Registration Dossier

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Diss Factsheets

Administrative data

Description of key information

oral: 

subacute (OECD 422): NOAEL = 1000 mg/kg bw (ERBC 2022)

inhalation

NOAEC = 5 mg/m3 (Short-Termin inhalation study with recovery and BALF)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov 2021 to Oct 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature
Species:
rat
Strain:
Wistar
Remarks:
Wistar Hannover rat
Details on species / strain selection:
The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC in water, softened by reverse osmosis
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Low-level dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Medium-level dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
High-level dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from subacute oral toxicity studies with structurally related pigments. 1000 mg/kg bw is the maximimum dose prescribed in the OECD testing guideline.
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes

BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum, dams were also weighed on days 1, 4, 7, 13 post partum and just before to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy: the clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices), changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
- Pups: all pups found dead in the cage were examined for external and internal abnormalities, all culled pups sacrificed at day 4 post partum were subjected to an external examination, all live pups sacrificed on day 14 post partum were examined for external abnormalities, gonads were inspected from all pups in order to confirm the sex previously determined by external examination

ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation

HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed

The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups
Other examinations:
PARTURITION AND GESTATION LENGTH:
- Parturition check from day 20 - 25 post coitum, three rimes a day during working day or twice daily during weekends and Public Holidays
- Females which did not give birth after 25 days of post coitum period were sacrificed shortly after
- Gestation length calculated as the time between the day of successful mating (day 0 post coitum) and the day of birth
- Day of birth was defined as day 0 post partum

PUPS IDENTIFICATION, WEIGHT AND OBSERVATIONS:
- As soon as possible after parturition was considered complete (day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified
- Live pups were individually weighed on days 1, 4, 7 and 13 post partum
- Pups dying during the lactation period were weighed before the despatch to necropsy
- Observation was performed once daily for all litters from day 0 post partum until termination
- After culling, all pups were sacrificed with the dams on Day 14 post partum

ANOGENITAL DISTANCE (AGD):
- For each pup measured on day 1 post partum
- AGD was normalized to the cube root of body weight collected on day 1 post partum

NIPPLE COUNT CHECK:
- presence of nipples/areolae in male pups was checked on day 13 post partum

REPRODUCTIVE INDICES:
- Males: copulation index, fertility index
- Females: copulatory index, fertility index, pre-natal loss, post-natal loss at day 0, 4 and 13 post partum
- Pre coital interval
- Sex ratio of litter
Statistics:
Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain for male and female animals were comparable between treated and control groups through the study. The statistically significant decrease in body weight gain recorded in high dose males and mid-dose females were considered incidental.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both genders during the study.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
he statistically significant differences between contrTol and treated animals (neutrophils in males and platelets in females) were recorded only in animals dosed at 300 mg/kg/day, therefore they were considered to be incidental.
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Description (incidence and severity):
The determination of T4 and TSH was performed on:
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically significantly lower than controls in parental males (31%). This is due to 2 control animals, which showed values above the range of historical data, therefore the differences observed has no biological/toxicological significance.

The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control groups. Copulatory index and fertility index did not show any treatment-related differences.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity was set to 1000 mg/kg bw/day.
Executive summary:

Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats. The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 1), 100 mg/kg bw/d (test group 2), 300 mg/kg bw/d (test group 3) and 1000 mg/kg bw/d (test group 4). 0.5% carboxymethylcellulose served as vehicle, control animals were dosed daily with the vehicle only. All doses were administered at a constant volume of 10 mL/kg body weight. For males, the duration of treatment covered a 2-week premating period and pairing with females until the day before necropsy (a total of 39 days). Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprised from 51 to 55 days.

No test-item related mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females. No differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological (including coagulation and blood clotting time) or clinical chemistry parameters. Thyroid hormone evaluation in parental animals and pups sacrificed on day 14 post partum did not show treatment-related changes. No treatment-related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls. No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation. No treatment-related changes were observed in reproductive and developmental parameters.

Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show changes. No differences in the anogenital distance (normalised value) and no nipple were seen between control and treated groups both for male and female pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect.

Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 1000 mg/kg bw/day for male and female Wistar rats.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr 22 - Oct 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
yellow powder
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks (when supplied)
- Weight at study initiation (means):ca 265g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in
the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest,
Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks
(Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switze rland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%) 45 - 65
- Air changes (per hr): 15
Photoperiod (hrs dark / hrs light):: 12h/12h
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 0.66 - <= 0.77 µm
Geometric standard deviation (GSD):
245
Remarks on MMAD:
All measurements of particle size resulted in MMADs between 0.66 and 0.77 µm with GSDs between 2.06 and 2.90. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 90.0 % and 97.6 %.

SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter.

The SMPS showed very high particle count concentrations in all concentrations. The geometric mean count diameters were between 266 nm and 295 nm.
Details on inhalation exposure:
For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed
with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The con
centration was adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.In these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.

The particle size analysis was carried out with a cascade impactor with the following equipment:
• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA)
• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA)
• Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA)
• Sampling probe internal diameter 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/metal collecting disc and backup filter before and after sampling.The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.

To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH& Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. During the exposure period, one measurement per concentration with 10 repeats each were performed.

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations.
Duration of treatment / exposure:
6h for 5 days
Frequency of treatment:
daily
Dose / conc.:
4.98 mg/m³ air (analytical)
Remarks:
SD 0.27
Dose / conc.:
20 mg/m³ air (analytical)
Remarks:
SD 1.0
Dose / conc.:
60.4 mg/m³ air (analytical)
Remarks:
SD 2.1
No. of animals per sex per dose:
10 (five for sacrifice after exposure and 5 for sacrifice after recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results of short-term inhalation studies with other inert organic pigments
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical bioche Particlesmistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks



Positive control:
not applicable
Observations and examinations performed and frequency:
A check for moribund or dead animals was carried out twice per day on working days. A check for moribund or dead animals was carried out once per day on weekends and holidays.

The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.During exposure only a group wise examination was possible.

The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.

Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible
Sacrifice and pathology:
Clinical pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined
Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins,Triglycerides,Cholesterol
Bronchoalveolar lavage fluid (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline.
Parameters and methods of cytological examination in BAL: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase
Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin

Necropsy
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes werelavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage,
the animals were necropsied and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder


Extend of histological processing and sub-sequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal)






Other examinations:
Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
Statistics:
Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.

Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.
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 examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In males of test group 3 (60 mg/m3) absolute neutrophil counts were significantly higher compared to controls. The values were slightly above the historical control range (males, neutrophils 0.53-1.01 Giga/L). however, total white blood (WBC) counts as well as all other differential blood cell fractions were not changed. Therefore, this isolated alteration of absolute neutrophil counts in males of test group 3 was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).

After the three-week recovery period, no changes of absolute neutrophil counts were observed. In males of test group 2 (20 mg/m3) absolute and relative, large unstained cell (LUC) counts were significantly higher compared to study controls, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
Clinical biochemistry findings:
no effects observed
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significant increase of absolute and relative lung weight in animals of test group 3 (60 mg/m³) and the relative lung weight in test group 2 (20 mg/m³) is regarded as treatment-related and correlates with histopathological findings.
(See tables below). Findings were reversible within the recovery period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Two animals of test group 3 (60 mg/m³) showed a yellow discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related.
After recovery, three animals of test group three showed a yellow discoloration, and so did one animal of the low dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the lungs animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to moderate hyperplasia/hypertrophy of bronchioli (mainly small and terminal bronchi and the bronchio-alveolar transition were affected). Furthermore, an increase of alveolar histiocytes was observed which contained yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³) and a minimal infiltration of intra alveolar neutrophils in test group 3 animals (60 mg/m³). Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above-mentioned yellow particles within their cytoplasm. These findings were regarded to be treatment-related.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes. In the mediastinal lymph nodes of test groups 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed the same yellow particles as described for the lungs. These findings were regarded to be treatment-related.


Recovery findings:
Comparable to the main groups yellow particles within minimally increased numbers of histiocytes were observed in treated recovery animals of test groups 2 and 3 (20 and 60 mg/m³). The histiocytes tended to accumulate in the area of the bronchio-alveolar transition. Macrophages with particles within the BALT were observed in animals of test group 2 and 3 (20 and 60 mg/m³) as well as particles within alveolar septae in test group 3, only. Alveolar histiocytes containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³) but were not increased in number.
A single animal (No. 39) of test group 3 (60 mg/m³) still showed hyperplasia/hypertrophy bronchiole, neutrophilic infiltrates and cellular debris intra alveolar in addition. It was regarded to be degenerating histiocytes as also free yellow particles were observed. All these findings were regarded to be treatment-related. The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal lymph nodes of all treated test groups macrophages were observed that revealed comparable particles as described for the lungs. They were also found as agglomerated macrophages with particles. These findings were regarded to be treatment-related.

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:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar lavage fluid (BAL)
After the administration period in BAL of males of test group 3 (60 mg/m3) total cell counts as well as above all absolute and relative neutrophil cell and absolute eosinophil cell counts were increased (absolute eosinophil counts not statistically significantly). Additionally, a moderate increase of absolute and relative lymphocyte as well as absolute macrophage, monocyte and epithelial cell counts could be observed. In BAL of males of test group 2 (20 mg/m3) total cell counts were not changed, but absolute and relative neutrophil, lymphocyte counts as well as absolute monocyte cell counts were already increased. Relative macrophage counts in BAL of males in test groups 2 and 3 were decreased. These alterations were regarded as treatment related and adverse.

In males of test group 2 (20 mg/m3) absolute eosinophil counts were also increased, but not statistically significantly. However, the mean value was within the historical control range (males, absolute eosinophils 0.00-0.21 cn/µL BAL). Therefore, this change was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell count values in BAL were back in the normal range. In males of test group 11 (5 mg/m3) relative lymphocyte counts were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

In BAL of males in test group 3 (60 mg/m³) N-acetyl--D-glucosaminidase (NAG) activities were also significantly increased, but the values were within the historical control range (males, NAG 10-79 nkat/L). The same was true for significantly increased total protein levels in BAL of males in test group 2 (20 mg/m3) as well as in GGT activities in BAL of males in test group 1 (5 mg/m3)( males, total protein 18-50 mg/L, GGT 25-64 nkat/L). Therefore, these changes were regarded as incidental and not treatment related.
After the three-week recovery period, all BAL parameter values were back in the normal range.

During the exposure period the animals all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur. Moreover, substance-like discoloration of the fur was observed in all animals of the high concentration of the test substancewerewed substance-like discoloration of the fur on study day 6.
Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
Dose descriptor:
NOEC
Remarks:
systemic effects
Effect level:
>= 60 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: only findings in BALF
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes

Table 1: Test concentration and particle size distribution

Target concentration

Measurement

MMAD (µm)

GSD

Fraction < 3 µm

5 mg/m³

1

0.77

2.90

90.0 %

2

0.70

2.41

95.0 %

20 mg/m³

1

0.71

2.22

96.4 %

2

0.66

2.67

93.9 %

60 mg/m³

1

0.72

2.06

97.6 %

2

0.85

2.51

91.6 %

Table 2: Particle count distribution measured by SMPS

Target concentration

Total count concentration (N/cm³)

Geometric mean diameter
(nm)

Geometric standard deviation

5

103723

266

1.75

20

340464

295

1.75

60

840216

288

1.75

Table 3 Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure).

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr. 12

20 mg/m3

Gr. 13

60 mg/m3

Total Cells

1.1

1.2

3.3**

0.6

1.1

0.7

Eosinophils

3.1

10.4

24.4

0.1

0.3

0.4

Lymphocytes

2.0

2.8*

7.9**

1.5

1.0

1.0

Macrophages

1.1

1.1

2.3**

0.6

1.1

0.7

Neutrophils

0.7

5.6**

27.8**

1.3

1.3

2.1

Monocytes

1.3

3.1*

9.9**

8.9

0.0

2.8

Epithelial cells

0.8

0.4

4.1*

0.9

2.4

0.2

One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01: + increase could not be calculated because of zero activity in controls

Table 4: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr.2 2

20 mg/m3

Gr. 13

60 mg/m3

Total Protein

1.0

1.3*

1.9**

1.3

1.4

1.1

GGT

1.2**

2.4**

3.9**

1.0

1.3

1.0

LDH

1.0

1.1

2.3**

0.9

1.3

1.0

ALP

1.2

1.9**

2.8**

1.1

1.0

1.2

NAG

1.2

1.3

2.2*

1.1

1.3

0.9

GGT =g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = N-Acetyl-b--D-glucosaminidase, One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01

Table 5: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr. 12

20 mg/m3

Gr. 13

60 mg/m3

CINC-1/IL-8

1.5*

4.1**

4.2**

1.1

1.4*

1.3

Osteopontin

1.1

1.1

4.3**

1.3

1.4

2.5*

.BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1

one-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01

Table 6a: Absolute and relative organ weights (main group)

Test group

(mg/m³)

01

(5)

02

(20)

03

(60)

Lungs, absolute

(and relative)

+8.8%

(+ 3.3%)

+20.9%

(+12.6%)*

+24.6%*

(+21.8%)*

*p <= 0.05; **p <= 0.01

Table 6b Relative changes of absolute organ weights (recovery group).

 

Male animals

Test group

(mg/m³)

11

(5)

12

(20)

13

(60)

Brain

+8.8%**

+4.8%**

+3.5%

Heart

+18.1%**

+5.8%

+15.0%*

Spleen

+18.6%

+11.6%

+34.5%**

NB All mean relative weight parameters did not show significant differences when compared to the control group

Table 7a: Histopathology ( Incidence and severity of histological findings in the lungs of main group animals)

Lungs

Male animals

Test group

(mg/m³)

00

(0)

01

(5)

02

(20)

03

(60)

No. of animals

5

5

5

5

Hyperplasia/hypertrophy, bronchioli, (m)f

0

0

5

5

·       Grade 1

 

 

3

1

·       Grade 2

 

 

2

2

·       Grade 3

 

 

 

2

Histiocytosis alveolar with particles, (m)f

0

0

5

5

  • Grade 1

 

 

5

 

  • Grade 2

 

 

 

5

Particles within histiocytes*

0

5

0

0

Balt: macrophages with particles (m)f

0

0

4

4

·       Grade 1

 

0

4

4

Infiltrate neutrophilic, (m)f

0

0

0

4

·       Grade 1

 

 

 

4

Table 7b Incidence and severity of histological findings in the lungs of recovery group animals

Lungs

Male animals

Test group

(mg/m³)

01

(0)

11

(5)

12

(20)

13

(60)

No. of animals

5

5

5

5

Hyperplasia/hypertrophy, bonchioli

0

0

0

1

·       Grade 1

 

 

 

1

Debris, cellular, (m)f

0

0

0

1

·       Grade 1

 

 

 

1

Histiocytosis alveolar with particles, (m)f

0

0

5

5

  • Grade 1

 

 

5

5

Macrophages with particles, interstitial (m)f

0

0

0

2

  • Grade 1

 

 

 

2

Particles within histiocytes*

0

5

0

0

Balt:[WT1] macrophages with particles (m)f

0

0

1

3

·       Grade 1

 

 

1

2

·       Grade 2

 

 

 

1

Infiltrate neutrophilic, (m)f

0

0

0

1

·       Grade 1

 

 

 

1

Executive summary:

Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.

 After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³. 

 Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3.. The systemic NOAEC is above 60 mg/m³ (high concentration group).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Apr 22 - Oct 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Principles of method if other than guideline:
5-day dust inhalation study in rats (with bronchoalveolar lavage, 3 weeks recovery period)
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
yellow powder
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH; 97633 Sulzfeld
- Age at study initiation: about 7 weeks (when supplied)
- Weight at study initiation (means):ca 265g
- Housing: The rats were housed together (up to 5 animals per cage) in Polysulfon cages (H-Temp [P SU]) supplied by TECNIPLAST, Hohenpeißenberg, Germany (floor area about 2065 cm2). Bedding in
the Polycarbonate cages were Type Lignocel fibres, dust-free bedding, supplied by SSNIFF, Soest,
Germany. Dust-free wooden bedding was used in this study. For enrichment wooden gnawing blocks
(Typ NGM E-022), supplied by Abedd Lab. and Vet. Service GmbH, Vienna, Austria, were added.
- Diet: Mouse/rat laboratory diet “GLP”, 10 mm pellets (Provimi Kliba SA, Kaiseraugst, Basel Switze rland), ad libitum.
- Water: Tap water, ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%) 45 - 65
- Air changes (per hr): 15
Photoperiod (hrs dark / hrs light):: 12h/12h
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
clean air
Mass median aerodynamic diameter (MMAD):
>= 0.66 - <= 0.77 µm
Geometric standard deviation (GSD):
245
Remarks on MMAD:
All measurements of particle size resulted in MMADs between 0.66 and 0.77 µm with GSDs between 2.06 and 2.90. The calculated mass fractions of particles below 3 µm aerodynamic size ranged between 90.0 % and 97.6 %.

SMPS showed different geometric mean concentration than those measured by cascade impactor measurement. Major reason is that this geometric mean referred to count distribution, while cascade impactor measurement measured mass-based aerodynamic diameter.

The SMPS showed very high particle count concentrations in all concentrations. The geometric mean count diameters were between 266 nm and 295 nm.
Details on inhalation exposure:
For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage, mixed with conditioned air, and passed via the cyclonic separator and glass tube into the inhalation system

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Generator systems: Solid particle generators (brush-generator), Aerosol mixing tube (Stainless steel), Glass cyclonic separators
- Generation procedure: The test substance was used unchanged. By means of dust generators the substance to be tested is generated into dust aerosols using compressed air in a mixing stage, mixed
with conditioned air and passed into the inhalation systems via cyclonic separators. For each concentration, a solid particle generator (brush-generator) wias used for generating the dust. The con
centration was adjusted by varying the piston feed and by varying the brush rotation. For each concentration the dust aerosol was generated with the dust generator and compressed air inside a mixing stage mixed with conditioned dilution air and passed via the cyclonic separator into the inhalation system.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentrations of the inhalation atmospheres in test groups 1 - 3 were analyzed by gravimetry. This method was applicable because the test item possessed extremely low vapor pressure. Daily means were calculated based on 3 measured samples per concentration and exposure. From the daily mean values of each concentration, mean concentrations and standard deviations for the entire study were derived.In these groups, the constancy of concentrations in each chamber was continuously monitored using scattered light photometers.

The particle size analysis was carried out with a cascade impactor with the following equipment:
• Stack sampler Marple 298 (New Star Environmental, Inc., Roswell, Georgia 30075, USA)
• Vacuum compressed air pump (Millipore Corporation, Billerica, MA 01821, USA)
• Limiting orifice 3 L/min (Millipore Corporation, Billerica, MA 01821, USA)
• Sampling probe internal diameter 6.9 mm
• Balance Sartorius MSA 6.6S-000-DF (Sartorius AG, Göttingen, Germany)
Sampling for particle size analyses:Pre-weighed metal collecting discs and a backup particle filter were placed into the cascade impactor and two samples were taken in each concentration at a sampling velocity of 1.25 m/sec. from the breathing zones of the animals.
The amount of dust deposited by each stage in mg was calculated from the difference between the weight of the filter/metal collecting disc and backup filter before and after sampling.The deposits in the probe and the wall losses in the impactor were also determined as difference of the total mass increase of the impactor and the sum of masses on the collecting discs and backup filter.

To determine the particle size distribution in the submicrometer range, each test atmosphere was measured with the Scanning Mobility Particle Sizer (SMPS; Grimm Aerosol Technik GmbH& Co KG, Ainring, Germany). The SMPS system comprises an Electrostatic Classifier (Model Vienna U-DMA) which separates the particles into known size fractions, and a Condensation Particle Counter (CPC) which measures particle count concentrations. The DMA was equipped with Am-241 neutralizer. During the exposure period, one measurement per concentration with 10 repeats each were performed.

Real time surveillance of the inhalation atmospheres with scattered light photometers generally proved the constancy of each concentration throughout the daily exposures.
The air flows were constantly maintained in the desired range. An air change of about 65 to 67 times per hour can be calculated by dividing the supply air flow through the volume of each inhalation system. Daily mean relative humidities in the inhalation systems ranged between 33.7 and 49.6 %. Daily mean temperatures in the inhalation systems ranged between 20.6 and 22.1 °C. These values were within guideline recommendations.
Duration of treatment / exposure:
6h for 5 days
Frequency of treatment:
daily
Dose / conc.:
4.98 mg/m³ air (analytical)
Remarks:
SD 0.27
Dose / conc.:
20 mg/m³ air (analytical)
Remarks:
SD 1.0
Dose / conc.:
60.4 mg/m³ air (analytical)
Remarks:
SD 2.1
No. of animals per sex per dose:
10 (five for sacrifice after exposure and 5 for sacrifice after recovery)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Results of short-term inhalation studies with other inert organic pigments
- Rationale for animal assignment: random
- Fasting period before blood sampling for clinical bioche Particlesmistry: overnight
- Rationale for selecting satellite groups: Clearance of inert particles by lung macrophages is known to take time
- Post-exposure recovery period in satellite groups: 3 weeks



Positive control:
not applicable
Observations and examinations performed and frequency:
A check for moribund or dead animals was carried out twice per day on working days. A check for moribund or dead animals was carried out once per day on weekends and holidays.

The clinical observation was performed on each animal at least three times (before, during and after exposure) on exposure days and once a day during pre-exposure and post exposure observation days. On exposure-free weekends and post exposure observation weekends, no clinical observation was performed. Signs and findings were recorded for each animal.During exposure only a group wise examination was possible.

The animals were weighed prior to the pre-exposure period (study day -5), at the start of the exposure period (study day 0), at the end of the exposure period (study day 4), as well as on the study days 5, 12, 19 and 26.

Food consumption was determined once over the exposure period (study day 0 – study day 4), during the post-exposure period weekly and calculated as mean food consumption in grams per animal and day.The animals were maintained in social-housing cages, with 5 animals per cage, during the whole study period. Therefore, the food consumption was determined cage-wise. The food consumption per animal and day was calculated by dividing food consumption of the day of a respective cage by the 5 animals per cage. As the animals of each test group were housed in only two cages per sex, no statistical evaluation of food consumption is possible
Sacrifice and pathology:
Clinical pathology
In the morning blood was taken from the retro-bulbar venous plexus from fasted animals. The animals were anaesthetized using isoflurane. The blood sampling procedure and subsequent analysis of blood samples were carried out in a randomized sequence (the list of randomization instructions was compiled with a computer).
The assays of blood and serum parameters were performed under internal laboratory quality control conditions with reference controls to assure reliable test results.
The results of clinical pathology examinations were expressed in International System (SI) units. The following parameters of the animals were examined
Clinical chemistry: Alanine aminotransferase, Aspartate aminotransferase, Alkaline phosphatase,-Glutamyltransferase, Inorganic phosphate, Calcium, Urea, Creatinine, Glucose, Total bilirubin, Total protein, Albumin, Globulins,Triglycerides,Cholesterol
Bronchoalveolar lavage fluid (BAL): The animals designated for lung lavage were killed by exsanguination from aorta abdominalis and vena cava under Narcoren® anesthesia. The lung was lavaged by two instillations of physiologic saline.
Parameters and methods of cytological examination in BAL: Total cell count, Macrophages, Polymorphonuclear neutrophils, Lymphocytes, Eosinophils, Monocytes, Epithelial, Gamma−Glutamyltransferase, Protein, Lactate dehydrogenase, Alkaline phosphatase, N-acetyl-Beta-Glucosaminidase
Cytokines in BAL: Rat monocyte chemoattractant protein-1 (rat MCP-1), Rat cytokine-induced neutrophil chemoattractant-1 level (rat CINC-1/IL-8), Rodent osteopontin

Necropsy
The animals were sacrificed under pentobarbital anesthesia by exsanguination from the abdominal aorta and vena cava. Afterwards, the thorax was opened, the right lung lobes werelavaged, whereas the left lung lobe was ligated during lavage. Immediately after lung lavage,
the animals were necropsied and assessed by gross pathology.

The following weights were determined in all animals sacrificed on schedule:
1. Anesthetized animals (terminal body weight)
2. Adrenal glands (fixed)
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Lungs
9. Spleen
10. Testes
11. Thymus (fixed)
12. Thyroid glands (with parathyroid glands) (fixed)
All paired organs were weighed together (left and right).

The following organs or tissues were fixed in 4% neutral buffered formaldehyde solution:
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain with olfactory bulb
5. Epididymides
6. Esophagus
7. Eyes with optic nerve
8. Heart
9. Kidneys
10. Larynx/pharynx
11. Liver
12. Lungs
13. Lymph nodes (tracheobronchial and mediastinal lymph nodes)
14. Nose (nasal cavity)
15. Seminal vesicles
16. Spinal cord (cervical, thoracic and lumbar cord)
17. Spleen
18. Stomach (forestomach and glandular stomach)
19. Testes
20. Thyroid glands
21. Thymus
22. Trachea
23. Urinary bladder


Extend of histological processing and sub-sequent microscopical examinations in main group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal), nasal cavity (4 levels), trachea and in recovery group animals: all gross lesions, larynx (3 level), lungs, lymph nodes (tracheobronchial, mediastinal)






Other examinations:
Lung lavage: The animals intended for lung lavage were sacrificed under pentobarbitone anesthesia by exsanguination from the abdominal aorta and vena cava. The right lung was lavaged in situ with physiological saline, whereas the left lung was ligated during this procedure.
Statistics:
Body weight, body weight change: Comparison of each group with the control group was performed using DUNNETT test (two-sided) for the hypothesis of equal means
Blood parameters: For parameters with bidirectional changes: Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting pvalue was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the hypothesis of equal medians
For parameters with unidirectional changes: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
BALF: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians
Organ weights: Non-parametric one-way analysis using the Kruskal-Wallis test (two-sided). If the resulting p-value was equal to or less than 0.05, a pair-wise comparison of each dose group with the control group was performed using the Wilcoxon test (two-sided) for the hypothesis of equal medians.

Terminal body weight: Comparison of each group with the control group was performed using the Dunnett test (two-sided) for the hypothesis of equal means.
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 examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
In males of test group 3 (60 mg/m3) absolute neutrophil counts were significantly higher compared to controls. The values were slightly above the historical control range (males, neutrophils 0.53-1.01 Giga/L). however, total white blood (WBC) counts as well as all other differential blood cell fractions were not changed. Therefore, this isolated alteration of absolute neutrophil counts in males of test group 3 was regarded as treatment related but non-adverse (ECETOC Technical Report No. 85, 2002).

After the three-week recovery period, no changes of absolute neutrophil counts were observed. In males of test group 2 (20 mg/m3) absolute and relative, large unstained cell (LUC) counts were significantly higher compared to study controls, but the change was not dose dependent. Therefore, this alteration was regarded as incidental and not treatment related.
Clinical biochemistry findings:
no effects observed
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The significant increase of absolute and relative lung weight in animals of test group 3 (60 mg/m³) and the relative lung weight in test group 2 (20 mg/m³) is regarded as treatment-related and correlates with histopathological findings.
(See tables below). Findings were reversible within the recovery period.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Two animals of test group 3 (60 mg/m³) showed a yellow discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related.
After recovery, three animals of test group three showed a yellow discoloration, and so did one animal of the low dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
In the lungs animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to moderate hyperplasia/hypertrophy of bronchioli (mainly small and terminal bronchi and the bronchio-alveolar transition were affected). Furthermore, an increase of alveolar histiocytes was observed which contained yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³) and a minimal infiltration of intra alveolar neutrophils in test group 3 animals (60 mg/m³). Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above-mentioned yellow particles within their cytoplasm. These findings were regarded to be treatment-related.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes. In the mediastinal lymph nodes of test groups 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed the same yellow particles as described for the lungs. These findings were regarded to be treatment-related.


Recovery findings:
Comparable to the main groups yellow particles within minimally increased numbers of histiocytes were observed in treated recovery animals of test groups 2 and 3 (20 and 60 mg/m³). The histiocytes tended to accumulate in the area of the bronchio-alveolar transition. Macrophages with particles within the BALT were observed in animals of test group 2 and 3 (20 and 60 mg/m³) as well as particles within alveolar septae in test group 3, only. Alveolar histiocytes containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³) but were not increased in number.
A single animal (No. 39) of test group 3 (60 mg/m³) still showed hyperplasia/hypertrophy bronchiole, neutrophilic infiltrates and cellular debris intra alveolar in addition. It was regarded to be degenerating histiocytes as also free yellow particles were observed. All these findings were regarded to be treatment-related. The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal lymph nodes of all treated test groups macrophages were observed that revealed comparable particles as described for the lungs. They were also found as agglomerated macrophages with particles. These findings were regarded to be treatment-related.

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:
not examined
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Bronchoalveolar lavage fluid (BAL)
After the administration period in BAL of males of test group 3 (60 mg/m3) total cell counts as well as above all absolute and relative neutrophil cell and absolute eosinophil cell counts were increased (absolute eosinophil counts not statistically significantly). Additionally, a moderate increase of absolute and relative lymphocyte as well as absolute macrophage, monocyte and epithelial cell counts could be observed. In BAL of males of test group 2 (20 mg/m3) total cell counts were not changed, but absolute and relative neutrophil, lymphocyte counts as well as absolute monocyte cell counts were already increased. Relative macrophage counts in BAL of males in test groups 2 and 3 were decreased. These alterations were regarded as treatment related and adverse.

In males of test group 2 (20 mg/m3) absolute eosinophil counts were also increased, but not statistically significantly. However, the mean value was within the historical control range (males, absolute eosinophils 0.00-0.21 cn/µL BAL). Therefore, this change was regarded as incidental and not treatment related.
After the three-week recovery period all altered cell count values in BAL were back in the normal range. In males of test group 11 (5 mg/m3) relative lymphocyte counts were significantly increased, but the change was not dose dependent. Therefore, it was regarded as incidental and not treatment related.

In BAL of males in test group 3 (60 mg/m³) N-acetyl--D-glucosaminidase (NAG) activities were also significantly increased, but the values were within the historical control range (males, NAG 10-79 nkat/L). The same was true for significantly increased total protein levels in BAL of males in test group 2 (20 mg/m3) as well as in GGT activities in BAL of males in test group 1 (5 mg/m3)( males, total protein 18-50 mg/L, GGT 25-64 nkat/L). Therefore, these changes were regarded as incidental and not treatment related.
After the three-week recovery period, all BAL parameter values were back in the normal range.

During the exposure period the animals all animals of the mid and high concentration (20 and 60 mg/m³) showed substance-contaminated fur. Moreover, substance-like discoloration of the fur was observed in all animals of the high concentration of the test substancewerewed substance-like discoloration of the fur on study day 6.
Dose descriptor:
LOAEC
Remarks:
local effects
Effect level:
5 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: min. infiltration of neutrophils within bronchiolar epithelium, hyptrophy/hyperplasia of terminal bronchi (terminal and small), hyperplasia of type II pneumocytes, increased cellularity in the mediastinal lymph nodes. Findings in BALF
Dose descriptor:
NOEC
Remarks:
systemic effects
Effect level:
>= 60 mg/m³ air (analytical)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: only findings in BALF
Critical effects observed:
yes
Lowest effective dose / conc.:
5 mg/m³ air (analytical)
System:
respiratory system: upper respiratory tract
Organ:
lungs
Treatment related:
yes
Dose response relationship:
yes

Table 1: Test concentration and particle size distribution

Target concentration

Measurement

MMAD (µm)

GSD

Fraction < 3 µm

5 mg/m³

1

0.77

2.90

90.0 %

2

0.70

2.41

95.0 %

20 mg/m³

1

0.71

2.22

96.4 %

2

0.66

2.67

93.9 %

60 mg/m³

1

0.72

2.06

97.6 %

2

0.85

2.51

91.6 %

Table 2: Particle count distribution measured by SMPS

Target concentration

Total count concentration (N/cm³)

Geometric mean diameter
(nm)

Geometric standard deviation

5

103723

266

1.75

20

340464

295

1.75

60

840216

288

1.75

Table 3 Changes in mean absolute cell counts in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure).

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr. 12

20 mg/m3

Gr. 13

60 mg/m3

Total Cells

1.1

1.2

3.3**

0.6

1.1

0.7

Eosinophils

3.1

10.4

24.4

0.1

0.3

0.4

Lymphocytes

2.0

2.8*

7.9**

1.5

1.0

1.0

Macrophages

1.1

1.1

2.3**

0.6

1.1

0.7

Neutrophils

0.7

5.6**

27.8**

1.3

1.3

2.1

Monocytes

1.3

3.1*

9.9**

8.9

0.0

2.8

Epithelial cells

0.8

0.4

4.1*

0.9

2.4

0.2

One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01: + increase could not be calculated because of zero activity in controls

Table 4: Changes in mean total protein and enzyme levels in BAL (x-fold of concurrent control) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr.2 2

20 mg/m3

Gr. 13

60 mg/m3

Total Protein

1.0

1.3*

1.9**

1.3

1.4

1.1

GGT

1.2**

2.4**

3.9**

1.0

1.3

1.0

LDH

1.0

1.1

2.3**

0.9

1.3

1.0

ALP

1.2

1.9**

2.8**

1.1

1.0

1.2

NAG

1.2

1.3

2.2*

1.1

1.3

0.9

GGT =g-Glutamyl-transferase; LDH = Lactate dehydrogenase; ALP = Alkaline phosphatase;

NAG = N-Acetyl-b--D-glucosaminidase, One-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01

Table 5: Changes in antigen levels in BAL (x-fold of concurrent control means) on study day 5 (1 day after last exposure) and study day 30 (3 weeks after last exposure)

Analyte

Study day 5

Study day 30

 

Gr. 1

5 mg/m3

Gr. 2

20 mg/m3

Gr. 3

60 mg/m3

Gr. 11

5 mg/m3

Gr. 12

20 mg/m3

Gr. 13

60 mg/m3

CINC-1/IL-8

1.5*

4.1**

4.2**

1.1

1.4*

1.3

Osteopontin

1.1

1.1

4.3**

1.3

1.4

2.5*

.BALF = Broncho-alveolar lavage fluid; CINC-1/IL-8 = cytokine-induced neutrophil chemoattractant-1

one-sided Wilcoxon-test: * : p<=0.05; ** : p<=0.01

Table 6a: Absolute and relative organ weights (main group)

Test group

(mg/m³)

01

(5)

02

(20)

03

(60)

Lungs, absolute

(and relative)

+8.8%

(+ 3.3%)

+20.9%

(+12.6%)*

+24.6%*

(+21.8%)*

*p <= 0.05; **p <= 0.01

Table 6b Relative changes of absolute organ weights (recovery group).

 

Male animals

Test group

(mg/m³)

11

(5)

12

(20)

13

(60)

Brain

+8.8%**

+4.8%**

+3.5%

Heart

+18.1%**

+5.8%

+15.0%*

Spleen

+18.6%

+11.6%

+34.5%**

NB All mean relative weight parameters did not show significant differences when compared to the control group

Table 7a: Histopathology ( Incidence and severity of histological findings in the lungs of main group animals)

Lungs

Male animals

Test group

(mg/m³)

00

(0)

01

(5)

02

(20)

03

(60)

No. of animals

5

5

5

5

Hyperplasia/hypertrophy, bronchioli, (m)f

0

0

5

5

·       Grade 1

 

 

3

1

·       Grade 2

 

 

2

2

·       Grade 3

 

 

 

2

Histiocytosis alveolar with particles, (m)f

0

0

5

5

  • Grade 1

 

 

5

 

  • Grade 2

 

 

 

5

Particles within histiocytes*

0

5

0

0

Balt: macrophages with particles (m)f

0

0

4

4

·       Grade 1

 

0

4

4

Infiltrate neutrophilic, (m)f

0

0

0

4

·       Grade 1

 

 

 

4

Table 7b Incidence and severity of histological findings in the lungs of recovery group animals

Lungs

Male animals

Test group

(mg/m³)

01

(0)

11

(5)

12

(20)

13

(60)

No. of animals

5

5

5

5

Hyperplasia/hypertrophy, bonchioli

0

0

0

1

·       Grade 1

 

 

 

1

Debris, cellular, (m)f

0

0

0

1

·       Grade 1

 

 

 

1

Histiocytosis alveolar with particles, (m)f

0

0

5

5

  • Grade 1

 

 

5

5

Macrophages with particles, interstitial (m)f

0

0

0

2

  • Grade 1

 

 

 

2

Particles within histiocytes*

0

5

0

0

Balt:[WT1] macrophages with particles (m)f

0

0

1

3

·       Grade 1

 

 

1

2

·       Grade 2

 

 

 

1

Infiltrate neutrophilic, (m)f

0

0

0

1

·       Grade 1

 

 

 

1

Executive summary:

Inhalation exposure of rats to 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.

 After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³. 

 Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) for local effects could was 5 mg/m3.. The systemic NOAEC is above 60 mg/m³ (high concentration group).

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
5 mg/m³
Study duration:
subacute
Species:
rat

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

The effects of the test item on subacute oral toxicity as well as male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, when administered daily by oral gavage to Wistar Hannover rat (OECD 422, GLP) (ERBC 2022). Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, up to a total of 35/36 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, up to at least 51 days. No treatment-related mortalities occurred during the study. One control female was sacrificed for humane reasons on Day 22 post coitum due to difficulties in parturition. No treatment-related clinical signs were recorded. No treatment-related signs of neurotoxicity and no treatment-related differences in body weight, body weight gain and food consumption were observed in treated animals, compared to the controls. No treatment-related changes were observed in haematological and clinical chemistry parameters. No changes considered treatment-related were observed in thyroid hormone levels in parental animals or in pups at Days 4 and 14 post partum determinations. No treatment-related anomalies were noted in the oestrous cycle, copulatory and fertility indices of the treated females, when compared to controls. Parturition, lactation, implantation, litter data and sex ratio did not show any changes which could be considered adverse. No significant differences in the anogenital distance was recorded in pups on Day 1 post partum and no nipples were seen between control and treated groups of male pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect. No treatment-related changes were detected at post mortem examination in treated animals, when compared with controls. There were no test item-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides). On the basis of these results the dosage of 1000 mg/kg/day is considered the NOAEL (No Observed Adverse Effect Level) for systemic toxicity, fertility and reproduction parameters for F0 males, F0 females and F1 pups.

The purpose of the short-term inhalation study (BASF 2022) 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 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.For this purpose, nine-week-old male Wistar rats (5 rats per main group and 5 rats per post-exposure observation group) were nose only exposed to fresh air (control group) or dust of the test substance at concentrations of 5, 20, and 60 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. On half of t he 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.

 

Inhalation exposure of rats to 5, 20 or 60 mg/m³ on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and monocyte and macrophage counts in bronchoalveolar lavage, while relative macrophage count was reduced in lavage fluid. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, minimal infiltration of neutrophils within bronchiolar epithelium was observed, as well as hyptrophy/hyperplasia of bronchioli. The absolute and relative lung weights were increased at the high concentration. Similar findings were also observed at 20 mg/m³ with reduced severity.

 After the post-exposure period of 3 weeks, the effects resolved partly at the high concentration of 60 mg/m³ and was fully recovered at the mid concentration of 20 mg/m³. 

 Thus, under current study conditions, a no observed adverse effect concentration (NOAEC) forlocal effects could was 5 mg/m3.. The systemic NOAEC is above 60 mg/m³ (high concentration group).

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on repeated dose toxicity, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.