, Cholesterol,

URINALYSIS: No

BRONCHO-ALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis: End of exposure or end of recovery
- Dose groups that were examined: all
- Number of animals: all
- Parameters: total & diff. cell count; protein, GGT, LDH, alk. Phosphatase, NAG;
Antigens: rat MCP-1, rat CINC-1/IL-8, M-CSF, osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In control group, test group 1 and 2, no clinical signs of toxicity were observed during the whole study period. In test group 3, substance contaminated fur was observed in individuals after the exposure on single days during the exposure period. This finding was considered treatmentrelated, but not adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The lungs of all animals of test group 3 (30 mg/m³) of the final sacrifice group revealed an orange discoloration, the lung of one animal and the mediastinal lymph nodes of two animals
of test group 3 (30 mg/m³) of the recovery groups showed a yellow discoloration. These findings were regarded to be treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the lungs of treated animals of test groups 2 and 3 (10 and 30 mg/m³), there was an increase in number of alveolar macrophages, which contained brownish particles within their cytoplasm.
An increase in number with increasing concentration was observed. Whereas in animals of test group 1 (3 mg/m³) the number of histiocytes was not increased but they still contained brownish particles in their cytoplasm when present.
In addition, two animals of test group 3 (30 mg/m³) revealed a minimal hypertrophy/hyperplasia mainly of the terminal bronchi. All animals of test groups 2 and 3 (10 and 30 mg/m³) showed the same particles observed in alveolar histiocytes within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related.
In the mediastinal and tracheobronchial lymph node, in all males of test group 3 (30 mg/m³) the same particles described for the lungs were observed within histiocytes within the lymph node. This finding was regarded to be treatment-related.
After the recovery period in test group 2 and 3 (10 and 30 mg/m³) still a minimal to slight increase in alveolar histiocytes containing brownish particles within their cytoplasm was observed. In test group 1 (3 mg/m³) the number of histiocytes was not increased when compared to control, but still brownish particles were present within the cytoplasm. Almost all treated animals revealed the same particles within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related.
The mediastinal lymph nodes of two animals that showed macroscopically discoloration were correlated microscopically to the occurrence of the same particles as observed in the lungs within histiocytes in the lymph nodes.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)

Cytology in BAL: No treatment-related changes regarding cytology parameters in BAL were observed.

Enzymes and protein in BAL
No treatment-related adverse changes regarding BAL enzyme activities and total protein levels in BAL were observed.

Antigens in BAL
No treatment-related changes among cytokine levels in BAL were observed.

Key result

Dose descriptor:

NOAEC

Effect level:

> 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: Bronchoalveolar lavage fluid (BAL)

Key result

Critical effects observed:

no

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ Pigment Yellow 83 opaque on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract.
Some adaptive non-adverse findings comprising hypertrophy/hyperplasia of the broncholar epithelia as well as histiocytosis with particles within histiocytes were observed in histology, in absence of any changes in bronchoalveolar lavage. The changes in broncholar epithelium fully regressed, histocytosis partly regressed after 3-week recovery period.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³ for Pigment Yellow 83 opaque.

Executive summary:

To determine potential pulmonary irritation and systemic toxicity of Pigment Yellow 83 opaque, a 5-day inhalation study with recovery groups was carried out with dust aerosols. The examinations were mainly focused on potential effects on pulmonary inflammation and reversibility.

Male Wistar rats were head-nose exposed to respirable dusts on 6 hours per day, on 5 consecutive days. The target concentrations were 3, 10 and 30 mg/m³. Concurrent control groups were exposed to conditioned air. The test atmospheres were produced with brush particle generators. The dust concentration was determined by gravimetrical measurements. Particle size was determined twice using a Marple 298 cascade impactor (Part II). In addition, an aerodynamic Particle Sizer, an optical particle counter and a scanning mobility particle sizer were used in order to characterize particles in the micrometer and sub-micrometer range.

During the study, the animals were monitored for mortality and clinical signs of toxicity. Body weights were determined twice weekly. After the last exposure (study day 4) and after a recovery period of 21 days 3 animals per group and time point were sacrificed and underwent gross necropsy.

Selected organs were weighed, a broad set of organs and tissues were preserved and the respiratory tract were examined histologically. On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters were examined in blood.

After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Results

Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³) (Main and

recovery group animals):

• No test substance-related adverse effects.

Conclusion

Inhalation exposure of rats to up 30 mg/m³ Pigment Yellow 83 opaque on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some adaptive non-adverse findings comprising hypertrophy/hyperplasia of the broncholar epithelia as well as histiocytosis with particles within histiocytes were observed in histology, in absence of any changes in bronchoalveolar lavage. The changes in broncholar epithelium fully regressed, histocytosis partly regressed after 3-week recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³ for Pigment Yellow 83 opaque.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

yes

Remarks:

reduced exposure period, males only

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced exposure period, males only

GLP compliance:

not specified

Remarks:

The study was performed in a GLP certified institute according to standard methods and procedures but no GLP-compliance statement is included.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: ca 240 g

- Housing:
- Diet (e.g. ad libitum): ad lib. except during exposure
- Water (e.g. ad libitum): ad lib. except during exposure
- Acclimation period: 13 d

DETAILS OF FOOD AND WATER QUALITY:
The rats were housed together (up to 5 animals per cage) in Typ 2000P ca. 2065 cm2
(polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used
in this study (the present supplier is documented in the raw data). For enrichment wooden
gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna,
Austria and Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

> 0.4 - <= 0.6 µm

Details on inhalation exposure:

The nose-only exposure technique was preferably selected for this dust inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.
Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of t he final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume.
A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all t est groups were exposed for 6 hours on each workday over a time period suitable to reach 5 exposures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study means and standard deviations of test substance concentrations
Test group Target concentration (mg/m³) Measured concentration (mg/m³) Nominal concentration (mg/m³) Effectiveness of generation (%)
Mean SD
0 - - - - -
1 3 3,2 0,4 4,1 78,0
2 10 10,0 0,5 11,1 90,1
3 30 30,1 2,5 36,6 82,2

Duration of treatment / exposure:

6h x 5 days + 3 weeks recovery

Frequency of treatment:

daily

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

16 males

Control animals:

yes, concurrent vehicle

Details on study design:

Nine week old male Wistar rats (16 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule: before, during and after exposure period (3/day)

BODY WEIGHT: Yes
- Time schedule for examinations: start & end of exposure period, then 2x weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters: WBC, RBC, HGB, MCV, HCT, MCHC, PLT, diff. count, Retic.

CLINICAL CHEMISTRY: Yes
- Parameters: ALAT, ASAT, GGT, ALP, phosphate, Ca, urea, creatinine, Glucose, Bilirubin, Albumin, Triglycerides, Protein, Globuline, Cholesterol,

URINALYSIS: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis: end of exposure or end of recovery
- Dose groups that were examined: all
- Number of animals: all
- Parameters: total & diff. cell count; protein, GGT, LDH, alk. Phosphatase, NAG;
Antigens: rat MCP-1, rat CINC-1/IL-8, M-CSF, osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the exposure period, absolute and relative monocyte counts of group 1 were
lower compared to those of the control group. This was also true for absolute monocyte counts
of group 3. However, the changes were not dose-dependent and therefore they were regarded
as incidental and not treatment-related.
After three weeks of recovery, in groups 2 and 3 red blood cell counts were higher compared
to controls. This was the only changed parameter in these animals at this date and therefore,
it was regarded as maybe treatment-related but not adverse

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period in groups 1 and 3 cholesterol levels were higher
compared to controls. Cholesterol values were not dose-dependently changed. The cholesterol
mean in group 3 was slightly above the historical control range (cholesterol 1.53-2.17 mmol/L,
PART III, Supplement). However, this was the only changed parameter in these individuals at
this date. Therefore, this alteration was regarded as probably treatment-related but not
adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the lungs of all treated animals, there was an increase in number of alveolar macrophages, which contained golden-brown particles within their cytoplasm. An increase in number with increasing concentration was observed. In addition, all animals of test group 2 and 3 (10 and 30 mg/m³) revealed a minimal to slight hypertrophy/hyperplasia mainly of the terminal bronchi. One male of test group 2 (10 mg/m³) and all males of test group 3 (30 mg/m³) revealed minimal infiltration of neutrophils within the bronchiolar epithelium which was regarded to be treatment related and adverse. Almost all animals showed the same particles observed in alveolar histiocytes within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatmentrelated
but not to be adverse.
Mediastinal and tracheobronchial lymph nodes in all males of test group 3 (30 mg/m³) showed the same particles described for the lungs were observed within histiocytes within the lymph node. This finding was regarded to be treatment-related
Nasal cavity in group 3 animals contained the same golden-brown particles as observed in the lungs on the surface of the nasal mucosa. This was regarded to be treatment-related.
In the trachea of one test group 3 (30 mg/m³) animal minimal golden-brown particles were observed within the submucosa in the region of the carina. This finding was regarded to be treatment-related.

After the recovery period in test group 2 and 3 (10 and 30 mg/m³) still a minimal to slight increase in alveolar histiocytes containing golden-brown particles within their cytoplasm was observed. In test group 1 (3 mg/m³) the number of histiocytes was not increased when compared to control, but still golden-brown particles were present within the cytoplasm. All treated animals revealed the same particles within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related but not to be adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)

Cytology in BAL: At the end of the administration period, absolute and relative lymphocyte, neutrophil and monocyte counts in the BALF were increased in group 3, although this did not lead to an alteration of the total cell counts in BALF. The mentioned relative cell counts were increased in favor of relative macrophages counts in this test group. After the three weeks recovery period, cell counts were back in the range of the controls.

Enzymes and protein in BAL
No treatment-related adverse changes regarding BAL enzyme activities and total protein levels in BAL were observed. After one week of inhalation lactate dehydrogenase (LDH) and γ-glutamyl transferase (GGT) activities in group 3 were slightly higher compared to controls, but the changes were minimal (< 2fold) and therefore they were regarded as treatment-related but not adverse. After the recovery period, all enzyme activities were in the control range.

Antigens in BAL
After the administration period in group 3 monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels were slightly increased.
CINC-1/IL-8 values were already higher in group 2, but the levels of the latter parameter were not dose-dependently changed and therefore, this change was regarded as incidental and not treatment-related.

Key result

Dose descriptor:

NOAEC

Effect level:

> 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: Bronchoalveolar lavage fluid (BAL)

Key result

Critical effects observed:

no

Conclusions:

Inhalation exposure of rats to 30 mg/m³ Pigment Yellow 83 transparent on 5 consecutive days caused increased absolute and relative lymphocyte, neutrophils and monocyte counts in bronchoalveolar lavage. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed. Neutrophil infiltration was also observed at 10 mg/m³. All effects were reversible within 3 weeks exposure-free recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 3 mg/m³ for Pigment Yellow 83 transparent. The systemic NOAEC is above 30 mg/m³ (high concentration group). Target organ is the lung.

Executive summary:

The purpose of this 5 day-inhalation study with 3 weeks recovery period was to determine the pulmonary toxicity in rats using a short term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of Pigment Yellow 83

transparent was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.

Nine week old male Wistar rats (16 rats per concentration group) were exposed nose only to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days.

Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

During the exposure period the target concentrations were reached. The particle size resulted in MMADs between 0.4 and 0.6 μm with GSDs around 3. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Test group 3 (30 mg/m³), main group

• Increased absolute and relative lymphocyte, neutrophil and monocyte cell counts in the BALF

• Increased monocyte chemoattractant protein-1 (MCP-1) levels in the BALF

• Minimal infiltration of neutrophils within bronchiolar epithelium of all animals in test group.

Test group 2 (10 mg/m³), main group

• Minimal infiltration of neutrophils within bronchiolar epithelium of one animal in test group;

Test group 1 (3 mg/m³), main group

• No treatment-related effects

Recovery group animals: Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³)

• No treatment-related effects

The no observed adverse effect concentration (NOAEC) for local effects was 3 mg/m³ for Pigment Yellow 83 transparent. The systemic NOAEC is above 30 mg/m³ (high concentration group). Target organ is the lung.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

yes

Remarks:

reduced exposure period, males only

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced exposure period, males only

GLP compliance:

yes (incl. QA statement)

Remarks:

The study was performed in a GLP certified institute according to standard methods and procedures but no GLP-compliance statement is included.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 9 weeks
- Weight at study initiation: ca 240 g

- Housing:
- Diet (e.g. ad libitum): ad lib. except during exposure
- Water (e.g. ad libitum): ad lib. except during exposure
- Acclimation period: 13 d

DETAILS OF FOOD AND WATER QUALITY:
The rats were housed together (up to 5 animals per cage) in Typ 2000P ca. 2065 cm2
(polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used
in this study (the present supplier is documented in the raw data). For enrichment wooden
gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna,
Austria and Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

> 0.3 - <= 0.4 µm

Details on inhalation exposure:

The nose-only exposure technique was preferably selected for this dust inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.
Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of t he final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume.
A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all t est groups were exposed for 6 hours on each workday over a time period suitable to reach 5 exposures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study means and standard deviations of test substance concentrations
Test group Target concentration (mg/m³) Measured concentration (mg/m³) Nominal concentration (mg/m³) Effectiveness of generation (%)
Mean SD
0 - - - - -
1 3 3,3 0,9
2 10 10, 3 0,7
3 30 31,4 4,2

Duration of treatment / exposure:

6h x 5 days + 3 weeks recovery

Frequency of treatment:

daily

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

8 males (main:5; recovery: 3)

Control animals:

yes, concurrent vehicle

Details on study design:

Nine week old male Wistar rats (16 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule: before, during and after exposure period (3/day)

BODY WEIGHT: Yes
- Time schedule for examinations: start & end of exposure period, then 2x weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters: WBC, RBC, HGB, MCV, HCT, MCHC, PLT, diff. count, Retic.

CLINICAL CHEMISTRY: Yes
- Parameters: ALAT, ASAT, ALP, GGT, phosphate, Ca, urea, creatinine, Glucose, Bilirubin, Albumin, Triglycerides, Protein, Globuline, Cholesterol,

URINALYSIS: No

BRONCHO-ALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis: End of exposure or end of recovery
- Dose groups that were examined: all
- Number of animals: all
- Parameters: total & diff. cell count; protein, GGT, LDH, alk. Phosphatase, NAG;
Antigens: rat MCP-1, rat CINC-1/IL-8, M-CSF, osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In control group, test group 1 and 2, no clinical signs of toxicity were observed during the whole study period. In test group 3, substance contaminated fur was observed in individuals after the exposure on single days during the exposure period. This finding was considered treatmentrelated, but not adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The lungs of all animals of test group 3 (30 mg/m³) of the final sacrifice group revealed an orange discoloration, the lung of one animal and the mediastinal lymph nodes of two animals
of test group 3 (30 mg/m³) of the recovery groups showed a yellow discoloration. These findings were regarded to be treatment-related.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the lungs of treated animals of test groups 2 and 3 (10 and 30 mg/m³), there was an increase in number of alveolar macrophages, which contained brownish particles within their cytoplasm.
An increase in number with increasing concentration was observed. Whereas in animals of test group 1 (3 mg/m³) the number of histiocytes was not increased but they still contained brownish particles in their cytoplasm when present.
In addition, two animals of test group 3 (30 mg/m³) revealed a minimal hypertrophy/hyperplasia mainly of the terminal bronchi. All animals of test groups 2 and 3 (10 and 30 mg/m³) showed the same particles observed in alveolar histiocytes within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related.
In the mediastinal and tracheobronchial lymph node, in all males of test group 3 (30 mg/m³) the same particles described for the lungs were observed within histiocytes within the lymph node. This finding was regarded to be treatment-related.
After the recovery period in test group 2 and 3 (10 and 30 mg/m³) still a minimal to slight increase in alveolar histiocytes containing brownish particles within their cytoplasm was observed. In test group 1 (3 mg/m³) the number of histiocytes was not increased when compared to control, but still brownish particles were present within the cytoplasm. Almost all treated animals revealed the same particles within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related.
The mediastinal lymph nodes of two animals that showed macroscopically discoloration were correlated microscopically to the occurrence of the same particles as observed in the lungs within histiocytes in the lymph nodes.
All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)

Cytology in BAL: No treatment-related changes regarding cytology parameters in BAL were observed.

Enzymes and protein in BAL
No treatment-related adverse changes regarding BAL enzyme activities and total protein levels in BAL were observed.

Antigens in BAL
No treatment-related changes among cytokine levels in BAL were observed.

Key result

Dose descriptor:

NOAEC

Effect level:

> 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: Bronchoalveolar lavage fluid (BAL)

Key result

Critical effects observed:

no

Conclusions:

Inhalation exposure of rats to up 30 mg/m³ Pigment Yellow 83 opaque on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract.
Some adaptive non-adverse findings comprising hypertrophy/hyperplasia of the broncholar epithelia as well as histiocytosis with particles within histiocytes were observed in histology, in absence of any changes in bronchoalveolar lavage. The changes in broncholar epithelium fully regressed, histocytosis partly regressed after 3-week recovery period.
Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³ for Pigment Yellow 83 opaque.

Executive summary:

To determine potential pulmonary irritation and systemic toxicity of Pigment Yellow 83 opaque, a 5-day inhalation study with recovery groups was carried out with dust aerosols. The examinations were mainly focused on potential effects on pulmonary inflammation and reversibility.

Male Wistar rats were head-nose exposed to respirable dusts on 6 hours per day, on 5 consecutive days. The target concentrations were 3, 10 and 30 mg/m³. Concurrent control groups were exposed to conditioned air. The test atmospheres were produced with brush particle generators. The dust concentration was determined by gravimetrical measurements. Particle size was determined twice using a Marple 298 cascade impactor (Part II). In addition, an aerodynamic Particle Sizer, an optical particle counter and a scanning mobility particle sizer were used in order to characterize particles in the micrometer and sub-micrometer range.

During the study, the animals were monitored for mortality and clinical signs of toxicity. Body weights were determined twice weekly. After the last exposure (study day 4) and after a recovery period of 21 days 3 animals per group and time point were sacrificed and underwent gross necropsy.

Selected organs were weighed, a broad set of organs and tissues were preserved and the respiratory tract were examined histologically. On study days 7 and 28, blood was sampled from 5 rats/group and time point. Clinical chemistry parameters, hematology parameters were examined in blood.

After blood sampling the animals underwent bronchoalveolar lavage. Lavage fluid was examined for cytological and biochemical parameters including selected antigens.

Results

Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³) (Main and

recovery group animals):

• No test substance-related adverse effects.

Conclusion

Inhalation exposure of rats to up 30 mg/m³ Pigment Yellow 83 opaque on 5 consecutive days did not lead to any treatment-related adverse effects within the respiratory tract. Some adaptive non-adverse findings comprising hypertrophy/hyperplasia of the broncholar epithelia as well as histiocytosis with particles within histiocytes were observed in histology, in absence of any changes in bronchoalveolar lavage. The changes in broncholar epithelium fully regressed, histocytosis partly regressed after 3-week recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) was 30 mg/m³ for Pigment Yellow 83 opaque.

Reference 2

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (28-Day (Subacute) Inhalation Toxicity Study

Deviations:

yes

Remarks:

reduced exposure period, males only

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.8 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

reduced exposure period, males only

GLP compliance:

not specified

Remarks:

The study was performed in a GLP certified institute according to standard methods and procedures but no GLP-compliance statement is included.

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl:WI(Han)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River
- Females (if applicable) nulliparous and non-pregnant: not applicable
- Age at study initiation: ca. 7 weeks
- Weight at study initiation: ca 240 g

- Housing:
- Diet (e.g. ad libitum): ad lib. except during exposure
- Water (e.g. ad libitum): ad lib. except during exposure
- Acclimation period: 13 d

DETAILS OF FOOD AND WATER QUALITY:
The rats were housed together (up to 5 animals per cage) in Typ 2000P ca. 2065 cm2
(polysulfone cages) supplied by TECNIPLAST, Germany. Dust-free wooden bedding was used
in this study (the present supplier is documented in the raw data). For enrichment wooden
gnawing blocks (Typ NGM E-022), supplied by Abedd® Lab. and Vet. Service GmbH, Vienna,
Austria and Play Tunnel, large (Art. 14153); PLEXX b.v., Elst, Netherlands were added.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

clean air

Mass median aerodynamic diameter (MMAD):

> 0.4 - <= 0.6 µm

Details on inhalation exposure:

The nose-only exposure technique was preferably selected for this dust inhalation study to minimize fur contamination of the animals with the substance, which cannot be avoided during whole-body exposure. Fur contamination may lead to an additional dermal and oral uptake (animals preen as their fur becomes contaminated). Thus an estimation of an nominal dose, taken up by the animals and its correlation to a toxic effect becomes more difficult.
Furthermore, by using the dynamic mode of operation with a low-volume chamber, the equilibrium characteristic of this exposure technique is favorable: t99 (the time to reach 99% of t he final target concentration) is shorter as compared to whole-body chambers with a higher chamber volume.
A positive pressure was maintained inside the exposure systems by adjusting the air flow of the exhaust air system. This ensured that the aerosol in the breathing zones of the animals was not diluted by laboratory air.
In order to accustom the animals to exposure they were treated with supply air under conditions comparable to exposure on two days before start of exposure (pre-exposure period). Then all t est groups were exposed for 6 hours on each workday over a time period suitable to reach 5 exposures.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Study means and standard deviations of test substance concentrations
Test group Target concentration (mg/m³) Measured concentration (mg/m³) Nominal concentration (mg/m³) Effectiveness of generation (%)
Mean SD
0 - - - - -
1 3 3,2 0,4 4,1 78,0
2 10 10,0 0,5 11,1 90,1
3 30 30,1 2,5 36,6 82,2

Duration of treatment / exposure:

6h x 5 days + 3 weeks recovery

Frequency of treatment:

daily

Dose / conc.:

0 mg/m³ air (nominal)

Dose / conc.:

3 mg/m³ air (nominal)

Dose / conc.:

10 mg/m³ air (nominal)

Dose / conc.:

30 mg/m³ air (nominal)

No. of animals per sex per dose:

16 males

Control animals:

yes, concurrent vehicle

Details on study design:

Nine week old male Wistar rats (16 rats per concentration group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m3 (low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes / No / Not specified
- Time schedule: before, during and after exposure period (3/day)

BODY WEIGHT: Yes
- Time schedule for examinations: start & end of exposure period, then 2x weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: all
- Parameters: WBC, RBC, HGB, MCV, HCT, MCHC, PLT, diff. count, Retic.

CLINICAL CHEMISTRY: Yes
- Parameters: ALAT, ASAT, GGT, ALP, phosphate, Ca, urea, creatinine, Glucose, Bilirubin, Albumin, Triglycerides, Protein, Globuline, Cholesterol,

URINALYSIS: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes / No / Not specified
- Time schedule for analysis: end of exposure or end of recovery
- Dose groups that were examined: all
- Number of animals: all
- Parameters: total & diff. cell count; protein, GGT, LDH, alk. Phosphatase, NAG;
Antigens: rat MCP-1, rat CINC-1/IL-8, M-CSF, osteopontin

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the exposure period, absolute and relative monocyte counts of group 1 were
lower compared to those of the control group. This was also true for absolute monocyte counts
of group 3. However, the changes were not dose-dependent and therefore they were regarded
as incidental and not treatment-related.
After three weeks of recovery, in groups 2 and 3 red blood cell counts were higher compared
to controls. This was the only changed parameter in these animals at this date and therefore,
it was regarded as maybe treatment-related but not adverse

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At the end of the administration period in groups 1 and 3 cholesterol levels were higher
compared to controls. Cholesterol values were not dose-dependently changed. The cholesterol
mean in group 3 was slightly above the historical control range (cholesterol 1.53-2.17 mmol/L,
PART III, Supplement). However, this was the only changed parameter in these individuals at
this date. Therefore, this alteration was regarded as probably treatment-related but not
adverse.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the lungs of all treated animals, there was an increase in number of alveolar macrophages, which contained golden-brown particles within their cytoplasm. An increase in number with increasing concentration was observed. In addition, all animals of test group 2 and 3 (10 and 30 mg/m³) revealed a minimal to slight hypertrophy/hyperplasia mainly of the terminal bronchi. One male of test group 2 (10 mg/m³) and all males of test group 3 (30 mg/m³) revealed minimal infiltration of neutrophils within the bronchiolar epithelium which was regarded to be treatment related and adverse. Almost all animals showed the same particles observed in alveolar histiocytes within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatmentrelated
but not to be adverse.
Mediastinal and tracheobronchial lymph nodes in all males of test group 3 (30 mg/m³) showed the same particles described for the lungs were observed within histiocytes within the lymph node. This finding was regarded to be treatment-related
Nasal cavity in group 3 animals contained the same golden-brown particles as observed in the lungs on the surface of the nasal mucosa. This was regarded to be treatment-related.
In the trachea of one test group 3 (30 mg/m³) animal minimal golden-brown particles were observed within the submucosa in the region of the carina. This finding was regarded to be treatment-related.

After the recovery period in test group 2 and 3 (10 and 30 mg/m³) still a minimal to slight increase in alveolar histiocytes containing golden-brown particles within their cytoplasm was observed. In test group 1 (3 mg/m³) the number of histiocytes was not increased when compared to control, but still golden-brown particles were present within the cytoplasm. All treated animals revealed the same particles within the bronchus associated lymphoid tissue (BALT). These findings were regarded to be treatment-related but not to be adverse. All other findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid (BAL)

Cytology in BAL: At the end of the administration period, absolute and relative lymphocyte, neutrophil and monocyte counts in the BALF were increased in group 3, although this did not lead to an alteration of the total cell counts in BALF. The mentioned relative cell counts were increased in favor of relative macrophages counts in this test group. After the three weeks recovery period, cell counts were back in the range of the controls.

Enzymes and protein in BAL
No treatment-related adverse changes regarding BAL enzyme activities and total protein levels in BAL were observed. After one week of inhalation lactate dehydrogenase (LDH) and γ-glutamyl transferase (GGT) activities in group 3 were slightly higher compared to controls, but the changes were minimal (< 2fold) and therefore they were regarded as treatment-related but not adverse. After the recovery period, all enzyme activities were in the control range.

Antigens in BAL
After the administration period in group 3 monocyte chemoattractant protein-1 (MCP-1) and cytokine-induced neutrophil chemoattractant-1 (CINC-1/IL-8) levels were slightly increased.
CINC-1/IL-8 values were already higher in group 2, but the levels of the latter parameter were not dose-dependently changed and therefore, this change was regarded as incidental and not treatment-related.

Key result

Dose descriptor:

NOAEC

Effect level:

> 30 mg/m³ air

Based on:

test mat.

Sex:

male

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

dermal irritation

gross pathology

haematology

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

other: Bronchoalveolar lavage fluid (BAL)

Key result

Critical effects observed:

no

Conclusions:

Inhalation exposure of rats to 30 mg/m³ Pigment Yellow 83 transparent on 5 consecutive days caused increased absolute and relative lymphocyte, neutrophils and monocyte counts in bronchoalveolar lavage. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed. Neutrophil infiltration was also observed at 10 mg/m³. All effects were reversible within 3 weeks exposure-free recovery period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 3 mg/m³ for Pigment Yellow 83 transparent. The systemic NOAEC is above 30 mg/m³ (high concentration group). Target organ is the lung.

Executive summary:

The purpose of this 5 day-inhalation study with 3 weeks recovery period was to determine the pulmonary toxicity in rats using a short term bioassay including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid and pathological examination of the lung. The No Observed Adverse Effect Concentration (NOAEC) after 5 days inhalation exposure to dust of Pigment Yellow 83

transparent was determined. In addition, recovery group animals were examined after an exposure-free period of 3 weeks to detect any reversibility or progression of potential toxic effects.

Nine week old male Wistar rats (16 rats per concentration group) were exposed nose only to fresh air (control group) or dust of the test substance at concentrations of 3, 10, and 30 mg/m³ (low, mid, and high concentration) for 6 hours per day and 5 days.

Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3 week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.

During the exposure period the target concentrations were reached. The particle size resulted in MMADs between 0.4 and 0.6 μm with GSDs around 3. The calculated mass fractions of particles below 3 μm aerodynamic size is greater than 90 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

Test group 3 (30 mg/m³), main group

• Increased absolute and relative lymphocyte, neutrophil and monocyte cell counts in the BALF

• Increased monocyte chemoattractant protein-1 (MCP-1) levels in the BALF

• Minimal infiltration of neutrophils within bronchiolar epithelium of all animals in test group.

Test group 2 (10 mg/m³), main group

• Minimal infiltration of neutrophils within bronchiolar epithelium of one animal in test group;

Test group 1 (3 mg/m³), main group

• No treatment-related effects

Recovery group animals: Test group 3 (30 mg/m³), test group 2 (10 mg/m³), test group 1 (3 mg/m³)

• No treatment-related effects

The no observed adverse effect concentration (NOAEC) for local effects was 3 mg/m³ for Pigment Yellow 83 transparent. The systemic NOAEC is above 30 mg/m³ (high concentration group). Target organ is the lung.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

30 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

reliable

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable chronic toxicity study is available, conducted with an appropriate species and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a sub-chronic toxicity study (90 days) does not need to be conducted because a reliable chronic toxicity study is available, conducted with an appropriate species and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

In the absence of any evidence for species specific effects or modes of action the observations reported for animals are regarded as relevant for humans.

Justification for classification or non-classification

As no adverse effects were observed in repeated dose studies performed with Pigment Yellow 83 it is not classified for specific target organ toxicity – repeated exposure or repeated dose toxicity according to Regulation (EC) No 1272/2008.