Registration Dossier

Administrative data

Description of key information

90-d Inhalation study (OECD413, GLP): NOAEL 7.29 mg/m3 (actual concentration)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
9 August 2012 - 16 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, the Netherlands
- Age at study initiation: 7-9 weeks
- Weight at study initiation:
Range finding study: male - 236 g, female - 163 g
Main study: male - 222 g, female - 169 g
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany), separated by sex, five rats per cage
- Diet (e.g. ad libitum): ad libitum, except during exposure and the fasting period before scheduled sacrifice
- Water (e.g. ad libitum): ad libitum, except during exposure and the fasting period before scheduled sacrifice
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 22 August 012 (arrival) To: 29 January to 1 February 2013
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: low-dose: 1.41 µm (± 0.08)
mid-dose: 1.74 µm (± 0.30)
high-dose: 1.46 µm (± 0.05)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only exposure units consisting of a cylindrical column, surrounded by a transparent cylinder
- Method of holding animals in test chamber: animals were secured in plastic animal holders
- Source and rate of air: at least 1 L/min for each rat
- Method of conditioning air: flow of humidified air driving an educator (Cheng et al., 1989) and air flow added by suction
- System of generating particulates/aerosols: test material was aerosolized using a turntable dust feeder
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 30-70% humidity, positive pressure in central column and slightly negative pressure in outer cylinder
- Air flow rate: at least 1 L/min for each rat
- Air change rate: continous supply of fresh test atmosphere
- Method of particle size determination: Cascade impactor

TEST ATMOSPHERE
- Brief description of analytical method used:gravimetric analysis

VEHICLE: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical verification for the verification of test material identity and properties was not performed in this study. The test concentrations were verified by means of gravimetric analysis.

A Certificate of Analysis on the total Crystalline Silica content of the batch used in the 90-day inhalation toxicity study is attached as a pdf file. The content of total Crystalline Silica is < 1% (w/w%), meaning that the content of respitable Crystalline Silica is also below 1% (w/w%).
Duration of treatment / exposure:
6 hours per day, 5 days per week, for a 93-day study period (total of 65 exposure days).
Frequency of treatment:
Daily, 5 days per week, 6 h per day
Remarks:
Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3.33 mg/m3
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
7.29 mg/m3
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
10.25 mg/m3
Basis:
other: actual concentration
No. of animals per sex per dose:
Range-finding study: 5 males and 5 females per dose
Main study: 10 males and 10 females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: A range finding study was performed with 0, 3, 10, and 30 mg/m3. Based on observed results the doses for the main study were chosen.
- Rationale for animal assignment (if not random): computer randomisation proportionally to body weight
Positive control:
No positive control group was included.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes,
- Time schedule: Daily
- Cage side observations included: a detailed list of clinical signs, annexed to the study report

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and halfway through the 6-hr exposure period (group-wise)

BODY WEIGHT: Yes
- Time schedule for examinations:
Range-finding study: Before start of exposure (male day -1, female day -2), prior to exposure (day 0), twice weekly thereafter (day 3, 7, 10), and prior to sacrifice (day 14)
Main study: Before start of exposure (male day -2 or -1, female day -6 or -5), prior to exposure (day 0), twice weekly during first month (day 3/4, 7, 10/11, 14, 17/18, 21, 24/25, 28), weekly thereafter (day 35, 39 (male only), 42, 49, 56, 63, 70, 77, 84, 91, 93/95)), and fasted bodyweights prior to sacrifice (day 94/96).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/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

OPHTHALMOSCOPIC EXAMINATION: Yes, main study only
- Time schedule for examinations and examined dose groups: Prior to start of treatment (all animals), towards the end of treatment (Control and high-dose group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment period
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined:
Haemoglobin concentration (Hb)
Packed Cell Volume (PCV)
Red Blood Cell count (RBC)
reticulocytes
Total White Blood Cell count (WBC)
Differential White Blood Cells
Prothrombin time
Thrombocyte count (platelet count)
- Calculated parameters:
Mean Corpuscular volume (MCV)
Mean Corpuscular Haemoglobin (MCH)
Mean Corpuscular Haemoglobin Concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment period
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined:
Alkaline phosphatase activity (ALP)
Aspartate aminotransferase activity (ASAT)
Alanine aminotransferase activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Fasting glucose
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phopholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Estrus cycle evaluation, Sperm analysis and Bronchoalveolar lavage and measurements

ESTRUS CYCLE EVALUATION
Daily in the week prior to sacrifice (including day of sacrifice) vaginal smears were made to evaluate oestrus cycle length and normality. Only smears of control and high-dose animals were evaluated.

SPERM ANALYSIS
Epididymal sperm was derived from the left cauda epididymis and motility was measured with the Hamilton Thorne Integrated Visual Optical System (IVOS), which was also used for sperm count. Furthermore, a smear of each male of the control group and high-dose group were analysed.
At necropsy, testicular sperm count was performed and daily sperm production was calculated. Evaluation of homogenization resistant spermatids was performed in the control and high-dose groups.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
At necropsy, the lungs of all animals were lavaged according to a standardized method. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (γ-GT) were determined. Furthermore, total white blood cell numbers were counted using a Coulter Counter. The number of viable cells and differential cells were determined using an acridine orange/ethidium bromide staining method in combination with fluorescent microscopic evaluation.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The following organs were weighed in the Range finding study:
Heart
Adrenals
Kidneys
Liver
Spleen
Testes
Lungs with trachea
Trachea-bronchial lymph nodes
- The following organs were weighed in the Main study:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Testes
Thymus
Thyroids
Left lung lobe
Ovaries
Uterus
Epididymus

HISTOPATHOLOGY: Yes
- Range finding study:
Left lung lobe (control and high-dose group)
Tracheobronchal lymph nodes (control and high-dose group)
- Main study:
Adrenals
Aorta
Axillary lymph nodes
Brain (brain stem, cerebrum and cerebellum)
Caecum
Colon
Epididymes
Eyes (with optic nerve)
Exorbital lachrymal glands
Femur with joint
Heart
Kidneys
Liver
Left lung lobe/trachea/larynx
Mammary glands (females)
Cervical lymph nodes
Nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth)
Nerve peripheral (sciatic nerve)
Oesophagus
Olfactory bulb
Ovaries
Pancreas
Parathyroids
Pharynx
Parotid salivary glands
Pituitary
Prostate
Rectum
Seminal vesicles with coagulation glands
Skeletal muscle (thigh)
Skin (flank)
Small intestines (duodenum, ileum, and jejunum)
Spinal cord (three levels)
Spleen
Sternum with bone marrow
Stomach (glandular, non-glandular)
Sublingual salivary glands
Submaxillary salivary glands
Testes
Thymus
Thyroid
Tongue
Tracheobronchial (mediastinal) lymph nodes
Ureter
Urethra
Urinary bladder
Uterus (with cervix)

All preserved tissues of all animals of the control and high-dose groups were examined histopathologically (by light microscopy). In addition, the lungs and the tracheobronchial lymph nodes were also examined in animals of the low- and mid-dose groups.
Other examinations:
No other examinations performed
Statistics:
- Post-treatment body weight data: AnCova and Dunnett's Test.
- Pre-treatment body weight data, haematology, clincial chemistry, broncho alveolar lavage data, and organ weight: Generalised Anova/Ancova Test and Kruskal-Wallis or Dunnett's test
- Food consumption: Sunnett's multiple comparison test
- Estrus cyclicity: Fisher's exact test or Kruskal-Wallis nonparamtric ANOVA followed by Mann-Whitney U test
- Sperm parameters: ANOVA dollowed by Dunnett's multiple comparison test or Kruskal/Wallis non parametric ANOVA followed by Mann-Whitney U test
- Incidences of histopathological changes: Fisher's exact probability test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose animals showed increased absolute weight of the left lung. High and mid-dose animals showed an increased relative weight of the left lung.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
17/20 low-dose, 18/20 mid-dose and 19/20 high-dose animals showed gray/black discoloration/patches of the lungs. 12/20 low-dose, 17/20 mid-dose and 20/20 high-dose animals showed dark/black discoloration of the tracheobronchial lymph nodes.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At microscopic observation the lungs of all animals exposed to the low-, mid- and high-dose showed accumulation of black pigment in the lungs and also in the tracheobronchial lymph nodes (except in one low-dose female).
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment related effects observed.

BODY WEIGHT AND WEIGHT GAIN
No treatment related effects observed.

FOOD CONSUMPTION
No treatment related effects observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects observed.

HAEMATOLOGY
No treatment related effects observed.

CLINICAL CHEMISTRY
No treatment related effects observed.

ORGAN WEIGHTS
High dose animals showed increased absolute weight of the left lung. High and mid-dose animals showed an increased relative weight of the left lung.

GROSS PATHOLOGY
17/20 low-dose, 18/20 mid-dose and 19/20 high-dose animals showed gray/black discoloration/patches of the lungs. 12/20 low-dose, 17/20 mid-dose and 20/20 high-dose animals showed dark/black discoloration of the tracheobronchial lymph nodes.

HISTOPATHOLOGY: NON-NEOPLASTIC
At microscopic observation the lungs of all animals exposed to the low-, mid- and high-dose showed accumulation of black pigment in the lungs (majority in alveolar macrophages) and also in the tracheobronchial lymph nodes (except in one low-dose female). Small amounts of pigment were present as free particles, mostly in the alveoli, sometimes also in the bronchiolar lumen. In the tracheobronchial lymph nodes the black pigment was present as free particles as well as within macrophages. In general, the amount of pigment in the lungs and tracheobronchial lymph nodes was in proportion to the exposure levels. One control female had an adenocarcinoma of the mammary gland, but otherwise the histopathological changes in the other organs and tissues were unremarkable. They were common findings in rats of this strain and age or occurred as individual chance findings, unrelated to the treatment. The study pathologist concluded that: ”the microscopic findings show a normal physiological response of the animals by macrophages attempting to clear the particles from the respiratory tract”. The observed microscopic change in alveolar macrophages is therefore considered physiological and not pathological.

HISTOPATHOLOGY: NEOPLASTIC
No treatment related effects observed

OTHER FINDINGS
ESTRUS CYCLE EVALUATION
One control animal could not be judged because there was no full cycle determined. The number of acyclic females, the mean length of the longest cycle and the number of females with a prolonged estrus period were comparable among the groups.

SPERM ANALYSIS
Epididymal sperm motility. A significant decrease was found in curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) in high-dose group animals compared. This was mainly due to three animals which had relatively low values for these parameters. All high-dose animals had VCL levels within the historical control range. Only one animal had a ALH level below the historical control range. The decrease in VCL and ALH was not supported by histopathological changes. Therefore, this change was considered an incidental finding. No significant differences between groups were observed for other parameters.

Epididymal sperm count. No statistically significant effects on epididymal sperm counts were observed.

Testicular sperm count. Statistical analysis of parenchymal tissue weight indicated a significant difference between high-dose animals and controls. However, as no significant effect was observed on testes weight, this difference can be attributed to the preparation procedure and was not considered test material related. No effect on the number of spermatozoa per gram testicular parenchyma or on daily sperm production was observed.

Epididymal sperm morphology. One smear of a control animal contained a large number of malformed sperm cells. No statistically significant effect on sperm morphology was observed.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
Significant increases in all biochemical and cellular parameters were observed in bronchoalveolar lavage fluid. ALP was increased in a dose-dependent manner, and was statistically significant in the mid and high-dose group. LDH was increased in a dose-dependent manner, and was statistically significant for the high-dose group. NAG was significantly increased in male animals of the high-dose group. Protein was significantly increased in all animals of the high-dose group.Total number of cells significantly increased in male animals of the high-dose group. Absolute number of viable cells significantly increased in animals of the high-dose group. Absolute and relative number of neutrophils significantly increased in all exposed animals in a dose dependent manner. Absolute number of lymphocytes significantly increased in males of the high-dose group and females of the low- and mid-dose group. Relative number of lymphocytes was significantly increased in males of the low-and high-dose group and females of the mid- and high-dose group. Relative number of macrophages was significantly decreased in all exposed animals. No treatment related effects were observed on the absolute or relative number of eosinophils.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 7.29 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight lung inflammatory changes based on minimal increases in pulmonary neutrophils.
Key result
Critical effects observed:
no

A sub-chronic (13-week) inhalation toxicity study with Activated Carbon – High Density Skeleton in rats.

Tabular data on biochemical and relative cellular Broncho Alveolar Lavage analyses (Tables 1 - 4) are given below. Tabular data on absolute cellular BAL analyses and on histopathology of lungs, lung-draining lymph nodes and sex organs (Tables 5 - 12) are given in a separate pdf attachment (Tables 5-12_BAL_Histopath_Estrus_Sperm analysis.pdf).

 

Table 1. Bronchoalveolar lavage: biochemical determinations in males

 

 

Lavage ALP (U/L)

LavageGGT (U/L)

Lavage LDH (U/L)

LavageNAG (U/L)

Lavage Protein

(mg/L)

 

 

 

 

 

 

 

control

Mean

18.4¹

1.540⁴

42.9¹

2.589⁴

105.0⁴

 

SD

4.6

0.709

21.8

0.707

34.5

 

N

10

10

10

10

10

3 mg/m3

Mean

25.3d²

3.180dd³

30.9

3.013

89.0

 

SD

6.7

0.487

6.5

0.856

18.8

 

N

10

10

10

10

10

 

 

 

 

 

 

 

7 mg/m3

Mean

39.2dd³

3.490dd³

32.0

2.755

96.6

 

SD

9.4

0.918

7.6

0.751

19.0

 

N

10

10

10

10

10

 

 

 

 

 

 

 

10 mg/m3

Mean

46.2dd³

4.470dd³

74.0dd³

4.038dd³

140.4d²

 

SD

10.3

0.884

25.8

1.246

31.9

 

N

10

10

10

10

10

1 [L,aa - Automatic Transformation: Log, Group Factor Test: Anova p < 0.01]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

4 [I,aa - Automatic Transformation: Identity (None), Group Factor Test: Anova p < 0.01]

 


Table 2. Bronchoalveolar lavage: biochemical determinations in females

 

 

Lavage ALP (U/L)

Lavage GGT (U/L)

Lavage LDH (U/L)

Lavage NAG(U/L)

Lavage Protein (mg/L)

control

Mean

26.5¹

3.720⁴

54.4 L,a⁵

4.651 I⁶

141.4 R,k⁷

 

SD

9.5

1.127

26.7

2.027

60.8

 

N

10

10

10

10

10

 

 

 

 

 

 

 

3 mg/m3

Mean

36.6

5.740 dd³

70.1

4.331

130.1

 

SD

9.6

1.613

30.8

1.235

16.2

 

N

10

10

10

10

10

 

 

 

 

 

 

 

7 mg/m3

Mean

40.7 d²

6.150 dd³

68.2

4.205

137.9

 

SD

10.6

1.990

30.6

0.939

21.1

 

N

10

10

10

10

10

 

 

 

 

 

 

 

10 mg/m3

Mean

56.7 dd³

6.730 dd³

97.8 dd³

5.038

171.2 d⁸

 

SD

12.4

1.084

27.7

1.496

34.3

 

N

10

10

10

10

10

1 [I,aa - Automatic Transformation: Identity (None), Group Factor Test: Anova p < 0.01]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

4 [L,aa - Automatic Transformation: Log, Group Factor Test: Anova p < 0.01]

5 [L,a - Automatic Transformation: Log, Group Factor Test: Anova p < 0.05]

6 [I - Automatic Transformation: Identity (None)]

7 [R,k - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.05]

8 [d - Test: Dunnett Non-Parametric 2 Sided p < 0.05]


 

Table 3. Bronchoalveolar lavage: relative cell count in males

 

 

Lavage Total-C (10E6/L)

Lavage Viable-C (%)

Lavage Mon/Macr (%)

Lavage Neutroph (%)

Lavage Eosinoph (%)

Lavage Lympho (%)

 

 

 

 

 

 

 

 

control

Mean

1.820 R,k¹

91.10 I³

99.650⁴

0.350⁴

0.000 R⁶

0.000⁴

 

SD

0.681

5.04

0.530

0.530

0.000

0.000

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3 mg/m3

Mean

1.820

92.10

97.750 dd⁵

1.950 dd⁵

0.000

0.300 d²

 

SD

0.140

6.47

1.532

1.423

0.000

0.350

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

7 mg/m3

Mean

1.600

90.40

90.500 dd⁵

9.350 dd⁵

0.000

0.150

 

SD

0.596

6.48

3.771

3.852

0.000

0.337

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

10 mg/m3

Mean

2.630 d²

90.90

80.050 dd⁵

19.150 dd⁵

0.000

0.800 dd⁵

 

SD

0.918

2.69

8.786

8.560

0.000

0.715

 

N

10

10

10

10

10

10

1 [R,k - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.05]

2 [d -Test: Dunnett Non-Parametric 2 Sided p < 0.05]

3 [I - Automatic Transformation: Identity (None)]

4 [R,kk - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.01]

5 [dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01]

6 [R - Automatic Transformation: Rank]

Table 4. Bronchoalveolar lavage: relative cell count in females

 

 

Lavage Total-C (10E6/L)

Lavage Viable-C (%)

Lavage Mon/Macr (%)

Lavage Neutroph (%)

Lavage Eosinoph (%)

Lavage Lympho (%)

 

 

 

 

 

 

 

 

control

Mean

1.350 L¹

89.20 R²

99.650³

0.250³

0.050 R²

0.050³

 

SD

0.460

9.00

0.337

0.354

0.158

0.158

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3 mg/m3

Mean

1.410

88.00

98.100 dd⁴

1.850 dd⁴

0.000

0.050

 

SD

0.498

7.63

1.630

1.582

0.000

0.158

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

7 mg/m3

Mean

1.810

93.00

92.400 dd⁴

7.000 dd⁴

0.000

0.600 dd⁴

 

SD

1.060

6.11

3.596

3.266

0.000

0.568

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

10 mg/m3

Mean

2.200

94.60

81.850 dd⁴

17.250 dd⁴

0.000

0.950 dd⁴

 

SD

1.054

1.90

6.429

6.413

0.000

0.438

 

N

10

10

10

10

10

10

1 [L - Automatic Transformation: Log]

2 [R- Automatic Transformation: Rank]

3 [R,kk - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.01]

4 [dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01]

Conclusions:
In a sub-chronic toxicity study performed in accordance with OECD 413 and under GLP conditions, male and female rats were exposed to Activated Carbon – High Density Skeleton (AC-HDS) for 13 weeks, 6 hours per day and 5 days per week, nose-only. A NOAEL of 7.29 mg/m3 (actual concentration) was observed based on slight lung inflammatory changes based on increased pulmonary neutrophils in the high-dose group (10.25 mg/m3, actual concentration) .
Executive summary:

In a sub-chronic toxicity study performed in accordance with OECD 413 and under GLP conditions, male and female rats were exposed to Activated Carbon – High Density Skeleton (AC-HDS) for 13 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.33, 7.29, and 10.25 mg/m3(actual concentrations), and a control group that was exposed to clean air was included. The dose concentrations approximately correspond to daily doses of 0, 0.18, 0.39 and 0.55 mg/kg body weight/day.

The 90-day study was preceded by a 14-day Dose Range finding study in which male and female rats were exposed to AC-HDS for 2 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.1, 10.8, and 30.4 mg/m3(actual concentrations). A control group was exposed to clean air.

 

No treatment related clinical abnormalities, ocular changes, effects on body weight, food consumption, and estrus cycle were observed during the study. Furthermore, no treatment-related effects on hematology, clinical chemistry, and sperm parameters were observed. The only observed effect was minimal lung inflammation at the lowest dose, as indicated by minimal, but statistically significant increases in biochemical and cellular parameters in bronchoalveolar lavage fluid. These effects are confirmed microscopically by more active alveolar macrophages, which remained however within the normal physiological range. The study pathologist concluded that: "the microscopic findings show a normal physiological response of the animals by macrophages attempting to clear the particles from the respiratory tract". The observed microscopic change in alveolar macrophages is therefore considered physiological and not pathological. Based on the absence of other indications of lung toxicity (e.g. cytotoxicity and histopathological changes), it seems justified to consider the observed increased macrophage-mediated response to inhaled respirable AC-HDS as a physiological response of little, if any, toxicological significance.

 

As effects were observed at all concentration levels, no NOEL was established in the study report. The LOEL was considered to be 3.33 mg/m3 for male and female rats in the study report. However, based on comparison with other sub-chronic inhalation studies in rats with a similar inert particulate test substance in which lung particle overload was studied, it can be concluded that the 3.33 mg/m3 and 7.29 mg/m3 dose levels are most probably below the threshold for lung particle overload, hence covering a dose range relevant for extrapolation to human hazard. Furthermore, no pulmonary cytotoxicity is seen at the 3.33 mg/m3 and the 7.29 mg/m3 dose levels and the very slight lung inflammatory changes based on minimal increases in pulmonary neutrophils seen at the 3.33 mg/m3 and 7.29 mg/m3 dose levels are considered responses within the normal physiological range.

 

Based on the above the NOAEL for AC-HDS is considered to be 7.29 mg/m3.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
10.25 mg/m³
Study duration:
subchronic
Species:
rat

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:
9 August 2012 - 16 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Remarks:
Performed under GLP
Qualifier:
according to
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories, the Netherlands
- Age at study initiation: 7-9 weeks
- Weight at study initiation:
Range finding study: male - 236 g, female - 163 g
Main study: male - 222 g, female - 169 g
- Fasting period before study: No
- Housing: Macrolon cages with a bedding of wood shavings (Lignocel, Rettenmaier, Rosenberg, Germany), separated by sex, five rats per cage
- Diet (e.g. ad libitum): ad libitum, except during exposure and the fasting period before scheduled sacrifice
- Water (e.g. ad libitum): ad libitum, except during exposure and the fasting period before scheduled sacrifice
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 45-65%
- Air changes (per hr): 10 per hr
- Photoperiod (hrs dark / hrs light): 12 hrs dark/12 hrs light

IN-LIFE DATES: From: 22 August 012 (arrival) To: 29 January to 1 February 2013
Route of administration:
inhalation: dust
Type of inhalation exposure:
nose/head only
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: low-dose: 1.41 µm (± 0.08)
mid-dose: 1.74 µm (± 0.30)
high-dose: 1.46 µm (± 0.05)
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: Nose-only exposure units consisting of a cylindrical column, surrounded by a transparent cylinder
- Method of holding animals in test chamber: animals were secured in plastic animal holders
- Source and rate of air: at least 1 L/min for each rat
- Method of conditioning air: flow of humidified air driving an educator (Cheng et al., 1989) and air flow added by suction
- System of generating particulates/aerosols: test material was aerosolized using a turntable dust feeder
- Temperature, humidity, pressure in air chamber: 22 ± 2°C, 30-70% humidity, positive pressure in central column and slightly negative pressure in outer cylinder
- Air flow rate: at least 1 L/min for each rat
- Air change rate: continous supply of fresh test atmosphere
- Method of particle size determination: Cascade impactor

TEST ATMOSPHERE
- Brief description of analytical method used:gravimetric analysis

VEHICLE: Not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical verification for the verification of test material identity and properties was not performed in this study. The test concentrations were verified by means of gravimetric analysis.

A Certificate of Analysis on the total Crystalline Silica content of the batch used in the 90-day inhalation toxicity study is attached as a pdf file. The content of total Crystalline Silica is < 1% (w/w%), meaning that the content of respitable Crystalline Silica is also below 1% (w/w%).
Duration of treatment / exposure:
6 hours per day, 5 days per week, for a 93-day study period (total of 65 exposure days).
Frequency of treatment:
Daily, 5 days per week, 6 h per day
Remarks:
Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.
Remarks:
Doses / Concentrations:
3.33 mg/m3
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
7.29 mg/m3
Basis:
other: actual concentration
Remarks:
Doses / Concentrations:
10.25 mg/m3
Basis:
other: actual concentration
No. of animals per sex per dose:
Range-finding study: 5 males and 5 females per dose
Main study: 10 males and 10 females per dose
Control animals:
yes, sham-exposed
Details on study design:
- Dose selection rationale: A range finding study was performed with 0, 3, 10, and 30 mg/m3. Based on observed results the doses for the main study were chosen.
- Rationale for animal assignment (if not random): computer randomisation proportionally to body weight
Positive control:
No positive control group was included.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes,
- Time schedule: Daily
- Cage side observations included: a detailed list of clinical signs, annexed to the study report

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily and halfway through the 6-hr exposure period (group-wise)

BODY WEIGHT: Yes
- Time schedule for examinations:
Range-finding study: Before start of exposure (male day -1, female day -2), prior to exposure (day 0), twice weekly thereafter (day 3, 7, 10), and prior to sacrifice (day 14)
Main study: Before start of exposure (male day -2 or -1, female day -6 or -5), prior to exposure (day 0), twice weekly during first month (day 3/4, 7, 10/11, 14, 17/18, 21, 24/25, 28), weekly thereafter (day 35, 39 (male only), 42, 49, 56, 63, 70, 77, 84, 91, 93/95)), and fasted bodyweights prior to sacrifice (day 94/96).

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/animal/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

OPHTHALMOSCOPIC EXAMINATION: Yes, main study only
- Time schedule for examinations and examined dose groups: Prior to start of treatment (all animals), towards the end of treatment (Control and high-dose group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment period
- Anaesthetic used for blood collection: Yes (pentobarbital)
- Animals fasted: Yes (overnight)
- How many animals: all animals
- Parameters examined:
Haemoglobin concentration (Hb)
Packed Cell Volume (PCV)
Red Blood Cell count (RBC)
reticulocytes
Total White Blood Cell count (WBC)
Differential White Blood Cells
Prothrombin time
Thrombocyte count (platelet count)
- Calculated parameters:
Mean Corpuscular volume (MCV)
Mean Corpuscular Haemoglobin (MCH)
Mean Corpuscular Haemoglobin Concentration (MCHC)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment period
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined:
Alkaline phosphatase activity (ALP)
Aspartate aminotransferase activity (ASAT)
Alanine aminotransferase activity (ALAT)
Gamma glutamyl transferase activity (GGT)
Total protein
Albumin
Ratio albumin to globulin
Urea
Creatinine
Fasting glucose
Bilirubin (total)
Cholesterol (total)
Triglycerides
Phopholipids
Calcium (Ca)
Sodium (Na)
Potassium (K)
Chloride (Cl)
Inorganic phosphate

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: Estrus cycle evaluation, Sperm analysis and Bronchoalveolar lavage and measurements

ESTRUS CYCLE EVALUATION
Daily in the week prior to sacrifice (including day of sacrifice) vaginal smears were made to evaluate oestrus cycle length and normality. Only smears of control and high-dose animals were evaluated.

SPERM ANALYSIS
Epididymal sperm was derived from the left cauda epididymis and motility was measured with the Hamilton Thorne Integrated Visual Optical System (IVOS), which was also used for sperm count. Furthermore, a smear of each male of the control group and high-dose group were analysed.
At necropsy, testicular sperm count was performed and daily sperm production was calculated. Evaluation of homogenization resistant spermatids was performed in the control and high-dose groups.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
At necropsy, the lungs of all animals were lavaged according to a standardized method. Total protein, alkaline phosphatase (ALP), lactate dehydrogenase (LDH), N-acetylglucosaminidase (NAG), gamma-glutamyltransferase (γ-GT) were determined. Furthermore, total white blood cell numbers were counted using a Coulter Counter. The number of viable cells and differential cells were determined using an acridine orange/ethidium bromide staining method in combination with fluorescent microscopic evaluation.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- The following organs were weighed in the Range finding study:
Heart
Adrenals
Kidneys
Liver
Spleen
Testes
Lungs with trachea
Trachea-bronchial lymph nodes
- The following organs were weighed in the Main study:
Adrenals
Brain
Heart
Kidneys
Liver
Spleen
Testes
Thymus
Thyroids
Left lung lobe
Ovaries
Uterus
Epididymus

HISTOPATHOLOGY: Yes
- Range finding study:
Left lung lobe (control and high-dose group)
Tracheobronchal lymph nodes (control and high-dose group)
- Main study:
Adrenals
Aorta
Axillary lymph nodes
Brain (brain stem, cerebrum and cerebellum)
Caecum
Colon
Epididymes
Eyes (with optic nerve)
Exorbital lachrymal glands
Femur with joint
Heart
Kidneys
Liver
Left lung lobe/trachea/larynx
Mammary glands (females)
Cervical lymph nodes
Nasopharyngeal tissue (with nasal associated lymphoid tissue and teeth)
Nerve peripheral (sciatic nerve)
Oesophagus
Olfactory bulb
Ovaries
Pancreas
Parathyroids
Pharynx
Parotid salivary glands
Pituitary
Prostate
Rectum
Seminal vesicles with coagulation glands
Skeletal muscle (thigh)
Skin (flank)
Small intestines (duodenum, ileum, and jejunum)
Spinal cord (three levels)
Spleen
Sternum with bone marrow
Stomach (glandular, non-glandular)
Sublingual salivary glands
Submaxillary salivary glands
Testes
Thymus
Thyroid
Tongue
Tracheobronchial (mediastinal) lymph nodes
Ureter
Urethra
Urinary bladder
Uterus (with cervix)

All preserved tissues of all animals of the control and high-dose groups were examined histopathologically (by light microscopy). In addition, the lungs and the tracheobronchial lymph nodes were also examined in animals of the low- and mid-dose groups.
Other examinations:
No other examinations performed
Statistics:
- Post-treatment body weight data: AnCova and Dunnett's Test.
- Pre-treatment body weight data, haematology, clincial chemistry, broncho alveolar lavage data, and organ weight: Generalised Anova/Ancova Test and Kruskal-Wallis or Dunnett's test
- Food consumption: Sunnett's multiple comparison test
- Estrus cyclicity: Fisher's exact test or Kruskal-Wallis nonparamtric ANOVA followed by Mann-Whitney U test
- Sperm parameters: ANOVA dollowed by Dunnett's multiple comparison test or Kruskal/Wallis non parametric ANOVA followed by Mann-Whitney U test
- Incidences of histopathological changes: Fisher's exact probability test
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
High dose animals showed increased absolute weight of the left lung. High and mid-dose animals showed an increased relative weight of the left lung.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
17/20 low-dose, 18/20 mid-dose and 19/20 high-dose animals showed gray/black discoloration/patches of the lungs. 12/20 low-dose, 17/20 mid-dose and 20/20 high-dose animals showed dark/black discoloration of the tracheobronchial lymph nodes.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
At microscopic observation the lungs of all animals exposed to the low-, mid- and high-dose showed accumulation of black pigment in the lungs and also in the tracheobronchial lymph nodes (except in one low-dose female).
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment related effects observed.

BODY WEIGHT AND WEIGHT GAIN
No treatment related effects observed.

FOOD CONSUMPTION
No treatment related effects observed.

OPHTHALMOSCOPIC EXAMINATION
No treatment related effects observed.

HAEMATOLOGY
No treatment related effects observed.

CLINICAL CHEMISTRY
No treatment related effects observed.

ORGAN WEIGHTS
High dose animals showed increased absolute weight of the left lung. High and mid-dose animals showed an increased relative weight of the left lung.

GROSS PATHOLOGY
17/20 low-dose, 18/20 mid-dose and 19/20 high-dose animals showed gray/black discoloration/patches of the lungs. 12/20 low-dose, 17/20 mid-dose and 20/20 high-dose animals showed dark/black discoloration of the tracheobronchial lymph nodes.

HISTOPATHOLOGY: NON-NEOPLASTIC
At microscopic observation the lungs of all animals exposed to the low-, mid- and high-dose showed accumulation of black pigment in the lungs (majority in alveolar macrophages) and also in the tracheobronchial lymph nodes (except in one low-dose female). Small amounts of pigment were present as free particles, mostly in the alveoli, sometimes also in the bronchiolar lumen. In the tracheobronchial lymph nodes the black pigment was present as free particles as well as within macrophages. In general, the amount of pigment in the lungs and tracheobronchial lymph nodes was in proportion to the exposure levels. One control female had an adenocarcinoma of the mammary gland, but otherwise the histopathological changes in the other organs and tissues were unremarkable. They were common findings in rats of this strain and age or occurred as individual chance findings, unrelated to the treatment. The study pathologist concluded that: ”the microscopic findings show a normal physiological response of the animals by macrophages attempting to clear the particles from the respiratory tract”. The observed microscopic change in alveolar macrophages is therefore considered physiological and not pathological.

HISTOPATHOLOGY: NEOPLASTIC
No treatment related effects observed

OTHER FINDINGS
ESTRUS CYCLE EVALUATION
One control animal could not be judged because there was no full cycle determined. The number of acyclic females, the mean length of the longest cycle and the number of females with a prolonged estrus period were comparable among the groups.

SPERM ANALYSIS
Epididymal sperm motility. A significant decrease was found in curvilinear velocity (VCL) and amplitude of the lateral head displacement (ALH) in high-dose group animals compared. This was mainly due to three animals which had relatively low values for these parameters. All high-dose animals had VCL levels within the historical control range. Only one animal had a ALH level below the historical control range. The decrease in VCL and ALH was not supported by histopathological changes. Therefore, this change was considered an incidental finding. No significant differences between groups were observed for other parameters.

Epididymal sperm count. No statistically significant effects on epididymal sperm counts were observed.

Testicular sperm count. Statistical analysis of parenchymal tissue weight indicated a significant difference between high-dose animals and controls. However, as no significant effect was observed on testes weight, this difference can be attributed to the preparation procedure and was not considered test material related. No effect on the number of spermatozoa per gram testicular parenchyma or on daily sperm production was observed.

Epididymal sperm morphology. One smear of a control animal contained a large number of malformed sperm cells. No statistically significant effect on sperm morphology was observed.

BRONCHOALVEOLAR LAVAGE AND MEASUREMENTS
Significant increases in all biochemical and cellular parameters were observed in bronchoalveolar lavage fluid. ALP was increased in a dose-dependent manner, and was statistically significant in the mid and high-dose group. LDH was increased in a dose-dependent manner, and was statistically significant for the high-dose group. NAG was significantly increased in male animals of the high-dose group. Protein was significantly increased in all animals of the high-dose group.Total number of cells significantly increased in male animals of the high-dose group. Absolute number of viable cells significantly increased in animals of the high-dose group. Absolute and relative number of neutrophils significantly increased in all exposed animals in a dose dependent manner. Absolute number of lymphocytes significantly increased in males of the high-dose group and females of the low- and mid-dose group. Relative number of lymphocytes was significantly increased in males of the low-and high-dose group and females of the mid- and high-dose group. Relative number of macrophages was significantly decreased in all exposed animals. No treatment related effects were observed on the absolute or relative number of eosinophils.
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 7.29 mg/m³ air
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Slight lung inflammatory changes based on minimal increases in pulmonary neutrophils.
Key result
Critical effects observed:
no

A sub-chronic (13-week) inhalation toxicity study with Activated Carbon – High Density Skeleton in rats.

Tabular data on biochemical and relative cellular Broncho Alveolar Lavage analyses (Tables 1 - 4) are given below. Tabular data on absolute cellular BAL analyses and on histopathology of lungs, lung-draining lymph nodes and sex organs (Tables 5 - 12) are given in a separate pdf attachment (Tables 5-12_BAL_Histopath_Estrus_Sperm analysis.pdf).

 

Table 1. Bronchoalveolar lavage: biochemical determinations in males

 

 

Lavage ALP (U/L)

LavageGGT (U/L)

Lavage LDH (U/L)

LavageNAG (U/L)

Lavage Protein

(mg/L)

 

 

 

 

 

 

 

control

Mean

18.4¹

1.540⁴

42.9¹

2.589⁴

105.0⁴

 

SD

4.6

0.709

21.8

0.707

34.5

 

N

10

10

10

10

10

3 mg/m3

Mean

25.3d²

3.180dd³

30.9

3.013

89.0

 

SD

6.7

0.487

6.5

0.856

18.8

 

N

10

10

10

10

10

 

 

 

 

 

 

 

7 mg/m3

Mean

39.2dd³

3.490dd³

32.0

2.755

96.6

 

SD

9.4

0.918

7.6

0.751

19.0

 

N

10

10

10

10

10

 

 

 

 

 

 

 

10 mg/m3

Mean

46.2dd³

4.470dd³

74.0dd³

4.038dd³

140.4d²

 

SD

10.3

0.884

25.8

1.246

31.9

 

N

10

10

10

10

10

1 [L,aa - Automatic Transformation: Log, Group Factor Test: Anova p < 0.01]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

4 [I,aa - Automatic Transformation: Identity (None), Group Factor Test: Anova p < 0.01]

 


Table 2. Bronchoalveolar lavage: biochemical determinations in females

 

 

Lavage ALP (U/L)

Lavage GGT (U/L)

Lavage LDH (U/L)

Lavage NAG(U/L)

Lavage Protein (mg/L)

control

Mean

26.5¹

3.720⁴

54.4 L,a⁵

4.651 I⁶

141.4 R,k⁷

 

SD

9.5

1.127

26.7

2.027

60.8

 

N

10

10

10

10

10

 

 

 

 

 

 

 

3 mg/m3

Mean

36.6

5.740 dd³

70.1

4.331

130.1

 

SD

9.6

1.613

30.8

1.235

16.2

 

N

10

10

10

10

10

 

 

 

 

 

 

 

7 mg/m3

Mean

40.7 d²

6.150 dd³

68.2

4.205

137.9

 

SD

10.6

1.990

30.6

0.939

21.1

 

N

10

10

10

10

10

 

 

 

 

 

 

 

10 mg/m3

Mean

56.7 dd³

6.730 dd³

97.8 dd³

5.038

171.2 d⁸

 

SD

12.4

1.084

27.7

1.496

34.3

 

N

10

10

10

10

10

1 [I,aa - Automatic Transformation: Identity (None), Group Factor Test: Anova p < 0.01]

2 [d - Test: Dunnett 2 Sided p < 0.05]

3 [dd - Test: Dunnett 2 Sided p < 0.01]

4 [L,aa - Automatic Transformation: Log, Group Factor Test: Anova p < 0.01]

5 [L,a - Automatic Transformation: Log, Group Factor Test: Anova p < 0.05]

6 [I - Automatic Transformation: Identity (None)]

7 [R,k - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.05]

8 [d - Test: Dunnett Non-Parametric 2 Sided p < 0.05]


 

Table 3. Bronchoalveolar lavage: relative cell count in males

 

 

Lavage Total-C (10E6/L)

Lavage Viable-C (%)

Lavage Mon/Macr (%)

Lavage Neutroph (%)

Lavage Eosinoph (%)

Lavage Lympho (%)

 

 

 

 

 

 

 

 

control

Mean

1.820 R,k¹

91.10 I³

99.650⁴

0.350⁴

0.000 R⁶

0.000⁴

 

SD

0.681

5.04

0.530

0.530

0.000

0.000

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3 mg/m3

Mean

1.820

92.10

97.750 dd⁵

1.950 dd⁵

0.000

0.300 d²

 

SD

0.140

6.47

1.532

1.423

0.000

0.350

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

7 mg/m3

Mean

1.600

90.40

90.500 dd⁵

9.350 dd⁵

0.000

0.150

 

SD

0.596

6.48

3.771

3.852

0.000

0.337

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

10 mg/m3

Mean

2.630 d²

90.90

80.050 dd⁵

19.150 dd⁵

0.000

0.800 dd⁵

 

SD

0.918

2.69

8.786

8.560

0.000

0.715

 

N

10

10

10

10

10

10

1 [R,k - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.05]

2 [d -Test: Dunnett Non-Parametric 2 Sided p < 0.05]

3 [I - Automatic Transformation: Identity (None)]

4 [R,kk - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.01]

5 [dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01]

6 [R - Automatic Transformation: Rank]

Table 4. Bronchoalveolar lavage: relative cell count in females

 

 

Lavage Total-C (10E6/L)

Lavage Viable-C (%)

Lavage Mon/Macr (%)

Lavage Neutroph (%)

Lavage Eosinoph (%)

Lavage Lympho (%)

 

 

 

 

 

 

 

 

control

Mean

1.350 L¹

89.20 R²

99.650³

0.250³

0.050 R²

0.050³

 

SD

0.460

9.00

0.337

0.354

0.158

0.158

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

3 mg/m3

Mean

1.410

88.00

98.100 dd⁴

1.850 dd⁴

0.000

0.050

 

SD

0.498

7.63

1.630

1.582

0.000

0.158

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

7 mg/m3

Mean

1.810

93.00

92.400 dd⁴

7.000 dd⁴

0.000

0.600 dd⁴

 

SD

1.060

6.11

3.596

3.266

0.000

0.568

 

N

10

10

10

10

10

10

 

 

 

 

 

 

 

 

10 mg/m3

Mean

2.200

94.60

81.850 dd⁴

17.250 dd⁴

0.000

0.950 dd⁴

 

SD

1.054

1.90

6.429

6.413

0.000

0.438

 

N

10

10

10

10

10

10

1 [L - Automatic Transformation: Log]

2 [R- Automatic Transformation: Rank]

3 [R,kk - Automatic Transformation: Rank, Group Factor Test: Kruskal-Wallis p < 0.01]

4 [dd - Test: Dunnett Non-Parametric 2 Sided p < 0.01]

Conclusions:
In a sub-chronic toxicity study performed in accordance with OECD 413 and under GLP conditions, male and female rats were exposed to Activated Carbon – High Density Skeleton (AC-HDS) for 13 weeks, 6 hours per day and 5 days per week, nose-only. A NOAEL of 7.29 mg/m3 (actual concentration) was observed based on slight lung inflammatory changes based on increased pulmonary neutrophils in the high-dose group (10.25 mg/m3, actual concentration) .
Executive summary:

In a sub-chronic toxicity study performed in accordance with OECD 413 and under GLP conditions, male and female rats were exposed to Activated Carbon – High Density Skeleton (AC-HDS) for 13 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.33, 7.29, and 10.25 mg/m3(actual concentrations), and a control group that was exposed to clean air was included. The dose concentrations approximately correspond to daily doses of 0, 0.18, 0.39 and 0.55 mg/kg body weight/day.

The 90-day study was preceded by a 14-day Dose Range finding study in which male and female rats were exposed to AC-HDS for 2 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.1, 10.8, and 30.4 mg/m3(actual concentrations). A control group was exposed to clean air.

 

No treatment related clinical abnormalities, ocular changes, effects on body weight, food consumption, and estrus cycle were observed during the study. Furthermore, no treatment-related effects on hematology, clinical chemistry, and sperm parameters were observed. The only observed effect was minimal lung inflammation at the lowest dose, as indicated by minimal, but statistically significant increases in biochemical and cellular parameters in bronchoalveolar lavage fluid. These effects are confirmed microscopically by more active alveolar macrophages, which remained however within the normal physiological range. The study pathologist concluded that: "the microscopic findings show a normal physiological response of the animals by macrophages attempting to clear the particles from the respiratory tract". The observed microscopic change in alveolar macrophages is therefore considered physiological and not pathological. Based on the absence of other indications of lung toxicity (e.g. cytotoxicity and histopathological changes), it seems justified to consider the observed increased macrophage-mediated response to inhaled respirable AC-HDS as a physiological response of little, if any, toxicological significance.

 

As effects were observed at all concentration levels, no NOEL was established in the study report. The LOEL was considered to be 3.33 mg/m3 for male and female rats in the study report. However, based on comparison with other sub-chronic inhalation studies in rats with a similar inert particulate test substance in which lung particle overload was studied, it can be concluded that the 3.33 mg/m3 and 7.29 mg/m3 dose levels are most probably below the threshold for lung particle overload, hence covering a dose range relevant for extrapolation to human hazard. Furthermore, no pulmonary cytotoxicity is seen at the 3.33 mg/m3 and the 7.29 mg/m3 dose levels and the very slight lung inflammatory changes based on minimal increases in pulmonary neutrophils seen at the 3.33 mg/m3 and 7.29 mg/m3 dose levels are considered responses within the normal physiological range.

 

Based on the above the NOAEL for AC-HDS is considered to be 7.29 mg/m3.

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

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Sub-chronic inhalation study with activated carbon

The key study is a sub-chronic toxicity study performed in accordance with OECD 413, male and female rats were exposed to Activated Carbon – High Density Skeleton (AC-HDS) for 13 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.33, 7.29, and 10.25 mg/m3(actual concentrations), and a control group that was exposed to clean air was included. The dose concentrations approximately correspond to daily doses of 0, 0.18, 0.39 and 0.55 mg/kg body weight/day.

The 90-day study was preceded by a 14-day Dose Range finding study in which male and female rats were exposed to AC-HDS for 2 weeks, 6 hours per day and 5 days per week, nose-only. Exposure concentrations were 3.1, 10.8, and 30.4 mg/m3(actual concentrations). A control group was exposed to clean air.

 

No treatment related clinical abnormalities, ocular changes, effects on body weight, food consumption, and estrus cycle were observed during the study. Furthermore, no treatment-related effects on hematology, clinical chemistry, and sperm parameters were observed. The only observed adverse effect was minimal lung inflammation at the lowest dose, as indicated by minimal, but statistically significant increases in biochemical and cellular parameters in bronchoalveolar lavage fluid. These effects are confirmed microscopically by more active alveolar macrophages, which remained however within the normal physiological range. The study pathologist concluded that: "the microscopic findings show a normal physiological response of the animals by macrophages attempting to clear the particles from the respiratory tract". The observed microscopic change in alveolar macrophages is therefore considered physiological and not pathological.


Severity and toxicological relevance of the effects

 

Lung particle overload

Sub-chronic exposure to AC-HDS did not result in any systemic toxicity up to a concentration of 10.25 mg/m3. The substance-related changes were confined to the lungs and their physiological nature and low severity are discussed in relation to the general responses to respired poorly soluble “inert” dust of low toxicity. Inhaled respirable Poorly Soluble Particles (PSP) will elicit a physiological response by the body to clear these PSPs from the pulmonary system. The alveolar macrophages play the most prominent role in the pulmonary clearance or retention of deposited particles. This is a general physiological defense mechanism. The recruitment of alveolar macrophages and neutrophils in the defense against inhaled respirable PSPs shows the same characteristics as early lung inflammation. However, to a certain extent this cellular change can be considered a physiological form of inflammation, without toxicological significance. The increase in pulmonary macrophages will most probably remain in the physiological range when the macrophage-mediated defense mechanisms are not “overwhelmed”, i.e. when lung particle overload is not (yet) evident.

Elder et al.(2005) stated that lung overload is characterized by altered retention kinetics, which occurs in rodents when mass burdens reach 1–3 mg particles/g lung tissue. In their studies with carbon black, particle overload was achieved in rats and mice exposed to 7 and 50 mg/m3of High Surface area Carbon black (HSCb) for 90 days. Rats exposed to 1 mg/m3of HSCb for 90 days did not show lung overload, which was further evidenced by unaltered retention half times at this dose level. These results indicate that the threshold for lung particle overload in rats lies between 1 and 7 mg/m3for HSCb. The clearance of particulates from the deep lungs is mainly mediated by pulmonary macrophages, which should remain functional and present at adequate numbers to ensure sufficient defense capacity in the lungs. It is generally accepted that the percentage of pulmonary macrophages in BAL fluid is considered normal when it is larger than 85% (Meyer et al., 2012). In the sub-chronic toxicity study with AC-HDS, the percentages of macrophages were > 90% at the dose levels of 3.33 and 7.29 mg/m3(see Table 2), indicating a normal capacity and an intact defense mechanism.

It should be noted that several studies with various PSPs (e,g., titanium dioxide, carbon black, coal dust and talc) have shown that rats are especially sensitive to the phenomenon of lung overload (ILSI, 2000). Although in the present 90-day study retention kinetics were not determined, lung overload is most probably not reached at 3 mg/m3and 7 mg/m3, because of the minimal and physiological nature of the lung changes observed and the absence of any histopathological changes at dose levels up to 10 mg/m3.

 

In December 2013, ECETOC prepared an extensive evaluation of the scientific literature on Poorly Soluble Particles (PSP) and lung overload (ECETOC TR122, 2013). In this document ECETOC concludes that using an assessment factor of 1 for intraspecies differences is reasonable for PSP. In this case the ECETOC guidance is justified, because: (1) "activated carbon" may be considered a PSP of low toxicity; (2) only minimal lung inflammatory changes within the physiological range were seen in the 90-day inhalation toxicity study in rats; (3) "activated carbon" appears not to give any significant systemic bioavailability; (4) the minimal lung effects were "port-of-entry" effects (i.e. local effects). The ECETOC report concludes that for concentration dependent local effects for PSP, an intraspecies AF of 1 is considered sufficient. Therefore, an assessment factor for intraspecies variation of 1 is used for the derivation of the DNEL for AC-HDS and AC-LDS.

 

Pulmonary cytotoxicity

Lactate dehydrogenase (LDH) is a “household” enzyme that can be used as a general marker for cytotoxicity. For this reason the marker was determined in the Broncho Alveolar Lavage (BAL) fluid of the rats exposed to AC-HDC for 90 days. LDH was increased significantly in males and females of the high-dose group only. The LDH activity was increased to maximally 98 U/L (see Tables 1 and 2 below), whereas control values ranged from 43 to 78 U/L.

Warheit (2013) treated rats once with fine or ultra-fine TiO2 particles by intratracheal instillation and showed at 1 mg/kg bwt (corresponding to approximately twice the daily dose of AC-HDS in the 10 mg/m3group) similar LDH levels (ca. 100 U/L) in BAL fluid which, were not different from control values 24 h after treatment. Intratracheal treatment with TiO2 particles at 5 mg/kg bwt (ca. 10 times the AC-HDS inhalation dose at 10 mg/m3) did also not result in higher LDH levels in most cases. Based on this information, it can be concluded that the pulmonary cytotoxic effect of AC-HDS after sub-chronic repeated exposure is very limited at 10 mg/m3and absent at 3 and 7 mg/m3.

 

 

Table 1 - 14-day inhalation Range Finding study with AC-HDS: summary of parameters related to local lung effects

Male rats

%Neutrophils in BAL fluid

No.Neutrophils in BAL fluid (10E6/mL)

LDH in BAL fluid (U/L)

%Macrophages in BAL fluid

No.Macrophages in BAL fluid (10E6/mL)

Controls

0.4

0.002

77

99.4

0.67

3.1 mg/m3

0.6

0.005

46

98.8

0.89

10.8 mg/m3

1.0

0.009

44

98.7

0.89

31.4 mg/m3

6.6*

0.055*

52

92.7

0.78

Female rats

%Neutrophils in BAL fluid

No.Neutrophils in BAL fluid (10E6/mL)

LDH in BAL fluid (U/L)

%Macrophages in BAL fluid

No.Macrophages in BAL fluid (10E6/mL)

Controls

0.2

0.001

78

99.8

0.48

3.1 mg/m3

0.4

0.001

66

99.6

0.34

10.8 mg/m3

1.2

0.005

49

98.6

0.45

31.4 mg/m3

9.8*

0.062*

47

90.1

0.56

 

 

Table 2 - 90-day inhalation study with AC-HDS: summary of parameters related to local lung effects

Male rats

%Neutrophils in BAL fluid

No.Neutrophils in BAL fluid (10E6/mL)

LDH in BAL fluid (U/L)

%Macrophages in BAL fluid

No.Macrophages in BAL fluid (10E6/mL)

Controls

0.35

0.006

43

99.7

1.81

3.33 mg/m3

1.95*

0.036*

31

97.8*

1.78

7.29 mg/m3

9.35*

0.139*

32

90.5*

1.46

10.25 mg/m3

19.15*

0.487*

74*

80.0*

2.12

Female rats

%Neutrophils in BAL fluid

No.Neutrophils in BAL fluid (10E6/mL)

LDH in BAL fluid (U/L)

%Macrophages in BAL fluid

No.Macrophages in BAL fluid (10E6/mL)

Controls

0.25

0.002

54

99.7

1.35

3.33 mg/m3

1.85*

0.027*

70

98.1*

1.38

7.29 mg/m3

7.00*

0.109*

68

92.4*

1.69

10.25 mg/m3

17.25*

0.418*

98*

81.9*

1.76

 

 

Pulmonary inflammation

PMN (neutrophils) in BAL fluid can be used as indicator of lung inflammation. It is a very sensitive marker that consistently shows the defensive response to increased exposure to PSPs by inhalation. Table 3 below compares the percentage neutrophils in BAL fluid and lung histopathology between AC-HDS and carbon black (Carter et al., 2006) after 90-day exposure by inhalation. It demonstrates that the change in neutrophils is much less severe with AC-HDS than with carbon black at 7 mg/m3. It also shows that the histopathological changes indicative of more severe lung inflammation and epithelial changes seen with carbon black at 7 mg/m3are absent with AC-HDS at the same and higher exposure levels. Moreover, Table 4 shows that in the study with carbon black, the changes in BAL fluid neutrophils slowly recover, most pronounced at the 7 mg/m3 level at 11 months post exposure. The presence of neutrophils in BAL fluid is very dynamic. A single intratracheal instillation of physiological saline in rats results in a rapid increase of percentage pulmonary neutrophils in BAL fluid to 10% - 15%, which subsequently recovers to normal values within one week (Warheit, 2013).

 


Table 3 – Comparison of AC-HDS with Carbon Black: % Neutrophils in BAL fluid and lung histopathology observed 1 day after 90-day exposure by inhalation

 

% Neutrophils in BAL fluid

Lung histopathology1

Female rats

AC-HDS

Carbon Black

AC-HDS

Carbon Black

Controls

0.25

0.48

-

-

1 mg/m3

 

0.83

 

-

3 mg/m3

1.85*

 

-

 

7 mg/m3

7.00*

17.60*

-

+

10 mg/m3

17.25*

 

-

 

50 mg/m3

 

47.95*

 

++

 

1Lung histopathology: (+): present; (++): more severely present. The principal exposure-related lung lesions were centered primarily in the centriacinar regions of the lungs with the most extensive epithelial and inflammatory responses in the alveolar ducts and the immediately surrounding alveolar parenchyma. These lesions were characterized by :

a.    the accumulation of large numbers of markedly hypertrophic and highly vacuolated alveolar macrophages laden with HSCb;

b.    an associated mixed inflammatory cell influx composed of neutrophils and lesser numbers of mononuclear cells located in the alveolar walls, alveolar air spaces, and perivascular interstitium; and

c.    hyperplasia and hypertrophy of alveolar type II cells

 

 

 

Table 4 - Carbon Black:

recovery of % Neutrophils in BAL after a 90-day exposure by inhalation period

Female rats

1 day post exposure

3 months post exposure

11 months post exposure

Controls

0.48

0.15

0.46

1 mg/m3

0.83

0.24

0.32

7 mg/m3

17.60*

16.52*

2.84*

50 mg/m3

47.95*

33.52*

26.24*

 

Other available information

A repeated dose inhalation study published by Gross and Nau (1967) is included in the dossier as a supporting study. In this study, guinea pigs, rats and mice were exposed to aerosols of both lignite and activated carbon (no information on substance identity according to the REACH requirements) for one work year (seven hours per day, five days per week, for one year).The tested concentration of AC-HDS (as Steam Activated Carbon) averaged 8.12 mg/m3. Unexposed control animals were kept in regular chambers and did not receive treatment.

 

Local rather than systemic effects were observed. Pathological studies revealed extensive deposition of dust in alveoli, both free and within alveolar macrophages. Furthermore, extensive peribronchial and interstitial accumulations of the carbon dust with minimal fibrosis were noted. No treatment-related clinical signs or mortality were reported. The findings of this study are in accordance with the characteristics of an “inert” or nonfibrogenic dust reaction. The tested substance was steam activated carbon with a silica content of ≥ 6.5%. From the results of this study it can be concluded that the silica in the activated carbon is not biologically available and therefore cannot exert its effects. This may be related to enclosure of the silica in the activated carbon matrix.

 

The results of the Gross and Nau (1967) study indicate that exposure to activated carbon will not result in systemic effects, but local effects in the lung may occur after long-term exposure.

 

Conclusion on severity and toxicological relevance of the effects

 

Based on the absence of other indications of lung toxicity (e.g. cytotoxicity and histopathological changes), it seems justified to consider the observed increased macrophage-mediated response to inhaled respirable AC-HDS in the 90-day toxicity study as a physiological response of little, if any, toxicological significance.

As effects were observed at all concentration levels, no NOEL could be established. The LOEL was considered to be 3.33 mg/m3for male and female rats. However, the following considerations lead to the derivation of the NOAEL for AC-HDS:

·        Based on comparison with the study by Elder et al. (2005), it can be concluded that the 3.33 mg/m3and 7.29 mg/m3dose levels are most probably below the threshold for lung particle overload, hence covering a dose range relevant for extrapolation to human hazard;

·        No pulmonary cytotoxicity is seen at the 3.33 mg/m3and the 7.29 mg/m3dose levels;

·        The very slight lung inflammatory changes based on minimal increases in pulmonary neutrophils seen at the 3.33 mg/m3and 7.29 mg/m3dose levels are considered responses within the normal physiological range.

 

Based on the above the NOAEL for AC-HDS is considered to be 7.29 mg/m3. This value will be used for the derivation of the DNEL.

References

 

·      Elder, A., Gelein, R., Finkelstein, J.N., Driscoll, K.E., Harkema, J., Oberdörster, G..(2005). Effects of subchronically inhaled Carbon Black in three species. I. Retention kinetics, lung inflammation, and histopathology. Toxicological Sciences, 88(2), 614-629.

·      Meyer, K.C., Raghu, G., Baughman, R.P., Brown, K.K., Costabel, U., du Bois, R.M., Drent, M., Haslam, P.L., Kim D.S., Nagai, S., Rottoli, P., Saltini, C., Selman, M., Strange, C., Wood, B. (2012). An official American Thoracic Society Clinical practice Guideline: The clinical utility of bronchoalveolar lavage cellular analysis in interstitial lung disease. American Thoracic Society Document. January 2012.

·      ILSI, 2000. Risk Science Institute Workshop: the relevance of the rat lung response to particle overload for human risk assessment. Inhalation Toxicology 12, 1–17.

- ECETOC TRA 122, Poorly soluble particles/lung overload. Technical Report No. 122, European Center for Ecotoxicology and Toxicology of Chemicals, December 2013

·      Carter, J.M., Corson, N., Driscoll, K.E., Elder, A., Finkelstein, J.N., Harkema, J.N., Gelein, R., Wade-Mercer, P., Nguyen, K., Oberdorster, G., (2006).A comparative dose-related response of several key pro- and antiinflammatory mediators in the lungs of rats, mice, and hamsters after subchronic inhalation of carbon black. Journal of Occupational and Environmental Medicine, 48(12), 1265-1278.

·      Warheit, D.B. (2013). How to measure hazards/risks following exposures to nanoscale or pigment-grade titanium dioxide particles. Toxicology Letters, 220, 193-204.

·      Gross and Nau (1967). Lignite and the Derived Steam-Activated Carbon - The pulmonary response to their dusts. Arch Env Health, 14(3): 450-460.

 


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
The key study was a subchronic inhalation study that was performed according to OECD guidelines and under GLP conditions.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
The key study was a subchronic inhalation study that was performed according to OECD guidelines and under GLP conditions.

Justification for classification or non-classification

In order to evaluate whether classification of AC-HDS and AC-LDS is justified, the LOAEL and the nature and severity of the observed toxicological effect seen at this LOAEL has to be taken into account. It must be determined whether the observed LOAEL after 90-days of exposure to AC-HDS represents an effective dose level (ED) that induces significant/severe target organ toxicity in rats. The results from the 90-day study show the following:

 

·        No morbidity or death resulting from repeated 90-days exposure were observed;

·        No functional changes in the central or peripheral nervous systems or any other organ systems were observed after 90-days of exposure;

·        No adverse changes in clinical biochemistry or haematology parameters were observed;

·        No organ damage nor any adverse macroscopical changes were noted at necropsy and no adverse changes were observed at subsequent microscopic examination;

·        No adverse changes were seen in bronchoalveolar lavage fluid at concentrations of 0.003 and 0.007 mg/L/6h/day and only minimal indication of lung inflammation was seen at a concentration of 0.01 mg/L/6h/day, as indicated by minimal increases in biochemical and cellular parameters in bronchoalveolar lavage fluid. None of these changes were accompanied by histopathology in the lungs. Moreover, it is not clear if lung particle overload plays a role at the LOAEL of 0.01 mg/L/6h/day and what the relevance of lung overload is to the human situation.

 

The LOAEL (0.01 mg/L/6h/day) from the 90-day inhalation toxicity study with AC-HDS is below the Guidance Values (GV) for Category 1 and 2, possibly warranting classification. Below it is evaluated whether the changes seen at the LOAEL of AC-HDS are severe enough to warrant STOT RE classification.

The changes in bronchoalveolar lavage parameters observed at the LOAEL of 0.01 mg/L/6h/day were of minimal severity and were not accompanied by any histopathological lesions in the lungs. The observed cellular and biochemical changes in BAL fluid at 0.01 mg/L/6h/day are indicative of early lung inflammation, but are considered at the borderline of normal physiological response and minimal adverse response. Therefore, the toxicological significance is considered as very minor.

Although the LOAEL is below the Guidance Values warranting STOT RE1 or STOT RE2 classification, it seems justified not to classify AC-HDS for Specific Target Organ Toxicity following repeated exposure, because of the minimal nature of the observed effects. In December 2013, ECETOC prepared an extensive evaluation of the scientific literature on Poorly Soluble Particles (PSP) and lung overload (ECETOC TR122, 2013). Review of this document did not change the conclusion on non-classification for "activated carbon". Since the results for sub-chronic repeated dose toxicity with AC-HDS will be read across to AC-LDS, the non-classification will also apply to AC-LDS.