Tetraethyl orthosilicate

Endpoint summary

Description of key information

The key repeated dose toxicity data set for tetraethyl orthosilicate (CAS No. 78-10-4, EC No. 201-083-8) is comprised of the key oral study for its condensed hydrolysis product polysilicic acid (equivalent to synthetic amorphous silica [SAS], CAS No. 112926-00-8, EC No. 231-545-4) and the key inhalation study for tetraethyl orthosilicate.

 

In the key repeated dose oral toxicity study conducted according to OECD Test Guideline 408 and in compliance with GLP (Kim et al. 2014, reliability score 1), the systemic NOAELs for two particle sizes of SAS (20 and 100 nm) in the Sprague-Dawley rat were concluded to be ≥2000 mg/kg bw/day.

 

In the key repeated inhalation study conducted in a manner similar to OECD 412 (no information on GLP status, Omae et al. 1995, reliability score 2), the LOAEC for tetraethyl orthosilicate was 50 ppm vapour (approximately 0.4 mg/L) in male mice after a 2- or 4-week exposure, based primarily on histopathological (inflammatory) findings in the nasal mucosa and with some haematological changes noted. Renal histopathology was observed at 100 ppm (approximately 0.9 mg/L). A NOAEC was not identified. As discussed in the endpoint summary, the renal and nasal cavity findings are considered related to a physical effect of the condensed hydrolysis product polysilicic acid, and not to tetraethyl orthosilicate toxicity.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

No data

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Reliability 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Orient Bio, Korea
- Age at study initiation: 6 weeks old at purchase
- Weight at study initiation: 187.9-205.3 grams for males and 143.3-167.1 grams for females
- Fasting period before study: No
- Housing: Two animals per cage were housed in stainless steel wire cages
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5-23.3°C
- Humidity (%): 42.0-51.4 %
- Air changes (per hr): 10-15 per hour
- Photoperiod (hrs dark / hrs light): 12 hours dark / 12 hours light

IN-LIFE DATES: No data

Route of administration:

oral: gavage

Vehicle:

other: Distilled water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Colloidal silica NPs with 20 nm and 100 nm diameters were diluted in distilled water and each of the tested doses were formulated by dilution of the highest dose with distilled water. Working samples of each dose formulation were prepared daily during the study. The formulated test particles were homogenised by vortexing just prior to administration.

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

20 nm synthetic amorphous silica particles

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

20 nm synthetic amorphous silica particles

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

20 nm synthetic amorphous silica particles

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

100 nm synthetic amorphous silica particles

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

100 nm synthetic amorphous silica particles

Dose / conc.:

2 000 mg/kg bw/day (actual dose received)

Remarks:

100 nm synthetic amorphous silica particles

No. of animals per sex per dose:

Control and 2000 mg/kg bw/day: 15 (includes 5 animals for the recovery group);
500 and 1000 mg/kg bw/day: 10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: A 14-day dose range-finding study was conducted where 5 male and 5 female rats were randomly distributed to each dose groups so the final mean body weight was approximately equal. The highest dose was selected to 2000 mg/kg bw/day, middle dose to 1000 mg/kg bw/day and the low dose to 500 mg/kg bw/day. A negative control group was included where the animals received distilled water. Clinical signs and body weight were monitored throughout the study whereas gross pathology was examined on the scheduled necropsy day. No toxicity was observed at the highest dose tested, therefore, the main study used the same highest dose of 2000 mg/kg bw/day.
- Post-exposure recovery period in satellite groups: Control and highest dose group had 5 extra animals each, which were observed for a recovery period of two weeks following the 90-day treatment period.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed once daily after treatment for well-being, mortality and clinical signs of toxicity (no further detail).

DETAILED CLINICAL OBSERVATIONS: It is not clear how detailed the observations were as it was not described in the publication.

BODY WEIGHT: Yes
- Time schedule for examinations: Prior to the administration of the test substance and thereafter, once a week.

FOOD CONSUMPTION: Yes
g food per rat was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Daily, calculated by difference between supplied amounts and remaining amounts measured the next day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Last week of 90-day treatment period
- Dose groups that were examined: Control and highest dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: The night before scheduled necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: The night before scheduled necropsy
- Animals fasted: Yes
- How many animals: All animals
- Parameters checked in table 1 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: For five animals per group of each sex during the last week of the 90-day treatment period. Total urine volume was calculated from the urine collected over a 24-h period.
- Metabolism cages used for collection of urine: Yes, for five animals from the control and highest dose group over a 3-h sampling period.
- Animals fasted: No data
- Parameters checked in Table 1 were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 2), on moribund or dead animals, and on all animals at the end of the 90-day exposure period.
HISTOPATHOLOGY: Yes (see Table 2), tissues from the control and highest dose group were examined histopathologically.

Statistics:

Body weight values, feed and water consumption data, haematological data, blood biochemistry data, and organ weight values were analysed for homogeneity of variance using Levene’s test. One-way analysis of variance was performed to evaluate the significance of differences. If the variance was homogeneous and a significant difference was identified, Scheffe’s multiple comparison test was performed as a post hoc test. If the variance was not homogeneous, the data were analysed using Dunnett’s T3 test. Analysis of data from the recovery groups was performed using the Student’s t-test. All analyses were performed using Statistical Package for the Social Sciences version 19.0 software (SPSS Inc, Chicago, IL, USA).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Irrespective of particle size, no treatment-related clinical signs occurred during the experimental period for rats of either sex. A few occurrences of salivation, loss of fur and wound scratching were observed in the 2000 mg/kg bw/day group for 20 nm silica. However, due to the sporadic nature and no dose-dependency, the study authors concluded that these observations were not related to treatment.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in any dose groups.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Regardless of SiO2 particle size, there were no statistically significant differences in body weight between treated rats and their respective control groups for either males or females.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

There were sporadic increases in food consumption for 20 nm silica and sporadic increases in food consumption for 100 nm silica. However, these differences were not considered as treatment-related.

Food efficiency:

not examined

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

effects observed, non-treatment-related

Description (incidence and severity):

There were sporadic decreases in water consumption for 20 nm and 100 nm silica, however, these were not considered to be treatment-related.

Ophthalmological findings:

not specified

Description (incidence and severity):

The results were not reported although ophthalmoscopic examination was performed.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In the high dose recovery group for 20 nm silica, a statistically significant increase in lymphocyte counts was observed compared to the control recovery group. There were no findings for the groups treated with 100 nm silica and therefore, the findings were not considered as treatment-related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

No differences between treated and control animals were observed for 20 nm silica. For females of the high dose recovery group treated with 100 nm silica, aspartate aminotransferase and creatine kinase concentrations were statistically significantly decreased compared with the controls.

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in urinalysis parameters in any treated group compared with the respective control groups for either 20 nm or 100 nm silica.

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Relative liver weight was statistically significantly decreased whereas the absolute and relative lung weights were statistically significantly increased in males of the 2000 mg/kg bw/day dose group exposed to 20 nm silica compared to the control group. A statistically significantly increase in absolute weights of kidney, lung and submandibular glands and a statistically significant increase in the relative weights of kidneys and lungs were observed in the male 2000 mg/kg bw/day recovery group exposed to 100 nm silica. Females in the high dose recovery group had statistically significantly decreased absolute and relative ovary weights. The study authors concluded that due to the sporadic nature of the organ weight findings, the observations were not treatment related. No further details are available.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Pale yellowish discoloration on the posterior surface of the left lateral lobe of the liver was observed in one male animal from the 20 nm silica 500 mg/kg bw/day group. With regard to 100 nm silica, a small-sized right testis and epididymis were observed in one male from the 500 mg/kg group. A light yellow discoloration of the left lateral lobe of the liver (about 1 mm diameter), and a light yellow-coloured cyst with adjacent fat near the right kidney were also observed in another male in the 1000 mg/kg group. In addition, a small-sized left ovary was observed in one female from the 2000 mg/kg recovery group. However, no dose-dependency occurred and the effects were not considered to be related to treatment.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Granulomatous inflammation was observed in one male rat from the 1000 mg/kg bw/day group and two from the 2000 mg/kg bw/day group treated with 20 nm silica. Chronic bronchioalveolar inflammation was observed in one male rat from the 2000 mg/kg bw/day group treated with 20 nm silica. There were no adverse findings from the groups treated with 100nm silica. These lesions were observed in four cases in total and were minimal to mild in severity.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

>= 2 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: No adverse observed effects were observed

Critical effects observed:

no

Table 1 - Organ weights and organ weight to body weight ratios of male rats in the 90-day gavage study (main group) of 20 nm SiO2 nanoparticles*

	0 (vehicle control)	500 mg/kg bw/day	1000 mg/kg bw/day	2000 mg/kg bw/day
Number of animals	10	10	10	10
Male
Necropsy body weight	565.9±43.4	553.7±45.1	529.1±48.4	526.6±48.9
Liver
Absolute (g)	14.70±1.90	13.65±1.26	13.11±1.94	12.59±1.71
Relative (%)	2.59±0.18	2.47±0.14	2.47±0.16	2.38±0.11^a

*Organ (absolute) weights and body weights are given in grams: organ weight to body (relative) weights are given as mg organ weight/g body weight (mean ± standard deviation); ^aP<0.05 significantly different from control, by Scheffe’s test.

Table 2 - Organ weights and organ weight to body weight ratios for the recovery group in the 90-day gavage study of SiO2 nanoparticles*

	0 (vehicle control)	2000 mg/kg bw/day
Number of animals	5	5
SiO2 (20 nm)
Male
Necropsy body weight	569.8±38.4	545.9±35.0
Lung
Absolute (g)	1.61±0.07	1.81±0.12^a
Relative (%)	0.28±0.02	0.33±0.02^a
SiO2 (100 nm)
Male
Necropsy body weight	577.4±60.5	603.3±22.0
Kidney
Absolute (g)	3.27±0.21	3.69±0.21^a
Relative (%)	0.57±0.03	0.61±0.02^a
Lung
Absolute (g)	1.62±0.09	1.93±0.08^a
Relative (%)	0.28±0.02	0.32±0.02^a
Submaxillary gland
Absolute (g)	0.78±0.04	0.96±0.12^a
Relative (%)	0.14±0.01	0.16±0.02
Female
Necropsy body weight	303.9±47.8	304.9±16.9
Ovary
Absolute	0.094±0.009	0.066±0.022^a
Relative	0.0314±0.0040	0.0217±0.0069^a

*Organ (absolute) weights and body weights are given in grams: organ weight to body (relative) weights are given as mg organ weights/g body weight (mean ± standard deviation); ^aP<0.05, significantly different from control by Scheffe’s test.

Conclusions:

In a 90-day oral repeated dose toxicity study, conducted according to OECD Test Guideline 408 and in compliance with GLP (reliability score 1), the NOAELs for 20 nm and 100 nm colloidal silica particles) were ≥2000 mg/kg bw/day in Sprague-Dawley rats based on the authors' conclusion of no adverse effects observed. Condensed polysilicic acid (equivalent to synthetic amorphous silica) is the primary hydrolysis product of tetraethyl orthosilicate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch score of 1, based on the repeated dose oral (reliability score 1) data for tetraethyl orthosilicate and its condensed hydrolysis product SAS

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

limited examinations, males only, limited information on exposure conditions.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

ICR

Remarks:

SPF grade

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: Four weeks
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: Five per transparent plastic cages with stainless steel wire mesh ceiling
- Diet (e.g. ad libitum): Pelleted rodent food (CRF-1), ad libitum
- Water (e.g. ad libitum): Distilled water, ad libitum
- Acclimation period: One week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60
- Air changes (per hr): Not specified, filtered air, ventilated
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: Not specified

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Chamber: Tightly sealed 500 Lstainless steel exposure chamber
- Tetraethyl orthosilicate was supplied from an organic solvent vapour generator

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

monitored at 10 minute intervals by means of GC

Duration of treatment / exposure:

6 hours/day for 2 or 4 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

50 ppm (analytical)

Dose / conc.:

100 ppm (analytical)

No. of animals per sex per dose:

10M

Control animals:

other: filtered room air

Details on study design:

- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Not specified
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post-exposure recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Behaviour and external appearance checked daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed on Monday, Wednesday, and Friday prior to exposure

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in Table 1 below were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in Table 1 below were determined

URINALYSIS: Yes
- Time schedule for collection of urine: Every Friday night
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in Table 1 below were determined

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE
The mice were sacrificed by exsanguination 2 days after the final exposure.

GROSS PATHOLOGY: Yes
As described, their organs were examined grossly.

HISTOPATHOLOGY: Yes
The tissues identified in Table 2 below were subjected to microscopic examination.

Statistics:

Student's t test or Welch's methods were adopted for the statistical test of difference between means of the effects indices.

Clinical signs:

no effects observed

Description (incidence and severity):

Immediately after being exposed on each day, most mice began to perform face-washing movements and lick the lower abdomen for short periods more frequently than non-exposed mice. Finding is considered non-adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were observed between the exposed and non-exposed groups in body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

RBC, Hb and Ht values were lower in exposed mice than in non-exposed mice, mixed statistical results (see Table 3 below). The proportion of neutrophils were significantly higher at 50 and 100 ppm in the 2-week study, but not in the 4-week. The neutrophil count (not shown) was significantly higher at 100 ppm in the 4-week study.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Kidney weights were slightly lower at 50 and 100 ppm, after 2- or 4-weeks of exposure (not significant). No trends reported for lung, liver, or spleen weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

“Organs were examined,” but no findings reported.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidney, 2/10 mice exposed to 100 ppm for 2 weeks and 2/10 mice exposed to 100 ppm for 4 weeks developed histopathological lesions (tubulo-interstitial nephritis). The lesions were wedge-shaped and extended from the cortex into the medulla. In the affected regions, small numbers of neutrophils and mononuclear inflammatory cells had infiltrated interstitial tissue, and some tubules were dilated, exhibiting atrophy and obliteration. No kidney lesions were observed in mice exposed to 50 ppm.

Inflammation of the nasal mucosa was observed in almost all the mice (9/10 to 10/10) exposed to 50 ppm or 100 ppm, for 2 or 4 weeks. Exudates containing inflammatory cells and necrotic epithelial cells were observed in the nasal cavity, inflammatory cell infiltration in submucosal tissue, and eosinophilic hyaline droplets predominantly in the olfactory epithelium. The findings were slightly more severe in the mice exposed to 100 ppm.

In the bone marrow, increases in the M: E ratio were observed, no further details provided.

No lesions were observed in the liver, lungs, respiratory tract, spleen, pancreas, thymus, thyroid, or cornea.

Key result

Dose descriptor:

NOAEC

Effect level:

< 50 ppm (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Nasal cavity histopathology (inflammation) and haematology findings, noting renal histopathology at 100 ppm

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm (analytical)

System:

urinary

Organ:

kidney

Treatment related:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Concentrations of tetraethyl orthosilicate (Mean +/- SD):
Exposure level of 100 ppm:
2 weeks: 100 +/- 4
4 weeks: 101 +/- 5

Exposure level of 50 ppm:
2 weeks: 51 +/- 3
4 weeks: 51 +/- 3

 

Table 3 Summary of haematological findings following two and four weeks exposure.

 

Exposure Concentration (ppm)	Two week exposure			Four week exposure
	100	50	0	100	50	0
RBC (x10000/µ l)	823± 60*	866± 51	895± 60	840± 39	815± 36*	859± 35
Hg (g/dl)	-	-	-	14.1± 0.5*	13.7± 0.5**	14.7± 0.6
Ht (%)	42.9± 3.2**	45.1± 2.7	47.5± 3.2	43.1± 1.4	42.3± 1.6**	44.6± 1.8
WBC (x100 µ l)	-	-	-	36± 13	21± 6	28± 14
Neutrophil (%)	30± 10**	27± 5*	13± 7	34± 9	24± 11	25± 18
Lymphocyte (%)	68± 10**	72± 5**	85± 7	64± 9	75± 12	74± 19

 

Table 4 Number of mice with significant histopathological findings on the kidney and nasal cavity.

 

Exposure concentration (ppm)	100		50		0
Exposure duration	2	4	2	4	2	4
Kidney TIN	2	2	0	0	0	0
Nasal cavity
SIN	10	10	7	10	0	0
All positive findings	10	10	9	10	0	0

TIN: tubulo-interstitial nephritis.

SIN: submucosal infiltration of neutrophilic leukocytes.

Conclusions:

In the key repeated inhalation study which was similar to OECD 412 (no information on GLP status), the LOAEC for tetraethyl orthosilicate was 50 ppm vapour (ca. 0.4 mg/L) in male mice after a 2- or 4-week exposure, based primarily on histopathological (inflammatory) findings in the nasal mucosa and with some haematological changes noted. Histopathology findings in the kidney were observed at 100 ppm (ca. 0.9 mg/L) when mice were exposed over 2 or 4 weeks. Kidney weights were slightly lower at 50 and 100 ppm after 2 or 4 weeks, but this renal result was not significant. A NOAEC is not identified. The published study is well documented, meets generally accepted scientific principles, is acceptable for assessment, and thus is assigned reliability score 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

426 mg/m³

Study duration:

subacute

Species:

mouse

Quality of whole database:

Klimisch score 2, based on repeated dose inhalation (reliability score 2) data for tetraethyl orthosilicate

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

not specified

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

yes

Remarks:

limited examinations, males only, limited information on exposure conditions.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

ICR

Remarks:

SPF grade

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan, Inc.
- Age at study initiation: Four weeks
- Weight at study initiation: Not specified
- Fasting period before study: Not specified
- Housing: Five per transparent plastic cages with stainless steel wire mesh ceiling
- Diet (e.g. ad libitum): Pelleted rodent food (CRF-1), ad libitum
- Water (e.g. ad libitum): Distilled water, ad libitum
- Acclimation period: One week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 60
- Air changes (per hr): Not specified, filtered air, ventilated
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: Not specified

Route of administration:

inhalation: vapour

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Chamber: Tightly sealed 500 Lstainless steel exposure chamber
- Tetraethyl orthosilicate was supplied from an organic solvent vapour generator

TEST ATMOSPHERE
- Brief description of analytical method used: GC
- Samples taken from breathing zone: Not specified

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

monitored at 10 minute intervals by means of GC

Duration of treatment / exposure:

6 hours/day for 2 or 4 weeks

Frequency of treatment:

5 days/week

Dose / conc.:

50 ppm (analytical)

Dose / conc.:

100 ppm (analytical)

No. of animals per sex per dose:

10M

Control animals:

other: filtered room air

Details on study design:

- Dose selection rationale: Not specified
- Rationale for animal assignment (if not random): Not specified
- Rationale for selecting satellite groups: No satellite groups
- Post-exposure recovery period in satellite groups: No post-exposure recovery period

Positive control:

None

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Behaviour and external appearance checked daily

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: Weighed on Monday, Wednesday, and Friday prior to exposure

FOOD CONSUMPTION: No

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Not specified
- Anaesthetic used: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in Table 1 below were determined

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Not specified
- Animals fasted: Not specified
- How many animals: Not specified
- Parameters checked in Table 1 below were determined

URINALYSIS: Yes
- Time schedule for collection of urine: Every Friday night
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Not specified
- Parameters checked in Table 1 below were determined

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

SACRIFICE
The mice were sacrificed by exsanguination 2 days after the final exposure.

GROSS PATHOLOGY: Yes
As described, their organs were examined grossly.

HISTOPATHOLOGY: Yes
The tissues identified in Table 2 below were subjected to microscopic examination.

Statistics:

Student's t test or Welch's methods were adopted for the statistical test of difference between means of the effects indices.

Clinical signs:

no effects observed

Description (incidence and severity):

Immediately after being exposed on each day, most mice began to perform face-washing movements and lick the lower abdomen for short periods more frequently than non-exposed mice. Finding is considered non-adverse.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No differences were observed between the exposed and non-exposed groups in body weight gain.

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

RBC, Hb and Ht values were lower in exposed mice than in non-exposed mice, mixed statistical results (see Table 3 below). The proportion of neutrophils were significantly higher at 50 and 100 ppm in the 2-week study, but not in the 4-week. The neutrophil count (not shown) was significantly higher at 100 ppm in the 4-week study.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Kidney weights were slightly lower at 50 and 100 ppm, after 2- or 4-weeks of exposure (not significant). No trends reported for lung, liver, or spleen weight.

Gross pathological findings:

no effects observed

Description (incidence and severity):

“Organs were examined,” but no findings reported.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidney, 2/10 mice exposed to 100 ppm for 2 weeks and 2/10 mice exposed to 100 ppm for 4 weeks developed histopathological lesions (tubulo-interstitial nephritis). The lesions were wedge-shaped and extended from the cortex into the medulla. In the affected regions, small numbers of neutrophils and mononuclear inflammatory cells had infiltrated interstitial tissue, and some tubules were dilated, exhibiting atrophy and obliteration. No kidney lesions were observed in mice exposed to 50 ppm.

Inflammation of the nasal mucosa was observed in almost all the mice (9/10 to 10/10) exposed to 50 ppm or 100 ppm, for 2 or 4 weeks. Exudates containing inflammatory cells and necrotic epithelial cells were observed in the nasal cavity, inflammatory cell infiltration in submucosal tissue, and eosinophilic hyaline droplets predominantly in the olfactory epithelium. The findings were slightly more severe in the mice exposed to 100 ppm.

In the bone marrow, increases in the M: E ratio were observed, no further details provided.

No lesions were observed in the liver, lungs, respiratory tract, spleen, pancreas, thymus, thyroid, or cornea.

Key result

Dose descriptor:

NOAEC

Effect level:

< 50 ppm (analytical)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: Nasal cavity histopathology (inflammation) and haematology findings, noting renal histopathology at 100 ppm

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

100 ppm (analytical)

System:

urinary

Organ:

kidney

Treatment related:

yes

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

50 ppm (analytical)

System:

respiratory system: upper respiratory tract

Organ:

nasal cavity

Treatment related:

yes

Concentrations of tetraethyl orthosilicate (Mean +/- SD):
Exposure level of 100 ppm:
2 weeks: 100 +/- 4
4 weeks: 101 +/- 5

Exposure level of 50 ppm:
2 weeks: 51 +/- 3
4 weeks: 51 +/- 3

 

Table 3 Summary of haematological findings following two and four weeks exposure.

 

Exposure Concentration (ppm)	Two week exposure			Four week exposure
	100	50	0	100	50	0
RBC (x10000/µ l)	823± 60*	866± 51	895± 60	840± 39	815± 36*	859± 35
Hg (g/dl)	-	-	-	14.1± 0.5*	13.7± 0.5**	14.7± 0.6
Ht (%)	42.9± 3.2**	45.1± 2.7	47.5± 3.2	43.1± 1.4	42.3± 1.6**	44.6± 1.8
WBC (x100 µ l)	-	-	-	36± 13	21± 6	28± 14
Neutrophil (%)	30± 10**	27± 5*	13± 7	34± 9	24± 11	25± 18
Lymphocyte (%)	68± 10**	72± 5**	85± 7	64± 9	75± 12	74± 19

 

Table 4 Number of mice with significant histopathological findings on the kidney and nasal cavity.

 

Exposure concentration (ppm)	100		50		0
Exposure duration	2	4	2	4	2	4
Kidney TIN	2	2	0	0	0	0
Nasal cavity
SIN	10	10	7	10	0	0
All positive findings	10	10	9	10	0	0

TIN: tubulo-interstitial nephritis.

SIN: submucosal infiltration of neutrophilic leukocytes.

Conclusions:

In the key repeated inhalation study which was similar to OECD 412 (no information on GLP status), the LOAEC for tetraethyl orthosilicate was 50 ppm vapour (ca. 0.4 mg/L) in male mice after a 2- or 4-week exposure, based primarily on histopathological (inflammatory) findings in the nasal mucosa and with some haematological changes noted. Histopathology findings in the kidney were observed at 100 ppm (ca. 0.9 mg/L) when mice were exposed over 2 or 4 weeks. Kidney weights were slightly lower at 50 and 100 ppm after 2 or 4 weeks, but this renal result was not significant. A NOAEC is not identified. The published study is well documented, meets generally accepted scientific principles, is acceptable for assessment, and thus is assigned reliability score 2.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

426 mg/m³

Species:

mouse

Quality of whole database:

Klimisch score 2, based on repeated dose inhalation (reliability score 2) data for tetraethyl orthosilicate

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

OVERVIEW

In contact with water, tetraethyl orthosilicate (CAS 78-10-4, EC No. 201-083-8) hydrolyses rapidly, with a hydrolysis half-life of 0.11 hr (7 min), 4.4 hr and 0.22 hr (13 min) at pH 4, 7 and 9 respectively and at 25 °C to form monosilicic acid. Monosilicic acid exists only in dilute aqueous solutions and, following neat or aqueous-based tetraethyl orthosilicate dosing, would readily condense at concentrations above approximately 100 - 150 mg/L as 'SiO2 equivalent' to give a dynamic equilibrium between the monomer, oligomers and the condensed insoluble polysilicic acid. However, in the presence of a non-polar vehicle (e.g., corn oil in the OECD Test Guideline 422 study [CIT Safety & Health Research Laboratories 2005], see discussion of this study under “study findings” below), hydrolysis of tetraethyl orthosilicate would be delayed, resulting in higher (g/L) levels monosilicic acid in the small intestine and therefore exposure of the kidneys to artificially high levels of monosilicic acid (which would immediately condense to insoluble polysilicic acid within the kidneys). The non-silanol product of tetraethyl orthosilicate hydrolysis is ethanol.

 

The repeated dose oral toxicity data set for tetraethyl orthosilicate is comprised of:

- Key OECD Test Guideline 408 study for the condensed hydrolysis product polysilicic acid (equivalent to SAS) (Kim et al. 2014, reliability score 1)

- Supporting OECD Test Guideline 422 with tetraethyl orthosilicate (CIT Safety & Health Research Laboratories 2005, reliability score 1)

- Supporting 7-day range finding study (CIT Safety & Health Research Laboratories 2006, reliability score 2)

- Multiple additional supporting studies with tetraethyl orthosilicate which are reliability score 4. These studies are non-guideline (or not specified), and the GLP status is not specified.

The repeated dose inhalation toxicity data set for tetraethyl orthosilicate is comprised of:

- Key study similar to OECD Test Guideline 412 study for tetraethyl orthosilicate (Omae et al. 1995, reliability score 2)

- Supporting study similar to OECD Test Guideline 412 with tetraethyl orthosilicate (Nakashima et al. 1994, reliability score 2)

- Multiple additional supporting studies with tetraethyl orthosilicate which are reliability score 4. These studies either pre-date OECD guidelines and GLP, or the studies are either non-guideline (or not specified) and the GLP status is not indicated.

 

As mentioned, the repeated dose oral toxicity data for the tetraethyl orthosilicate condensed hydrolysis product synthetic amorphous silica (SAS) is read across to tetraethyl orthosilicate. The basis for this approach is presented in detail under “basis for SAS read-across below.” In summary, the analysis indicates that the tetraethyl orthosilicate final condensed hydrolysis product polysilicic acid is considered equivalent to SAS. Furthermore, following neat or aqueous-based tetraethyl orthosilicate dosing, insoluble polysilicic acid will predominate in the small intestine and the concentration of soluble monosilicic acid (expressed as ‘SiO2 equivalent’) is expected to be comparable to the level expected after SAS administration.

 

A summary for the non-silane hydrolysis product ethanol is available (OECD SIDS Document UNEP Publication; SIDS Initial Assessment Report for SIAM 19; Berlin, Germany 19-22 October 2004, www.inchem.org/). Generally, in repeat dose studies in animals with ethanol very large doses are used, and often specific endpoints relating to known effects in humans are the primary focus of such studies. However, adverse effects on the liver have been noted in animals but only at very high doses >8000 mg/kg bw/day. Ethanol is not classified for repeated dose toxicity in Annex VI of Regulation (EC) No 1272/2008.

 

ORAL ROUTE

 

SYSTEMIC EFFECTS

 

BASIS FOR SAS READ-ACROSS

The overall basis of the read-across of repeated dose oral toxicity data from SAS to tetraethyl orthosilicate is two-fold, including first the equivalency of the tetraethyl orthosilicate final condensed hydrolysis product polysilicic acid and SAS. Secondly, after neat or aqueous-based tetraethyl orthosilicate dosing and after SAS dosing, the concentration of monosilicic acid in the small intestine is expected to be similar (100 - 150 mg/L as 'SiO2 equivalent'). However, as discussed further under “study findings” below, the presence of a corn oil vehicle (OECD Test Guideline 422 study) would significantly delay the hydrolysis of tetraethyl orthosilicate and result in artificially high concentrations of soluble monosilicic acid in the kidneys which would immediately condense to the insoluble polysilicic acid within this organ.

 

Under aqueous conditions, the tetraethyl orthosilicate hydrolysis product monosilicic acid condenses (gels) to insoluble polysilicic acid (equivalent to SAS) at concentrations higher than 100-150 mg/L ‘SiO2 equivalent’ in water (Holleman-Wiberg, 2001). At low pH (e.g., in the stomach), the data indicate a similar range of 120-150 mg/L ‘SiO2 equivalent’. The process of soluble monosilicic acid condensation to insoluble polysilicic acid reflects a dynamic continuum, takes time, and is dependent upon multiple factors:

•          Concentration

•          pH

•          Temperature

•          Presence of disaggregating substances such as solvent (e.g., ethanol)

•          Ionic strength (will increase with salt / dissolved solids)

 

The variation of pH across the gastrointestinal (GI) tract is a critical factor, with these residence time versus pH data available (rat GI pH for mouth and upper stomach estimated herein):

	Human residence time	Human pH	Rat residence time	Rat pH
Mouth (saliva)	1 minute	6.5-7.5	N/A	Probably 6.5 -7.5
Upper stomach	30-60 minutes	4.0-6.5	N/A	Probably 5.1-6.5
Lower stomach	60-180 minutes	1.5-4.0	N/A	3.1-5.1
Duodenum	30-60 minutes	7.0-8.5	N/A	6.9
Small intestine [Jejunum & Ileum]	60-300 minutes	4.0-7.0	N/A	7.4
Large intestine [Cecum and Colon]	10 hours to days	4.0-7.0	N/A	6.4-6.8

 

At very high concentrations and over time, polysilicic acid will condense to silicon dioxide (SiO2).

 

Monosilicic acid and polysilicic acid are naturally occurring substances which are ubiquitous in the environment. Soluble monosilicic acid is the major bioavailable form of silicon and plays an important role in the biogeochemical cycle of silicon (ECETOC, 2006). Typical background concentrations of monosilicic acid in the environment are up to 75 mg/L ‘SiO2 equivalent’ in river water and up to 14 mg/L ‘SiO2 equivalent’ in seawater (Iler, 1979).

 

The literature gives various values for the solubility of silicic acid, determined indirectly as ‘SiO2 equivalent’ because the soluble species cannot be directly measured:

 

The solubility of monosilicic acid according to Alexander et al. (1954) at 25°C:

·  150 mg/L ‘SiO2 equivalent’ at pH 2.0 and pH 3.0 a

·  130 mg/L ‘SiO2 equivalent’ at pH 4.2 a

·  110 mg/L ‘SiO2 equivalent’ at pH 5.7

·  100 mg/L ‘SiO2 equivalent’ at pH 7.7 b

·  490 mg/L ‘SiO2 equivalent’ at pH 10.3

·  1120 mg/L ‘SiO2 equivalent’ at pH 10.6

The solubility of monosilicic acid according to Goto and Okura (1953) at 25 °C:

·  120 mg/L ‘SiO2 equivalent’ at pH 2.0 a

·  150 mg/L ‘SiO2 equivalent’ at pH 7.0 b

 

The solubility of monosilicic acid according to Elmer and Nordberg (1958) at neutral pH:

·  170 mg/L ‘SiO2 equivalent’ at 35°C

·  270 mg/L ‘SiO2 equivalent’ at 65°C

·  465 mg/L ‘SiO2 equivalent’ at 95°C

 

a Data at low pH, similar to the stomach

b Data at neutral pH, similar to the duodenum and parts of the intestine

 

Under aqueous conditions (following ingestion of tetraethyl orthosilicate, or after neat or aqueous-based gavage dosing) and based on physicochemical properties, tetraethyl orthosilicate will rapidly hydrolyse to soluble monosilicic acid will occur and the monomer will start to condense to form insoluble polysilicic acid. This condensation will start to occur when the concentration of monosilicic acid reaches approximately 100-150 mg/L ‘SiO2 equivalent’ in the gastric juice, with 150 mg/L selected as the representation value.

 

Gel times for a colloidal silica product (Ludox) [30% SiO2, 0.3% Na2O with 15 nm particle size] at various pH values (Shell 1962) are available:

pH	When HCl added
pH 9.8	very stable
pH 8.0	330 hours
pH 7.0	8 hours
pH 6.0	3 hours
pH 5,0	3 hours
pH 4.0	4 hours
pH 3.0	27 hours
pH 2.0	265 hours

 

Following dosing with tetraethyl orthosilicate by oral gavage, partitioning will occur between the dose vehicle (if used) and the aqueous environment in the stomach.

For neat or aqueous-based gavage dosing, the ‘SiO2 equivalent’ dose level is first estimated as:

 

Dose level (mg/kg bw/day, as ' SiO2 equivalent')

= dose concentration (i.e., solubility, in mg/L) x dosing volume (in L) / body weight (in kg)

= 0.8 mg/kg bw/day ' SiO2 equivalent'

 

Where:

Dose concentration = 150 mg/L, as ‘SiO2 equivalent’

Estimated aqueous dosing volume = 1.5 mL = 0.0015 L per day

Body weight of rat = 0.3 kg

 

Therefore, based on the representative monosilicic acid solubility of 150 mg/L, the maximum ' SiO2 equivalent' dose level that could be administered in practice to ensure exposure mainly to monosilicic acid in the stomach of experimental animals is approximately 0.8 mg/kg bw/day or less.

 

To estimate the corresponding maximum dose for tetraethyl orthosilicate, a correction for molecular weight is applied:

 

Dose level [tetraethyl orthosilicate]

= Dose level [SiO2 equivalent] x MW [tetraethyl orthosilicate] / MW [SiO2]

= 3 mg/kg bw/day

 

Where:

Dose level [SiO2 equivalent] = 0.8 mg/kg bw/day

MW [tetraethyl orthosilicate] = 208.33 g/mol

MW [SiO2] = 60.08 g/mol

 

Therefore, the maximum dose level of tetraethyl orthosilicate (neat or in an aqueous vehicle) that could theoretically be administered to ensure exposure mainly to monosilicic acid is approximately 3 mg/kg bw/day. This dose level is well below the typical dosing levels for such studies (100 to 1000 mg/kg bw/day). This comparison suggests that after neat or aqueous-based tetraethyl orthosilicate administration, the predominant hydrolysis product present in the small intestine will be the condensed and insoluble hydrolysis product polysilicic acid which is equivalent to SAS. Thus, it is appropriate to use toxicology data on SAS to address the potential for oral toxicity of tetraethyl orthosilicate, with these SAS data specifically relevant to the tetraethyl orthosilicate Chemical Safety Report (CSR).

 

As discussed for the OECD Test Guideline 422 study (see “study findings” below), a corn oil vehicle would delay tetraethyl orthosilicate hydrolysis such that artificially high (g/L) concentrations of soluble monosilicic acid would occur in the small intestine, ultimately resulting in exposure of the kidneys to high concentrations of the insoluble polysilicic acid. In contrast, such renal exposures would not be expected after neat or aqueous tetraethyl orthosilicate dosing or after SAS dosing. The lack of significant renal findings (sporadic, no dose-response, no positive histopathology; author conclusion of a systemic NOAEL at the highest dose tested) in the SAS OECD Test Guideline 408 study (distilled water vehicle) supports this conclusion.

 

ORAL ROUTE

 

SYSTEMIC EFFECTS

 

STUDY FINDINGS

In the key 90-day oral repeated dose toxicity study conducted according to OECD Test Guideline 408 (including observations and examinations) and in compliance with GLP (Kim et al., 2014, reliability score 1), Sprague-Dawley rats were exposed daily to 20 or 100 nm of two forms of synthetic amorphous silica (described as SAS and NM-202; differing in particle size and specific surface area) in doses of 500, 1000 or 2000 mg/kg bw/day, respectively, via oral gavage for 90 days (10 animals/sex/group). The particles were described as either 20 or 100 nm in diameter. Extra animals were included in the control (received distilled water only) and highest dose groups to allow for a two-week post-exposure recovery period. Satellite groups included the control group and the highest dose groups and were observed for 14 days following the initial 90 days of exposure.

 

Irrespective of particle size, no treatment-related clinical signs occurred during the experimental period for rats of either sex. A few occurrences of salivation, loss of fur and wound scratching occurred in the 2000 mg/kg bw/day group for 20 nm silica. However, there were no dose-dependency and the findings occurred spontaneously and therefore, were not considered related to treatment. No treatment-related differences in mortality, body-weight changes, food or water consumption, haematological findings, clinical biochemistry nor urinalysis were observed.

 

Sporadic organ weight effects were observed. Relative liver weight was statistically significantly decreased whereas the absolute and relative lung weights were statistically significantly increased in males of the 2000 mg/kg bw/day dose group exposed to 20 nm silica compared to the control group. A statistically significantly increase in absolute weights of kidney, lung and submandibular glands and a statistically significant increase in the relative weights of kidneys and lungs were observed in the male 2000 mg/kg bw/day recovery group exposed to 100 nm silica. Females in the high dose recovery group had statistically significantly decreased absolute and relative ovary weights. The study authors concluded that, due to the sporadic nature of the organ weight findings, the observations were not treatment-related.

 

The histopathology findings lacked a dose-response to treatment, were sporadic, and/or were minimal to mild in nature. Pale yellowish discolouration on the posterior surface of the left lateral lobe of the liver was observed in one male animal from the 20 nm silica 500 mg/kg bw/day group. With regard to 100 nm silica, a small sized right testis and epididymis were observed in one male from the 500 mg/kg bw/day group. A light yellow discoloration of the left lateral lobe of the liver (about 1 mm diameter) and a light yellow-coloured cyst with adjacent fat near the right kidney were also noted in another male in the 1000 mg/kg bw/day group. In addition, a small-sized left ovary was observed in one female from the 2000 mg/kg bw/day recovery group. However, no dose-dependency occurred and the effects were therefore not considered to be related to treatment. The histopathology examination revealed granulomatous inflammation in one male rat from the 1000 mg/kg bw/day group and two from the 2000 mg/kg bw/day group treated with 20 nm silica. Chronic bronchioalveolar inflammation was observed in one male rat from the 2000 mg/kg bw/day group treated with 20 nm silica. There were no adverse findings from the groups treated with 100 nm silica. These lesions were observed in four cases in total and were minimal to mild in severity.

Based on an overall conclusion of no observed adverse effects, the systemic NOAELs for 20 nm (described as SAS) and 100 nm (described as NM-202) colloidal silica particles were concluded to be ≥2000 mg/kg bw/day in Sprague-Dawley rats (Kim et al. 2014).

 

In a supporting study conducted according to OECD Test Guideline 422 and in compliance with GLP (CIT Safety & Health Research Laboratories 2005, reliability score 1), tetraethyl orthosilicate was administered by oral gavage in corn oil to Sprague-Dawley rats at 10, 50 and 100 mg/kg bw/day. Observations and examinations followed OECD Test Guideline 422. The systemic NOAEL was 50 mg/kg bw/day for females and 10 mg/kg bw/day for males, based primarily on adverse effects on the kidneys (tubular nephropathy). However, as discussed below, these effects are considered a consequence of the corn oil dosing.

 

Hydrolysis of a substance will not occur until it is released from the organic phase. With a corn oil vehicle, the bulk of the release of tetraethyl orthosilicate from the non-polar organic phase (corn oil) to the aqueous phase and its subsequent hydrolysis would be delayed until the pH is raised to about pH 7 by the addition of bile in the duodenum. At this elevated pH, some of the corn oil fatty acids would be neutralised to sodium salts which would then act as anionic surfactants. As a result, the remaining corn oil would form an emulsion of small droplets. The creation of this emulsion would immediately release the tetraethyl orthosilicate into the aqueous phase in the duodenum. Only then could the hydrolysis of tetraethyl orthosilicate to monosilicic acid and ethanol occur.

 

Once released from the corn oil into the duodenum, tetraethyl orthosilicate would hydrolyse rapidly to monosilicic acid. The rate of hydrolysis would be regulated by the pH. The resulting concentration of monosilicic acid in the duodenum and then small intestine would be controlled by the rate of condensation to insoluble polysilicic acid over time. Since the condensation process would take a number of hours at neutral pH, most of the insoluble polysilicic acid would form in the small intestine after corn oil dosing. Correspondingly, such dosing would result in artificially high (g/L) levels of soluble monosilicic acid in the small intestine.

 

In contrast, after dosing with SAS in water (e.g., OECD 408 study), the concentration of monosilicic acid in the small intestine would not exceed the SAS solubility level of 100 to 150 mg/L ‘as SiO2 equivalent.’

 

A lower concentration of soluble monosilicic acid in the small intestine also would be expected after neat or aqueous-based dosing of tetraethyl orthosilicate as compared to a corn oil vehicle. In this case, hydrolysis would rapidly initiate in the upper stomach (not the duodenum) under the mildly acidic conditions (pH 4.0-6.5) and would be almost completely hydrolysed by the time it reaches the more acidic lower stomach (pH 1.5-4). The hydrolysis half-life has been determined to be 7 minutes at pH 4.0. Thus, monosilicic acid would start to undergo condensation in the lower stomach, a process that would be completed when the pH is raised to neutral in the duodenum by the addition of bile. As a result, the concentration of soluble monosilicic acid would approach 100-150 mg/L ‘SiO2 equivalent’ when it enters the small intestine. This level of monosilicic acid contrasts starkly to the g/L levels that would be observed when dosing tetraethyl orthosilicate in a corn oil vehicle. Further, the concentrations of monosilicic acid in the small intestine after neat or aqueous-based tetraethyl orthosilicate dosing would be more realistic as well as more comparable to those seen after SAS administration (again, in the 100-150 mg/L ‘SiO2 equivalent’ range).

 

From the small intestine, the soluble monosilicic acid would then pass into the blood stream. However, any polysilicic acid formed in the duodenum or small intestine would be insoluble and would not be absorbed into blood.

 

Monosilicic acid in the blood stream would pass to the kidneys, where the removal of water would cause the monosilicic acid to immediately condense into insoluble polysilicic acid.

 

Overall, the renal findings in the tetraethyl orthosilicate OECD Test Guideline 422 study with a corn oil vehicle are considered a consequence of the artificially high levels of polysilicic acid formation within the kidneys. That is, the corn oil dosing would have delayed tetraethyl orthosilicate hydrolysis (until the duodenum) and resulted in high concentrations of monosilicic acid in the small intestine, absorption of more monosilicic acid from the small intestine into blood, and the exposure of the kidneys to high levels of the monosilicic acid which would immediately condense to insoluble polysilicic acid within the kidneys.

 

In a supporting 7-day oral range-finding study conducted in compliance with GLP (CIT Safety & Health Research Laboratories 2006, reliability score 2), tetraethyl orthosilicate was administered via oral gavage in corn oil to Sprague-Dawley rats at 200, 600, and 1000 mg/kg bw/day. The study was undertaken to provide the basis for dose selection in the OECD Test Guideline 422 study. A systemic NOAEL was not determined because toxicity (body weight and food consumption reduction, effects on the kidney, prostate, and seminal vesicles) was observed at the lowest dose tested (200 mg/kg bw/day). The renal findings are consistent with the supporting OECD Test Guideline 422 findings. However, as discussed in detail for the OECD Test Guideline 422 study above, the renal findings are regarded as a result of the corn oil dosing.

 

INHALATION ROUTE

 

SYSTEMIC AND LOCAL EFFECTS

In the key repeated inhalation study conducted in a manner similar to OECD 412 (no information on GLP status, Omae et al. 1995, reliability score 2), male mice were exposed to tetraethyl orthosilicate at 50 or 100 ppm vapour for 2 or 4 weeks, with sacrifice for half the mice 1 day after the end of exposure and, for the other half, after a 2-week observation period.

Red blood cell, haemoglobin, and haematocrit values were lower in exposed mice than in non-exposed mice, with mixed statistical significance. The proportion of neutrophils were significantly higher at 50 and 100 ppm in the 2-week study, but not in the 4-week. The neutrophil count was significantly higher at 100 ppm in the 4-week study.

Kidney weights were slightly lower at 50 and 100 ppm after 2 or 4 weeks, but this renal result was not significant.

 

Histopathology findings in the kidney were observed at 100 ppm (approximately 0.9 mg/L) when mice were exposed over 2 or 4 weeks. In the kidney, 2/10 mice exposed to 100 ppm for 2 weeks and 2/10 mice exposed to 100 ppm for 4 weeks developed histopathological lesions (tubulointerstitial nephritis, TIN). The lesions were wedge-shaped and extended from the cortex into the medulla. In the affected regions, small numbers of neutrophils and mononuclear inflammatory cells had infiltrated interstitial tissue, and some tubules were dilated, exhibiting atrophy and obliteration. No kidney lesions were observed in mice exposed to 50 ppm.

 

In the nasal mucosa, histopathology findings (inflammatory) were observed in almost all the mice (9/10 to 10/10) exposed to 50 ppm or 100 ppm, for 2 or 4 weeks. Exudates containing inflammatory cells and necrotic epithelial cells were observed in the nasal cavity, inflammatory cell infiltration in submucosal tissue, and eosinophilic hyaline droplets predominantly in the olfactory epithelium. The findings were slightly more severe in the mice exposed to 100 ppm.

 

In the bone marrow, increases in the M: E ratio were observed, no further details provided.

The LOAEC for systemic and local effects was 50 ppm vapour (approximately 0.4 mg/L) in male mice after a 2- or 4-week exposure, based on histopathological (inflammatory) findings in the nasal mucosa and haematological changes. Renal histopathology was observed at 100 ppm (approximately 0.9 mg/L). A NOAEC was not identified. However, both the renal and nasal cavity findings are considered related to a physical effect of the insoluble polysilicic acid, and not a toxic effect of tetraethyl orthosilicate. First, the relative humidity maintained during the study (60%) makes it more likely that some fraction of the tetraethyl orthosilicate had already hydrolysed to monosilic acid prior to mouse inhalation. In addition, inflammation occurred in the nasal cavity in the absence of other lung findings. This observation suggests the formation of sufficient insoluble polysilicic acid within the nose to cause inflammation.

 

Further, similar to the suggested oral OECD Test Guideline 422 mechanism of effect, any monosilicic acid absorbed through the nasal mucosa into blood would be transported to the kidneys and then immediately condense to polysilicic acid when the water is removed, again suggesting a physical effect of insoluble polysilicic acid in this organ.

 

In a supporting repeated inhalation study conducted in a manner similar to OECD 412 (no information on GLP status, Nakashima et al. 1994, reliability score 2), male mice were exposed to tetraethyl orthosilicate at 200 ppm vapour for 2 or 4 weeks (at 60% relative humidity), with sacrifice 1 day after the end of exposure. Body weight gain was suppressed during exposure for 2 or 4 weeks but, in the 2-week exposure group, recovered by the end of observation. The kidney of almost all exposed mice developed TIN), 8/10 and 9/10 mice after 2- and 4-week exposures, respectively, across the two sacrifice timepoints. In the nasal mucosa, histopathology findings were seen in all mice (10/10) killed 1 day after exposure, across the two exposure durations. The systemic and local effect LOAECs for tetraethyl orthosilicate were 200 ppm vapour (approximately 1.7 mg/L) in mice after a 2- or 4-week exposure, based on histopathological findings in the kidney (including TIN) and in the nasal mucosa (inflammatory). No NOAEC values identified. The renal and nasal cavity histopathology results support the key study findings. However, as discussed for the Omae et al. 1995 study, both the renal and nasal cavity findings are considered related to a physical effect of the insoluble polysilicic acid rather than tetraethyl orthosilicate toxicity.

Justification for classification or non-classification

Based on the available data for the tetraethyl orthosilicate condensed hydrolysis product polysilicic acid (equivalent to SAS), tetraethyl orthosilicate does not require classification for specific target organ toxicity following repeated exposures according to Regulation (EC) No. 1272/2008.