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Administrative data

Description of key information

A 90 day oral study in rats, NOAEL 3737.9 mg/kg (highest concentration tested) no treatment related effects.  No dermal repeat dose studies have been considered neccssary. Repeat dose inhalation studies performed using either the substance itself or amoprhous or crystalline silica demonstrate that, although amorphous silica does cause a transient inflammatory response, crystalline silica at lower time- or dose-exposure leads to much more persistent (and consequently much more severe) pulmonary toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEL
3 737.9 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Additional information

Read-Across Justification for the inhalation route

Kieselguhr soda ash flux-calcined is a UVCB substance, the main constituents of which are amorphous silica and crystalline silica, principally in the form of cristobalite. The percentages of each type of silica may vary between 20-70 %w/w and 4-80%w/w, respectively, for the amorphous and crystalline forms. The toxicological properties via the inhalation route of both forms are well described, both in animal studies and in human epidemiological studies and may be used to predict the effects of exposure to kieselguhr soda ash flux-calcined via the inhalation route and to support the available data for this substance.

Summary of available data for repeated dose toxicity via inhalation

In a study performed according to OECD 413, Wistar rats were exposed to amorphous silica (Aerosil 200 at concentrations of 0 (control) 1.3, 5.9 or 31 mg SiO2/m3or Sipernat 22 at 35 mg/m3) for 6h/d, 5 d/w for 13 weeks. Additional animals were exposed to crystalline silica (quartz) at 58.5 mg/m3as a positive control [Reuzel et al 1991]. The primary particle size was calculated theoretically from electron microscopic photographs (range <6 – 45 nm). However, it is important to note that primary particles do not exist as individual free units, but only as aggregates and agglomerates. Because of technical problems, the aerodynamic aggregate/agglomerate size distribution in the test atmospheres was not determined. The approximate maximum of the geometric aggregate/agglomerate size distribution of Aerosil 200 was estimated to be at 10 μm, based on a normal photography technique. Reliable analytical data on the actual experimental particle size distribution in the test chamber are not available and technically difficult to obtain. Ten male and ten female rats per dose were sacrificed directly after termination of exposure. 50 additional animals were saved for examinations at 13, 26, 39, and 52 weeks post-exposure.

During exposure, there was a concentration-related increase in the respiration rate of animals exposed to Aerosil 200. The respiration rate quickly returned to normal when the exposure was ended. At the end of the exposure period, body weight gain was 5-10% lower in males exposed to 31 mg Aerosil 200/m3or 35 mg Supernat 22S/m3. The body weight of quartz exposed rats was not affected during the exposure period. After a 13 week post exposure period body weights had returned to normal in all groups exposed to the amorphous silicas. However, the quartz-exposed rats showed a slightly progressive reduction in weight gain throughout the post exposure period. At the end of the exposure period, neutrophilic leucocyte counts were higher in most groups than in controls, but the elevation was only statistically significant in rats exposed to 31 mg Aerosil 200/m3or to quartz. These counts returned to normal in rats exposed to Aerosil 200 within 13 weeks of termination of the exposure. In quartz-exposed rats the neutrophilic leucocyte counts increased during the first 13 week after the end of the exposures and remained high during the whole post-exposure period. Red blood cell counts, haemoglobin content and packed cell volumes slightly increased in males exposed to 31 mg Aerosil 200/m3or quartz by the end of the exposure period. After 13 weeks of non-exposure, these parameters had returned to normal in all animals exposed to amorphous silica. The males exposed to quartz continued to show high red blood cell values throughout the observation period. From week 13 after exposure, alanine aminotransferase activity increased in quartz-exposed rats. In males the 50-90% increase observed was significant compared to controls. Alkaline phosphatase activity increased in rats exposed to quartz 52 weeks after the exposure period. The remaining haematological and biochemical parameters that were examined did not show differences related to treatment. Urine analyses were essentially negative.

At the end of the exposure period statistically significant increases in both absolute and relative lung weight were observed in all treated groups compared to controls except for the 1.3 mg Aerosil 200/m3group. Lung weight returned to normal in all amorphous silica groups by week 26 post exposure. However, in the quartz treated group it increased progressively to two or three times higher than controls. The only other organ that showed increased weight was the thymus in quartz-exposed rats. This increase had disappeared by week 39 after the end of exposure.

Lung collagen content of all exposed groups was higher than controls at the end of the exposure period and was statistically significant in all groups except females exposed to 1.3 mg Aerosil 200/m3or to quartz. During the post exposure period the levels decreased in all groups exposed to amorphous silicas but after one year were only comparable to control levels in rats exposed to 1.3 mg Aerosil 200/m3or 35 mg Sipernat 22S/m3. In quartz exposed rats the collagen content was only slightly higher than controls but increased markedly during the post exposure period.

Silicon levels in the lungs of males (except those exposed to Aerosil 200) increased up to week 26 (and up to week 39 in the case of quartz). This effect was not observed in females. Most of the rats exposed to amorphous silica or quartz and killed at the end of the exposure period had swollen and spotted lungs with a spongy consistency and/or irregular surface and enlarged lung-associated lymph nodes. There changes were most pronounced in the group exposed to quartz. At week 26 after exposure the gross changes had disappeared in all groups exposed to the amorphous silicas, however the gross lesions remained throughout the whole post exposure period in rats exposed to quartz. Microscopic changes were mainly observed in the lungs. Changes in rats killed at the end of the exposure period comprised slight to severe accumulation of alveolar macrophages, intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes in the alveolar spaces and an increased septal cellularity, seen as an increase in the number of Type II pneumocytes and macrophages within the alveolar walls. In general, the most severe changes were observed in rats exposed to 31 mg Aerosil 200/m3or quartz. No recovery from lung lesions was observed in quartz-exposed rats whereas in amorphous silica exposed rats the changes disappeared partly or completely. Accumulations of alveolar macrophages were still found 52 weeks after the end of exposure in rats exposed to quartz or 31 mg Aerosil 200/m3. Accumulation of intra-alveolar granular material, cellular debris and polymorphonuclear leucocytes were occasionally found in the group exposed to 31 mg Aerosil 200/m3and in all quartz-exposed rats. The rats exposed to Sipernat 22S recovered completely from the slight increases in septal cellularity observed at the end of the exposure period. A lesser degree of recovery was seen in rats exposed to Aerosil 200 and no recovery was observed in the rats exposed to quartz. Focal interstitial fibrosis was first observed 13 weeks after exposure in all exposed groups. During the post exposure period it disappeared in rats exposed to Sipernat 22S but became more severe in rats exposed to 31 mg Aerosil 200/m3or to quartz.

In a 28-day repeat dose inhalation study performed according to OECD TG 412 under GLP, kieselguhr soda ash flux-calcined was administered to Wistar rats (20 animals/sex/dose) by nose-only, flow-past inhalation for a period of 5 days/week (6 hours/day) for 4 consecutive weeks at aerosol concentrations of 0.044, 0.207 and 0.700 mg/L air [Schuler 2011].

The reversibility or progression of any test item related effects or any delayed toxicity was assessed during a 9-week treatment-free recovery period following the treatment period in selected animals of all groups. Sub-groups of male and female animals served for determination of broncho-alveolar lavage fluid (BALF) parameters on day 28 of treatment or were kept for a 9-week off-dose period. Mortality, clinical signs, body weight, food consumption, organ weights, macroscopic and microscopic findings were recorded and ophthalmoscopic examinations and clinical laboratory investigations were performed. BALF samples were collected after the end of treatment and after recovery.

No premature deaths and no clinical signs were observed except for a slight and transient effect on body weight gain at the high dose. A dose-dependent increase in lung weights was recorded from the low dose to the high dose at the end of the treatment. A further increase in lung weights and lymph nodes had increased in size at the end of the recovery period. An increase in spleen, adrenals and liver weights at the high-dose at the end of the recovery period was observed but these findings were not considered as treatment-related in absence of microscopic changes. In the alveoli, histiocytosis was observed increasing in incidence and severity from the low dose to the high dose at the end of the treatment. A progression in incidence and severity was also observed to occur after the recovery period. The presence of the test item was detected in the alveoli of the animals of the mid and high dose groups at the end of the treatment period. This was observed to persist at least until the end of the recovery period and additionally it was observed in 2/10 animals of the low dose recovery group.

In the observations on the peribronchial/perivascular zone, lymphoid hyperplasia was observed in the high dose group animals at the end of the treatment period. There was a progression in severity after the recovery period and additionally it was observed in some mid-dosed animals of the recovery group. The occurrence of microgranuloma and fibrosis was observed at the end of the treatment-free period. For the tracheobronchial lymph nodes, histiocytic granuloma was observed increasing in severity from the low dose to the high dose at the end of the treatment. There was a progression in incidence and severity as shown by the occurrence of fibrosis after the recovery period.

There were no adverse effects detected in organs other than the lungs. Some adaptive responses may have been induced in the liver, adrenals and spleen in response to the irritation and damage to the lung tissue.

On the basis of the microscopic findings, the lungs and tracheobronchial lymph nodes were considered as target organs and a NOAEL could not be established.

It should be noted that the initial sample used in the 28-day study contained 45% of cristobalite and approximately 55% of amorphous silica. The respirable fraction was 3.8% including 1.8% crystalline silica. In order to be compliant with the OECD TG 412 (which recommends the generation of aerosols with mass median aerodynamic diameters ranging from 1 to 3 μm), the test material was micronized for its use in the 28-day study to allow that the majority of material/particles could reach the deep lung (alveoli). The test method of processing of the material resulted in the content of respirable crystalline silica being increased from 1.8% to approximately 45%, which is approximately double the worse-case level found in commercially produced material. Under these conditions, the toxicity observed in the study material reflects and confirms the health hazards of extremely high concentrations of the crystalline silica. However, as stated above, the study material is not reflective of the toxicity of, or risk factor associated with, the much lower concentration of crystalline silica contained in commercially available product, the registered substance.

A5-day inhalation toxicity study [Schuler 2010] was performed on Soda-ash flux-calcined kieselguhr as a range-finding study for a 28-day inhalation toxicity study. The dose levels used were 0, 0.18, 0.58, and 1.57 mg/L of air. No premature deaths and no clinical signs were observed although there was some minor body weight loss at the high dose. There was an increase in lung weights at mid and high dose levels. Histological examinations showed alveolar histiocytosis, increasing in incidence and severity from the low dose to the high dose. Microgranulomas and amorphous material were also observed in the alveoli at the high dose. Based on these data, aerosol concentrations of 0.050, 0.20 and 0.80 mg/L air were considered to be suitable target concentrations for the 28-day inhalation study.

Fischer 344 rats were exposed via inhalation to synthetic amorphous silica (Aerosil 200, pyrogenic type) at a concentration of 50 mg SiO2/m3for 13 weeks, followed recovery for 3 or 8 months. Additional rats were exposed to crystalline silica (cristobalite) at 3 mg/ m3as a positive control [Johnstonet al,2000]. The mass median diameter of amorphous silica particles was 0.81 µm, i.e. close to 100 % respirable, whilst for the cristobalite it was 1.3 µm.

 

Dose selection was such as to apply a highly inflammatory but not lethal dose in either case. Besides on histopathology of the lung tissue, cytotoxicity, and SiO2 analysis, the study was focussed on the examination of the inflammation response expressed by specific sensitive cellular and biochemical inflammation markers in the broncho-alveolar lavage fluid. The broncho-alveolar lavage fluid analysis included: the elucidation of mutagenic events in isolated alveolar type-II cells, cell-type analysis and profile analysis (cytometry), inflammatory cytokine gene expression (MIP-2), and immunohistochemistry for DNA damage (terminal transferase dUTP nick-end-labeling = TUNEL staining).

The concentration of amorphous silica increased quickly during the first 6.5 weeks of exposure in the lungs (to approximately 0.76 mg SiO2/lung) but only slowly after the second half of the exposure period (plateau phase, steady state at about 0.88 mg SiO2/lung). Despite the much lower exposure concentration, the pulmonary level of crystalline silica increased steadily from about 0.34 mg SiO2/lung (after 6.5 weeks) and to approximately 0.82 mg SiO2/lung after 13 weeks.

 

During recovery, the amorphous silica lung burden disappeared rapidly from lung tissue down to about 15 % of the final lung burden in a first phase (during 12-weeks post-exposure) with an estimated elimination half-life of 4 weeks, and decreased further to about 6 % in a second phase (during subsequent 20 weeks). The overall elimination half-life over 32 weeks post-exposure is estimated to be 8 weeks. This indicates that for amorphous silica a rapid removal phase is followed by a delayed phase for clearing the residual SiO2from the lung. On the other hand, crystalline silica persisted in the lung with no substantial decrease during the post-exposure period.

 

Fibrosis was present in the alveolar septae (based on Gormor´s trichrome staining). Fibrosis subsided during recovery in the case of amorphous silica, but persisted for crystalline silica. Broncho-alveolar lavage (BAL) analysis showed mean cell number in the lavage increased at a factor of about 5 to 15 vs. control, comprising of more than 50 % polymorphous neutrophil cells (PMN) and some 2% lymphocytes, while the control lavages only contained less than 1 % of either cell type. Protein content and enzyme activities (lactate dehydrogenase and glucuronidase) were markedly higher than under control conditions. All BAL markers approached normal levels after 13 weeks post-exposure in the amorphous silica group, but a few still showed minimal increases. Gene expression of MIP-2 (inflammatory cytokine gene) showed that significant expression after 13 weeks was observed in silica-exposed rats (amorphous as well as crystalline). Minimal or no detectable MIP-2 m-RNA expression was found in control rats. After 8 months recovery, minimal expression was left in rat lungs exposed to amorphous silica, whereas a high level of MIP-2 mRNA was still detectable in rat lungs exposed to crystalline silica.

In conclusion, the data available demonstrate that, although amorphous silica does cause a transient inflammatory response, crystalline silica at lower time- or dose-exposure leads to much more persistent (and consequently much more severe) pulmonary toxicity.

 

 

 

 

Justification for classification or non-classification

Inhalation:

In the case of crystalline silica (quartz, cristabloite and tridymite), although there is no harmonised EU classification for this group of substances under the former European Dangerous Substances Directive, it has been the practice for many years to self classify and label crystalline silica flours as harmful with the label Xn and the risk phrases R48/20. Harmful: danger of serious damage to health by prolonged exposure through inhalation.

The CLP Article 8.6 specifies that “Tests that are carried out for the purposes of this Regulation shall be carried out on the substance or mixture in the form (s) or physical state(s) in which the substance or mixture is placed on the market and in which it can be reasonably expected to be used". In addition the ECHA Guidance to the CLP Regulation published on 13 July 2009 mentions that “for human health different forms (e.g. particle sizes, coating) or physical states may result in different hazardous properties of a substance or mixture in use” and therefore they may be classified differently. It is therefore justifiable to consider quartz and cristobalite in their respirable form – hereafter named as “RCS” ‐ for the purpose of classification.

It is considered to classify respirable quartz and respirable cristobalite as STOT RE 1 for the silicosis hazard.

Consequently, mixtures and substances containing RCS, whether in the form of an identified impurity additive or individual constituent, shall be classified according to CLP as:

STOT RE 1: if the RCS concentration is equal to, or greater than 10%;

STOT RE 2: if the RCS concentration is between 1 and 10%.

In the case the RCS content in mixtures and substances is below 1%, no classification is required.

As kiselguhr soda ash flux calcined comes in a variety of different grades/forms containing variable amount of RCS each grade will have to be classified accordingly as described above.