Quaternary ammonium compounds, bis(hydrogenated tallow alkyl)dimethyl, chlorides, reaction products with polyethylenepolyamines and tall-oil fatty acids, humates hydrochlorides

Endpoint summary

Description of key information

There were no key oral or dermal repeated dose studies conducted because these study types are not warranted or appropriate.  Repeated dose oral toxicity studies were not appropriate because oral exposure for workers is not relevantan no exposure of the general population is expected. Repeated dose dermal toxicity studies were not warranted because of the physicochemical properties of organolignite. In a subchronic repeated dose toxicity study (OECD 413) via inhalation exposure no NOAEC could be established. A lung overload, i.e. an inflammatory response and correlated changes in haematology and histopathology parameters was observed in all dose groups. This effect is considered to be a local effect. The systemic effects are secondary effects. Therefore, 40 µg/L (40 mg/m³) is considered as local LOAEC and for systemic effects the NOAEL is established as 400 µg/L (400 mg/m³). In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation exposure, the NOAEC was established at 300 mg/m³ (i.e., the high dose) based on the lack of adverse local and systemic effects observed.

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

Data waiving:

exposure considerations

Justification for data waiving:

a short-term toxicity study does not need to be conducted because relevant human exposure can be excluded as based on the provided thorough and rigorous exposure assessment

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: inhalation

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

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)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Limited, Margate, Kent, UK
- Age at study initiation: ca 6-7 weeks
- Weight at study initiation: 201-227 g males; 163-195 g females
- Housing: Housed in polycarbonate cages with stainless steel grid tops and solid bottoms. Animals were initially housed 2/cage by sex. Males were individually housed a few days prior to mating. After mating, males were re-housed with original cage mates.
- Diet (e.g., ad libitum): Rat and Mouse Breeder Diet No. 3 (Expanded) provided ad libitum
- Water (e.g., ad libitum): Public water supply provided ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-23°C
- Humidity (%): 40-70%
- Air changes (per hr): 10 air changes per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark / 12 hrs light

Route of administration:

inhalation: dust

Type of inhalation exposure:

nose only

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: Gravimetric particle size distribution measurement
Treatment Group 1: Air Control
Treatment Group 2: MMAD <4.45; GSD 2.123
Treatment Group 3: MMAD <4.85; GSD 2.043
Treatment Group 4: MMAD <5.11; GSD 2.153

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Modular snout only flow through system
- Method of holding animals in test chamber: Rats were restrained in clear, tapered, polycarbonate tubes fitted with a back-stop appropriately adjusted to prevent the animals from turning in the tubes.
- Rate of air: ~20 L/min
- Method of conditioning air: Not reported
- System of generating particulates/aerosols: Rotating brush generator device
- Temperature, humidity, pressure in air chamber: 17.0-25.2 ºC, 0.7-43.7 %, pressure not reported
- Method of particle size determination: Particle size was measured by a cascade impactor located and sealed in a port in the animals’ breathing zone; particle size distribution was determined by plotting the cumulative percentage (by mass) of particles smaller than the cut point of each impactor stage against the logarithm of each stage cut point. The mass median aerodynamic diameter (MMAD) and geometric standard deviation (GSD) of the test aerosols were derived by Probit analysis.
- Treatment of exhaust air: Chamber air was exhausted via a double filter system with a fiber glass coarse prefilter and a HEPA ultimate particulate filter.

TEST ATMOSPHERE
- Brief description of analytical method used: The aerosol concentration of the test aerosols in the animals’ breathing zone was measured
gravimetrically for Groups 2 to 4 at regular intervals throughout each daily exposure period. Aerosol concentrations were also measured and assessed prior to initiation of inhalation exposure to ensure concentrations were within target ranges. The test aerosols were sampled with filter media placed in a filter holder with a sampling system in place. Filters were weighed before and after sampling, and the volume of air sampled was recorded.
- Samples taken from breathing zone: Yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Comparison of the nominal and achieved gravimetric aerosol concentrations showed good correlation throughout the study. Overall, the system efficiency 14 to 23%, 12 to 25 % and 15 to 26% for Groups 2, 3, and 4 respectively. The achieved concentrations were 100%, 102% and 107% of target for Groups 2, 3 and 4, respectively.

Duration of treatment / exposure:

Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

Frequency of treatment:

6 hr per day, 7 days per week

Dose / conc.:

5 mg/m³ air (nominal)

Dose / conc.:

50 mg/m³ air (nominal)

Dose / conc.:

300 mg/m³ air (nominal)

No. of animals per sex per dose:

10 animals/sex/dose

Control animals:

yes

Details on study design:

Dose selection rationale: Doses were selected based on the results of a 14-Day Dose Range Finding Study (supporting study) with organolignite.
Rationale for animal assignment (if not random): Cages were allocated by the use of randomly sequenced numbers in such a way that each complete rack contained representatives from all treatment groups.

Positive control:

The study did not include a positive control.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and weekly thereafter
- Cage side observations included: posture/condition (i.e., prostration, lethargy, writhing, circling, breathing abnormalities, gait abnormalities, tremor, fasciculation, convulsions, biting, vocalizations, and piloerection); ease of removal from the cage; body temperature; condition of the eyes (i.e., pupillary function, miosis, mydriasis, exophthalmos, encrustation, and lacrimation) and coat; presence of salivation, and overall ease of handling.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: One week prior to treatment and daily thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. Thereafter, males were weighed twice weekly at the start of dosing until the end of the study. Females were weighed twice weekly at the start of dosing and daily from mating to termination.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

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

WATER CONSUMPTION: No
- Time schedule for examinations: Not reported

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: One week prior to treatment for both sexes. An additional evaluation was conducted at Week 4 for males and shortly prior to sacrifice for females
- Dose groups that were examined: All dose levels

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Samples were obtained at Week 4 for males and on Day 4 of lactation for females
- Anaesthetic used for blood collection: No data
- Animals fasted: No
- How many animals: First 5 males per group; first 5 females per group that reared litter to Day 3 of lactation
- Parameters examined included: red and white blood cell count; hemoglobin concentration; hematocrit; mean cell volume; red blood cell width and volume; mean cell hemoglobin and mean cell hemoglobin concentration; reticulocyte count; platelet count; absolute neutriphil, lymphocyte, monocyte, eosinophil, and basophil count; large unstained cells; activated partial thromboplastin time; fibrogen; and prothrombin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Samples were obtained at Week 4 for males and on Day 4 of lactation for females
- Animals fasted: No
- How many animals: First 5 males per group; first 5 females per group that reared litter to Day 3 of lactation
- Parameters examined included: urea; blood urea nitrogen; total bile acids; glucose; aspartate aminotransferase; alanine aminotransferase; alkaline phosphatase; creatine phosphokinase; lactate dehydrogenase; sodium; potassium; chloride; total protein; albumin; globulin; albumin/globulin ratio; cholesterol; creatinine; total bilirubin; calcium; inorganic phosphate

URINALYSIS: No data

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 4 for males and during lactation for females
- Dose groups that were examined: 5 males per group and first 5 females per group that reared litter to Day 3 of lactation
- Battery of functions tested: / grip strength / motor activity / pain perception / landing foot splay / other physical and/or functional abnormalities

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 1)
HISTOPATHOLOGY: Yes; tissues identified in table 1 (except bone marrow smears) were processed from 5 males and 5 females from the control and high-dose groups.

Other examinations:

Appropriate reproductive and developmental endpoints were evaluated. Discussion of these endpoints is provided in the Results section of the main Combined Study Report.

Statistics:

The following endpoints were analyzed using the 'F Max' test: selected body weight and food consumption data; all hematology, coagulation, and clinical chemistry; and selected functional observational battery and motor activity data. Homogeneous variances were analyzed using a parametric ANOVA. Pairwise comparisons were made using Fisher's F protected LSD method via Student's t test only if the overall F test was significant. Heterogeneous variances were initially transformed using the log or square root. If variances remained heterogeneous, then a Kruskal-wallis non-parametric ANOVA was used and pairwise compassions were made using chi squared protection.

Organ weight data were analyzed using the methods noted above and through analysis of covariance (ANCOVA) using terminal body weight as the covariate. Organ weights were also analyzed as a percentage of terminal body weight.

Clinical signs:

effects observed, treatment-related

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not specified

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL SIGNS AND MORTALITY: Increased incidence of staining around the head, nose, eyes, muzzle, and ears for all treated animals. Mid- and high-dose animals also had staining on their coats. Staining is considered due to dark color of test material.

HAEMATOLOGY: Measured endpoints in females were comparable to control values for all dose levels. There was a statistically significant increase in white blood cells (p<0.001), neutrophils (p<0.01), monocytes (p,0.01), basophils (p<0.001) and large unclassified cells (p<0.05) in 0.3-mg/L males. Mid-dose males also exhibited a slight increase in basophils; however, this effect was not statistically significant and was considered to be incidental. Statistically-significant increases in the number of white blood cells (p<0.05), monocytes (p<0.05), and basophils (p<0.05) were observed in low-dose males. Because of the lack of effect observed at the mid dose, these effects were considered not to be treatment-related.

CLINICAL CHEMISTRY: In high-dose males, there was a decrease in LDH levels that were considered incidental. All other clinical chemistry parameters for both males and females at all dose levels were similar to the control values.

ORGAN WEIGHTS: At the high-dose, a statistically significant increase in both absolute and relative lung weight was observed in high-dose males and females. At the mid-dose, there was a statistically significant increase in absolute lung weight for both males and females; however, only females exhibited a statistically significant increase in relative lung weight. No effects on organ weights were observed at the low dose for both males and females.

GROSS PATHOLOGY: There was an increased incidence of dark discoloration of the lungs for all high-dose males and females and dark discoloration of the bronchial lymph node for high-dose males (10/10) and females (9/10). High-dose females also showed an increased incidence of pale foci on the lungs (5/10) and dark discoloration of the mediastinal lymph node (5/10). Mid-dose males (8/10) and females (8/10) showed an increased incidence of dark discoloration of the lungs.

HISTOPATHOLOGY: NON-NEOPLASTIC: At the high-dose, the following treatment-related findings were noted: bronchiolo-alveolar pigmented macrophages/hyperplasia; multifocal alveolar foamy macrophage accumulation; and a higher severity and incidence of mononuclear cell infiltration. In the bronchial lymph node there were pigmented macrophages and increased cellularity. In the mediastinal lymph node, there were pigmented macrophages. A microscopic examination was not conducted at 0.05 and 0.005 mg/L.

Dose descriptor:

NOAEC

Effect level:

300 mg/m³ air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No local, systemic or reproductive/developmental adverse effects were observed at the high dose.

Critical effects observed:

no

Conclusions:

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation, the NOAEC was established at 300 mg/m3 (equivalent to 0.3 mg/L; i.e., the high dose) based on the lack of adverse effects.

Executive summary:

The purpose of this study was to assess whether inhalation exposure of rats to the test article, organolignite, was associated with any systemic toxicity, and to provide information on effects on reproduction and/or development. The study also placed an emphasis on neurological effects as a specific endpoint to identify any neurotoxic potential of the test article. 

Sprague-Dawley rats were randomised into 3 exposure groups (i.e., 5, 50, or 300 mg/m³) and one control group (air exposure only), each group contained 10 males and 10 females. The males were treated daily for 2 weeks prior to mating, then through mating and until the day prior to necropsy (ca 4 weeks of treatment). Females were treated daily for 2 weeks prior to mating, throughout mating and up to Day 19 of gestation (ca 6 weeks). Females were maintained until Days 4-7 of lactation.

 

The following parameters and end points were evaluated in this study: clinical signs, body weights, body weight changes, food consumption, ophthalmology, detailed functional tests and observations, clinical pathology parameters (haematology, coagulation and clinical chemistry), mating and pregnancy performance, fertility, maternal care and pup performance (litter survival and pup weights), gross necropsy findings, organ weights and histopathological examinations.

 

With regards to clinical signs, there was an increase in the incidence of dark staining around the head, nose, eyes, muzzle, ears and coat in all organolignite treated groups. These signs were all observed immediately after dosing and were a direct result of being exposed to organolignite, which is dark in color, on the inhalation dosing chamber. There were no treatment related effects at any dose level on body weight gains, food consumption, ophthalmic examinations or functional observational battery parameters.

 

There were no statistically significant changes to the haematology and coagulation parameters in the females treated at 300 mg/m³, compared to the controls. The females ceased dosing in late gestation to allow littering, and therefore, it is possible that any changes to the white blood cell types had started to recover by the time blood samples were collected. There were no changes to the clinical chemistry parameters in males or females that were associated with treatment.

 

At 300 mg/m³, there was an increase in some of the white blood cell types in the males (white blood cells (p < 0.001), neutrophils (p < 0.01), monocytes (p < 0.01), basophils (p < 0.001) and large unclassified cells (p<0.05). At 5 mg/m³, there was an increase in the number of white blood cells (p < 0.05), monocytes (p < 0.05) and basophils (p < 0.05). There were no observed changes at 50 mg/m³. As there was no similar pattern at 50 mg/m³, and the increases were small, these differences were not considered to be biologically significant and were not considered to constitute an adverse effect.

 

At necropsy, dark discolouration of the lungs was noted in males and females treated at 50 and 300 mg/m³. However, this observation is considered to be associated with the dark color of the test article and is not considered to constitute an adverse effect. Other necropsy findings at 300 mg/m³ included pale foci on the lungs, dark discolouration of the bronchial lymph nodes and dark discolouration of the mediastinal lymph nodes. Lymph node discolouration is consistent with clearance. Dark discolouration of the skin was also noted. At 300 mg/m³, there was an increase in lung weights in males and females that was still statistically significant (p<0.001) after adjustment for body weight. At 50 mg/m³, there was an increase in absolute lung weights in males and females, however, after adjustment for body weight, this increase only achieved statistical significance in females (p<0.001). The increased lung weight in females was not considered to constitute an adverse effect.

 

Microscopic findings at 300 mg/m³ included pigmented macrophages in the lung, which is expected following inhalation administration of a dark pigmented material; multifocal alveolar macrophage accumulation is also associated with this phagocytosis process. At this dose level, areas of bronchiolo-alveolar hyperplasia were characterised by hypertrophic alveolar Type II pneumocyte cells forming a single layer outlining alveolar walls. However, there was no evidence of associated tissue damage such as necrosis, degeneration or fibrosis. Nor was there any evidence of focal proliferation of epithelial cells, which might be indicative of a pre-neoplastic lesion. Both bronchiolo-alveolar hyperplasia, and the slightly

higher incidence of mononuclear cell infiltration, represent a local response to the pigment and pigmented macrophages. Observed bronchiole-alveolar hyperplasia was a local pulmonary response to the test article and based on the microscopic findings was considered not to be an adverse change after 4 or 6 weeks of exposure. In conclusion, under the conditions of this study, the No Observed Adverse Effect Concentration (NOAEC) for adults was considered to be 300 mg/m³ (equivalent to 0.3 mg/L). The change in the white blood cell parameters in the males did not show a dose-response relationship and is considered a reversible effect, therefore, this effect was considered not to be adverse.

Reference 2

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-12 - 2020-02-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Details on species / strain selection:

The rat is an animal model commonly utilized in toxicity studies like the OECD 413 guideline and was the rodent species used in the toxicity testing program for organolignite. In addition, a historical database of this species is available for comparative evaluation and prior studies have been conducted in rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, USA
- Females (if applicable) nulliparous and non-pregnant: not provided
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: Males: 254 - 323 g; Females: 176 - 227 g (range)
- Fasting period before study: overnight fasting before necropsy/blood sampling for clinical pathology
- Housing: Nominally 2 animals of the same sex per cage
- Diet: Teklad Global 16% Protein Rodent Diet (certified), 2016C ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants in the feed and water that were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 18 Apr 2019 To: 12 Sep 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 2.3 µm

Geometric standard deviation (GSD):

3.02

Remarks on MMAD:

The MMAD given above is the maximum range over all groups. The GSD is the maximum observed.
Values per group are as follows:
Group 2: MMAD Range: 1.0 - 1.7 µm, GSD Range: 1.75 - 2.81
Group 3: MMAD Range: 1.1 - 1.9 µm, GSD Range: 1.78 - 3.02
Group 4: MMAD Range: 1.1 - 2.3 µm, GSD Range: 1.80 - 2.68

The MMAD values for all samples collected showed that the delivered particle size was respirable to the rats. The wider than anticipated range of MMAD was attributed to test item wear to the internal components of the jet mills. Jet mills were replaced as needed to ensure particle distribution remained in the range 1 – 3 µm equivalent aerodynamic diameter.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through (ADG) snout-only exposure system
- Method of holding animals in test chamber: Restraints (molded clear thermoplastic tubes). The animals were acclimated to the method of restraint over a 3-day period immediately preceding the first test item exposure.
- Source and rate of air: dry air from in-house compressed air system; Exposure Day 1 - 48: 60.0 L/minute, Exposure Day 49 - 66: 41.0 L/minute
- System of generating particulates/aerosols: Mark I Wright Dust Feed (WDF)
- Temperature, humidity, pressure in air chamber: 22 °C, 44% humidity
- Air flow rate: 20L/minute (aerosol generation)
- Method of particle size determination: cascade impaction
- Treatment of exhaust air: filtered locally

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples were collected as follows: Filter type - glass fiber filter, held in an open face filter holder; Sample flow - 2.0 L/minute; Sample volume - measured by timed flowmeter; Sample frequency - minimum of 3 samples/group/exposure; Sample location - representative animal exposure position, alternated weekly throughout study; Sample analysis - gravimetric - groups 1 to 4; Sample disposition - filters discarded after gravimetric weights collected
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Group 2: Target: 40 µg/L, Achieved: 40 µg/L
Group 3: Target: 100 µg/L, Achieved: 105 µg/L
Group 4: Target: 400 µg/L, Achieved: 397 µg/L
Overall mean achieved concentration for Group 2 was on target. Group 3 mean concentration was 5% above target and Group 4 was 1% below target.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.04 mg/L air (nominal)

Dose / conc.:

0.1 mg/L air (nominal)

Dose / conc.:

0.4 mg/L air (nominal)

No. of animals per sex per dose:

10 (main group)
10 (control and high dose group for 6 week recovery period)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure levels for organolignite were selected based on the results of previously conducted inhalation toxicity studies. A repeated exposure inhalation toxicity study in rats was conducted at 0.5, 1.0, 2.0 mg/L for 14 days. Clinical signs such as gasping were only seen at the highest exposure level. Animals at the lower exposure levels were unaffected thus the no observed adverse effect concentration (NOAEC) from this study was determined to be 1.0 mg/L (1000 µg/L). An OECD TG 422 combined repeated exposure inhalation toxicity study with a reproduction/developmental toxicity-screening test was conducted in rats at 0.005, 0.05 and 0.3 mg/L for 4 weeks of exposure. No clinical signs were seen thus the no observed adverse effect concentration (NOAEC) from this study was determined to be 0.3 mg/L (300 µg/L). On the basis of these prior studies, the exposure levels of 40, 100 and 400 µg/L were selected for this study and are expected to be tolerated for 13 weeks of exposures.

- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice pretest and weekly (once every 7 days post-exposure on exposure days during treatment and recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest and twice weekly during the first 4 weeks of treatment period and weekly (once every 7 days) from Week 5 through the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was measured (weighed) weekly, beginning one week prior to treatment.

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and at the end of exposures (Week 13)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals per group
- Parameters checked: Haemoglobin (HGB), Haematocrit (HCT), Red blood cell count (RBC), Platelet count (PLT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Red cell distribution width (RDW), Total white blood cell count (WBC), Reticulocyte count (RETIC), Differential white blood cell count: Neutrophils (ANEU), Lymphocytes (ALYM), Eosinophils (AEOS), Basophils (ABASO), Monocytes (AMONO), Large unstained cells (ALUC); and Prothrombin time (PT) and Activated partial thromboplastin time (APTT) .

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: all animals per group
- Parameters checked: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALKP), Blood urea nitrogen (BUN), Creatinine (CREAT), Glucose (GLU), Cholesterol (CHOL), Triglycerides (TRIG), Total protein (TP), Albumin (ALB), Total bilirubin (TBILI), Sodium (NA+), Potassium (K+), Chloride (Cl-), Calcium (Ca++) and Inorganic phosphorus (PHOS).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at termination
- Dose groups that were examined: all
- Number of animals: all
- Parameters checked: Total white blood cell count (TWBC), Total protein (TP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALKP), Differential white blood cell count: Macrocytes (MACRO), Neutrophils (NEUT), Lymphocytes (LYMPH), Eosinophils (EOS), Monocytes (MONO) and Basophils (BASO).

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 in "any other information on materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see table 1 in "any other information on materials and methods incl. tables")

Other examinations:

Organs were weighed for all animals at the scheduled necropsy intervals (see table 1 in "any other information on materials and methods incl. tables").

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
body weight
body weight change from interval to interval
cumulative body weight change from baseline
food consumption
haematology
coagulation
clinical chemistry
organ weights
The following comparisons were made:
Group 1 vs 2, 3 and 4
The parameters to analyze were identified as either continuous (greater than 6 distinct values), discrete (between 3 and 6 distinct values) or binary (2 distinct values).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weight gains were seen in males exposed to ≥ 40 µg/L but not in a clearly exposure-level related pattern. For instance, body weight gains from Day 1 to Day 91 was reduced in males by 21.6%, 14.4% and 17.0% in 40, 100 and 400 µg/kg bw/day groups compared with controls. At the end of the recovery period the male animals treated with 400 µg/kg bw/day had still a reduced body weight (- 15.0%) compared to the controls. This effects are not considered to be toxicolgical relevant as no clear exposure-level reated pattern was observed.
Decreased body weight gains over the whole treatment period (Day 1 to Day 91) were seen only in females exposed to 400 µg/L (-13.3%). Comparing the body weights of 400 µg/L females on Day 91 with the controls a non-significant decrease of 4.8% was observed. Therefore, these changes were test item-related but not considered adverse. A recovery in body weight gain was seen after the end of the exposures. For details see table 2 and 3 in "any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased food consumption was seen in males exposed to ≥ 40 µg/L but not in a clearly exposure-level related pattern. Decreased food consumption was seen in females exposed to 400 µg/L but not continuosly over the treatment period. Significant decrease was only observed on Day 22-29, 29-36, 43-50. Therefore the decrease in food consumption in males and females were test item-related but not considered adverse. A recovery in food consumption was seen after the end of the exposures. For details see table 4 in "any other information on results incl. tables".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related haematology changes were observed in male animals of the 100 and 400 µg/L dose groups as well as in females in all dose groups.
Males: Neutrophils were 1.43- and 1.4-fold increased (statistical significant) in males in dose group 100 and 400 µg/L, respectively, but without corresponding increase of white blood cells. In the recovery group the white blood cells were significantly increased in the 400 µg/L dose group by 1.24-fold and the neutrophils by 1.49-fold. These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.07-, 1.06- and 1.05-fold which are considered test substance-related.

Females: Neutrophils were 2.65-, 2.79- and 3.47-fold increased (statistically significant) in females in dose group 40, 100 and 400 µg/L, respectively. These changes corresponded to an significant increase of white blood cells (1.6-, 1.29- and 1.47-fold). In the recovery group the white blood cells were not increased anymore but the neutrophils in the 400 µg/L dose group were still significantly increased 2.5-fold compared to the conrols. These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.08-, 1.07- and 1.08-fold which are considered test substance-related..
See table 5 and 5a/b in "any other information on results incl. tables" for details.

There were no test item-related coagulation changes at any exposure tested.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main group:
There were no test item-related clinical chemistry changes at any exposure tested.
There were minimal to mild increases in aspartate and alanine aminotransferase activities in one female at each exposure level that were considered not test item-related as they lacked an exposure level relationship and there were no microscopic correlates in the liver or skeletal muscles.

Recovery group:
There were no test item-related clinical chemistry changes following a 6-week recovery period in animals exposed to 400 µg/L.

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):

Statistically significant higher absolute and relative lung and bronchi weights were present in males and females exposed to ≥40 µg/L compared to controls. Higher lung weights were related to increased pigment/pigmented alveolar macrophages and inflammation observed microscopically. Higher lung weights did not recover and remained increased in a similar magnitude compared to the main group in males and females exposed to 400 µg/L following a 6-week exposure-free recovery period. See table 8 and 9 in "any other information on results incl. tables" for details.

All other organ weight changes in the terminal necropsy, statistically significant or not, were not considered organolignite-related because they were of low magnitude, independent of exposure level, and/or lacked a macroscopic or microscopic correlate.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormally colored (black) lungs were observed in 9/10, 10/10 and 10/10 males and females each in 40, 100 and 400 µg/L dose groups, respectively. Distended areas in the lungs were observed in 1/10, 3/10 and 4/10 males and 1/10, 1/10 and 2/10 females in the 40, 100 and 400 µg/L dose groups, respectively. Pale areas (white or tan) in the lungs were observed in 2/10, 5/10 and 4/10 males and 2/10, 7/10 and 3/10 females in the 40, 100 and 400 µg/L dose groups, respectively. These lung findings correlate to pigment/pigmented alveolar macrophages and/or inflammation microscopically. No recovery was observed for abnormally colored (black) lungs and (white or tan) in pale areas in the lungs. All animals in the recovery group showed black lungs and 9/10 males and 6/10 females showed (white or tan) pale areas in the lungs. Therefore, these effects are considered treatment-related and adverse.

Abnormally colored (black) and enlarged tracheobronchial and thymic lymph nodes were also observed in males and females exposed to organolignite at ≥40 µg/L with the following incidence rate:
Tracheobronchial lymph nodes: Abnormal colored (black): 8/10, 9/10 and 9/10 males and 9/10, 8/10 and 10/10 females in 40, 100 and 400 µg/L dose groups respectively. Enlarged: 8/10, 9/10 and 9/10 males and 8/10, 9/10 and 10/10 females in 40, 100 and 400 µg/L dose groups respectively.
Thymic lymph nodes: Abnormal colored (black): 6/10, 7/10 and 8/10 males and 8/10, 7/10 and 8/10 females in 40, 100 and 400 µg/L dose groups respectively. Enlarged: 5/10, 6/10 and 7/10 males and 7/10, 6/10 and 8/10 females in 40, 100 and 400 µg/L dose groups respectively.
For both tissues tracheobronchial and thymic lymph nodes the observed effects was still observed in the recovery group.

All above mentioned effecs correlated to pigmented macrophages and/or increased paracortical cellularity microscopically and thus are considered treatment-related and adverse.
See tables 10 to 15 in "any other information on results incl. tables" for details.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings related to organolignite were observed in the lungs and bronchi, tracheobronchial and thymic lymph nodes, nose/turbinates, larynx, tracheal bifurcation, and trachea at ≥40 µg/L.
Terminal necropsy:
Lungs and Bronchi:
Pigment/pigmented alveolar macrophages (moderate to severe) were present in all male and female rats exposed at ≥40 µg/L, with exposure-related increases in incidence and severity. Alveoli were diffusely expanded by large numbers of variably pigmented macrophages characterized by light tan intracytoplasmic speckling to dark brown cytoplasmic contents. Extracellular dark brown pigment was also multifocally present within alveoli. Pigmented material was interpreted to be organolignite. Pigment/pigmented alveolar macrophages were accompanied by bronchioloalveolar inflammation (minimal to marked) characterized by neutrophilic infiltrates, necrotic cellular debris in airways, type II pneumocyte hyperplasia, perivascular and peribronchiolar lymphocytes, and increased bronchus-associated lymphoid tissue. Microscopic findings in the lungs and bronchi correlated with higher lung weights and abnormal (black) coloration, distention, and/or pale (white or tan) areas observed macroscopically. See table 16 in "any other information on results incl. tables" for details.
No recovery of pigment/pigmented macrophages and inflammation in male and female rats of the recovery groups were observed. These findings were present in comparable incidence and severity following the recovery period compared to main group animals, with increased incidences of marked inflammation at recovery compared to the terminal phase. Microscopic findings in the lungs and bronchi correlated with higher lung weights and abnormal (black) coloration and/or pale (white or tan) areas observed macroscopically. See table 23 in "any other information on results incl. tables" for details.

Tracheobronchial and Thymic Lymph Nodes:
Organolignite-related increased paracortical cellularity (minimal to marked) and intrasinusoidal pigmented macrophages (minimal to moderate) were present at ≥ 40 µg/L, with exposure-related increases in incidence and severity. Increased paracortical cellularity and pigmented macrophages correlated with macroscopically enlarged lymph nodes and abnormal (black) coloration, respectively. See tables 17 and 18 in "any other information on results incl. tables" for details.
No recovery in paracortical cellularity and intrasinusoidal pigmented macrophages in male and female rats of the recovery group were observed. These findings were present in comparable incidence and severity following the recovery period. Increased paracortical cellularity and pigmented macrophages correlated with macroscopically enlarged lymph nodes and abnormal (black) coloration, respectively. See tables 24 and 25 in "any other information on results incl. tables" for details.

Nose/Turbinates
Organolignite-related olfactory epithelial degeneration (minimal to marked) was present in the nose/turbinates of rats exposed to ≥ 100 µg/L, with exposure-related increases in incidence and severity. One 40 µg/L female had minimal olfactory epithelial degeneration, but this was considered within background range incidence and severity. Olfactory epithelial degeneration was characterized by disorganization of cell layers, attenuation, loss of cilia, bleb formation, and increased intercellular spaces. Respiratory epithelial degeneration (minimal to slight) was present in few male (1/10 at 100 µg/L and 5/9 at 400 µg/L) and female (0/10 at 100µg/L and 3/10 at 400 µg/L) rats exposed to ≥100 µg/L. This finding was similarly characterized by disorganization, attenuation, and loss of cilia and was infrequently accompanied by mixed cell inflammation (minimal) and/or squamous metaplasia (minimal to slight). Areas of epithelial degeneration (olfactory and/or respiratory) often contained mucosal, submucosal or luminal accumulations of extracellular pigment/pigmented macrophages (minimal to moderate). See table 19 in "any other information on results incl. tables" for details.
No recovery in olfactory epithelial degeneration in male and female rats of the recovery group were observed. It was accompanied by olfactory epithelial inflammation in two rats. Also, no recovery in pigment/pigmented macrophages was observed in males and females of the recovery group. Both olfactory epithelial degeneration and pigment/pigmented macrophages were present in comparable incidence and severity following the recovery period. Partially recovery in respiratory epithelial degeneration and inflammation were observed and were both present in fewer animals and/or at lesser severity compared to main group animals. Squamous metaplasia of respiratory epithelium was not present in animals of the recovery group, consistent with complete recovery of this finding. See table 26 in "any other information on results incl. tables" for details.

Larynx
Organolignite-related epithelial alteration (minimal to slight) and/or squamous metaplasia (minimal to slight) were present in the larynx of rats exposed to ≥40 µg/L, with exposure-related increases in incidence and/or severity. Epithelial alteration and squamous metaplasia mostly occurred along the base of the epiglottis within the anterior (level I) larynx. Epithelial alteration, a pre-metaplastic finding, was characterized by focal loss of cilia and epithelial flattening that did not fulfill the criteria of squamous metaplasia. Squamous metaplasia was a more focally extensive to diffuse finding characterized by 3 to 4 cell layers of flattened, stratified epithelium with desquamation and/or keratinization. Pigment/pigmented macrophages (minimal to slight) were present in the lumen and/or submucosa at ≥ 40 µg/L. See table 20 in "any other information on results incl. tables" for details.
In the recovery group epithelial alteration was not present and incidences and severities in male and female rats suggested that squamous metaplasia in male rats had recovered to epithelial alteration in some male animals of the recovery group. It is also noteworthy that incidence and severity was comparable between organolignite-exposed males of the recovery group and control females of the recovery group. Squamous metaplasia was not present in male recovery rats and showed a lower incidence female rats of the recovery group. Squamous metaplasia was present at decreased incidence and severity in females of the recovery group compared to females of the main group. Pigment/pigmented macrophages were not observed in males of the recovery group and the incidence was reduced females of the recovery group compared to the main group. See table 27 in "any other information on results incl. tables" for details.

Tracheal Bifurcation/Trachea
Organolignite-related pigment/pigmented macrophages (minimal) were present in rats exposed to ≥ 40 µg/L, with some exposure-related increases in incidence. Pigment/pigmented macrophages were present in the tracheal lumen, submucosa, and/or serosa/adventitia. See tables 21 and 22 in "any other information on results incl. tables" for details.
No recovery in pigment/pigmented macrophages in male and females rats of the recovery group was observed. These findings were present in comparable incidence and severity following the recovery period compared to main group animals. See tables 28 and 29 in "any other information on results incl. tables" for details.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage

Test item-related bronchoalveolar fluid changes at all exposures included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also, at all dose levels there were increases in total protein and lactate dehydrogenase activities. At 400 µg/L, there was increases in alkaline phosphatase activities. All of these changes were considered test item-related and appeared to be generally dose level dependent. These changes correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse at ≥ 40 µg/L. See table 6 in "any other information on results incl. tables" for details.
Test item-related bronchoalveolar fluid changes in the recovery group of treated rats compared to control rats included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also in treated animals of the recovery group, there were increases in total protein, lactate dehydrogenase, and alkaline phosphatase activities. These changes correlated with the microscopic findings of inflammation in the respiratory tract in the recovery animals. For details see table 7 in "any other information on results incl. tables".

Key result

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

0.04 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.4 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects observed up to the highest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.04 mg/L air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 2. Body weight - group mean values (g)

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-5

1-8

1-12

1-15

1-19

1-22

1-26

1-29

1-36

1-43

1-50

1-57

Statistics test

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Control / male

Mean SD N

29 5.1 20

39 8.2 20

65 11.4 20

77 12.9 20

99 15.0 20

109 17.3 20

127 20.1 20

137 21.9 20

157 24.2 20

173 28.0 20

190 30.7 20

205 31.8 20

40 µg/L / male

Mean SD N

24* 5.2 10

31 8.2 10

50* 11.6 10

60** 13.6 10

80* 16.0 10

86** 17.0 10

99** 18.0 10

105** 20.0 10

121** 22.8 10

135** 20.6 10

150** 24.7 10

159** 24.8 10

100 µg/L / male

Mean SD N

25* 3.5 10

36 6.3 10

60* 11.6 10

68** 12.0 10

90* 16.2 10

96** 17.0 10

112** 20.5 10

117** 22.8 10

135** 26.9 10

153** 28., 10

161 32.3 10

178** 32.0 10

400 µg/L / male

Mean SD N

23** 4.5 20

33* 7.4 20

55** 8.6 20

62** 11.5 20

83** 13.3 20

90** 15.0 20

105** 16.8 20

113** 18.4 20

132** 21.3 20

143** 23.1 20

157** 25.5 20

168** 28.3 20

  

Table 2. (continued) Body weight - group mean values (g)

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-64

1-71

1-78

1-85

1-91

1-R1

1-R8

1-R15

1-R22

1-R29

1-R36

Statistics test

Wi

Wi

Wi

Wi

Wi

Tt

Tt

Tt

Tt

Tt

Tt

Control / M

Mean SD N

220 34.0 20

233 33.9 20

246 36.5 20

255 39.4 20

264 41.6 20

261 40.1 10

301 42.7 10

322 44.0 10

346 47.7 10

364 49.3 10

379 51.3 10

40 µg/L / M

Mean SD N

171** 26.5 10

187** 26.1 10

193** 26.8 10

201** 26.9 10

207** 28.7 10

100 µg/L / M

Mean SD N

190** 33.2 10

202** 34.4 10

211** 36.4 10

219** 41.3 10

226** 43.0 10

400 µg/L / M

Mean SD N

180** 27.4 20

190** 29.8 20

197** 32.1 20

207** 32.7 20

219** 33.5 20

205** 31.9 10

244** 34.5 10

268** 33.4 10

297** 33.5 10

308** 36.3 10

322** 22.8 10

Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01

Table 3. Body weight - group mean values (g)

Group/Sex		Change	Change	Change	Change	Change	Change	Change	Change	Change	Change	Change	Change
		1-5	1-8	1-12	1-15	1-19	1-22	1-26	1-29	1-36	1-43	1-50	1-57
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
Control / female	Mean SD N	11 4.1 20	12 4.3 20	20 4.9 20	22 7.4 20	31 8.5 20	33 7.3 20	40 8.3 20	45 5.9 20	51 10.6 20	56 15.5 20	60 10.5 20	67 13.5 20
40 µg/L / female	Mean SD N	11 3.0 10	16* 4.6 10	26 4.2 10	26 5.3 10	33 7.3 10	36 3.5 10	43 4.8 10	48 4.8 10	56 6.6 10	61 7.9 10	66 6.7 10	72 7.9 10
100 µg/L / female	Mean SD N	13 3.6 10	18* 7.7 10	23 6.4 10	27 3.8 10	35 6.4 10	35 5.8 10	40 4.5 10	41 6.9 10	46 6.8 10	57 9.0 10	60 8.3 10	62 11.3 10
400 µg/L / female	Mean SD N	9 3.7 20	15* 5.0 20	23 7.7 20	24 7.6 20	33 8.7 20	33 8.5 20	39 10.3 20	42 9.8 20	48 11.7 20	55 13.7 20	56 14.8 20	60 15.8 20

 

Table 3. (continued) Body weight - group mean values (g)

Day

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-64

1-71

1-78

1-85

1-91

1-R1

1-R8

1-R15

1-R22

1-R29

1-R36

Statistics test

Wi

Wi

Wi

Wi

Wi

Tt

Tt

Tt

Tt

Tt

Tt

Control / female

Mean SD N

70 14.9 20

76 14.5 20

80 17.7 20

87 18.4 20

90 18.9 20

85 12.2 10

108 13.5 10

116 14.8 10

128 16.3 10

138 19.8 10

145 25.0 10

40 µg/L / female

Mean SD N

75 9.5 10

80 12.6 10

85 13.9 10

88 12.3 10

90 12.8 10

100 µg/L / female

Mean SD N

70 12.4 10

71 10.4 10

75 13.7 10

77 14.2 10

84 14.0 10

400 µg/L / female

Mean SD N

65 15.7 20

69 15.7 20

71 16.6 20

77 17.9 20

78* 18.8 20

65* 19.4 10

87** 18.8 10

96* 18.1 10

108* 20.5 10

116* 24.4 10

118* 24.4 10

 Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01; *: p < 0.05

Table 4. Food consumption – group mean value (g/animal/day)

		Day
Group/Sex		Ra8-Ra14	1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-91	R1-R8	R8-R15	R15-R22	R22-R29	R29-R36	R36-R41
Statisticstest		Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Tt	Tt	Tt	Tt	Tt	Tt
Control / male	Mean	27	26	26	26	27	27	27	27	27	27	26	26	26	26	30	32	32	32	33	33
	SD	1.5	1.3	1.5	1.7	2.0	1.6	2.2	2.2	2.3	2.6	1.9	2.3	2.4	2.4	2.6	2.5	2.9	2.5	3.1	2.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5
40 µg/L / male	Mean	25	24	24	24	25	25	25	24**	24*	24	25	23*	24	24
	SD	1.2	1.4	1.4	1.6	1.5	1.8	1.0	1.3	0.7	0.7	0.9	0.5	0.5	0.7
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
100 µg/L / male	Mean	27	26	25	25	26	26	26	25**	25*	25	26	25*	24	25
	SD	1.8	1.9	1.9	1.7	2.2	2.3	1.6	2.1	1.8	1.6	1.5	1.9	2.7	2.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
400 µg/L / male	Mean	27	24*	24*	25	26	25*	25*	24**	24**	25*	25*	24**	24*	25	27	29	28	29	31	31
	SD	1.4	1.4	1.6	1.5	1.7	1.4	1.8	1.4	1.6	1.8	1.7	1.7	1.8	1.8	1.7	1.6	1.9	1.5	1.9	1.4
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5

Table 4 (continued). Food consumption – group mean value (g/animal/day)

		Day
Group/Sex		Ra8-Ra14	1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-91	R1-R8	R8-R15	R15-R22	R22-R29	R29-R36	R36-R41
Statisticstest		AV	Wi	Wi	Wi	Wi	Wi	Wi	Sh	Sh	Sh	Wi	Wi	Wi	Wi	Tt	Tt	Tt	Tt	Tt	Tt
Control / female	Mean	17	17	17	17	18	18	18	18	17	17	17	17	26	26	30	32	32	32	33	33
	SD	0.6	0.6	0.5	1.0	1.2	0.9	1.3	0.6	1.0	1.0	1.2	1.7	2.4	2.4	2.6	2.5	2.9	2.5	3.1	2.7
	N	10	10	10	10	9	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5
40 µg/L / female	Mean	17	18	17	18	18	19	19	18	18	19	18	17	24	24
	SD	1.0	1.0	1.0	0.6	0.7	0.7	0.7	1.2	0.3	1.1	1.0	0.8	0.5	0.7
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
100 µg/L / female	Mean	16	18	16	16	16*	17	17	17	16	17	16	16	24	25
	SD	1.1	1.7	1.4	0.9	0.7	0.5	1.0	0.6	0.9	0.9	1.0	1.2	2.7	2.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
400 µg/L / female	Mean	17	17	16	17	17*	17*	17	17*	17	17	17	17	24*	25	27	29	28	29	31	31
	SD	0.8	0.7	0.8	0.8	0.8	0.9	1.2	3.4	3.1	3.2	2.1	2.8	1.8	1.8	1.7	1.6	1.9	1.5	1.9	1.4
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-test where appropriate 

Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01; *: p < 0.05

Table 5. Test item-related hematology changes in rats exposed for 13 weeks

Sex	M	M	M	F	F	F
Exposure (µg/L)	40	100	400	40	100	400
HGB	-	-	1.07**	-	-	1.08**
HCT	-	-	1.06*	-	-	1.07**
RBC	-	-	1.05**	-	-	1.08**
WBC	-	-	-	1.60*	1.29*	1.47*
Neutrophils	-	1.43*	1.40*	2.65**	2.79**	3.47**
HGB: Hemoglobin; HCT: Hematocrit; RBC: Red Blood Cells; WBC: Total White Blood Cells Changes expressed as fold change versus concurrent control. * = Statistically significant at p < 0.05 and ** = Statistically significant at p < 0.01 - : no test article-related change

Table 5a. Haematology - group mean values - main group

Group /Sex		HGB	HCT	RBC	RETIC	PLT	MCV	MCH	MCHC	RDW	WBC	ANEU	ALYM	AMONO	AEOS	ABASO	ALUC
		g/dL	%	x10^6/µL	x10^9/L	x10^3/µL	fL	pg	g/dL	%	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL
Control / male	Mean	15.0	45.1	8.52	194.5	963	52.9	17.6	33.2	13.2	10.49	1.87	7.93	0.41	0.13	0.03	0.12
	SD	0.78	2.34	0.267	23.41	75.7	1.77	0.62	0.34	1.20	3.070	0.806	2.280	0.104	0.037	0.016	0.074
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
40 µg/L / male	Mean	15.5	46.7	8.96	168.2	910	52.2	17.4	33.2	12.9	9.83	1.76	7.45	0.34	0.14	0.03	0.11
	SD	0.49	1.25	0.356	26.42	53.0	1.64	0.60	0.43	0.62	1.316	0.456	1.076	0.093	0.039	0.009	0.063
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 µg/L / male	Mean	15.2	46.1	8.68	208.4	970	53.1	17.5	33.0	13.3	12.34	2.68*	9.01	0.41	0.12	0.04	0.09
	SD	0.88	2.30	0.410	130.83	149.2	1.34	0.56	0.75	1.97	2.150	0.837	1.733	0.127	0.043	0.011	0.044
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / male	Mean	16.1**	47.6*	8.97**	187.9	912	53.1	17.9	33.8	13.5	12.07	2.62*	8.70	0.44	0.16	0.04	0.12
	SD	0.54	2.05	0.372	40.74	97.6	1.59	0.77	1.15	1.38	2.515	0.570	1.998	0.085	0.057	0.016	0.062
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Control / female	Mean	14.4	42.5	7.85	163.0	968	54.2	18.4	33.8	11.6	6.78	0.66	5.72	0.23	0.08	0.01	0.08
	SD	0.47	1.44	0.219	31.71	122.0	1.32	0.49	0.36	0.41	1.795	0.280	1.571	0.129	0.047	0.008	0.050
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
40 µg/L / female	Mean	14.7	43.5	7.95	134.0	923	54.7	18.5	33.9	11.6	10.87*	1.75**	8.45	0.39	0.13	0.03	0.12
	SD	0.74	1.99	0.335	22.60	115.8	1.61	0.73	0.49	0.52	4.478	0.594	3.881	0.135	0.065	0.025	0.047
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 µg/L / female	Mean	14.6	43.2	8.03	166.1	975	53.8	18.2	33.9	11.7	8.74*	1.84**	6.39	0.30	0.10	0.02	0.10
	SD	0.44	1.40	0.247	24.48	137.5	1.48	0.51	0.58	0.63	2.047	0.529	1.565	0.079	0.041	0.008	0.062
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / female	Mean	15.5**	45.5**	8.45**	135.9*	956	53.8	18.3	34.0	11.4	9.94*	2.29**	7.10	0.30	0.15**	0.02	0.09
	SD	0.40	1.29	0.304	29.55	110.6	0.93	0.43	0.41	0.25	2.955	0.603	2.361	0.123	0.063	0.009	0.070
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Table 5b. Haematology - group mean vlaues - recovery group

Group /Sex		HGB	HCT	RBC	RETIC	PLT	MCV	MCH	MCHC	RDW	WBC	ANEU	ALYM	AMONO	AEOS	ABASO	ALUC
		g/dL	%	x10^6/µL	x10^9/L	x10^3/µL	fL	pg	g/dL	%	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL
Control / male	Mean	15.2	48.2	8.85	195.9	738	54.5	17.2	31.5	14.0	9.36	1.76	6.98	0.38	0.15	0.05	0.06
	SD	0.42	1.51	0.350	24.29	132.7	1.20	0.46	0.31	1.01	1.011	0.662	1.061	0.060	0.029	0.012	0.025
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / male	Mean	15.3	48.2	8.99	186.7	703	53.8	17.0	31.6	13.8	11.56*	2.62*	8.24	0.41	0.14	0.06	0.09*
	SD	0.98	2.59	0.515	28.31	155.9	3.56	1.44	0.72	1.34	2.692	0.859	1.878	0.087	0.030	0.033	0.044
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Control / female	Mean	14.6	44.8	8.04	138.8	774	55.8	18.2	32.6	11.9	6.48	0.82	5.29	0.22	0.09	0.03	0.05
	SD	0.71	2.27	0.462	29.87	118.7	1.71	0.74	0.40	0.41	1.573	0.251	1.364	0.069	0.032	0.012	0.022
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / female	Mean	15.2	47.2*	8.32	143.2	743	56.8	18.3	32.2	12.0	7.54	2.05**	5.06	0.27	0.10	0.02	0.04
	SD	0.63	2.15	0.442	31.88	88.4	1.59	0.57	0.49	0.60	1.574	0.194	1.410	0.106	0.023	0.009	0.016
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Table 6. Test item-related bronchoalveolar fluid changes in rats exposed for 13 weeks

Sex	M	M	M	F	F	F
Exposure (µg/L)	40	100	400	40	100	400
TWBC	6.50a	3.87**	9.85**	4.03*	11.52**	21.48**
Total Protein	3.40**	5.10**	11.40**	4.13**	9.00**	17.38**
LDH	3.10*	2.79*	19.13**	1.89	4.17	20.37**
ALKP	-	-	2.47**	-	-	2.85**
TWBC: Total White Blood Cells; LDH: Lactate Dehydrogenase; ALKP: Alkaline Phosphatase Changes expressed as fold change versus concurrent control. * = Statistically significant at p < 0.05 and ** = Statistically significant at p < 0.01 a Animal No. 2042 only and calculated as individual animal value versus concurrent control mean. - : no test article-related change

Table 7: Test item-related bronchoalveolar fluid changes in rats at the end of a 6-week recovery period

Sex	M	F
Exposure (µg/L)	400	400
TWBC	1.95	3.37**
Total Protein	9.40**	13.57**
LDH	8.93	2.41
ALKP	4.09	2.73
TWBC: Total White Blood Cells; LDH: Lactate Dehydrogenase; ALKP: Alkaline Phosphatase Changes expressed as fold change versus concurrent control. ** = Statistically significant at p < 0.01

Table 8. Test item-related organ weight changes (% difference relative to controls) in rats exposed to organolignite for 13 weeks

Group/sex	2M	3M	4M	2F	3F	4F
Exposure (µg/L)	40	100	400	40	100	400
Lungs and Bronchi
Absolute weight (%)	30a	74a	182a	51a	113a	209a
vs. body weight (%)	47a	84a	202a	46a	116a	210a
vs. brain weight (%)	36a	80a	187a	48a	117a	209a

a: Statistically significant difference between mean values for test item-treated and control group
M = Male F = Female

Table 9. Test item-related organ weight changes (% difference relative to controls) in rats at the end of 6 -week recovery period

Group/sex	4M	4F
Exposure (µg/L)	400	400
Lungs and Bronchi
Absolute weight (%)	186a	187a
vs. body weight (%)	211a	225a
vs. brain weight (%)	185a	189a

a: Statistically significant difference between mean values for test item-treated and control group
M = Male F = Female

Table 10. Test item-related macroscopic findings in the lungs in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	9	10	10	0	9	10	10
Distended	0	1	3	4	0	1	1	2
Pale area(s)	0	2	5	4	0	2	7	3
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

 

Table 11. Test item-related macroscopic findings in the tracheobronchial lymph nodes in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	8	9	9	0	9	8	10
Enlarged	0	8	9	9	0	8	9	10
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 12. Test item-related macroscopic findings in the thymic lymph nodes in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	6	7	8	0	8	7	8
Enlarged	0	5	6	7	0	7	6	8
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 13. Test item-related macroscopic findings in the lungs in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	10	0	10
Pale area(s)	0	9	0	6
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 14. Test item-related macroscopic findings in the tracheobronchial lymph nodes in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	8	0	10
Enlarged	0	7	0	7
Number of tissues examined	10	10	10	10

M = Male F = Female

Table 15. Test item-related macroscopic findings in the thymic lymph nodes in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	9	0	6
Enlarged	0	9	0	5
Number of tissues examined	10	10	10	10

M = Male F = Female

Table 16. Test item-related histopathological findings in the lungs and bronchi of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Alveolar Macrophages
Moderate	0	7	4	0	0	3	3	0
Marked	0	3	4	2	0	7	4	0
Severe	0	0	2	8	0	0	3	10
Total	0	10	10	10	0	10	10	10
Bronchioloalveolar Inflammation
Minimal	0	3	0	1	1	1	1	0
Slight	0	5	5	1	0	6	2	1
Moderate	0	2	4	7	0	2	7	6
Marked	0	0	1	1	0	1	0	3
Total	0	10	10	10	1	10	10	10
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 17: Test item-related histopthological findings in the tracheobronchial lymph nodes of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Increased Paracortical Cellularity
Minimal	0	2	3	2	0	2	5	2
Slight	0	4	4	3	0	4	2	2
Moderate	0	2	3	4	0	2	3	4
Marked	0	0	0	1	0	0	0	2
Total	0	8	10	10	0	8	10	10
Pigmented Macrophages
Minimal	0	4	3	0	0	6	4	4
Slight	0	5	6	6	0	4	5	4
Moderate	0	1	1	3	0	0	1	2
Total	0	10	10	9	0	10	10	10
Number of tissues examined	8	10	10	10	8	10	10	10

M = Male F = Female

Table 18. Test item-related histopathological findings in the thymic lymph nodes of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Increased Paracortical Cellularity
Minimal	0	3	2	1	0	3	2	0
Slight	0	1	3	1	0	3	2	4
Moderate	0	2	2	4	0	2	3	3
Marked	0	0	0	2	0	0	0	1
Total	0	6	7	8	0	8	7	8
Pigmented Macrophages
Minimal	0	0	0	0	0	3	2	0
Slight	0	6	4	2	0	4	3	3
Moderate	0	0	3	6	0	1	2	5
Total	0	6	7	8	0	8	7	8
Number of tissues examined	0	6	7	8	0	8	7	8

M = Male F = Female

 

Table 19. Test item-related histopathological

findings in the nose/turbinates of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Olfactory Epithelial Degeneration
Minimal	0	0	4	0	0	1	4	0
Slight	0	0	2	3	0	0	3	3
Moderate	0	0	0	6	0	0	0	6
Marked	0	0	0	0	0	0	0	1
Total	0	0	6	9	0	1	7	10
Respiratory Epithelial Degeneration
Minimal	0	0	1	3	0	0	0	1
Slight	0	0	0	2	0	0	0	2
Total	0	0	1	5	0	0	0	3
Respiratory Epithelial Inflammation
Minimal	0	0	1	2	0	0	0	0
Total	0	0	1	2	0	0	0	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	0	0	1	0	0	0	1
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	1	0	0	0	2
Pigment/Pigmented Macrophages
Minimal	0	0	1	3	0	0	1	3
Slight	0	0	0	2	0	0	0	7
Moderate	0	0	0	4	0	0	0	0
Total	0	0	1	9	0	0	1	10
Number of tissues examined	10	10	10	9	10	10	10	10

M = Male F = Female

 

Table 20. Test item-related histopathological findings in the larynx of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Epithelial Alteration
Minimal	0	0	0	1	1	1	0	0
Slight	0	2	3	0	0	4	0	0
Total	0	2	3	1	1	5	0	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	6	6	5	0	4	6	4
Slight	0	1	1	4	0	0	4	6
Total	0	7	7	9	0	4	10	10
Pigment/Pigmented Macrophages
Minimal	0	1	1	3	0	1	6	6
Slight	0	0	0	1	0	0	0	0
Total	0	1	1	4	0	1	6	6
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 21. Test item-related histopathological findings in the tracheal bifurcation of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Macrophages
Minimal	0	5	5	6	0	3	5	9
Total	0	5	5	6	0	3	5	9
Number of tissues examined	8	7	9	8	8	8	9	10

M = Male F = Female

 

Table 22. Test item-related histopathological findings in the trachea of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Macrophages
Minimal	0	2	2	5	0	3	3	5
Total	0	2	2	5	0	3	3	5
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 23. Test item-related histpathological findings in the lungs and bronchi of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Alveolar Macrophages
Marked	0	2	0	1
Severe	0	8	0	9
Total	0	10	0	10
Bronchioloalveolar Inflammation
Minimal	0	0	1	0
Moderate	0	5	0	3
Marked	0	5	0	7
Total	0	10	1	10
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 24. Test item-related histopathological findings in the tracheobronchial lymph nodes of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Increased Paracortical Cellularity
Minimal	0	6	0	5
Slight	0	2	0	1
Moderate	0	1	0	1
Marked	0	1	0	2
Total	0	10	0	9
Pigmented Macrophages
Minimal	0	1	0	0
Slight	0	6	0	7
Moderate	0	3	0	3
Total	0	10	0	10
Number of tissues examined	7	10	9	10

M = Male F = Female

 

Table 25. Test item-related histopathological findings in the thymic lymph nodes of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Increased Paracortical Cellularity
Minimal	0	1	0	2
Slight	0	2	0	2
Moderate	0	6	0	2
Marked	0	0	0	0
Total	0	9	0	6
Pigmented Macrophages
Slight	0	4	0	1
Moderate	0	4	0	4
Marked	0	1	0	1
Total	0	9	0	6
Number of tissues examined	0	9	0	6

M = Male F = Female

 

Table 26. Test item-related histopathological findings in the nose/turbinates of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Olfactory Epithelial Degeneration
Minimal	0	1	1	1
Slight	0	3	0	5
Moderate	0	5	0	3
Marked	0	0	0	1
Total	0	9	1	10
Olfactory Epithelial Inflammation
Slight	0	1	0	1
Total	0	1	0	1
Respiratory Epithelial Degeneration
Minimal	0	1	0	1
Total	0	1	0	1
Respiratory Epithelial Inflammation
Minimal	0	1	0	0
Total	0	1	0	0
Pigment/Pigmented Macrophages
Minimal	0	4	0	4
Slight	0	3	0	5
Moderate	0	2	0	0
Total	0	9	0	9
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 27. Test item-related histopathological findings in the larynx of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Epithelial Alteration
Minimal	0	1	1	0
Slight	0	2	3	0
Total	0	3	4	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	0	0	2
Total	0	0	0	2
Pigment/Pigmented Macrophages
Minimal	0	0	0	3
Total	0	0	0	3
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 28. Test item-related histopathological findings in the tracheal bifurcation of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Macrophages
Minimal	0	8	0	10
Total	0	8	0	10
Number of tissues examined	8	9	9	10

M = Male F = Female

Table 29. Test item-related histopathological findings in the trachea of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Macrophages
Minimal	0	7	0	8
Total	0	7	0	8
Number of tissues examined	10	10	10	10

M = Male F = Female

Conclusions:

In a subchronic repeated dose toxicity study (OECD 413) via inhalation a lung overload was observed resulting in an inflammatory response on the respiratory tract and correlated changes in hematology parameters. As these effects are considered to be local and the systemic effects are secondary effects a local NOAEC could not be established. A local LOAEC can be set to 40 µg/L. As systemic effects are considered secondary the systemic NOAEC is set to 400 mg/L.

Executive summary:

A subchronic repeated dose toxicity study according to OECD 413 and compliant to GLP was performed. Sprague-Dawley CD®rats (10 animals/sex/group) were exposed to target exposure levels of 0 (air only), 40, 100 or 400 µg/L test substance via nose only inhalation for 6 hours/day, 5 days/week for 13 consecutive weeks. Ten additional rats/sex/group in the control groups and 400 µg/L group were similarly exposed for 13 consecutive weeks and retained for a 6-week recovery period. At the end of the treatment and recovery phases, designated animals were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weights, food consumption, clinical pathology (termination and recovery), organ weights, bronchoalveolar lavage (BAL) analysis, macroscopic observations and microscopic pathology.

Overall mean achieved concentration for 40 µg/L group was on target. 100 µg/L group mean concentration was 5% above target and 40 µg/L group was 1% below target. The MMAD values for all samples collected showed that the delivered particle size was respirable to the rats. This study resulted in no test item-related effects on mortality, clinical observations, ophthalmology, coagulation and clinical chemistry. Decreased body weight gains and food consumption were seen at all exposure levels in the males and at the highest exposure levels in the females compared with control. These changes were test item-related but not considered adverse because the body weight gain and food consumption in males showed no dose-dependent pattern. In females the body weight gain and the food consumption was only occasionally observed. Recovery in these parameters were seen at the end of the recovery period.

Test item-related haematology changes at all exposure levels in females included increases in neutrophils with corresponding increases in total white blood cells. In males a neutrophil increase was only observed at 100 and 400 µg/L and without corresponding increase in total white blood cell count. In the recovery group the increase of neutrophils was still observed. These changes correlated with microscopic findings of inflammation in the respiratory tract that were considered adverse at ≥ 40 µg/L. At 400 µg/L, there were increases in red cell mass (haemoglobin, haematocrit, and red blood cells) in both sexes that were considered test item-related. In the recovery group no increase in red cell mass was observed. Inhalation exposures of 40, 100 or 400 µg/L organolignite in rats for 13 weeks resulted in test item-related findings in the lungs and bronchi, tracheobronchial and thymic lymph nodes, nose/turbinates, larynx, tracheal bifurcation, and/or trachea at ≥ 40 µg/L. Moderate pigment/pigmented macrophages and slight mixed inflammation in the lung at ≥ 40 µg/L was considered adverse since these findings could compromise pulmonary function and disrupt the ability to maintain respiratory homeostasis. Other adverse findings included slight olfactory and respiratory epithelial degeneration and accompanying inflammation in the nose. Remaining organolignite-related findings were considered non-adverse. Non-adversity was based on low severity grades, absence of morphologic damage, the expectation that these findings would not be harmful to the animals affected, and/or the presence of such findings at comparable severity in healthy control animals. Following a 6-week exposure-free recovery period, most test item-related findings were still observed at the same or lower magnitude as in the end of the treatment period in males and females exposed to organolignite at 400 µg/L. In the lungs and bronchi, pigment/pigmented macrophages and inflammation was still observed in the recover group. The incidence of markedly inflamed lungs and bronchi in males and females was increased in the recovery group compared to the main group suggesting that inflammation worsened in some animals during the 6-week recovery phase. In the tracheobronchial and thymic lymph nodes, pigmented macrophages were also still observed in the recovery group. No recovery in the nose, olfactory epithelial degeneration and pigmented/pigmented macrophages was observed in the recovery males and females, compared with the main group. Partial recovery in respiratory epithelial degeneration and inflammation was observed, while complete recovery in squamous metaplasia of respiratory epithelium in the nose was observed in males/females of the recovery group compared with the main group. In the larynx, epithelial alteration was observed to be normal in the recovery group in contrast with the main group, as did squamous metaplasia and pigment/pigmented macrophages in male rats. In females of the recovery group, squamous metaplasia and pigment/pigmented macrophages were observed with lower incidences compared to the main group. No recovery was observed in the tracheal bifurcation and trachea, pigment/pigmented macrophages. Lack of recovery in the aforementioned respiratory tissues was based on comparable incidence and severity of organolignite-related findings in 400 µg/L main group animals and recovery animals. Organolignite-related bronchoalveolar fluid changes at all exposures included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also, at all exposures there were increases in total protein and lactate dehydrogenase activities. At 400 µg/L, there were increases in alkaline phosphatase activities. Test item-related bronchoalveolar fluid changes following a 6-week recovery period in 400 µg/L rats included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. There were also increases in total protein, lactate dehydrogenase, and alkaline phosphatase activities. These bronchoalveolar lavage fluid results correlated with inflammatory changes in the lungs and bronchi observed in 400 µg/L recovery rats. Overall, the changes observed are consistent with a lung overload response, i.e., an inflammatory response in response to poorly soluble particles (PSPs). While lung overload and subsequent disease are a common phenomenon in the rat, the relevance of this finding to human disease risk is less clear (CLP Annex I, 3.9.2.8.1 e). In conclusion, organolignite exposures from 40 to 400 µg/L resulted in an inflammatory response on the respiratory tract and correlating changes in haematology parameters when rats were exposed for up to 13 consecutive weeks. Thus, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects was not established. The NOAEC for systemic effects was set to 400 µg/L because systemic effects were only observed as secondary effect of the lung overload.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

400 mg/m³

Study duration:

subchronic

Experimental exposure time per week (hours/week):

30

Species:

rat

Quality of whole database:

The quality of the whole database is high and meets the tonnage driven data requirements of REACH.

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2019-04-12 - 2020-02-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 413 (90-Day (Subchronic) Inhalation Toxicity Study

Version / remarks:

09 October 2017

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Remarks:

Crl:CD (SD)

Details on species / strain selection:

The rat is an animal model commonly utilized in toxicity studies like the OECD 413 guideline and was the rodent species used in the toxicity testing program for organolignite. In addition, a historical database of this species is available for comparative evaluation and prior studies have been conducted in rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Raleigh, USA
- Females (if applicable) nulliparous and non-pregnant: not provided
- Age at study initiation: approximately 8 weeks
- Weight at study initiation: Males: 254 - 323 g; Females: 176 - 227 g (range)
- Fasting period before study: overnight fasting before necropsy/blood sampling for clinical pathology
- Housing: Nominally 2 animals of the same sex per cage
- Diet: Teklad Global 16% Protein Rodent Diet (certified), 2016C ad libitum
- Water: tap water ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY: There were no known contaminants in the feed and water that were expected to interfere with the results of this study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 30 - 70
- Air changes (per hr): not provided
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 18 Apr 2019 To: 12 Sep 2019

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

snout only

Vehicle:

air

Mass median aerodynamic diameter (MMAD):

>= 1 - <= 2.3 µm

Geometric standard deviation (GSD):

3.02

Remarks on MMAD:

The MMAD given above is the maximum range over all groups. The GSD is the maximum observed.
Values per group are as follows:
Group 2: MMAD Range: 1.0 - 1.7 µm, GSD Range: 1.75 - 2.81
Group 3: MMAD Range: 1.1 - 1.9 µm, GSD Range: 1.78 - 3.02
Group 4: MMAD Range: 1.1 - 2.3 µm, GSD Range: 1.80 - 2.68

The MMAD values for all samples collected showed that the delivered particle size was respirable to the rats. The wider than anticipated range of MMAD was attributed to test item wear to the internal components of the jet mills. Jet mills were replaced as needed to ensure particle distribution remained in the range 1 – 3 µm equivalent aerodynamic diameter.

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Flow through (ADG) snout-only exposure system
- Method of holding animals in test chamber: Restraints (molded clear thermoplastic tubes). The animals were acclimated to the method of restraint over a 3-day period immediately preceding the first test item exposure.
- Source and rate of air: dry air from in-house compressed air system; Exposure Day 1 - 48: 60.0 L/minute, Exposure Day 49 - 66: 41.0 L/minute
- System of generating particulates/aerosols: Mark I Wright Dust Feed (WDF)
- Temperature, humidity, pressure in air chamber: 22 °C, 44% humidity
- Air flow rate: 20L/minute (aerosol generation)
- Method of particle size determination: cascade impaction
- Treatment of exhaust air: filtered locally

TEST ATMOSPHERE
- Brief description of analytical method used: Aerosol samples were collected as follows: Filter type - glass fiber filter, held in an open face filter holder; Sample flow - 2.0 L/minute; Sample volume - measured by timed flowmeter; Sample frequency - minimum of 3 samples/group/exposure; Sample location - representative animal exposure position, alternated weekly throughout study; Sample analysis - gravimetric - groups 1 to 4; Sample disposition - filters discarded after gravimetric weights collected
- Samples taken from breathing zone: yes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Group 2: Target: 40 µg/L, Achieved: 40 µg/L
Group 3: Target: 100 µg/L, Achieved: 105 µg/L
Group 4: Target: 400 µg/L, Achieved: 397 µg/L
Overall mean achieved concentration for Group 2 was on target. Group 3 mean concentration was 5% above target and Group 4 was 1% below target.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

6 hours/day, 5 days/week

Dose / conc.:

0.04 mg/L air (nominal)

Dose / conc.:

0.1 mg/L air (nominal)

Dose / conc.:

0.4 mg/L air (nominal)

No. of animals per sex per dose:

10 (main group)
10 (control and high dose group for 6 week recovery period)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The exposure levels for organolignite were selected based on the results of previously conducted inhalation toxicity studies. A repeated exposure inhalation toxicity study in rats was conducted at 0.5, 1.0, 2.0 mg/L for 14 days. Clinical signs such as gasping were only seen at the highest exposure level. Animals at the lower exposure levels were unaffected thus the no observed adverse effect concentration (NOAEC) from this study was determined to be 1.0 mg/L (1000 µg/L). An OECD TG 422 combined repeated exposure inhalation toxicity study with a reproduction/developmental toxicity-screening test was conducted in rats at 0.005, 0.05 and 0.3 mg/L for 4 weeks of exposure. No clinical signs were seen thus the no observed adverse effect concentration (NOAEC) from this study was determined to be 0.3 mg/L (300 µg/L). On the basis of these prior studies, the exposure levels of 40, 100 and 400 µg/L were selected for this study and are expected to be tolerated for 13 weeks of exposures.

- Fasting period before blood sampling for clinical biochemistry: overnight

Positive control:

none

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, general condition

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: twice pretest and weekly (once every 7 days post-exposure on exposure days during treatment and recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: twice pretest and twice weekly during the first 4 weeks of treatment period and weekly (once every 7 days) from Week 5 through the recovery period.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption was measured (weighed) weekly, beginning one week prior to treatment.

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

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: pretest and at the end of exposures (Week 13)
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals per group
- Parameters checked: Haemoglobin (HGB), Haematocrit (HCT), Red blood cell count (RBC), Platelet count (PLT), Mean corpuscular volume (MCV), Mean corpuscular haemoglobin (MCH), Mean corpuscular haemoglobin concentration (MCHC), Red cell distribution width (RDW), Total white blood cell count (WBC), Reticulocyte count (RETIC), Differential white blood cell count: Neutrophils (ANEU), Lymphocytes (ALYM), Eosinophils (AEOS), Basophils (ABASO), Monocytes (AMONO), Large unstained cells (ALUC); and Prothrombin time (PT) and Activated partial thromboplastin time (APTT) .

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination
- Animals fasted: Yes
- How many animals: all animals per group
- Parameters checked: Aspartate aminotransferase (AST), Alanine aminotransferase (ALT), Alkaline phosphatase (ALKP), Blood urea nitrogen (BUN), Creatinine (CREAT), Glucose (GLU), Cholesterol (CHOL), Triglycerides (TRIG), Total protein (TP), Albumin (ALB), Total bilirubin (TBILI), Sodium (NA+), Potassium (K+), Chloride (Cl-), Calcium (Ca++) and Inorganic phosphorus (PHOS).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

BRONCHOALVEOLAR LAVAGE FLUID (BALF): Yes
- Time schedule for analysis: at termination
- Dose groups that were examined: all
- Number of animals: all
- Parameters checked: Total white blood cell count (TWBC), Total protein (TP), Lactate dehydrogenase (LDH), Alkaline phosphatase (ALKP), Differential white blood cell count: Macrocytes (MACRO), Neutrophils (NEUT), Lymphocytes (LYMPH), Eosinophils (EOS), Monocytes (MONO) and Basophils (BASO).

LUNG BURDEN: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1 in "any other information on materials and methods incl. tables")
HISTOPATHOLOGY: Yes (see table 1 in "any other information on materials and methods incl. tables")

Other examinations:

Organs were weighed for all animals at the scheduled necropsy intervals (see table 1 in "any other information on materials and methods incl. tables").

Statistics:

All statistical analyses were carried out separately for males and females using the individual animal as the basic experimental unit.
The following data types were analyzed at each timepoint separately:
body weight
body weight change from interval to interval
cumulative body weight change from baseline
food consumption
haematology
coagulation
clinical chemistry
organ weights
The following comparisons were made:
Group 1 vs 2, 3 and 4
The parameters to analyze were identified as either continuous (greater than 6 distinct values), discrete (between 3 and 6 distinct values) or binary (2 distinct values).

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Decreased body weight gains were seen in males exposed to ≥ 40 µg/L but not in a clearly exposure-level related pattern. For instance, body weight gains from Day 1 to Day 91 was reduced in males by 21.6%, 14.4% and 17.0% in 40, 100 and 400 µg/kg bw/day groups compared with controls. At the end of the recovery period the male animals treated with 400 µg/kg bw/day had still a reduced body weight (- 15.0%) compared to the controls. This effects are not considered to be toxicolgical relevant as no clear exposure-level reated pattern was observed.
Decreased body weight gains over the whole treatment period (Day 1 to Day 91) were seen only in females exposed to 400 µg/L (-13.3%). Comparing the body weights of 400 µg/L females on Day 91 with the controls a non-significant decrease of 4.8% was observed. Therefore, these changes were test item-related but not considered adverse. A recovery in body weight gain was seen after the end of the exposures. For details see table 2 and 3 in "any other information on results incl. tables".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Decreased food consumption was seen in males exposed to ≥ 40 µg/L but not in a clearly exposure-level related pattern. Decreased food consumption was seen in females exposed to 400 µg/L but not continuosly over the treatment period. Significant decrease was only observed on Day 22-29, 29-36, 43-50. Therefore the decrease in food consumption in males and females were test item-related but not considered adverse. A recovery in food consumption was seen after the end of the exposures. For details see table 4 in "any other information on results incl. tables".

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related haematology changes were observed in male animals of the 100 and 400 µg/L dose groups as well as in females in all dose groups.
Males: Neutrophils were 1.43- and 1.4-fold increased (statistical significant) in males in dose group 100 and 400 µg/L, respectively, but without corresponding increase of white blood cells. In the recovery group the white blood cells were significantly increased in the 400 µg/L dose group by 1.24-fold and the neutrophils by 1.49-fold. These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.07-, 1.06- and 1.05-fold which are considered test substance-related.

Females: Neutrophils were 2.65-, 2.79- and 3.47-fold increased (statistically significant) in females in dose group 40, 100 and 400 µg/L, respectively. These changes corresponded to an significant increase of white blood cells (1.6-, 1.29- and 1.47-fold). In the recovery group the white blood cells were not increased anymore but the neutrophils in the 400 µg/L dose group were still significantly increased 2.5-fold compared to the conrols. These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.08-, 1.07- and 1.08-fold which are considered test substance-related..
See table 5 and 5a/b in "any other information on results incl. tables" for details.

There were no test item-related coagulation changes at any exposure tested.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Main group:
There were no test item-related clinical chemistry changes at any exposure tested.
There were minimal to mild increases in aspartate and alanine aminotransferase activities in one female at each exposure level that were considered not test item-related as they lacked an exposure level relationship and there were no microscopic correlates in the liver or skeletal muscles.

Recovery group:
There were no test item-related clinical chemistry changes following a 6-week recovery period in animals exposed to 400 µg/L.

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):

Statistically significant higher absolute and relative lung and bronchi weights were present in males and females exposed to ≥40 µg/L compared to controls. Higher lung weights were related to increased pigment/pigmented alveolar macrophages and inflammation observed microscopically. Higher lung weights did not recover and remained increased in a similar magnitude compared to the main group in males and females exposed to 400 µg/L following a 6-week exposure-free recovery period. See table 8 and 9 in "any other information on results incl. tables" for details.

All other organ weight changes in the terminal necropsy, statistically significant or not, were not considered organolignite-related because they were of low magnitude, independent of exposure level, and/or lacked a macroscopic or microscopic correlate.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Abnormally colored (black) lungs were observed in 9/10, 10/10 and 10/10 males and females each in 40, 100 and 400 µg/L dose groups, respectively. Distended areas in the lungs were observed in 1/10, 3/10 and 4/10 males and 1/10, 1/10 and 2/10 females in the 40, 100 and 400 µg/L dose groups, respectively. Pale areas (white or tan) in the lungs were observed in 2/10, 5/10 and 4/10 males and 2/10, 7/10 and 3/10 females in the 40, 100 and 400 µg/L dose groups, respectively. These lung findings correlate to pigment/pigmented alveolar macrophages and/or inflammation microscopically. No recovery was observed for abnormally colored (black) lungs and (white or tan) in pale areas in the lungs. All animals in the recovery group showed black lungs and 9/10 males and 6/10 females showed (white or tan) pale areas in the lungs. Therefore, these effects are considered treatment-related and adverse.

Abnormally colored (black) and enlarged tracheobronchial and thymic lymph nodes were also observed in males and females exposed to organolignite at ≥40 µg/L with the following incidence rate:
Tracheobronchial lymph nodes: Abnormal colored (black): 8/10, 9/10 and 9/10 males and 9/10, 8/10 and 10/10 females in 40, 100 and 400 µg/L dose groups respectively. Enlarged: 8/10, 9/10 and 9/10 males and 8/10, 9/10 and 10/10 females in 40, 100 and 400 µg/L dose groups respectively.
Thymic lymph nodes: Abnormal colored (black): 6/10, 7/10 and 8/10 males and 8/10, 7/10 and 8/10 females in 40, 100 and 400 µg/L dose groups respectively. Enlarged: 5/10, 6/10 and 7/10 males and 7/10, 6/10 and 8/10 females in 40, 100 and 400 µg/L dose groups respectively.
For both tissues tracheobronchial and thymic lymph nodes the observed effects was still observed in the recovery group.

All above mentioned effecs correlated to pigmented macrophages and/or increased paracortical cellularity microscopically and thus are considered treatment-related and adverse.
See tables 10 to 15 in "any other information on results incl. tables" for details.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Microscopic findings related to organolignite were observed in the lungs and bronchi, tracheobronchial and thymic lymph nodes, nose/turbinates, larynx, tracheal bifurcation, and trachea at ≥40 µg/L.
Terminal necropsy:
Lungs and Bronchi:
Pigment/pigmented alveolar macrophages (moderate to severe) were present in all male and female rats exposed at ≥40 µg/L, with exposure-related increases in incidence and severity. Alveoli were diffusely expanded by large numbers of variably pigmented macrophages characterized by light tan intracytoplasmic speckling to dark brown cytoplasmic contents. Extracellular dark brown pigment was also multifocally present within alveoli. Pigmented material was interpreted to be organolignite. Pigment/pigmented alveolar macrophages were accompanied by bronchioloalveolar inflammation (minimal to marked) characterized by neutrophilic infiltrates, necrotic cellular debris in airways, type II pneumocyte hyperplasia, perivascular and peribronchiolar lymphocytes, and increased bronchus-associated lymphoid tissue. Microscopic findings in the lungs and bronchi correlated with higher lung weights and abnormal (black) coloration, distention, and/or pale (white or tan) areas observed macroscopically. See table 16 in "any other information on results incl. tables" for details.
No recovery of pigment/pigmented macrophages and inflammation in male and female rats of the recovery groups were observed. These findings were present in comparable incidence and severity following the recovery period compared to main group animals, with increased incidences of marked inflammation at recovery compared to the terminal phase. Microscopic findings in the lungs and bronchi correlated with higher lung weights and abnormal (black) coloration and/or pale (white or tan) areas observed macroscopically. See table 23 in "any other information on results incl. tables" for details.

Tracheobronchial and Thymic Lymph Nodes:
Organolignite-related increased paracortical cellularity (minimal to marked) and intrasinusoidal pigmented macrophages (minimal to moderate) were present at ≥ 40 µg/L, with exposure-related increases in incidence and severity. Increased paracortical cellularity and pigmented macrophages correlated with macroscopically enlarged lymph nodes and abnormal (black) coloration, respectively. See tables 17 and 18 in "any other information on results incl. tables" for details.
No recovery in paracortical cellularity and intrasinusoidal pigmented macrophages in male and female rats of the recovery group were observed. These findings were present in comparable incidence and severity following the recovery period. Increased paracortical cellularity and pigmented macrophages correlated with macroscopically enlarged lymph nodes and abnormal (black) coloration, respectively. See tables 24 and 25 in "any other information on results incl. tables" for details.

Nose/Turbinates
Organolignite-related olfactory epithelial degeneration (minimal to marked) was present in the nose/turbinates of rats exposed to ≥ 100 µg/L, with exposure-related increases in incidence and severity. One 40 µg/L female had minimal olfactory epithelial degeneration, but this was considered within background range incidence and severity. Olfactory epithelial degeneration was characterized by disorganization of cell layers, attenuation, loss of cilia, bleb formation, and increased intercellular spaces. Respiratory epithelial degeneration (minimal to slight) was present in few male (1/10 at 100 µg/L and 5/9 at 400 µg/L) and female (0/10 at 100µg/L and 3/10 at 400 µg/L) rats exposed to ≥100 µg/L. This finding was similarly characterized by disorganization, attenuation, and loss of cilia and was infrequently accompanied by mixed cell inflammation (minimal) and/or squamous metaplasia (minimal to slight). Areas of epithelial degeneration (olfactory and/or respiratory) often contained mucosal, submucosal or luminal accumulations of extracellular pigment/pigmented macrophages (minimal to moderate). See table 19 in "any other information on results incl. tables" for details.
No recovery in olfactory epithelial degeneration in male and female rats of the recovery group were observed. It was accompanied by olfactory epithelial inflammation in two rats. Also, no recovery in pigment/pigmented macrophages was observed in males and females of the recovery group. Both olfactory epithelial degeneration and pigment/pigmented macrophages were present in comparable incidence and severity following the recovery period. Partially recovery in respiratory epithelial degeneration and inflammation were observed and were both present in fewer animals and/or at lesser severity compared to main group animals. Squamous metaplasia of respiratory epithelium was not present in animals of the recovery group, consistent with complete recovery of this finding. See table 26 in "any other information on results incl. tables" for details.

Larynx
Organolignite-related epithelial alteration (minimal to slight) and/or squamous metaplasia (minimal to slight) were present in the larynx of rats exposed to ≥40 µg/L, with exposure-related increases in incidence and/or severity. Epithelial alteration and squamous metaplasia mostly occurred along the base of the epiglottis within the anterior (level I) larynx. Epithelial alteration, a pre-metaplastic finding, was characterized by focal loss of cilia and epithelial flattening that did not fulfill the criteria of squamous metaplasia. Squamous metaplasia was a more focally extensive to diffuse finding characterized by 3 to 4 cell layers of flattened, stratified epithelium with desquamation and/or keratinization. Pigment/pigmented macrophages (minimal to slight) were present in the lumen and/or submucosa at ≥ 40 µg/L. See table 20 in "any other information on results incl. tables" for details.
In the recovery group epithelial alteration was not present and incidences and severities in male and female rats suggested that squamous metaplasia in male rats had recovered to epithelial alteration in some male animals of the recovery group. It is also noteworthy that incidence and severity was comparable between organolignite-exposed males of the recovery group and control females of the recovery group. Squamous metaplasia was not present in male recovery rats and showed a lower incidence female rats of the recovery group. Squamous metaplasia was present at decreased incidence and severity in females of the recovery group compared to females of the main group. Pigment/pigmented macrophages were not observed in males of the recovery group and the incidence was reduced females of the recovery group compared to the main group. See table 27 in "any other information on results incl. tables" for details.

Tracheal Bifurcation/Trachea
Organolignite-related pigment/pigmented macrophages (minimal) were present in rats exposed to ≥ 40 µg/L, with some exposure-related increases in incidence. Pigment/pigmented macrophages were present in the tracheal lumen, submucosa, and/or serosa/adventitia. See tables 21 and 22 in "any other information on results incl. tables" for details.
No recovery in pigment/pigmented macrophages in male and females rats of the recovery group was observed. These findings were present in comparable incidence and severity following the recovery period compared to main group animals. See tables 28 and 29 in "any other information on results incl. tables" for details.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Bronchoalveolar Lavage

Test item-related bronchoalveolar fluid changes at all exposures included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also, at all dose levels there were increases in total protein and lactate dehydrogenase activities. At 400 µg/L, there was increases in alkaline phosphatase activities. All of these changes were considered test item-related and appeared to be generally dose level dependent. These changes correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse at ≥ 40 µg/L. See table 6 in "any other information on results incl. tables" for details.
Test item-related bronchoalveolar fluid changes in the recovery group of treated rats compared to control rats included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also in treated animals of the recovery group, there were increases in total protein, lactate dehydrogenase, and alkaline phosphatase activities. These changes correlated with the microscopic findings of inflammation in the respiratory tract in the recovery animals. For details see table 7 in "any other information on results incl. tables".

Key result

Dose descriptor:

LOAEC

Remarks:

local

Effect level:

0.04 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Key result

Dose descriptor:

NOAEC

Remarks:

systemic

Effect level:

0.4 mg/L air (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no systemic adverse effects observed up to the highest dose tested

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

0.04 mg/L air (nominal)

System:

respiratory system: lower respiratory tract

Organ:

lungs

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 2. Body weight - group mean values (g)

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-5

1-8

1-12

1-15

1-19

1-22

1-26

1-29

1-36

1-43

1-50

1-57

Statistics test

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Wi

Control / male

Mean SD N

29 5.1 20

39 8.2 20

65 11.4 20

77 12.9 20

99 15.0 20

109 17.3 20

127 20.1 20

137 21.9 20

157 24.2 20

173 28.0 20

190 30.7 20

205 31.8 20

40 µg/L / male

Mean SD N

24* 5.2 10

31 8.2 10

50* 11.6 10

60** 13.6 10

80* 16.0 10

86** 17.0 10

99** 18.0 10

105** 20.0 10

121** 22.8 10

135** 20.6 10

150** 24.7 10

159** 24.8 10

100 µg/L / male

Mean SD N

25* 3.5 10

36 6.3 10

60* 11.6 10

68** 12.0 10

90* 16.2 10

96** 17.0 10

112** 20.5 10

117** 22.8 10

135** 26.9 10

153** 28., 10

161 32.3 10

178** 32.0 10

400 µg/L / male

Mean SD N

23** 4.5 20

33* 7.4 20

55** 8.6 20

62** 11.5 20

83** 13.3 20

90** 15.0 20

105** 16.8 20

113** 18.4 20

132** 21.3 20

143** 23.1 20

157** 25.5 20

168** 28.3 20

  

Table 2. (continued) Body weight - group mean values (g)

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-64

1-71

1-78

1-85

1-91

1-R1

1-R8

1-R15

1-R22

1-R29

1-R36

Statistics test

Wi

Wi

Wi

Wi

Wi

Tt

Tt

Tt

Tt

Tt

Tt

Control / M

Mean SD N

220 34.0 20

233 33.9 20

246 36.5 20

255 39.4 20

264 41.6 20

261 40.1 10

301 42.7 10

322 44.0 10

346 47.7 10

364 49.3 10

379 51.3 10

40 µg/L / M

Mean SD N

171** 26.5 10

187** 26.1 10

193** 26.8 10

201** 26.9 10

207** 28.7 10

100 µg/L / M

Mean SD N

190** 33.2 10

202** 34.4 10

211** 36.4 10

219** 41.3 10

226** 43.0 10

400 µg/L / M

Mean SD N

180** 27.4 20

190** 29.8 20

197** 32.1 20

207** 32.7 20

219** 33.5 20

205** 31.9 10

244** 34.5 10

268** 33.4 10

297** 33.5 10

308** 36.3 10

322** 22.8 10

Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01

Table 3. Body weight - group mean values (g)

Group/Sex		Change	Change	Change	Change	Change	Change	Change	Change	Change	Change	Change	Change
		1-5	1-8	1-12	1-15	1-19	1-22	1-26	1-29	1-36	1-43	1-50	1-57
Statistics test		Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi
Control / female	Mean SD N	11 4.1 20	12 4.3 20	20 4.9 20	22 7.4 20	31 8.5 20	33 7.3 20	40 8.3 20	45 5.9 20	51 10.6 20	56 15.5 20	60 10.5 20	67 13.5 20
40 µg/L / female	Mean SD N	11 3.0 10	16* 4.6 10	26 4.2 10	26 5.3 10	33 7.3 10	36 3.5 10	43 4.8 10	48 4.8 10	56 6.6 10	61 7.9 10	66 6.7 10	72 7.9 10
100 µg/L / female	Mean SD N	13 3.6 10	18* 7.7 10	23 6.4 10	27 3.8 10	35 6.4 10	35 5.8 10	40 4.5 10	41 6.9 10	46 6.8 10	57 9.0 10	60 8.3 10	62 11.3 10
400 µg/L / female	Mean SD N	9 3.7 20	15* 5.0 20	23 7.7 20	24 7.6 20	33 8.7 20	33 8.5 20	39 10.3 20	42 9.8 20	48 11.7 20	55 13.7 20	56 14.8 20	60 15.8 20

 

Table 3. (continued) Body weight - group mean values (g)

Day

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Change

Group /Sex

1-64

1-71

1-78

1-85

1-91

1-R1

1-R8

1-R15

1-R22

1-R29

1-R36

Statistics test

Wi

Wi

Wi

Wi

Wi

Tt

Tt

Tt

Tt

Tt

Tt

Control / female

Mean SD N

70 14.9 20

76 14.5 20

80 17.7 20

87 18.4 20

90 18.9 20

85 12.2 10

108 13.5 10

116 14.8 10

128 16.3 10

138 19.8 10

145 25.0 10

40 µg/L / female

Mean SD N

75 9.5 10

80 12.6 10

85 13.9 10

88 12.3 10

90 12.8 10

100 µg/L / female

Mean SD N

70 12.4 10

71 10.4 10

75 13.7 10

77 14.2 10

84 14.0 10

400 µg/L / female

Mean SD N

65 15.7 20

69 15.7 20

71 16.6 20

77 17.9 20

78* 18.8 20

65* 19.4 10

87** 18.8 10

96* 18.1 10

108* 20.5 10

116* 24.4 10

118* 24.4 10

 Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01; *: p < 0.05

Table 4. Food consumption – group mean value (g/animal/day)

		Day
Group/Sex		Ra8-Ra14	1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-91	R1-R8	R8-R15	R15-R22	R22-R29	R29-R36	R36-R41
Statisticstest		Av	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Wi	Tt	Tt	Tt	Tt	Tt	Tt
Control / male	Mean	27	26	26	26	27	27	27	27	27	27	26	26	26	26	30	32	32	32	33	33
	SD	1.5	1.3	1.5	1.7	2.0	1.6	2.2	2.2	2.3	2.6	1.9	2.3	2.4	2.4	2.6	2.5	2.9	2.5	3.1	2.7
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5
40 µg/L / male	Mean	25	24	24	24	25	25	25	24**	24*	24	25	23*	24	24
	SD	1.2	1.4	1.4	1.6	1.5	1.8	1.0	1.3	0.7	0.7	0.9	0.5	0.5	0.7
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
100 µg/L / male	Mean	27	26	25	25	26	26	26	25**	25*	25	26	25*	24	25
	SD	1.8	1.9	1.9	1.7	2.2	2.3	1.6	2.1	1.8	1.6	1.5	1.9	2.7	2.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
400 µg/L / male	Mean	27	24*	24*	25	26	25*	25*	24**	24**	25*	25*	24**	24*	25	27	29	28	29	31	31
	SD	1.4	1.4	1.6	1.5	1.7	1.4	1.8	1.4	1.6	1.8	1.7	1.7	1.8	1.8	1.7	1.6	1.9	1.5	1.9	1.4
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5

Table 4 (continued). Food consumption – group mean value (g/animal/day)

		Day
Group/Sex		Ra8-Ra14	1-8	8-15	15-22	22-29	29-36	36-43	43-50	50-57	57-64	64-71	71-78	78-85	85-91	R1-R8	R8-R15	R15-R22	R22-R29	R29-R36	R36-R41
Statisticstest		AV	Wi	Wi	Wi	Wi	Wi	Wi	Sh	Sh	Sh	Wi	Wi	Wi	Wi	Tt	Tt	Tt	Tt	Tt	Tt
Control / female	Mean	17	17	17	17	18	18	18	18	17	17	17	17	26	26	30	32	32	32	33	33
	SD	0.6	0.6	0.5	1.0	1.2	0.9	1.3	0.6	1.0	1.0	1.2	1.7	2.4	2.4	2.6	2.5	2.9	2.5	3.1	2.7
	N	10	10	10	10	9	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5
40 µg/L / female	Mean	17	18	17	18	18	19	19	18	18	19	18	17	24	24
	SD	1.0	1.0	1.0	0.6	0.7	0.7	0.7	1.2	0.3	1.1	1.0	0.8	0.5	0.7
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
100 µg/L / female	Mean	16	18	16	16	16*	17	17	17	16	17	16	16	24	25
	SD	1.1	1.7	1.4	0.9	0.7	0.5	1.0	0.6	0.9	0.9	1.0	1.2	2.7	2.2
	N	5	5	5	5	5	5	5	5	5	5	5	5	5	5
400 µg/L / female	Mean	17	17	16	17	17*	17*	17	17*	17	17	17	17	24*	25	27	29	28	29	31	31
	SD	0.8	0.7	0.8	0.8	0.8	0.9	1.2	3.4	3.1	3.2	2.1	2.8	1.8	1.8	1.7	1.6	1.9	1.5	1.9	1.4
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	5	5	5	5	5	5

Av: Pre-treatment comparison of all groups using Analysis of variance followed by pairwise t-test where appropriate 

Wi: treated groups compared with Control using Williams' test

Tt: Groups compared using t-test

**: p < 0.01; *: p < 0.05

Table 5. Test item-related hematology changes in rats exposed for 13 weeks

Sex	M	M	M	F	F	F
Exposure (µg/L)	40	100	400	40	100	400
HGB	-	-	1.07**	-	-	1.08**
HCT	-	-	1.06*	-	-	1.07**
RBC	-	-	1.05**	-	-	1.08**
WBC	-	-	-	1.60*	1.29*	1.47*
Neutrophils	-	1.43*	1.40*	2.65**	2.79**	3.47**
HGB: Hemoglobin; HCT: Hematocrit; RBC: Red Blood Cells; WBC: Total White Blood Cells Changes expressed as fold change versus concurrent control. * = Statistically significant at p < 0.05 and ** = Statistically significant at p < 0.01 - : no test article-related change

Table 5a. Haematology - group mean values - main group

Group /Sex		HGB	HCT	RBC	RETIC	PLT	MCV	MCH	MCHC	RDW	WBC	ANEU	ALYM	AMONO	AEOS	ABASO	ALUC
		g/dL	%	x10^6/µL	x10^9/L	x10^3/µL	fL	pg	g/dL	%	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL
Control / male	Mean	15.0	45.1	8.52	194.5	963	52.9	17.6	33.2	13.2	10.49	1.87	7.93	0.41	0.13	0.03	0.12
	SD	0.78	2.34	0.267	23.41	75.7	1.77	0.62	0.34	1.20	3.070	0.806	2.280	0.104	0.037	0.016	0.074
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
40 µg/L / male	Mean	15.5	46.7	8.96	168.2	910	52.2	17.4	33.2	12.9	9.83	1.76	7.45	0.34	0.14	0.03	0.11
	SD	0.49	1.25	0.356	26.42	53.0	1.64	0.60	0.43	0.62	1.316	0.456	1.076	0.093	0.039	0.009	0.063
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 µg/L / male	Mean	15.2	46.1	8.68	208.4	970	53.1	17.5	33.0	13.3	12.34	2.68*	9.01	0.41	0.12	0.04	0.09
	SD	0.88	2.30	0.410	130.83	149.2	1.34	0.56	0.75	1.97	2.150	0.837	1.733	0.127	0.043	0.011	0.044
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / male	Mean	16.1**	47.6*	8.97**	187.9	912	53.1	17.9	33.8	13.5	12.07	2.62*	8.70	0.44	0.16	0.04	0.12
	SD	0.54	2.05	0.372	40.74	97.6	1.59	0.77	1.15	1.38	2.515	0.570	1.998	0.085	0.057	0.016	0.062
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Control / female	Mean	14.4	42.5	7.85	163.0	968	54.2	18.4	33.8	11.6	6.78	0.66	5.72	0.23	0.08	0.01	0.08
	SD	0.47	1.44	0.219	31.71	122.0	1.32	0.49	0.36	0.41	1.795	0.280	1.571	0.129	0.047	0.008	0.050
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
40 µg/L / female	Mean	14.7	43.5	7.95	134.0	923	54.7	18.5	33.9	11.6	10.87*	1.75**	8.45	0.39	0.13	0.03	0.12
	SD	0.74	1.99	0.335	22.60	115.8	1.61	0.73	0.49	0.52	4.478	0.594	3.881	0.135	0.065	0.025	0.047
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
100 µg/L / female	Mean	14.6	43.2	8.03	166.1	975	53.8	18.2	33.9	11.7	8.74*	1.84**	6.39	0.30	0.10	0.02	0.10
	SD	0.44	1.40	0.247	24.48	137.5	1.48	0.51	0.58	0.63	2.047	0.529	1.565	0.079	0.041	0.008	0.062
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / female	Mean	15.5**	45.5**	8.45**	135.9*	956	53.8	18.3	34.0	11.4	9.94*	2.29**	7.10	0.30	0.15**	0.02	0.09
	SD	0.40	1.29	0.304	29.55	110.6	0.93	0.43	0.41	0.25	2.955	0.603	2.361	0.123	0.063	0.009	0.070
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Table 5b. Haematology - group mean vlaues - recovery group

Group /Sex		HGB	HCT	RBC	RETIC	PLT	MCV	MCH	MCHC	RDW	WBC	ANEU	ALYM	AMONO	AEOS	ABASO	ALUC
		g/dL	%	x10^6/µL	x10^9/L	x10^3/µL	fL	pg	g/dL	%	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL	x10^3/µL
Control / male	Mean	15.2	48.2	8.85	195.9	738	54.5	17.2	31.5	14.0	9.36	1.76	6.98	0.38	0.15	0.05	0.06
	SD	0.42	1.51	0.350	24.29	132.7	1.20	0.46	0.31	1.01	1.011	0.662	1.061	0.060	0.029	0.012	0.025
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / male	Mean	15.3	48.2	8.99	186.7	703	53.8	17.0	31.6	13.8	11.56*	2.62*	8.24	0.41	0.14	0.06	0.09*
	SD	0.98	2.59	0.515	28.31	155.9	3.56	1.44	0.72	1.34	2.692	0.859	1.878	0.087	0.030	0.033	0.044
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
Control / female	Mean	14.6	44.8	8.04	138.8	774	55.8	18.2	32.6	11.9	6.48	0.82	5.29	0.22	0.09	0.03	0.05
	SD	0.71	2.27	0.462	29.87	118.7	1.71	0.74	0.40	0.41	1.573	0.251	1.364	0.069	0.032	0.012	0.022
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10
400 µg/L / female	Mean	15.2	47.2*	8.32	143.2	743	56.8	18.3	32.2	12.0	7.54	2.05**	5.06	0.27	0.10	0.02	0.04
	SD	0.63	2.15	0.442	31.88	88.4	1.59	0.57	0.49	0.60	1.574	0.194	1.410	0.106	0.023	0.009	0.016
	N	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10	10

Table 6. Test item-related bronchoalveolar fluid changes in rats exposed for 13 weeks

Sex	M	M	M	F	F	F
Exposure (µg/L)	40	100	400	40	100	400
TWBC	6.50a	3.87**	9.85**	4.03*	11.52**	21.48**
Total Protein	3.40**	5.10**	11.40**	4.13**	9.00**	17.38**
LDH	3.10*	2.79*	19.13**	1.89	4.17	20.37**
ALKP	-	-	2.47**	-	-	2.85**
TWBC: Total White Blood Cells; LDH: Lactate Dehydrogenase; ALKP: Alkaline Phosphatase Changes expressed as fold change versus concurrent control. * = Statistically significant at p < 0.05 and ** = Statistically significant at p < 0.01 a Animal No. 2042 only and calculated as individual animal value versus concurrent control mean. - : no test article-related change

Table 7: Test item-related bronchoalveolar fluid changes in rats at the end of a 6-week recovery period

Sex	M	F
Exposure (µg/L)	400	400
TWBC	1.95	3.37**
Total Protein	9.40**	13.57**
LDH	8.93	2.41
ALKP	4.09	2.73
TWBC: Total White Blood Cells; LDH: Lactate Dehydrogenase; ALKP: Alkaline Phosphatase Changes expressed as fold change versus concurrent control. ** = Statistically significant at p < 0.01

Table 8. Test item-related organ weight changes (% difference relative to controls) in rats exposed to organolignite for 13 weeks

Group/sex	2M	3M	4M	2F	3F	4F
Exposure (µg/L)	40	100	400	40	100	400
Lungs and Bronchi
Absolute weight (%)	30a	74a	182a	51a	113a	209a
vs. body weight (%)	47a	84a	202a	46a	116a	210a
vs. brain weight (%)	36a	80a	187a	48a	117a	209a

a: Statistically significant difference between mean values for test item-treated and control group
M = Male F = Female

Table 9. Test item-related organ weight changes (% difference relative to controls) in rats at the end of 6 -week recovery period

Group/sex	4M	4F
Exposure (µg/L)	400	400
Lungs and Bronchi
Absolute weight (%)	186a	187a
vs. body weight (%)	211a	225a
vs. brain weight (%)	185a	189a

a: Statistically significant difference between mean values for test item-treated and control group
M = Male F = Female

Table 10. Test item-related macroscopic findings in the lungs in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	9	10	10	0	9	10	10
Distended	0	1	3	4	0	1	1	2
Pale area(s)	0	2	5	4	0	2	7	3
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

 

Table 11. Test item-related macroscopic findings in the tracheobronchial lymph nodes in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	8	9	9	0	9	8	10
Enlarged	0	8	9	9	0	8	9	10
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 12. Test item-related macroscopic findings in the thymic lymph nodes in rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Abnormal Colour (black)	0	6	7	8	0	8	7	8
Enlarged	0	5	6	7	0	7	6	8
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 13. Test item-related macroscopic findings in the lungs in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	10	0	10
Pale area(s)	0	9	0	6
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 14. Test item-related macroscopic findings in the tracheobronchial lymph nodes in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	8	0	10
Enlarged	0	7	0	7
Number of tissues examined	10	10	10	10

M = Male F = Female

Table 15. Test item-related macroscopic findings in the thymic lymph nodes in rats exposed to organolignite for 13 weeks followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Abnormal Colour (black)	0	9	0	6
Enlarged	0	9	0	5
Number of tissues examined	10	10	10	10

M = Male F = Female

Table 16. Test item-related histopathological findings in the lungs and bronchi of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Alveolar Macrophages
Moderate	0	7	4	0	0	3	3	0
Marked	0	3	4	2	0	7	4	0
Severe	0	0	2	8	0	0	3	10
Total	0	10	10	10	0	10	10	10
Bronchioloalveolar Inflammation
Minimal	0	3	0	1	1	1	1	0
Slight	0	5	5	1	0	6	2	1
Moderate	0	2	4	7	0	2	7	6
Marked	0	0	1	1	0	1	0	3
Total	0	10	10	10	1	10	10	10
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 17: Test item-related histopthological findings in the tracheobronchial lymph nodes of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Increased Paracortical Cellularity
Minimal	0	2	3	2	0	2	5	2
Slight	0	4	4	3	0	4	2	2
Moderate	0	2	3	4	0	2	3	4
Marked	0	0	0	1	0	0	0	2
Total	0	8	10	10	0	8	10	10
Pigmented Macrophages
Minimal	0	4	3	0	0	6	4	4
Slight	0	5	6	6	0	4	5	4
Moderate	0	1	1	3	0	0	1	2
Total	0	10	10	9	0	10	10	10
Number of tissues examined	8	10	10	10	8	10	10	10

M = Male F = Female

Table 18. Test item-related histopathological findings in the thymic lymph nodes of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Increased Paracortical Cellularity
Minimal	0	3	2	1	0	3	2	0
Slight	0	1	3	1	0	3	2	4
Moderate	0	2	2	4	0	2	3	3
Marked	0	0	0	2	0	0	0	1
Total	0	6	7	8	0	8	7	8
Pigmented Macrophages
Minimal	0	0	0	0	0	3	2	0
Slight	0	6	4	2	0	4	3	3
Moderate	0	0	3	6	0	1	2	5
Total	0	6	7	8	0	8	7	8
Number of tissues examined	0	6	7	8	0	8	7	8

M = Male F = Female

 

Table 19. Test item-related histopathological

findings in the nose/turbinates of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Olfactory Epithelial Degeneration
Minimal	0	0	4	0	0	1	4	0
Slight	0	0	2	3	0	0	3	3
Moderate	0	0	0	6	0	0	0	6
Marked	0	0	0	0	0	0	0	1
Total	0	0	6	9	0	1	7	10
Respiratory Epithelial Degeneration
Minimal	0	0	1	3	0	0	0	1
Slight	0	0	0	2	0	0	0	2
Total	0	0	1	5	0	0	0	3
Respiratory Epithelial Inflammation
Minimal	0	0	1	2	0	0	0	0
Total	0	0	1	2	0	0	0	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	0	0	1	0	0	0	1
Slight	0	0	0	0	0	0	0	1
Total	0	0	0	1	0	0	0	2
Pigment/Pigmented Macrophages
Minimal	0	0	1	3	0	0	1	3
Slight	0	0	0	2	0	0	0	7
Moderate	0	0	0	4	0	0	0	0
Total	0	0	1	9	0	0	1	10
Number of tissues examined	10	10	10	9	10	10	10	10

M = Male F = Female

 

Table 20. Test item-related histopathological findings in the larynx of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Epithelial Alteration
Minimal	0	0	0	1	1	1	0	0
Slight	0	2	3	0	0	4	0	0
Total	0	2	3	1	1	5	0	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	6	6	5	0	4	6	4
Slight	0	1	1	4	0	0	4	6
Total	0	7	7	9	0	4	10	10
Pigment/Pigmented Macrophages
Minimal	0	1	1	3	0	1	6	6
Slight	0	0	0	1	0	0	0	0
Total	0	1	1	4	0	1	6	6
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 21. Test item-related histopathological findings in the tracheal bifurcation of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Macrophages
Minimal	0	5	5	6	0	3	5	9
Total	0	5	5	6	0	3	5	9
Number of tissues examined	8	7	9	8	8	8	9	10

M = Male F = Female

 

Table 22. Test item-related histopathological findings in the trachea of rats exposed to organolignite for 13 weeks

Group/sex	1M	2M	3M	4M	1F	2F	3F	4F
Exposure (µg/L)	0	40	100	400	0	40	100	400
Pigment/Pigmented Macrophages
Minimal	0	2	2	5	0	3	3	5
Total	0	2	2	5	0	3	3	5
Number of tissues examined	10	10	10	10	10	10	10	10

M = Male F = Female

Table 23. Test item-related histpathological findings in the lungs and bronchi of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Alveolar Macrophages
Marked	0	2	0	1
Severe	0	8	0	9
Total	0	10	0	10
Bronchioloalveolar Inflammation
Minimal	0	0	1	0
Moderate	0	5	0	3
Marked	0	5	0	7
Total	0	10	1	10
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 24. Test item-related histopathological findings in the tracheobronchial lymph nodes of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Increased Paracortical Cellularity
Minimal	0	6	0	5
Slight	0	2	0	1
Moderate	0	1	0	1
Marked	0	1	0	2
Total	0	10	0	9
Pigmented Macrophages
Minimal	0	1	0	0
Slight	0	6	0	7
Moderate	0	3	0	3
Total	0	10	0	10
Number of tissues examined	7	10	9	10

M = Male F = Female

 

Table 25. Test item-related histopathological findings in the thymic lymph nodes of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Increased Paracortical Cellularity
Minimal	0	1	0	2
Slight	0	2	0	2
Moderate	0	6	0	2
Marked	0	0	0	0
Total	0	9	0	6
Pigmented Macrophages
Slight	0	4	0	1
Moderate	0	4	0	4
Marked	0	1	0	1
Total	0	9	0	6
Number of tissues examined	0	9	0	6

M = Male F = Female

 

Table 26. Test item-related histopathological findings in the nose/turbinates of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Olfactory Epithelial Degeneration
Minimal	0	1	1	1
Slight	0	3	0	5
Moderate	0	5	0	3
Marked	0	0	0	1
Total	0	9	1	10
Olfactory Epithelial Inflammation
Slight	0	1	0	1
Total	0	1	0	1
Respiratory Epithelial Degeneration
Minimal	0	1	0	1
Total	0	1	0	1
Respiratory Epithelial Inflammation
Minimal	0	1	0	0
Total	0	1	0	0
Pigment/Pigmented Macrophages
Minimal	0	4	0	4
Slight	0	3	0	5
Moderate	0	2	0	0
Total	0	9	0	9
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 27. Test item-related histopathological findings in the larynx of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Epithelial Alteration
Minimal	0	1	1	0
Slight	0	2	3	0
Total	0	3	4	0
Squamous Metaplasia of Respiratory Epithelium
Minimal	0	0	0	2
Total	0	0	0	2
Pigment/Pigmented Macrophages
Minimal	0	0	0	3
Total	0	0	0	3
Number of tissues examined	10	10	10	10

M = Male F = Female

 

Table 28. Test item-related histopathological findings in the tracheal bifurcation of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Macrophages
Minimal	0	8	0	10
Total	0	8	0	10
Number of tissues examined	8	9	9	10

M = Male F = Female

Table 29. Test item-related histopathological findings in the trachea of rats exposed to organolignite for 13 weeks and followed by a 6-week recovery period

Group/sex	1M	4M	1F	4F
Exposure (µg/L)	0	400	0	400
Pigment/Pigmented Macrophages
Minimal	0	7	0	8
Total	0	7	0	8
Number of tissues examined	10	10	10	10

M = Male F = Female

Conclusions:

In a subchronic repeated dose toxicity study (OECD 413) via inhalation a lung overload was observed resulting in an inflammatory response on the respiratory tract and correlated changes in hematology parameters. As these effects are considered to be local and the systemic effects are secondary effects a local NOAEC could not be established. A local LOAEC can be set to 40 µg/L. As systemic effects are considered secondary the systemic NOAEC is set to 400 mg/L.

Executive summary:

A subchronic repeated dose toxicity study according to OECD 413 and compliant to GLP was performed. Sprague-Dawley CD®rats (10 animals/sex/group) were exposed to target exposure levels of 0 (air only), 40, 100 or 400 µg/L test substance via nose only inhalation for 6 hours/day, 5 days/week for 13 consecutive weeks. Ten additional rats/sex/group in the control groups and 400 µg/L group were similarly exposed for 13 consecutive weeks and retained for a 6-week recovery period. At the end of the treatment and recovery phases, designated animals were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weights, food consumption, clinical pathology (termination and recovery), organ weights, bronchoalveolar lavage (BAL) analysis, macroscopic observations and microscopic pathology.

Overall mean achieved concentration for 40 µg/L group was on target. 100 µg/L group mean concentration was 5% above target and 40 µg/L group was 1% below target. The MMAD values for all samples collected showed that the delivered particle size was respirable to the rats. This study resulted in no test item-related effects on mortality, clinical observations, ophthalmology, coagulation and clinical chemistry. Decreased body weight gains and food consumption were seen at all exposure levels in the males and at the highest exposure levels in the females compared with control. These changes were test item-related but not considered adverse because the body weight gain and food consumption in males showed no dose-dependent pattern. In females the body weight gain and the food consumption was only occasionally observed. Recovery in these parameters were seen at the end of the recovery period.

Test item-related haematology changes at all exposure levels in females included increases in neutrophils with corresponding increases in total white blood cells. In males a neutrophil increase was only observed at 100 and 400 µg/L and without corresponding increase in total white blood cell count. In the recovery group the increase of neutrophils was still observed. These changes correlated with microscopic findings of inflammation in the respiratory tract that were considered adverse at ≥ 40 µg/L. At 400 µg/L, there were increases in red cell mass (haemoglobin, haematocrit, and red blood cells) in both sexes that were considered test item-related. In the recovery group no increase in red cell mass was observed. Inhalation exposures of 40, 100 or 400 µg/L organolignite in rats for 13 weeks resulted in test item-related findings in the lungs and bronchi, tracheobronchial and thymic lymph nodes, nose/turbinates, larynx, tracheal bifurcation, and/or trachea at ≥ 40 µg/L. Moderate pigment/pigmented macrophages and slight mixed inflammation in the lung at ≥ 40 µg/L was considered adverse since these findings could compromise pulmonary function and disrupt the ability to maintain respiratory homeostasis. Other adverse findings included slight olfactory and respiratory epithelial degeneration and accompanying inflammation in the nose. Remaining organolignite-related findings were considered non-adverse. Non-adversity was based on low severity grades, absence of morphologic damage, the expectation that these findings would not be harmful to the animals affected, and/or the presence of such findings at comparable severity in healthy control animals. Following a 6-week exposure-free recovery period, most test item-related findings were still observed at the same or lower magnitude as in the end of the treatment period in males and females exposed to organolignite at 400 µg/L. In the lungs and bronchi, pigment/pigmented macrophages and inflammation was still observed in the recover group. The incidence of markedly inflamed lungs and bronchi in males and females was increased in the recovery group compared to the main group suggesting that inflammation worsened in some animals during the 6-week recovery phase. In the tracheobronchial and thymic lymph nodes, pigmented macrophages were also still observed in the recovery group. No recovery in the nose, olfactory epithelial degeneration and pigmented/pigmented macrophages was observed in the recovery males and females, compared with the main group. Partial recovery in respiratory epithelial degeneration and inflammation was observed, while complete recovery in squamous metaplasia of respiratory epithelium in the nose was observed in males/females of the recovery group compared with the main group. In the larynx, epithelial alteration was observed to be normal in the recovery group in contrast with the main group, as did squamous metaplasia and pigment/pigmented macrophages in male rats. In females of the recovery group, squamous metaplasia and pigment/pigmented macrophages were observed with lower incidences compared to the main group. No recovery was observed in the tracheal bifurcation and trachea, pigment/pigmented macrophages. Lack of recovery in the aforementioned respiratory tissues was based on comparable incidence and severity of organolignite-related findings in 400 µg/L main group animals and recovery animals. Organolignite-related bronchoalveolar fluid changes at all exposures included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. Also, at all exposures there were increases in total protein and lactate dehydrogenase activities. At 400 µg/L, there were increases in alkaline phosphatase activities. Test item-related bronchoalveolar fluid changes following a 6-week recovery period in 400 µg/L rats included increases in total white blood cells with increases in neutrophil percentages and decreases in macrophage percentages. There were also increases in total protein, lactate dehydrogenase, and alkaline phosphatase activities. These bronchoalveolar lavage fluid results correlated with inflammatory changes in the lungs and bronchi observed in 400 µg/L recovery rats. Overall, the changes observed are consistent with a lung overload response, i.e., an inflammatory response in response to poorly soluble particles (PSPs). While lung overload and subsequent disease are a common phenomenon in the rat, the relevance of this finding to human disease risk is less clear (CLP Annex I, 3.9.2.8.1 e). In conclusion, organolignite exposures from 40 to 400 µg/L resulted in an inflammatory response on the respiratory tract and correlating changes in haematology parameters when rats were exposed for up to 13 consecutive weeks. Thus, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects was not established. The NOAEC for systemic effects was set to 400 µg/L because systemic effects were only observed as secondary effect of the lung overload.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

40 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

The quality of the whole database is high and meets the tonnage driven data requirements of REACH.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Data from a 13 -week repeated dose inhalation toxicity study indicate that organolignite results in a lung overload, i.e. an inflammatory response with correlating changes in haematology parameters and histopathology findings. These effects (with this severity) were not observed in a combined toxicity study with a reproduction/developmental toxicity screening test (OECD 422) or a 14 -day dose-range finding study. The results from the studies listed above are presented below in detail.

 

Subchronic repeated dose inhalation toxicity study (OECD 413)

A subchronic repeated dose inhalation toxicity study according to OECD 413 and compliant to GLP was performed. Sprague-Dawley CD®rats (10 animals/sex/group) were exposed to target exposure levels of 0 (air only), 40, 100 and 400 µg/L test substance via nose only inhalation for 6 h/day, 5 days/week, for 13 consecutive weeks. Ten additional rats/sex/group in the control group and 400 µg/L group were similarly exposed for 13 consecutive weeks and retained for a 6-week recovery period. At the end of the treatment and recovery phases, designated animals were euthanized and necropsied. Parameters evaluated during the study were: viability, clinical observations, ophthalmology, body weight, food consumption, clinical pathology (termination and recovery), organ weights, bronchoalveolar lavage (BAL) analysis, macroscopic assessment and microscopic pathology.

The overall mean achieved concentration for the 40 µg/L group was on target. The 100 µg/L group mean concentration was 5% above target and the 400 µg/L group was 1% below target. The MMAD values for all samples collected showed that the delivered particle size was respirable to the rats. This study resulted in no test item-related effects on mortality, clinical observations, ophthalmology, coagulation parameters and clinical chemistry parameters. Decreased body weight gains and food consumption were seen at all exposure levels in the males and at the highest exposure levels in the females compared with control. These changes were test item-related but not considered toxicologically relevant because the body weight gain and food consumption in males showed no dose-dependent pattern. At the end of the recovery period the male animals treated with 400 µg/kg bw/day had still a reduced body weight gain (- 15.0%) compared with the controls. This effect is not considered to be toxicologically relevant as the difference was minimal and the animals gained weight throughout the study, although slightly more slowly than the control. Decreased body weight gains over the whole treatment period (Day 1 to Day 91) were seen only in females exposed to 400 µg/L (-13.3%). Comparing the body weights of 400 µg/L females on Day 91 with the controls, a non-significant decrease of 4.8% was observed. Therefore, these changes were considered to be test item-related but not toxicologically relevant. A recovery in body weight gain was seen in the females during the recovery period. The neutrophil level was 1.43- and 1.4-fold increased (statistically significant) in males in dose group 100 and 400 µg/L, respectively, but without a corresponding increase of white blood cells. In the 400 µg/L recovery group the white blood cell level was significantly increased (1.24-fold), as was the neutrophil level (1.49-fold). These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.07-, 1.06- and 1.05-fold, which are considered test substance-related.

In the females, the neutrophils were 2.65-, 2.79- and 3.47-fold increased (statistically significant) in dose group 40, 100 and 400 µg/L, respectively. These changes corresponded to a significant increase of white blood cells (1.6-, 1.29- and 1.47-fold). In the recovery group the white blood cell level was comparable to that of the control, while the neutrophil level in the 400 µg/L dose group was still significantly increased 2.5-fold compared to the controls. These changes are considered treatment-related and adverse because they correlated with the microscopic findings of inflammation in the respiratory tract that were considered adverse. Furthermore, in the 400 µg/L dose group haemoglobin, haematocrit, and red blood cells were significantly increased by 1.08-, 1.07- and 1.08-fold, which was considered test substance-related. In the recovery group, no increase in red cell mass was observed.

Statistically significant higher absolute and relative lung and bronchi weights were present in males and females exposed to ≥40 µg/L compared to controls. Higher lung weights were related to increased pigment/pigmented alveolar macrophages and inflammation observed microscopically. Higher lung weights did not recover and remained increased in a similar magnitude compared to the main group in males and females exposed to 400 µg/L following a 6-week exposure-free recovery period. Inhalation exposures of 40, 100 or 400 µg/L organolignite in rats for 13 weeks resulted in test item-related histopathological findings in the lungs and bronchi, tracheobronchial and thymic lymph nodes, nose/turbinates, larynx, tracheal bifurcation, and/or trachea at ≥ 40 µg/L. the presence of moderate pigment/pigmented macrophages and slight mixed inflammation in the lung at ≥ 40 µg/L was considered adverse, since these findings could compromise pulmonary function and disrupt the ability to maintain respiratory homeostasis. Other adverse findings included slight olfactory and respiratory epithelial degeneration and accompanying inflammation in the nose. Remaining organolignite-related findings were considered non-adverse. This conclusion was based on low severity grades, absence of morphologic damage, the expectation that these findings would not be harmful to the animals affected, and/or the presence of such findings at comparable severity in healthy control animals.

Following a 6-week recovery period, most test item-related findings were still observed at the same or with reduced severity, compared with the end of the treatment period in males and females exposed to organolignite at 400 µg/L. In the lungs and bronchi, pigment/pigmented macrophages and inflammation was still observed in the recovery group. The incidence of markedly inflamed lungs and bronchi in males and females was increased in the recovery group compared to the main group, suggesting that inflammation worsened in some animals during the 6-week recovery phase. In the tracheobronchial and thymic lymph nodes, pigmented macrophages were still observed in the recovery group. No recovery in the nose, registered as olfactory epithelial degeneration and pigmented/pigmented macrophages, was observed in the recovery males and females, compared with the main group. Partial recovery in respiratory epithelial degeneration and inflammation was observed, while complete recovery in squamous metaplasia of respiratory epithelium in the nose was observed in males/females of the recovery group compared with the main group. In the larynx, the epithelial alteration observed in the main group had reversed and normalised in the recovery group, as had the squamous metaplasia and pigment/pigmented macrophages observed in male rats in the main group. In females of the recovery group, squamous metaplasia and pigment/pigmented macrophages were observed with lower incidences compared to the main group. No recovery was observed in the tracheal bifurcation and trachea, where pigment/pigmented macrophages were observed in the main group as well as the recovery group for males and females. Lack of recovery in the aforementioned respiratory tissues was based on comparable incidence and severity of organolignite-related findings in 400 µg/L main group animals and recovery animals.

Organolignite-related bronchoalveolar fluid changes at all exposure levels in males and females included increases in total white blood cells (significant at all dose levels with the exception of males administered 40 µg/L) with significant increases in neutrophil percentages and significant decreases in macrophage percentages. Also, at all exposure levels there were increases in total protein and lactate dehydrogenase activities. At 400 µg/L, there were significant increases in alkaline phosphatase activities. Test item-related bronchoalveolar fluid changes following a 6-week recovery period in 400 µg/L rats included increases in total white blood cells (significant in females) with significant increases in neutrophil percentages and significant decreases in macrophage percentages. There were also increases in total protein, lactate dehydrogenase, and alkaline phosphatase activities. These bronchoalveolar lavage fluid results correlated with inflammatory changes in the lungs and bronchi observed in 400 µg/L recovery rats.

Overall, the changes observed are consistent with a lung overload response, i.e., an inflammatory response in response to poorly soluble particles (PSPs). While lung overload and subsequent disease are a well-documented phenomenon in the rat, the relevance of this finding to human disease risk is less clear (CLP Annex I, 3.9.2.8.1 e). Furthermore, in the ECETOC Technical report No. 122 it is concluded that the rat represents a particularly sensitive model concerning the development of pulmonary non-neoplastic lesions and, a unique animal model with regard to lung neoplastic responses under conditions of lung overload. Humans are less sensitive to lung overload as epidemiological studies thus far have not been able to detect an association between occupational exposures to poorly soluble particles of low toxicity and an increased risk of lung cancer. Furthermore, no effects were seen on liver or kidney, which are typical sites of metabolism, indicating that systemic effects observed in this study are secondary to the local effects in the lung.

In conclusion, organolignite exposures from 40 to 400 µg/L resulted in an inflammatory response on the respiratory tract and correlating changes in haematology and histology parameters when rats were exposed for up to 13 consecutive weeks via inhalation. Thus, the No-Observed-Adverse-Effect-Concentration (NOAEC) for local effects was not established. The NOAEC for systemic effects was set to 400 µg/L because systemic effects were only observed as secondary effect of the lung overload.  

 

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422)

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422) via inhalation exposure, organolignite was administered to 10 Sprague-Dawley rats/sex/dose group by nose-only exposure at concentrations of 0, 5, 50, or 300 mg/m³ for 6 hours per day, 7 days/week. Males were treated daily 2 weeks prior to mating, throughout mating, and until the day prior to necropsy (ca. 4 weeks of treatment). Females were treated daily 2 weeks prior to mating, throughout mating, and up to Day 19 of gestation (ca. 6 weeks for treatment).

 

In high-dose males, there was a statistically significant increase in some white blood cell types. Statistically significant increases in select white blood cell parameters also were observed at the low dose for males; however, a similar pattern was not observed in mid-dose males. Measured haematology parameters for females for all dose levels were comparable to the control values. Therefore, these blood cell effects observed in males were not attributed to organolignite exposure. At necropsy, gross pathology results noted dark, discoloration of the lungs in mid- and high-dose males and females. At the high dose, males and females also exhibited a statistically significant increase in absolute and relative lung weight. At the mid-dose, there was a statistically significant increase in absolute lung weight for both males and females; however, only females exhibited a statistically significant increase in relative lung weight. The microscopic evaluation showed pigmented macrophages in the lung at the high dose for both sexes. At the high dose, areas of bronchiolo-alveolar hyperplasia characterized by hypertrophic alveolar Type II pneumocyte cells forming a single layer outlining alveolar walls also was observed. There was no evidence, however, of any associated tissue damage (i.e., necrosis, degeneration, or fibrosis). Additionally there was no evidence indicative of a pre-neoplastic lesion. Therefore, these effects were not considered adverse. No reproductive, developmental or pup care effects were observed in the study at any dose. Based on this study, the NOAEC is 300 mg/m³ (equivalent to 300 µg/L) because no adverse effects were observed at the highest dose administered.

 

14-day Dose Range Finding Study

A lack of systemic adverse effects after exposure via inhalation to organolignite was also observed in a 14 -day dose range finding (DRF) inhalation toxicity study, in which organolignite was administered to 5 Sprague-Dawley rats/sex/concentration by nose-only exposure at concentrations of 0, 500, 1000, or 2000 mg/m³ for 6 hours per day, 7 days/week for 2 weeks. The DRF study was conducted to help select doses for the combined inhalation toxicity study, and is provided as a supporting study in this dossier.

 

At the high-dose of 2000 mg/m³ (2.0 mg/L) intermittent wheezing and gasping were observed and were considered an adverse effect. Neutrophil values were higher for all treated groups when compared to the control values. Values reached statistical significance for all treated groups except for low-dose females, and there was a statistically significant dose-response relationship observed for males. Statistically significant increases in red blood cell count and statistically significant decreases in reticulocyte percentage occurred in high-dose males. Statistically significant increases in haematocrit values also were observed in mid- and high-dose males, as well as statistically significant increases in haemoglobin values in mid-dose males and high-dose males and females. The study authors concluded that the white blood cell and other measured cell types in the blood did not constitute an adverse effect in the context of this study. At the end of the treatment period at ≥ 500 mg/m³ microscopic findings were noted in the lung (bronchiolo-alveolar hyperplasia in both sexes, and mononuclear cell infiltration in females). However, because there was no associated tissue damage in the lungs (i.e., necrosis, degeneration, or fibrosis), the study authors concluded that these observations did not constitute an adverse effect in the context of this study. The only adverse effect observed in the study was the gasping and wheezing at the high exposure level. Thus, the no observed adverse effect level was considered to be 1000 mg/m³ (1.0 mg/L) for this study.

Justification for classification or non-classification

Key data from subchronic repeated dose inhalation toxicity studies indicate that organolignite produces a lung overload. The relevance of lung overload in animals to humans is currently not clear (CLP EU Regulation 1272/2008, Annex I, 3.9.2.8.1 (e)). Therefore, organolignite is not classified under CLP EU Regulation 1272/2008 for repeated dose toxicity.

Although there were no repeated dose toxicity studies identified for either oral or dermal exposure, acute toxicity data for both of these routes of exposure suggests that absorption is not significant for either route and that oral and dermal toxicity is not a significant cause for concern.