Activated Carbon - Low Density Skeleton

Repeated dose toxicity: inhalation

Administrative data

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 August 2012 - 16 October 2013

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Study was performed in accordance with OECD 413 and under GLP conditions. The test was performed with AC-HDS and read across to AC-LDS. The test is part of an Integrated Test Strategy (ITS) and its results will be used as such. The study was assigned a Klimisch 2 rating due to the read-across purpose, in accordance with the ECHA Practical guide #6 on the reporting of read-across in IUCLID.

Justification for type of information:

The justification for read across is attached as a separate document to the "Toxicological information" summary

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Activated Carbon - High Density Skeleton

EC Number:

931-328-0

Molecular formula:

C

IUPAC Name:

Activated Carbon - High Density Skeleton

Constituent 2

Reference substance name:

Carbon

EC Number:

231-153-3

EC Name:

Carbon

Cas Number:

7440-44-0

Molecular formula:

C

IUPAC Name:

Carbon

Details on test material:

- Name of test material (as cited in study report): Activated Carbon - High Density Skeleton
- Substance type: Black powder
- Physical state: Solid
- Analytical purity: Not applicable
- Lot/batch No.: Confidential information
- Expiration date of the lot/batch: approved for use until 3 August 2013
- Storage condition of test material: Ambient temperature

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system 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

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Remarks:

Doses / Concentrations:
0 mg/m3
Basis:
nominal conc.

Doses / concentrations 2

Remarks:

Doses / Concentrations:
3.33 mg/m3
Basis:
other: actual concentration

Doses / concentrations 3

Remarks:

Doses / Concentrations:
7.29 mg/m3
Basis:
other: actual concentration

Doses / concentrations 4

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.

Examinations

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

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

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.

Effect levels

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.

Target system / organ toxicity

Key result

Critical effects observed:

not specified

Any other information on results incl. tables

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]

Applicant's summary and conclusion

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.