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Administrative data

Endpoint:
short-term repeated dose toxicity: inhalation
Remarks:
OECD Guidelines for Testing of Chemicals No. 412 - “28-day (Sub-acute) inhalation toxicity study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
30 May 2022 to 20 September 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2022

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)
Version / remarks:
25.June 2018
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
EC Number:
271-178-7
EC Name:
N,N'-naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]
Cas Number:
68516-75-6
Molecular formula:
C44H26Cl4N6O4
IUPAC Name:
N,N'-naphthalene-1,5-diylbis{4-[(2,3-dichlorophenyl)diazenyl]-3-hydroxy-2-naphthamide}
Test material form:
solid: nanoform, no surface treatment
Details on test material:
Name of Test Item : PV Fast Brown RL
Chemical Name (IUPAC) : N,N`-Naphthalene-1,5-diylbis[4-[(2,3-dichlorophenyl)azo]3-hydroxynaphthalene-2-carboxamide]
Molecular formula : C44H26Cl4N6O4
Molecular weight : 844.55 g/mol
CAS No. : 68516-75-6
Physical Appearance (with color) : Brown powder
Storage Conditions : Ambient (21 to 29°C)

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Rat is the preferred laboratory rodent species for inhalation toxicity assessment and is also recommended by various regulatory guidelines.
Sex:
male/female
Details on test animals or test system and environmental conditions:
Animals were housed under standard laboratory
conditions in an environmentally monitored, air-conditioned room with adequate fresh air supply (12 to 15 air changes per hour), room temperature 19.6°C to 22.9°C and relative humidity 44% to 67%, with 12 hours fluorescent light and 12 hours dark cycle.

Administration / exposure

Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
1.82 - < 1.98 µm
Geometric standard deviation (GSD):
2.82
Details on inhalation exposure:
Inhalation exposure was conducted using a flow-past, nose-only dynamic inhalation exposure system supplied by CH Technologies, USA, with a provision of at least 12 air changes per hour. The exposure unit consisted of stackable exposure tiers with top and bottom sections or plates for introduction and exhaust of test item. Each tier has 12 exposure ports which were used for exposing up to 10 animals and sampling of test atmosphere. All parts (except O-ring seals) were constructed of stainless steel. The volume of each inner plenum of inhalation chamber was 0.76 liters (11 cm diameter and 8 cm height) that consisted of total 12 port hole (one tier).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Active ingredient content inhalation exposure atmosphere was determined by using UV-Vis spectrophotometer.
Duration of treatment / exposure:
Animals were exposed to aerosolized test item continuously for 6 hours per day, 5 days per week, for a total of 20 actual exposures, after equilibration period of the chamber concentration.
Frequency of treatment:
6 hours per day, 5 days per week
Doses / concentrationsopen allclose all
Dose / conc.:
0.03 mg/L air
Remarks:
G4
Dose / conc.:
0.01 mg/L air
Remarks:
G3
Dose / conc.:
0.003 mg/L air
Remarks:
G2
Dose / conc.:
0 mg/L air
Remarks:
G1
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
The selected animals were assigned to different treatment groups, as shown below:

Group mg/L
G1 + G1R Air only 0
G2 Low Dose 0.003
G3 Mid Dose 0.01
G4 + G4R High Dose 0.03

Group size: 5 M + 5 F
Exposure duration: 6 h x 5 days/ week x 4 weeks
R= Recovery

Examinations

Observations and examinations performed and frequency:
All the animals were observed for clinical signs and pre-terminal deaths at 1.5 hrs (±10 mins), 3 hrs (±10 mins), 4.5 hrs (±10 mins) and 6 hrs (±10 mins) during exposure period. Post-exposure clinical signs and 30 to 40 minutes and 1 hour (±10 mins) following exposure to the test chemical, thereafter once a day for clinical signs and twice daily for mortality.
Sacrifice and pathology:
On day 27, all the surviving animals of groups G1, G2, G3 and G4 was subjected to necropsy and detailed gross pathological examination. All the surviving animals from recovery groups (G1R and G4R) were subjected to necropsy and detailed gross pathological examination on day 83. The animals were fasted overnight (water was provided ad libitum) prior to scheduled necropsy. On the day of terminal sacrifice, the body weight of all the fasted animals were recorded prior to exsanguination. The animals were euthanized by intraperitoneal administration of sodium thiopentone (lethal dose) followed by exsanguination. The animals were sacrificed within 24 hours after the end of exposure period.
Necropsy was including an examination of external surfaces, external orifices, abdominal, thoracic and cranial cavities, organs and tissues.
Statistics:
The raw data were subjected to statistical analysis. The computer printout of the data (in the form of the appendix) was verified with the original raw data. After verification, the data were subjected to statistical analysis using SPSS Software version 27. Body weight, percent change in body weight with respect to Day 1, feed consumption, FOB, organ weight ratios, hematology, and clinical chemistry estimations, urine volume, pH, and specific gravity were subjected to statistical analysis. One-way ANOVA and t-test followed by Dunnett’s post-test was done for different treatment groups comparing with the control group data. All analyses and comparisons were evaluated at the 95% level of confidence (P<0.05). Statistically significant changes obtained from the aforementioned tests were designated by the superscripts in the summary table throughout the study report, as stated below:
*: Statistically significant (p<0.05).

Results and discussion

Results of examinations

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):
effects observed, non-treatment-related
Description (incidence and severity):
The observed statistically significant changes in food intake were not accompanied by any changes in mean body weight. Therefore, the observed changes in food intake were considered to be incidental.
Refer to Table 4
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
Variations noted at the end of recovery period are considered incidental as no such variations were noted at the end of treatment period or in other sex.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The noted changes are considered incidental as no such variations were noted at the end of treatment period or in the other sex at the end of recovery period.
Refer to Table 12
Endocrine findings:
not examined
Urinalysis findings:
effects observed, non-treatment-related
Description (incidence and severity):
The noted variations are considered incidental due to their isolated occurrences without any changes in any of the other parameters tested.
Refer to Table 13
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
The variation is considered as incidental as none of the other neuromuscular parameters were affected and similar changes were not noted in recovery females or at the end of treatment period.

Refer Tables 5, 6, 7 & 8
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
The variations noted in organ weight are considered incidental as they were not associated with any macroscopic finding and also no microscopic changes were noted at the high dose main group animals.
Refer to Table 14, 15 & 16
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
There were no gross pathological changes in the study. However, grossly, the brown colour of test item was retained on snout region of G3 and G4 group animals in both the sexes and this was attributable to the physical nature of the test item.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
In left lobe of the lungs, minimal to moderate, multifocal, variably sized, pigmented (brownish black) granular material was observed throughout the parenchyma of lung at low, mid, high and high dose recovery groups of rats in both the sexes. This pigment was distributed both in alveolar and bronchiolar spaces without any accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles as the physical appearance of test item was brown coloured powder. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the dose level of 0.03 mg/L. (Nikula KJ et. al., 2014).
Few microscopic findings observed in this study such as ultimobranchial cysts in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats (Elizabeth F. McInnes, 2012).

Effect levels

Key result
Dose descriptor:
NOAEC
Effect level:
0.03 mg/L air (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
behaviour (functional findings)
body weight and weight gain
clinical biochemistry
clinical signs
food consumption and compound intake
gross pathology
haematology
histopathology: non-neoplastic
mortality
ophthalmological examination
organ weights and organ / body weight ratios
serum/plasma biochemistry
urinalysis

Target system / organ toxicity

Key result
Critical effects observed:
no

Applicant's summary and conclusion

Conclusions:
Based on the observed results, it is concluded that the No Observed Adverse Effect Concentration (NOAEC) of the test item was found to be 0.03 mg/L when exposed for 6 hours/day, 5 days/week, for 4 weeks by flow-past nose-only inhalation route to Sprague Dawley rats. The nominal dose of 0.03 mg/L (6 hrs/day, 5 days/week) corresponded to an actual exposure concentration in males and females.
Executive summary:

The objective of this study was to determine the toxic potential of the test item, when administered for 6 hours/day, 5 days per week, for 4 consecutive weeks by flow-past nose-only inhalation route to Sprague Dawley rats. This study provides information on toxic effects, target organs, the possibility of cumulative effects, the reversibility of effects (after 56 days recovery period), and an estimate of the No Observed Adverse Effects Concentration (NOAEC).


A total of 60 (30 males + 30 females) healthy young Sprague Dawley rats were distributed to four groups viz., control (G1), low dose (G2), mid dose (G3), high dose (G4) and two recovery groups [(G1R (Air only) and G4R (high dose)]. Each main and recovery group comprised of 10 animals (5 males and 5 females). Animals allocated to Groups G2, G3 and G4/G4R were exposed to test item for 6 hours per day, 5 days per week, for 4 consecutive weeks, at a nominal target concentration of 0.003, 0.01 and 0.03 mg/L. Animals of the control group (G1/G1R) received air only inhalation for 6 hours/5 days per week for 4 consecutive weeks. The inhalation exposure of test item/air was achieved by a flow-past, nose-only dynamic inhalation exposure system.


All animals were observed once daily for clinical signs and twice daily for mortality.  Body weight was recorded before first exposure (day 1), twice during first two weeks and weekly once thereafter. Feed consumption was recorded weekly. Ophthalmoscopic examination was performed during the acclimatization period for all groups, and during Week 4 for main groups (G1 and G4), and during Week 12 for recovery groups (G1R and G4R). Neurological/Functional test was performed during Week 4 for G1 and G4 groups and during Week 12 for recovery group animals (G1R and G4R).


 At the end of treatment and recovery periods, all animals were fasted overnight (water was provided ad libitum), and the next day, blood, urine, and Broncho alveolar lavage fluid (BALF) samples were collected and analysed. Subsequently, the animals were sacrificed and subjected to gross pathological examination, and the organs were collected, weighed, and preserved. The tissues/organs in vehicle control and high dose group animals including recovery animals were subjected to histopathological examinations.


The data recorded for all exposure days relating to the chamber conditions like particle size, temperature, relative humidity, oxygen, and carbon dioxide concentrations determined during the exposure period were found within the specified range.


No treatment-related changes in body weight, percent change in body weight with respect to day 1, feed consumption and ophthalmoscopic examination were noted. No adverse effects were observed in the neurological/functional examination tests. No adverse effects were observed in haematology, clinical chemistry, BALF analysis and urinalysis parameters. However, few variations in differential counts in haematology and BALF fluid could be secondary effects due to accumulation of test item particles in lungs and considered non adverse. No toxicologically significant changes were observed in fasting body weight, absolute organ weight, or relative organ weight.


There were no gross pathological changes in the study. However, grossly, the brown colour of test item was retained on snout region of G3 and G4 group animals in both the sexes and this was attributable to the physical nature of the test item.


In left lobe of the lungs, minimal to moderate, multifocal, variably sized, pigmented (brownish black) granular material was observed throughout the parenchyma of lung at low, mid, high and high dose recovery groups of rats in both the sexes. This pigment was distributed both in alveolar and bronchiolar spaces without any accompanying inflammation or tissue destruction to the lung parenchyma. This pigment was suggestive of inhaled test item particles as the physical appearance of test item was brown coloured powder. In the absence of cellular changes to lung parenchyma, mere presence of pigment in lungs could be considered as non-adverse effect up to the dose effect up to the dose level of 0.03 mg/L. (Nikula KJ et. al., 2014).


Few microscopic findings observed in this study such as ultimobranchial cysts in thyroid gland, epithelial cysts in thymus and all other findings were considered incidental as they occurred randomly across the dose groups including concurrent controls and/or were expected for laboratory rats (Elizabeth F. McInnes, 2012).