2-ethoxy-2-methylpropane

Endpoint summary

Description of key information

Regarding oral exposure, results of chronic oral (drinking water) studies demonstrate alterations in clinical chemistry parameters in male and female rats with a NOAEL of 121 mg/kg bw/day. This NOAEL is quite conservative given the occurrence of reduced water consumption (and probable dehydration) noted in the higher treatment groups, an observation considered related to the unpleasant taste of ETBE.
The key studies for DNEL derivation for long term inhalation exposure are the 90 day and 2 year studies in rodents. The overall NOAEC was 500 ppm (2100 mg/m3) based on liver effects after sub-chronic exposure (mainly female mice, 1750 and 5000 ppm (7350 and 21000 mg/m3)) and reduced survival, slight haematological changes, altered clinical chemistry parameters and liver effects (increased relative weight, bile duct hyperplasia) (rats, 1500 and 5000 ppm (6300 and 21000 mg/m3)) after chronic exposure.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

121 mg/kg bw/day

Study duration:

chronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2 100 mg/m³

Study duration:

chronic

Species:

rat

Additional information

Results are available from two repeated oral toxicity studies in which male and female F344 rats received ETBE either by oral gavage at doses up to 400 mg/kg bw/day for 6-months (Chemicals Evaluation and Research Institute, 2008a), or via drinking water at received doses of approx. 540-560 mg/kg bw/day for up to 2-years (Japan Bioassay Research Centre, 2010a). The studies were GLP-compliant, and comparable in design to OECD Guideline 452 and OECD 453, respectively. There were no effects on survival in either study, however decreased spontaneous movement was noted after gavage (bolus) administration which persisted in the high dose group for several hours (results of functional observations were unremarkable). The key study for derivation of an oral DNEL for the general population is the 2-year drinking water investigation, where a NOAEL of 121 -171 mg/kg bw/day was obtained for males and females respectively, based on alterations in clinical chemistry parameters in the absence of any detectable histopathological change. Higher treatment levels were associated with an increased severity of chronic nephropathy, mineral deposition in the renal papilla and urothelial hyperplasia of the renal pelvis in males, however the toxicological significance of these findings is difficult to judge given a concurrent, marked (20-40%) reduction in water consumption. This is considered related to the unpleasant taste of ETBE, and would be expected to have lead to dehydration with unknown consequences for the clinical data underpinning the NOAEL. The NOAEL from this study is therefore considered quite conservative.

Repeated dose inhalation toxicity data are available for the rat covering durations of exposure of 28-days (IIT Research Institute, 1991), 90-days (CIIT, 1986a; Mitsubishi Chemical Safety Institute Ltd, 2008c) and 2-years duration (Japan Bioassay Research Center, 2010b). Sub-chronic inhalation toxicity data are available also for the mouse (CIIT, 1986c).

The available rat 28-day repeated inhalation toxicity study (OECD Guideline 412) showed that repeated exposure to high doses of ETBE (4000 ppm (16720 mg/m3)) produced transient signs of CNS depression and associated changes in body temperature and possibly hind limb splay (see section on neurotoxicity). Sub-chronic exposure of rats and mice according to EPA OTS 798.2450 (90-Day Inhalation Toxicity) Guideline or OECD 413 returned an overall NOAEC of 500 ppm (2100 mg/m3). Effects reported were: effects on testis (male rats, 1750 and 5000 ppm (7350 and 21000 mg/m3)), bone marrow congestion (female rats, 1750 and 5000 ppm) and liver effects (female rats, 5000 ppm; female mice, 1750 and 5000 ppm).

Chronic inhalation toxicity data are also available for the rat (Japan Bioassay Research Centre, 2010b), from a study following OECD Guideline 453 and performed under GLP. The principle findings included a reduction in survival (females, 1500 ppm), slight haematological changes (females, 5000 ppm), alterations in clinical chemistry values (both sexes, 5000 ppm), and liver effects (increased relative weight with bile duct hyperplasia in males, 5000 ppm) leading to an overall NOAEC of 500 ppm.

An increased incidence of renal nephropathy, cell proliferation and accumulation of protein (hyaline) droplets in proximal tubules reported in some studies for male rats exposed to ETBE was associated with positive immunoreactivity toward alpha-2u-globulin, suggesting the findings were specific to the male rat (accumulation of alpha-2u-globulin) and have no relevance for humans. Therefore, this effect is not taken into account for DNEL derivation.

Kidney histology slides from the 13-week rat inhalation toxicity study on ETBE together with renal tissue from the 2-year drinking water study (and the associated 13 week dose range-finding study) were reevaluated by Hard (2009) and by Research Pathology Associates (2011), respectively. The results of both assessments support the conclusion that ETBE treatment is associated with induction of a male rat-specific syndrome of alpha-2u-globulin nephropathy as a primary mode of action for kidney. The reevaluation also showed that ETBE exacerbates spontaneous chronic progressive nephropathy (CPN) with a dose-related increase of CPN foci. Neither of these two pathologic processes has a human counterpart and therefore neither is relevant for human risk assessment.

Regarding the relevance for human health of the critical effects, the following considerations have to be made:

- Testis effects:

Increases in the percentages of seminiferous tubules with spermatocyte degeneration were observed in Fischer 344 rats in 90 days study at ETBE exposure concentrations of 1750 and 5000 ppm (7350 and 21000 mg/m3). No effects were found at 500 ppm. ETBE did not have an effect on the frequency of stage of sperm development. As reported in the study report, the impact of the response of the testes would be best measured by sperm count, motility assessment and breeding studies.

In general, the testicular observations are common in Fischer 344 rats. This strain is an inbred strain of low fecundity and therefore unacceptable for use in reproduction studies designed to meet international regulatory requirements.

As a result of the observations in Fischer 344 rats in the study by CIIT (1996a), particular attention was paid to the male reproductive system, testis and epididymal evaluations in the performed reproductive/developmental toxicity dose-range finding study (oral dose levels of 0, 50, 250, 500 and 1000 mg/kg bw/day for 12 weeks) which was conducted in two strains of male rat: Fischer 344 (included for consistency with Medinsky et al., 1999) and Sprague Dawley (for compliance with regulatory testing requirements) (Centre International de Toxicologie , 2003). The detailed examination of the testes from F344 rats treated orally by gavage with ETBE for 12 weeks revealed minimal degeneration of the seminiferous tubules in 7/12 controls and 6/12 rats treated with 1000 mg/kg bw/day and minimal vacuolation of the seminiferous tubules in one Fischer 344 rat of each of these groups. In the Sprague-Dawley rats exposed in the same way, there was minimal desquamation of spermatocytes and minimal degeneration of seminiferous tubules in a single control rat. No effects were observed on sperm count and motility assessment in both strains of rats (Centre International de Toxicologie , 2003). Thus, the earlier report of effects of ETBE on the testes was not supported by these results with two strains of rats.

The inhalation exposures of 1750 and 5000 ppm in the CIIT (1996a) study were estimated to have provided received doses of about 555 and 1590 mg/kg bw/day for 5 days per week (e. g., 21.2 mg/l x 0.2 l/min x 0.26* x 360/0.25 kg** = 1587 mg/kg bw where* = pulmonary retention factor for ETBE; ** = mean body weight of male rats in the experiment) according to McGregor (2007). Using the REACH guidance, 1750 and 5000 ppm (7350 and 21000 mg/m3) are calculated to correspond to internal doses of 1066 and 3045 mg/kg bw/day (assuming an 6 hour rat respiratory volume of 0.29 m3/kg bw and 50% inhalation absorption). In either case the estimated oral doses for the 1750 ppm exposure (555 and 1066 mg/kg bw/day) are in the general range of the highest dose tested (1000 mg/kg bw/day) by Centre International de Toxicologie (2003) which failed to find evidence on testes and sperm.

Supporting information for disregarding the testes effects reported in Fischer 344 rats are the subsequent two-generation study and developmental toxicity study with Sprague Dawley rats (Centre International de Toxicologie , 2004a/2004b). No effects on fertility and no developmental toxicity were observed. No testicular changes were present in either the F0 or F1 generations of the 10-week premating segments of the two-generation study with Sprague-Dawley rats, in which there was particularly careful histological examination of the testes (Centre International de Toxicologie , 2004a). Male reproductive function was also unaffected in a one-generation reproduction study in which SD rats received ETBE by oral gavage at doses up 1000 mg/kg bw/day for 16 weeks (Safety Research Institute for Chemical Compounds, 2008).

Furthermore, de Peyster et al (2009) observed no testicular pathology in Fischer 344 rats after 14 days gavage exposure to 1800 mg/kg bw ETBE, nor were effects seen following exposure to ETBE via drinking water with received doses of approx. 540 mg/kg bw/day for up to 104 weeks (Japan Bioassay Research Center, 2010a); chronic inhalation exposure to 5000 ppm ETBE was also without effect in male 344 rats (Japan Bioassay Research Center, 2010b). No testis effects were observed in the available 13 week inhalation study with mice (CIIT, 1996b).

In conclusion, based on all available information, the observed increases in the percentages of seminiferous tubules with spermatocyte degeneration in Fischer 344 rats (reported in one study but not in others) is not considered a critical effect of ETBE for human risk assessment and this effect is therefore not taken into account for DNEL derivation.

- Liver effects:

In the rat and mouse inhalation studies conducted by CIIT (1986a/b/c), statistically increased liver weights were observed in rats at 1750 (males only) and 5000 ppm (both sexes) (equivalent to 7350 and 21000 mg/m3, respectively). In mice (mainly females), liver weight increase (absolute weight was measured; in the absence of an effect on body weight also the relative liver weight must have been increased), centrilobular hypertrophy and increased labelling indices (LI)) were observed at 1750 and 5000 ppm (7350 and 21000 mg/m3). The biological relevance of these changes is discussed in section 5.8.3. It is also the case that rodents are very sensitive to these effects which will not, therefore, be used for the purposes of DNEL derivation.

- Bone marrow congestion:

Although the bone marrow congestion was noted in female Fischer 344 rats following sub-chronic inhalation exposure to 1750 ppm and above (Chemical Industry Institute of Toxicology, 1996a; Medinsky et al., 1999a), the hematopoietic cell population appeared unaffected. The change identified in peripheral hematology (increased MCV) was not considered clinically significant and it is unlikely that bone marrow congestion has any clinical relevance. Nor was any comparable effect observed in male and female Fischer 344 rats following chronic inhalation exposure to 5000 ppm ETBE for up to 2 years (Japan Bioassay Research Center, 2010b).

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Chronic, reliable study. NOAEL based on significant, human relevant findings. No specific target organ. Critical end point are effects on clinical chemistry.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Chronic, reliable study. NOAEL based on significant, human relevant findings. No specific target organ. Critical end point are effects on clinical chemistry.

Justification for classification or non-classification

Based on the available data and in accordance to Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification is not necessary for repeated dose toxicity.