N,N'-(2-chloro-1,4-phenylene)bis[4-[(4-chloro-2-nitrophenyl)azo]-3-hydroxynaphthalene-2-carboxamide]

Endpoint summary

Description of key information

Oral: NOAEL >= 1000 mg/kg bw/day, rat, according to OECD422, GLP-compliant, K1, 2022

Inhalation: NOAEC = 5 mg/m3, rat, short-term inhalation study, GLP-compliant, K1, 2022

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 22 Nov 2021 to 03 Mar 2022

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same 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.: 0004501436
- Content 99.4%
- Retest date: 22 October 2022
- Appearance: brown 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: 11-12 weeks old
- Weight at acquisition: 181-209 g (females), 231-246 g (males)
- 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. 3 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: 22 Nov 2021 (allocation) To: 30 Jan 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 a preliminary non-GLP compliant study
- 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:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related clinical signs were noted in males and in females before the mating period and thereafter during post coitum and post partum periods.
One male receiving 300 mg/kg/day was isolated in cage for 5 days due to a presence of an injury on the head. During this period the same animal also showed teeth missing. Thereafter a recovery from these signs were noted. Corneal opacity, probably the result of trauma, was noted in one male dosed at 1000 mg/kg/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes in body weight and body weight gain were observed during the study in treated animals of both gender, when compared to controls.
The negative gain weight noted in all groups and sexes on the last measurement was due to the fact that the animals were placed under condition of food deprivation for blood collection.

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):

No treatment-related changes were recorded.
The statistically significant difference of platelets recorded between control and females dosed at 100 and 300 mg/kg/day was not dose-related, therefore it was considered to be incidental.
Coagulation: One females dosed at 1000mg/kg/day showed high blood clotting time. Due to the minimal incidence and the absence of changes in the other coagulation parameters, this finding was considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No treatment-related changes were recorded.
The differences statistically significant or not, observed between control and treated animals (alkaline phosphatase and aspartate aminotransferase in males, bile acids in females) were recorded in the low or medium groups only, therefore they were considered to be incidental.

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 and from samples from pups on Day 14 post partum.
No changes were recorded.

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

Description (incidence and severity):

No treatment-related changes in body weight and body weight gain were observed during the study in treated animals of both gender, when compared to controls. The negative gain weight noted in all groups and sexes on the last measurement was due to the fact that the animals were placed under condition of food deprivation for blood collection.
There were no treatment-related changes in terminal body weight and organ weights when compared to the controls. Any organ weight variations were not correlated in the range of expected spontaneous changes in rats of the same age and considered unrelated to treatment.

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, non-treatment-related

Description (incidence and severity):

Parturition: On day 28 post coitum, one control female showed unilateral total resorption on left uterine horn and was found not pregnant in the right one. One female of Group 3 (300 mg/kg/day) was found not pregnant at necropsy on day 29 post coitum. The number of pregnant females was: 10, 10, 9 and 10 for Groups 1, 2, 3, and 4 respectively. The number of females with live pups on day 14 post partum was 9 in the control group, 10 at 100 mg/kg/day, 9 at 300 mg/kg/day and 10 at 1000 mg/kg/day.
Reproduction indices: Copulatory and fertility indices were considered to be unaffected by treatment with the test item. Pre-natal loss (percentage) was similar between control and treated groups.
Litter data: No treatment-related differences were observed in total litter size, live litter size, mean pup loss and mean pup weights among the treated dams and the controls at birth and on days 1 and 4 post partum. On day 4 post partum, the increase in mean post-natal loss observed in Group 3 females, compared to the control, was attributed to one female. The litter size of this female was reduced (3 pups on day 1 vs 9 on the day of birth) by more than half compared to the day of birth. Litter weight and mean pups weight (for both sexes combined) of treated groups were similar with control. No differences in pup loss was noted between control and treated groups. No treatment-related differences were observed in sex ratio between treated and control litters at birth and on days 1, 4 and 13 post partum. No treatment-related clinical signs were observed in pups during the 14-day observation period. The litter of one female (Group 3 - 300 mg/kg/day) showed shortly after birth all pups cold to touch and then mostly were missing thereafter. No differences in the anogenital distance (value normalized to the cube root of bodyweight), performed on day 1 post partum, were seen between control and treated groups both for male and female pups. No differences were observed in the weight of thyroid in treated pups, when compared to controls. No significant findings were seen in pups killed on days 4 and 14 post partum. At necropsy, on day 14 post partum, no nipples were found in male pups to be retained.

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 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

Quality of whole database:

Valid without restriction

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:

Feb 8 2022 - August 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

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 255g
- 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):

>= 0.73 - <= 1.05 µm

Geometric standard deviation (GSD):

3

Remarks on MMAD:

The particle size resulted in MMADs between 0.51 and 0.77 µm with GSDs between 2.9 and 3.92 (Table 1). The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 88 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

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.:

5.1 mg/m³ air (analytical)

Remarks:

63853 particles per cm3 (measured by SMPS)

Dose / conc.:

20.1 mg/m³ air (analytical)

Remarks:

116362 particles per cm3 (measured by SMPS)

Dose / conc.:

59.7 mg/m³ air (analytical)

Remarks:

355121 particles per cm3 (measured by SMPS)

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:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.
During the exposure period the animals of the control group, showed no clinical signs and findings different from normal.
During the study two of the ten animals of test group 1 (5 mg/m3), seven of the ten animals of the test group 2 (20 mg/m3) and all the ten animals of test group 3 (60 mg/m3 ) showed test item
contaminated fur. Five of the ten animals of test group 3 (60 mg/m3) showed discolored fur up to day 19 of the post-exposure observation period. These findings were considered treatment-related, but not adverse.

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

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 (+17.6%) and relative (+14.2%) lung weight in animals of test group 3 (60 mg/m³) is regarded as treatment-related and correlates with histopathological findings. It was reversible within the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of test group 2 and 3 (20 and 60 mg/m³) showed a brown discoloration of the lungs as well as mediastinal and tracheobronchial (test group 3, only) lymph nodes. These findings were regarded to be treatment-related.
After recovery, only animals of test group 3 (60 mg/m³) showed a brown discoloration of the lungs. Animals of the same test group revealed a brown discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the base of the epiglottis of four animals of test group 3 (60 mg/m³) and two animals of test group 2 (20 mg/m³), a minimal focal area with flattened epithelium and loss of cilia was seen. This finding was regarded to be treatment related.

In the lungs an increase of alveolar histiocytes was observed which contained brown-yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³). Within the alveolar septae the same particles were observed either within pneumocytes, within histiocytes
located inside the septae and/or free within the interstitium. Additionally, animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to slight hyperplasia of pneumocyte type II cells, a hyperplasia/hypertrophy of bronchioli and a minimal to slight infiltration of intraalveolar neutrophils. Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen. Inside blood vessels occasionally single particle laden macrophages were observed. In test group 3 only, intraalveolar cellular debris was seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above mentioned brown-yellow particles within their cytoplasm. These findings were regarded to be treatment-related

Recovery findings:
In general, similar findings were observed in recovery animals as described for main group animals.
Comparable particles within histiocytes as described for the main group animals 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 broncho-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. Single macrophages containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³).
In the mediastinal and tracheobronchial lymph nodes of test group 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed comparable particles as described for the lungs. In test group 3 animals (60 mg/m³) an increase of agglomerated macrophages with particles was seen. These findings were regarded to be treatment-related.
One animal of test group 3 (60 mg/m³) revealed a minimal epithelial alteration in the larynx (levelI).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid: At the end of the administration period, absolute and relative neutrophil counts as well as absolute monocyte counts were increased in BAL in males of test groups 2 and 3 (20 and 60
mg/m3). In addition, males of test group 3 showed increased absolute lymphocyte and epithelial cell counts. These changes led to a concurrent significant increase in the total cell counts in
BAL in test groups 2 and 3, whereas relative macrophage counts were slightly, but significantly decreased. These alterations were regarded as treatment-related and adverse.
At the end of the administration period in BAL of males in test groups 2 and 3 (20 and 60 mg/m3), total protein levels (MTP BAL) as well as -Glutamyl-transferase (GGT BAL), lactate dehydrogenase (LDH BAL), and alkaline phosphatase (ALP BAL) activities were significantly
increased. Further, N-Acetyl-β-D-glucosaminidase (NAG BAL) activities were also significantly increased in males of test group 3. These changes were regarded as treatment-related and adverse.

At the end of the administration period, osteopontin levels were significantly increased in BAL of males in test groups 2 and 3 (20 and 60 mg/m3 ). This alteration was regarded as treatment-related and adverse.
After the three-week recovery period, absolute and relative neutrophil counts were still marginally increased, while relative macrophage counts were marginally decreased in BAL of males in test group 3 (60 mg/m3). Additionally, cytokine-induced neutrophil chemoattractant-1levels (IL-8 BAL) were marginally, but significantly increased. These findings were considered treatment-related and adverse.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal and tracheobronchial lymph nodes of all test groups macrophages were observed that revealed the same brown-yellow particles as described for the lungs. These findings were regarded to be treatment-related.

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: particle clearance by macrophages observed, which is the physiological process to remove particles and not an adverse reaction.

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.:

20 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Table1: Test concentration and particle size distribution

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		MMAD (µm)	GSD	Fraction of particles < 3 µm [%]
		Mean	SD
01	5.0	5.1	0.6	0.83 0.73	2.96 3.07	88.1 89.7
02	20.0	20.1	0.2	1.05 1.02	2.78 2.77	84.8 85.4
03	60.0	59.7	2.4	0.74 0.90	3.02 2.81	89.7 87.8

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 count concentration increased with ascending concentration. The geometric mean count diameters were between 353 nm and 373 nm. There were no difference between the test groups.

Table 2: 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 25 (3 weeks after last exposure).

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3		Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Cells	1.0	2.1*	6.0**		1.0	0.8	1.1
Eosinophils	1.8	6.9	9.8		0.7	0.0	0.6
Lymphocytes	0.7	2.5	15.0*		0.4	0.5	1.3
Macrophages	1.0	0.8	1.3		1.0	0.8	0.9
Neutrophils	2.0	67.3**	310.8**		0.8	1.5	3.5*
Monocytes	2.5	3.0*	21.4*		0.5	0.5	2.2
Epithelial cells	1.4	0.7	3.0*		0.7	0.3	0.8

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

Table 3 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 25 (3 weeks after last exposure)

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Protein	1.1	2.0**	3.3**	1.0	0.9	1.0
GGT BAL	1.2	3.1**	4.5**	1.2	0.9	1.3
LDH BAL	1.0	2.1**	4.8**	1.2	1.1	1.6
ALP BAL	1.0	2.8**	3.4**	1.1	1.0	1.1
NAG BAL	1.2	1.6	1.9**	0.6	0.4	0.9

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

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

Table 4:  Incidence of gross lesions in main group animals

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs
·       Discoloration	0	0	3	5
Mediastinal lymph nodes
·       Discoloration	0	0	2	4
Tracheobronchial lymph nodes
·       Discoloration	0	0	0	1

Table 5: Incidence and severity of histological findings in the larynx of main group animals

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Alteration, epithelial, (m)f	0	0	2	4
Grade 1			2	4

Table 6:  Incidence and severity of histological findings in the lungs of main group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Hyperplasia/hypertrophy, bronchioli, (m)f	0	0	4	5
·       Grade 1			4
·       Grade 2				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5
Grade 2				5
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Particles, alveolar septae, (m)f	0	0	3	5
·       Grade 1			3	1
·       Grade 2				4
Macrophages with particles, i.v.	0	0	1	4
·       Grade 1			1	4
Debris, cellular, (m)f	0	0	0	5
·       Grade 1				5
Balt: macrophages with particles (m)f	0	1	2	5
·       Grade 1		1	2	5

Table 7: Incidence and severity of histological findings in the mediastinal lymph nodes of main group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles, (m)f	0	1	5	5
Grade 1		1	4
Grade 2			1	5

Table 8: Incidence of gross lesions in recovery group animal

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs
·       Discoloration	0	0	0	2
Mediastinal lymph nodes
·       Discoloration	0	0	0	5

Table 9: Incidence and severity of histological findings in the lungs of recovery group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	0	5
·       Grade 1				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5	1
Grade 2				4
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	0	5
·       Grade 1				5
Particles, alveolar septae, (m)f	0	0	0	3
·       Grade 1				3
Macrophages with particles, i.v.	0	0	0	4
·       Grade 1				4
Balt: macrophages with particles (m)f	0	0	5	5
·       Grade 1			5	5

* was diagnosed as present

Table 10: Incidence and severity of histological findings in the mediastinal lymph nodes of recovery group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophage aggregates w particles, (m)f	0	0	4	5
Grade 1			4
Grade 2				5
Macrophages with particles, (m)f	0	0	2	0
Grade 1			2

Executive summary:

Inhalation exposure of rats to 60 mg/m³ Pigment Brown 23 on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and epithelia cell counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, absolute and relative lung weights were increased, minimal infiltration of neutrophils in the lungs, hyperplasia of type II pneumocytes, cellular debris, was observed in the lungs, as well as hyptrophy/hyperplasia of bronchioli. Particles were observed within alveolar septea in the lungs. Many of these findings were also observed at 20 mg/m³ with reduced severity. The effects did not resolve completely at 60 mg/m³ after 3 weeks post-exposure period. Those observed at 20 mg/m³ resolved completely within the postexposure period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ was the lung and the tracheobronchial lymph nodes.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

60 mg/m³

Study duration:

subacute

Species:

rat

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:

Feb 8 2022 - August 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

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 255g
- 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):

>= 0.73 - <= 1.05 µm

Geometric standard deviation (GSD):

3

Remarks on MMAD:

The particle size resulted in MMADs between 0.51 and 0.77 µm with GSDs between 2.9 and 3.92 (Table 1). The calculated mass fractions of particles below 3 µm aerodynamic size is greater than 88 %. Thus, the aerosols were highly respirable for rats and a very high proportion of the aerosol particles reached the lungs.

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.:

5.1 mg/m³ air (analytical)

Remarks:

63853 particles per cm3 (measured by SMPS)

Dose / conc.:

20.1 mg/m³ air (analytical)

Remarks:

116362 particles per cm3 (measured by SMPS)

Dose / conc.:

59.7 mg/m³ air (analytical)

Remarks:

355121 particles per cm3 (measured by SMPS)

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:

effects observed, treatment-related

Description (incidence and severity):

During the pre-exposure period the animals showed no clinical signs and findings different from normal.
During the exposure period the animals of the control group, showed no clinical signs and findings different from normal.
During the study two of the ten animals of test group 1 (5 mg/m3), seven of the ten animals of the test group 2 (20 mg/m3) and all the ten animals of test group 3 (60 mg/m3 ) showed test item
contaminated fur. Five of the ten animals of test group 3 (60 mg/m3) showed discolored fur up to day 19 of the post-exposure observation period. These findings were considered treatment-related, but not adverse.

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

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 (+17.6%) and relative (+14.2%) lung weight in animals of test group 3 (60 mg/m³) is regarded as treatment-related and correlates with histopathological findings. It was reversible within the recovery period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Animals of test group 2 and 3 (20 and 60 mg/m³) showed a brown discoloration of the lungs as well as mediastinal and tracheobronchial (test group 3, only) lymph nodes. These findings were regarded to be treatment-related.
After recovery, only animals of test group 3 (60 mg/m³) showed a brown discoloration of the lungs. Animals of the same test group revealed a brown discoloration of the mediastinal lymph nodes. These findings were regarded to be treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

At the base of the epiglottis of four animals of test group 3 (60 mg/m³) and two animals of test group 2 (20 mg/m³), a minimal focal area with flattened epithelium and loss of cilia was seen. This finding was regarded to be treatment related.

In the lungs an increase of alveolar histiocytes was observed which contained brown-yellow particles within their cytoplasm in test group 2 and 3 animals (20 and 60 mg/m³). Within the alveolar septae the same particles were observed either within pneumocytes, within histiocytes
located inside the septae and/or free within the interstitium. Additionally, animals of test group 3 (60 mg/m³) and 2 (20 mg/m³) revealed a minimal to slight hyperplasia of pneumocyte type II cells, a hyperplasia/hypertrophy of bronchioli and a minimal to slight infiltration of intraalveolar neutrophils. Within the BALT (Bronchio-alveolar lymphoid tissue) macrophages containing the above-mentioned particles were seen. Inside blood vessels occasionally single particle laden macrophages were observed. In test group 3 only, intraalveolar cellular debris was seen.
Test group 1 animals (5 mg/m³) showed no increase in histiocytes but the histiocytes that were present also revealed the above mentioned brown-yellow particles within their cytoplasm. These findings were regarded to be treatment-related

Recovery findings:
In general, similar findings were observed in recovery animals as described for main group animals.
Comparable particles within histiocytes as described for the main group animals 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 broncho-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. Single macrophages containing the particles in their cytoplasm were seen in animals of test group 1 (5 mg/m³).
In the mediastinal and tracheobronchial lymph nodes of test group 2 and 3 (20 and 60 mg/m³) macrophages were observed that revealed comparable particles as described for the lungs. In test group 3 animals (60 mg/m³) an increase of agglomerated macrophages with particles was seen. These findings were regarded to be treatment-related.
One animal of test group 3 (60 mg/m³) revealed a minimal epithelial alteration in the larynx (levelI).

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar lavage fluid: At the end of the administration period, absolute and relative neutrophil counts as well as absolute monocyte counts were increased in BAL in males of test groups 2 and 3 (20 and 60
mg/m3). In addition, males of test group 3 showed increased absolute lymphocyte and epithelial cell counts. These changes led to a concurrent significant increase in the total cell counts in
BAL in test groups 2 and 3, whereas relative macrophage counts were slightly, but significantly decreased. These alterations were regarded as treatment-related and adverse.
At the end of the administration period in BAL of males in test groups 2 and 3 (20 and 60 mg/m3), total protein levels (MTP BAL) as well as -Glutamyl-transferase (GGT BAL), lactate dehydrogenase (LDH BAL), and alkaline phosphatase (ALP BAL) activities were significantly
increased. Further, N-Acetyl-β-D-glucosaminidase (NAG BAL) activities were also significantly increased in males of test group 3. These changes were regarded as treatment-related and adverse.

At the end of the administration period, osteopontin levels were significantly increased in BAL of males in test groups 2 and 3 (20 and 60 mg/m3 ). This alteration was regarded as treatment-related and adverse.
After the three-week recovery period, absolute and relative neutrophil counts were still marginally increased, while relative macrophage counts were marginally decreased in BAL of males in test group 3 (60 mg/m3). Additionally, cytokine-induced neutrophil chemoattractant-1levels (IL-8 BAL) were marginally, but significantly increased. These findings were considered treatment-related and adverse.
The tracheobronchial lymph nodes showed similar findings as the mediastinal lymph nodes.
In the mediastinal and tracheobronchial lymph nodes of all test groups macrophages were observed that revealed the same brown-yellow particles as described for the lungs. These findings were regarded to be treatment-related.

Dose descriptor:

NOAEC

Effect level:

5 mg/m³ air (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: particle clearance by macrophages observed, which is the physiological process to remove particles and not an adverse reaction.

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.:

20 mg/m³ air (analytical)

System:

respiratory system: upper respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Table1: Test concentration and particle size distribution

Test group	Target concentration (mg/m³)	Measured concentration (mg/m³)		MMAD (µm)	GSD	Fraction of particles < 3 µm [%]
		Mean	SD
01	5.0	5.1	0.6	0.83 0.73	2.96 3.07	88.1 89.7
02	20.0	20.1	0.2	1.05 1.02	2.78 2.77	84.8 85.4
03	60.0	59.7	2.4	0.74 0.90	3.02 2.81	89.7 87.8

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 count concentration increased with ascending concentration. The geometric mean count diameters were between 353 nm and 373 nm. There were no difference between the test groups.

Table 2: 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 25 (3 weeks after last exposure).

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3		Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Cells	1.0	2.1*	6.0**		1.0	0.8	1.1
Eosinophils	1.8	6.9	9.8		0.7	0.0	0.6
Lymphocytes	0.7	2.5	15.0*		0.4	0.5	1.3
Macrophages	1.0	0.8	1.3		1.0	0.8	0.9
Neutrophils	2.0	67.3**	310.8**		0.8	1.5	3.5*
Monocytes	2.5	3.0*	21.4*		0.5	0.5	2.2
Epithelial cells	1.4	0.7	3.0*		0.7	0.3	0.8

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

Table 3 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 25 (3 weeks after last exposure)

Analyte	Study day 5			Study day 25
	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3	Gr. 1 5 mg/m^3	Gr. 2 20 mg/m^3	Gr. 3 60 mg/m^3
Total Protein	1.1	2.0**	3.3**	1.0	0.9	1.0
GGT BAL	1.2	3.1**	4.5**	1.2	0.9	1.3
LDH BAL	1.0	2.1**	4.8**	1.2	1.1	1.6
ALP BAL	1.0	2.8**	3.4**	1.1	1.0	1.1
NAG BAL	1.2	1.6	1.9**	0.6	0.4	0.9

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

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

Table 4:  Incidence of gross lesions in main group animals

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs
·       Discoloration	0	0	3	5
Mediastinal lymph nodes
·       Discoloration	0	0	2	4
Tracheobronchial lymph nodes
·       Discoloration	0	0	0	1

Table 5: Incidence and severity of histological findings in the larynx of main group animals

Larynx (level I)	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Alteration, epithelial, (m)f	0	0	2	4
Grade 1			2	4

Table 6:  Incidence and severity of histological findings in the lungs of main group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Hyperplasia/hypertrophy, bronchioli, (m)f	0	0	4	5
·       Grade 1			4
·       Grade 2				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5
Grade 2				5
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	5	5
·       Grade 1			5
·       Grade 2				5
Particles, alveolar septae, (m)f	0	0	3	5
·       Grade 1			3	1
·       Grade 2				4
Macrophages with particles, i.v.	0	0	1	4
·       Grade 1			1	4
Debris, cellular, (m)f	0	0	0	5
·       Grade 1				5
Balt: macrophages with particles (m)f	0	1	2	5
·       Grade 1		1	2	5

Table 7: Incidence and severity of histological findings in the mediastinal lymph nodes of main group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophages with particles, (m)f	0	1	5	5
Grade 1		1	4
Grade 2			1	5

Table 8: Incidence of gross lesions in recovery group animal

	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Lungs
·       Discoloration	0	0	0	2
Mediastinal lymph nodes
·       Discoloration	0	0	0	5

Table 9: Incidence and severity of histological findings in the lungs of recovery group animals

Lungs	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Hyperplasia type II pneumocytes, (m)f	0	0	0	5
·       Grade 1				5
Histiocytosis alveolar with particles, (m)f	0	0	5	5
Grade 1			5	1
Grade 2				4
Particles within histiocytes*	0	5	0	0
Infiltrate neutrophilic, (m)f	0	0	0	5
·       Grade 1				5
Particles, alveolar septae, (m)f	0	0	0	3
·       Grade 1				3
Macrophages with particles, i.v.	0	0	0	4
·       Grade 1				4
Balt: macrophages with particles (m)f	0	0	5	5
·       Grade 1			5	5

* was diagnosed as present

Table 10: Incidence and severity of histological findings in the mediastinal lymph nodes of recovery group animals

Mediastinal lymph nodes	Male animals
Test group (mg/m³)	0 (0)	1 (5)	2 (20)	3 (60)
No. of animals	5	5	5	5
Macrophage aggregates w particles, (m)f	0	0	4	5
Grade 1			4
Grade 2				5
Macrophages with particles, (m)f	0	0	2	0
Grade 1			2

Executive summary:

Inhalation exposure of rats to 60 mg/m³ Pigment Brown 23 on 5 consecutive days caused increased total cell count, increased absolute and relative lymphocytes, neutrophils and epithelia cell counts in bronchoalveolar lavage. Moreover, several biochemical parameters (protein concentration, enzyme activities and cytokine concentrations) were significantly increased in lavage fluid. Consistently, absolute and relative lung weights were increased, minimal infiltration of neutrophils in the lungs, hyperplasia of type II pneumocytes, cellular debris, was observed in the lungs, as well as hyptrophy/hyperplasia of bronchioli. Particles were observed within alveolar septea in the lungs. Many of these findings were also observed at 20 mg/m³ with reduced severity. The effects did not resolve completely at 60 mg/m³ after 3 weeks post-exposure period. Those observed at 20 mg/m³ resolved completely within the postexposure period. Thus, under current study conditions, the no observed adverse effect concentration (NOAEC) for local effects was 5 mg/m³. The systemic NOAEC is above 60 mg/m³ (high concentration group). Target organ was the lung and the tracheobronchial lymph nodes.

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

Repeated dose toxicity: oral

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 (European Research Biology Center, 2022). 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 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.

Repeated dose toxicity: inhalation

The purpose of the inhalation study 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, post-exposure observation 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 5.0, 20.0, and 60.0 mg/m³(low, mid, and high concentration) for 6 hours per day and 5 days. Body weight, mortality, and clinical observations were determined during the study. One half of the rats was examined at the end of the exposure period, whereas the other half was examined at the end of a 3-week post-exposure period by determining clinical pathology parameters including bronchoalveolar lavage with clinico-chemical and cytological evaluation of lavage fluid, organ weights and all histopathological changes.During the exposure period the target concentrations were reached.

The following substance-relative adverse findings were observed:

 Test group 3 (60 mg/m³), main group (sacrificed one day after the last exposure)

·      Increased total cell counts in BAL, absolute and relative neutrophil counts, absolute monocyte, lymphocyte, and epithelial cell counts in BAL

·      Decreased relative macrophage counts in BAL

·      Increased total protein levels andg-glutamyl-transferase (GGT BAL), lactate dehydrogenase (LDH_BAL), alkaline phosphatase (ALP BAL), andN-Acetyl-β-D-glucosaminidase (NAG BAL) activities in BAL

·      Increased osteopontin (OSP BAL) levels in BAL

·      Absolute and relative increase of lung weight

·      Macroscopically brown discoloration of the lungs in all animals

·      Slight alveolar histiocytosis with brown particles in the lungs of all animals

·      Slight infiltration of neutrophils in the lungs of all animals

·      Cellular debris (alveolar) in all animals

·      Slight hyperplasia of type II pneumocytes in the lungs of all animals

·      Slight hyperplasia/hypertrophy, bronchioli in all animals

·      Minimal to slight numbers of particles within alveolar septae in the lungs of all animals

Test group 3 (60 mg/m³), recovery group (sacrificed after 3 weeks post-rexposure)

·      Increased absolute and relative neutrophil counts in BAL

·      Decreased relative macrophage counts in BAL

·      Increasedcytokine-induced neutrophil chemoattractant-1 (IL-8 BAL) levels in BAL

·      Macroscopically brown discoloration of the lungs in two animals

·      Minimal to slight alveolar histiocytosis with brown particles in the lungs of all animals

·      Slight infiltration of neutrophils in the lungs of all animals

·      Slight hyperplasia of type II pneumocytes in the lungs of all animals

·      Minimal number of particles within alveolar septae in the lungs of three animals

Test group 2 (20 mg/m³), main group (sacrificed one day after the last exposure)

·      Increased total cell counts, absolute and relative neutrophil counts, and absolute monocyte counts in BAL

·      Increased total protein levels andg-glutamyl-transferase (GGT BAL), lactate dehydrogenase (LDH_BAL), and alkaline phosphatase (ALP BAL) activities in BAL

·      Macroscopically brown discoloration of the lungs in three animals

·      Minimal alveolar histiocytosis with brown particles in the lungs of all animals

·      Minimal infiltration of neutrophils in the lungs of all animals

·      Minimal hyperplasia of type II pneumocytes in the lungs of all animals

·      Minimal hyperplasia/hypertrophy, bronchioli in four animals

·      Minimal number of particles within alveolar septae in the lungs of three animals

 

Test group 2 (20 mg/m³), recovery group (sacrificed 3 weeks post-exposure)

·      No treatment-related adverse findings

Test group 1 (5 mg/m³), main group (sacrificed one day after the last exposure)

·      No treatment-related adverse findings

Test group 1 (5 mg/m³), recovery group (sacrificed 3 weeks post-exposure)

·      No treatment-related adverse findings

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 1272/2008. There were no  toxic effects at the highest dose tested (1000 mg/kg bw/day) upon subacute oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.