Tetrakis(2-butoxyethyl) orthosilicate

Endpoint summary

Description of key information

In a Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, conducted according to OECD TG 422 and in compliance with GLP, the reported NOAEL for tetrakis(2-butoxyethoxy) silane was 25 mg/kg bw/day for females and 100 mg/kg bw/day for males based on histomorphological adverse effects observed in mid and high dose females and high dose males (Eurofins, 2016).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

System:

haematopoietic

Organ:

blood

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

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

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, conducted according to OECD TG 422 and in compliance with GLP, the test item was administered daily to three groups (low, mid and high dose) of ten male and ten female rats for a treatment period of 54 days including pre-mating, mating, gestation and up to post-natal day 3 for females. Males were dosed up to day 28 of the study period. Recovery group animals which were observed for 14 days after the last dose administration, were included in order to allow a detection of possible delayed occurrence or persistence of toxic effects. The animals were tested with 25, 100, 250 mg/kg bw/day. The high dose of 250 mg/kg bw/day was reduced to 175 mg/kg bw/day from day 3 for 6 male and 6 female animals of the high dose group and all animals of the high dose recovery group or from day 2 for 4 male and 4 female animals of the high dose group. The test item was suspended in dried corn oil and test item formulation was freshly prepared every day.

During the administration period, the animals were observed each day for signs of toxicity and mortality. Animals that died were examined microscopically. Body weight and food consumption were measured weekly. Food consumption measurements were not taken during the mating period in female animals and the mating and post-mating period in male animals. In recovery group animals, food consumption was measured on the day corresponding to the body weight measurements. Blood samples were collected at terminal sacrifice from five randomly selected males and females from each group and were used for haematological and clinical biochemistry evaluations. Urinalysis was also performed on samples collected at terminal sacrifice from five males and five females randomly selected from each group. Functional observations including sensory reactivity to different stimuli, grip strength, motor activity assessment and other behaviour observations were performed on the week before treatment commencement and at the end of the study. After 14 days of treatment, animals were housed together for maximum of 14 days mating period in 1:1 ratio. Vaginal smears of females were used as proof of mating. When mating was confirmed, females were separated and housed individually. Each litter was examined immediately after delivery to establish number and sex of pups, stillbirths, live births, runts and presence of gross abnormalities. Live pups were were counted, sexed and weighed within 24 hours of parturition and on day 4 post-partum. Male animals were sacrificed on day 29 of the treatment period. Female animals and pups were sacrificed on post-natal day 4. One non-pregnant female was sacrificed on day 26 from the day of evidence of mating. Pups sacrificed on post natal day 4 and those found dead, were carefully examined for gross external abnormalities. Recovery group animals were sacrificed after the 14 -day recovery period. Full histopathological evaluation of the tissues was performed on five male and five female animals randomly selected from the control and the high dose groups, including one non-pregnant female. These examinations were extended to animals from all other dosage groups for treatment-related changes that were observed in the high dose group. Histopathological examinations were performed on animals from recovery group. Reproductive organs and accessory sex organs were examined in all animals. Gross lesions identified macroscopically were examined microscopically in all animals.

Treatment-related mortalities were observed in the high dose group at the initial dose of 250 mg/kg bw/day. One male from high dose group was found dead on pre-mating day 3 and it showed moderately reduced spontaneous activity a day before it was found dead. One male from high dose recovery group and one female from high dose group were euthanized due to overt toxicity on pre-mating day 7. Kyphosis, haematura, sunken flanks, moderate to severe piloerection and half eyelid closure were observed in both animals prior to euthanization. Additionally, reddish nasal discharge was observed in the male animals and moderate to severe salivation, dehydration and reduced spontaneous activity was seen in the female animal. These animals showed adverse clinical signs and weight loss, therefore the high dose was reduced to 175 mg/kg bw/day.

In the surviving males and females from the high dose and high dose recovery group, haematuria, reddish nasal discharge, slight to moderate piloerection, moving the bedding and reduced spontaneous activity were observed up to the end of premating days. Additionally, kyphosis and moderate salivation were observed in females. No clinical signs of toxicity were noted for the rest of the study period. No significant changes were observed during the weekly detailed clinical observations. No significant changes were noted during the functional observation battery test for neurological assessment. Statistically significantly lower food consumption and body weight gain were noted in high dose and high dose recovery animals during the first week of treatment with 250 mg/kg bw/day. After lowering the high dose to 175 mg/kg bw/day food consumption and body weight gain returned to normal.

No adverse effects were observed in total number of pups and mean number of live pups on PND 0. A statistically significantly lower number of live pups was recorded on PND 4 in the high dose group. Moreover, a markedly lower number of male and female live pups was noted on PND 4, but without statistical significance. Lower number of live pups during the lactation period was considered to be treatment-related. The dead pups were cold and did not show suckling, which was related to nursing behaviour of females and therefore, was considered to be secondary to maternal toxicity. Total litter weight was markedly lower in high dose pups on PND 4, which was attributed to higher pup mortality. Delivery and viability indices were lower in the high dose group, which was considered to be treatment-related.

In high dose males at the end of treatment, statistically significantly lower RBC, HB, HCT and MCHC and statistically significantly higher MCV and MCH were observed when compared to the corresponding control group. Statistically significant increase in MCV and MCH in the high dose recovery group males was also observed.

In females at the end of treatment, statistically significantly lower RBC were observed in the mid dose and high dose groups compared to the control. Statistically significantly higher MCV and MCH were observed in the high dose group compared to the control. Additionally, the reticulocyte count was higher in the female low dose, mid dose and high dose groups compared to the control in a dose response pattern, but without statistical significance. The mean reticulocyte count in the mid dose and high dose groups were above the range of historical control data. These changes could be due to possible bleeding during parturition. Moreover, this corresponded to a statistically significantly lower mean RBC in the mid dose and high dose groups and was related to test item treatment except in the low dose group where the mean RBC was not statistically significant and was considered non-adverse. This finding was related to test item treatment. The haematological findings in males and females at the end of treatment in the high dose group were due to haematotoxicity of test item as evident from the treatment-related pathological changes characterized by haemolysis-related findings including haemoglobinuric nephrosis and increased erythroid haemopoiesis as well as splenic infarction in survivors and decedents, and by the disseminated thrombosis in decedents. In both males and females, there were no effects of test- item on coagulation parameters including prothrombin time (PT) and activated prothrombin time (aPTT).

In both males and females (main and recovery groups), there were no effects of test item on coagulation parameters, serum biochemistry and urine parameters between the dose and corresponding control group. Black and marbled discoloration in kidneys, pale discoloration in liver, pituitary gland, adrenal glands and brain were observed in one male and one female animals that were euthanized on study day 7 for ethical reasons. In addition, pink discolouration in the testes was observed for the male animal. No macroscopic abnormalities were observed in one male animal that died on study day 2. Haemoglobin casts and/or tubular necrosis/degeneration were considered to be the cause for black and marbled discolouration of kidneys. Pale discoloration of spleen (no. 51) and adrenal glands (no. 87) were deemed to correlate microscopically with infarction of the spleen and increased vacuolation (fatty change) of zona fasciculata of the adrenal glands, respectively. Pink discoloration of testes (no. 51) was deemed to correlate microscopically with haemorrhage in the interstitium. It was considered that macroscopic findings in spleen, kidneys and testes corresponded to histologic lesions being attributed to treatment with the test item. In the survivors, small spleen was noted in two high dose males. Pale discolouration of the spleen was noted in another male. These changes correlate microscopically with infraction of the spleen caused by the test item. Fluid-filled uterus, red discolouration in the thymus, haemorrhage in the cecum, absent left testes and epididymis were also observed in the survivors, however these changes were normal physiological change, congenital anomaly, or incidental findings that were not treatment related.

In males at the end of the treatment, statistically significantly higher kidney weight relative to body weight was observed in the high dose group. In males at the end of recovery period, there was statistically significantly higher absolute kidney and liver weight, statistically significantly higher spleen weight (absolute and relative to body weight) and statistically significantly higher pituitary gland weight (absolute and relative to body weight) in the high dose recovery group compared to the control. In females at the end of the treatment, statistically significantly higher spleen weight relative to body weight was observed in the high dose group, which did not reverse after the 14-day recovery period. Statistically significantly higher spleen weight (absolute and relative to body weight) was noted in the high dose recovery group compared to the corresponding control. Additionally, in females, there were statistically significantly higher brain weights relative to body weight in the high dose group and statistically significantly higher liver weight relative to body weight in the mid dose group compared to the control. At the end of recovery, there was also statistically significantly higher absolute kidney weight compared to the corresponding controls. The observed changes in organ weight correlated histopathologically with observed structural changes in the kidneys and spleen of both sexes, and those were the changes related to haematotoxic effects of the test item.

Histopathological examinations showed that the test item produced haematotoxicity and the histopathological changes in high dose males and females of all dose groups were treatment-related. Two cases of granular casts and an increased degeneration/regenerationin the kidneys of females treated with 100 mg/kg bw/day were indicators of toxicity in kidneys. The granular casts that represent tubular cell necrosis are deemed to be of adverse nature and secondary to haemoglobinuric nephrosis.

Concentration analysis of formulation samples was measured in study weeks 1, 3, 5 and in last week for all dose groups. Mean recovery in the low dose, mid dose and high dose groups were 96%, 96% and 95% of nominal concentration, respectively. Homogeneity of formulation samples was investigated in study weeks 1 and 5 for low dose and high dose groups. Mean recovery measured in the low dose group was 99% and 100% of nominal value. Mean recovery measured in the high dose group was between 93% and 101%. The coefficient of variation (COV) of the different sampling locations (top, middle, bottom) was 1.4% and 4.4% in the low dose group and between 0.6% and 5.6% in the high dose group. All samples were homogenous, as COV was below or equal 10%.

In the Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test, conducted according to OECD TG 422 and in compliance with GLP, the reported NOAEL for tetrakis(2-butoxyethoxy) silane was 25 mg/kg bw/day for females and 100 mg/kg bw/day for males based on haematotoxic and histomorphological adverse effects observed in mid and high dose females and high dose males.

The adverse effects of the hydrolysis product, 2-butoxyethanol have been well characterised by numerous experimental animal and in vitro tests and apply to oral, dermal and inhalation exposures. As described in the toxicokinetic section, 2 -butoxyethanol is metabolised to 2-butoxy-acetic acid (BAA) in all studied species, including humans. However, there is a large species variation in their susceptibility to the haemolytic agent, BAA. Rats and mice are the most susceptible, rabbits less susceptible, and guinea pigs and humans the least susceptible. In fact, in vitro testing has shown that erythrocytes from humans are at least one order of magnitude less susceptible than erythrocytes from rats and mice to the haemolytic effects of BAA (EU SCHER, 2008; Burhan & Sullivan, 1993 - get this ref). Importantly, it has also been demonstrated that toxic but not lethal doses of 2-butoxyethanol provide protection against lethal doses administered within 1 -2 weeks. The hypothesis for this protection is that following the initial toxic dose of 2-butoxyethanol, which destroys erythrocytes, new erythrocytes are produced that are less susceptible to the haemolytic effects of the metabolite BAA. This protection reduces as the new cells age by 3-4 weeks following administration of the sublethal dose (Sivarao & Mehendale, 1994). There is no evidence for development of chronic toxicity independent of haemotoxicity.

The observed toxicity (haemotoxicity and secondary effects of haemotoxicity) in the OECD 422 study (Eurofins, 2016) on the registered substance is due to metabolism of the hydrolysis product, 2-butoxyethanol. There were no additional effects observed that could be due to the silanol hydrolysis product of the registered substance. The NOAEL from this OECD 422 study is in the same order of magnitude as a 13-week drinking water study (cited in EU Risk Assessment, 2008) on 2-butoxyethanol (69 and 82 mg/kg bw/day for males and females, respectively; the small difference in NOAEL is a result of the choice of doses studied in each test).

The other hydrolysis product of the registered substance is monosilicic acid, which will condense to form amorphous (poly)silicic acid). Amorphous (poly)silicic acid is equivalent to synthetic amorphous silica (SAS). Subchronic repeated dose toxicity, reproductive toxicity and developmental toxicity studies on SAS show that there is no hazard associated with this substance.

EU Risk Assessment Report on 2 -butoxyethanol (EGBE), CAS 111 -76 -2, Human Health Part, 2008.

Ghanayem B.I and Sullivan C.A. (1993) Assessment of the Haemolytic Activity of 2-Butoxyethanol and its Major Metabolite, Butoxyacetic Acid, in Various Mammals Including Humans. Human and Experimental Toxicology, Volume 12, 4, 305 -311.

Sivarao D.V. and Mehendale H.M. (1994) 2-Butoxyethanol autoprotection is due to resilience of newly formed erythrocytes to hemolysis. Archives of Toxicology, 69, 526.

Justification for classification or non-classification

Based on the available data for tetrakis(2-butoxyethyl) orthosilicate, classification for specific target organ toxicity (STOT)- repeated exposure, category 2 "H373: May cause damage to organs through prolonged or repeated exposure exposure" is required according to Regulation (EC) No 1272/2008. The target organ was identified as blood.