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Respiratory sensitisation

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Administrative data

respiratory sensitisation: in vivo
Type of information:
experimental study
Adequacy of study:
disregarded due to major methodological deficiencies
Study period:
not specified
other: not rated acc. to Klimisch
Rationale for reliability incl. deficiencies:
other: see "Remarks"
Publication on respiratory sensitisation with significant reporting and methodical deficiencies. This test system is most unusual and probably has not been used in chemical risk assessment before. The offspring that were tested for respiratory sensitisation never seemed to have inhaled carbon black, i.e. it is not clear if even a sensitisation to carbon black was measured in these animals. Lastly, the publication is not relevant for risk assessment since data on characterisation (particle size distribution) is lacking.

Data source

Reference Type:
Pulmonary exposure to particles during pregnancy causes increased neonatal asthma susceptibility
Fedulov, A.V. et al.
Bibliographic source:
Am J Respir Cell Mol Biol, 38: 57-67.

Materials and methods

Test guideline
no guideline followed
Principles of method if other than guideline:
Pregnant BALB/c mice received particle suspensions of carbon black (250 µg/mouse) or phosphate-buffered saline (vehicle control) intranasally at day 14 of pregnancy. Offspring of particle- or buffer-treated mothers were sensitized and aerosolized with ovalbumin (OVA), followed by assays of airway hyperresponsiveness and allergic inflammation.
GLP compliance:
not specified
not specified in the publication

Test material

Constituent 1
Chemical structure
Reference substance name:
Carbon black
EC Number:
EC Name:
Carbon black
Cas Number:
Molecular formula:
Test material form:
solid: particulate/powder
Details on test material:
not specified
Specific details on test material used for the study:
- Source (i.e. manufacturer or supplier) of test material: Dr. Ian Gilmour (U.S. E.P.A.) and Dr. Joseph Brain (Harvard University)

- Treatment of test material prior to testing (e.g. warming, grinding): particle samples were baked at 165 °C for 3 hours to eliminate endotoxin, aliquoted and stored frozen at -80 °C.

Test animals

Details on test animals or test system and environmental conditions:
- Source: Charles River Laboratories (Cambridge, MA)
- Housing: all mice were housed in a clean barrier facility

ENVIRONMENTAL CONDITIONS - independent pressure-gradient–enabled ventilation system.
- Temperature: 22 to 24°C
- Photoperiod (hrs dark / hrs light): 12/12

Test system

Route of induction exposure:
Route of challenge exposure:
other: phosphate-buffered saline (PBS) solution
250 µg/mouse
No. of animals per dose:
17 - 21 mice/group (not clearly specified in the publication)
Details on study design:
Pregnant female mice received the test item or the vehicle by intranasal insufflation on pregnancy day 14.

On Day 4 after birth, newborns from particle-exposed and normal control mother mice received a single intraperitoneal injection of 0.1 mL of 50 µg/mL ovalbumin (OVA, grade III; Sigma-Aldrich, St. Louis, MO) with alum. On Days 12 to 14 of life, these baby mice were exposed to aerosolized 3% OVA within individual compartments of a mouse pie chamber (Braintree Scientific, Braintree, MA) using a Pari IS2 nebulizer (Sun Medical Supply, Kansas City, KS) connected to air compressor (PulmoAID; DeVilbiss, Somerset, PA). After this challenge, the mice were subjected to pulmonary function and pathologic analysis.

During pulmonary function testing, airway responsiveness of mice to increasing concentrations of aerosolised methacholine was measured using whole body plethysmography (Buxco, Sharon, CT). Briefly, each mouse was placed in a chamber, and continuous measurements of box pressure/time wave were calculated via a connected transducer and associated computer data acquisition system. The main indicator of airflow obstruction, enhanced pause (Penh), which shows strong correlation in BALB/c mice with the airway resistance examined by standard evaluation methods, was calculated from the box waveform. After measurement of baseline Penh, aerosolized PBS or methacholine (MCh, acetyl-methylcholine chloride;) in increasing concentrations (6, 12, 25, 50, and 100 mg/mL) was nebulized through an inlet of the chamber for 1 minute, and Penh measurements were taken for 9 minutes after each dose. Penh values for the first 2 and the last 2 minutes after each nebulization were discarded, and the values for 5 minutes in between were averaged and used to compare results. Increased Penh was interpreted as evidence of increased airway hyperresponsiveness (AHR).

Animals were killed with sodium pentobarbital (Veterinary Laboratories, Lenexa, KS) and the animals were exsanguinated by cardiac puncture. The trachea was cannulated after blood collection. Bronchoalveolar lavage (BAL) was performed five times with 0.3 mL of sterile PBS instilled and harvested. Lavage fluid (recovery volume was ~ 90% of instilled) was collected and centrifuged, and the cell pellet was resuspended in 0.1 mL PBS. Total cell yield was quantified by hemocytometer. BAL differential cell counts were performed on cytocentrifuge slides prepared by centrifugation of samples (Cytospin 2; Shandon, Pittsburgh, PA). These slides were fixed in 95% methanol and stained with Diff-Quik (VWR, Boston, MA), a modified Wright-Giemsa stain, and a total of 200 cells were counted for each sample by microscopy. Macrophages, lymphocytes, neutrophils, and eosinophils were enumerated. After lavage, the lungs were instilled with 10% buffered formalin, removed, and fixed in the same solution. After paraffin embedding, sections for microscopy were stained with hematoxylin and eosin (H&E). For allergy responses, an index of pathologic changes in coded stained slides was derived by scoring the inflammatory cell infiltrates around airways and vessels for greatest severity (0, normal; 1, < 3 cell diameter thick; 2, 4–10 cells thick; 3, >10 cells thick) and overall prevalence (0, normal; 1, <25% of sample; 2, 25–50%; 3, 51–75%; 4, >75%). The index was calculated by multiplying severity by prevalence, with a maximum possible score of 9.

Data are presented as mean ± SEM. Data analysis was performed using Microsoft Excel from Microsoft Office 2003 Pro (Microsoft Corporation) and GraphPad Prism version 4.0 for Windows (GraphPad Software). Statistical significance was accepted when P < 0.05. To estimate significance of differences between groups in multiple comparisons ANOVA with Tukey’s Honest Significant Differences for unequal N post hoc test and Kruskal-Wallis test with Dunn’s post-test were used, as appropriate. For pairwise comparisons nonparametric Mann-Whitney U test was used. For repeated measurements in the plethysmography procedure we used repeated-measures ANOVA.
Challenge controls:
Please refer to the field "Details on study design" above.
Positive control substance(s):
not specified
Negative control substance(s):
not specified

Results and discussion

The offspring of pregnant mice treated with carbon black particles showed increased susceptibility to allergy, manifesting as increased airway hyperresponsiveness (Penh at 100 mg/mL Mch of 2.8 ± 0.3 in carbon black versus 1.0 ± 0.2 in PBS controls, P < 0.05) and allergic inflammation (bronchoalveolar lavage (BAL) eosinophilia was 10.7 ± 1.2% in carbon black versus 4.1 ± 1.0% in PBS controls).

Please also refer to the field "Attached background material".
Positive control results:
not specified
Negative control results:
not specified

Applicant's summary and conclusion

Interpretation of results:
other: publication cannot be used for chemical risk assessment.
According to the authors, neonates of mothers exposed to carbon black developed airway hyperresponsiveness and allergic inflammation, indicating that pregnancy exposure to carbon black caused increased asthma susceptibility in offspring.
This test system is most unusual and has not been used in chemicals risk assessment before. Furthermore, respiratory sensitizers within the definition of the CLP regulation/GHS are defined as substances "that will lead to hypersensitivity of the airways following inhalation of the substance. For respiratory sensitisation, the pattern of induction followed by elicitation phases is shared in common with skin sensitisation." (see CLP regulation (EC) 1272/2008, Annex 1, section 3.4). The offspring that were tested for respiratory sensitisation never seemed to have inhaled carbon black. As a consequence, it is considered doubtful whether any sensitisation to carbon black was actually verified in these animals.