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Toxicological information

Repeated dose toxicity: inhalation

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Administrative data

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

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
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

Test material

Constituent 1
Chemical structure
Reference substance name:
2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutyramide]
EC Number:
226-939-8
EC Name:
2,2'-[(3,3'-dichloro[1,1'-biphenyl]-4,4'-diyl)bis(azo)]bis[N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutyramide]
Cas Number:
5567-15-7
Molecular formula:
C36H32Cl4N6O8
IUPAC Name:
2,2'-[(3,3'-dichlorobiphenyl-4,4'-diyl)didiazene-2,1-diyl]bis[N-(4-chloro-2,5-dimethoxyphenyl)-3-oxobutanamide]
Test material form:
solid: bulk

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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

Examinations

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)

Results and discussion

Results of examinations

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.

Effect levels

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)

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

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.