Registration Dossier

Administrative data

Description of key information

DIBK:

A non-GLP oral repeated dose (90d) toxicity study using rats

2 non-GLP 6 week inhalation studies (rat and guinea pig)

A non-GLP 9 -day vapour inhalation study in rats

A non-GLP 14 -day inhalation study in rats

a non-GLP 10 -day dermal study in guinea pigs

Data on category members

MIBK:

A GLP 90 -day oral repeated dose study in rats

2 GLP Chronic toxicity/carcinogenicity studies via inhalation in rats and mice

Key value for chemical safety assessment

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Dose descriptor:
NOAEC
2 650 mg/m³
Species:
rat

Additional information

The repeated-dose toxicity of diisobutyl ketone (DIBK) can be assessed using the data available on DIBKitself (oral and Inhalation)and by making use of data on analogues (diisobutyl carbinol (DIBC), methyl isobutyl carbinol (MIBC) and methyl isobutyl ketone (MIBK)). This use of analogue data is considered acceptable based on the following justifications:

1) It can be demonstrated that DIBK and DIBC share the same metabolic pathway to that of MIBK and MIBC

2) The toxicity of all of these substances is very consistent and can be attributed to their common ADME characteristics, including a common metabolic pathway and structurally related metabolites.

 

A detailed justification for the grouping of these substances into a category for the purposes of read across is provided in the 'read across justification' attached to section 13 of the IUCLID dossier.

 

Within the group of substances the following data exist:

 

DIBK

Oral:

In a 90-day oral gavage study in rats at a dose of 2,000 mg/kg/day for 90 days absolute and relative liver weights, relative kidney weights, absolute and relative adrenal gland weights were statistically greater than controls. No gross pathological changes were detected. Histologically, compound related changes were evident in the stomach, liver and kidneys. Gastric changes due to direct contact with the solvent included hyperkeratosis with or without pseudoepitheliomatous hyperplasia. Hepatic changes included hepatocyte hypertrophy in all animals and hepatocyte vacuolation in one animal. However, no changes in the levels of serum liver enzymes were observed in clinical chemistry assessment indicating no tissue damage in the liver. Renal changes included hyalin droplet formation in the proximal tubular epithelium, regenerating tubular epithelium and tubular dilation with casts.

 

Dermal:

No valid or reliable data are available.

 

Inhalation:

Two studies of acceptable quality (Klimisch 2) are available in rats and one in guinea pigs. The only adverse effect observed in rats was hyaline droplet nephrosis in male rats. This effect is considered to be mediated by alpha 2µ globulin accumulation as demonstrated for MIBK (methyl isobutyl ketone) and therefore not considered to be relevant for human exposure.

 

DIBC

Oral:

Repeated dose toxicity and Reproductive/Developmental screening study (OECD 422). In a range finding study 5 rats/sex were given 0, 250, 500, 750, or 1,000 mg DIBC/kg via oral gavage for 14 days. Transiently increased salivation was noted in doses greater than or equal to 500 mg/kg; increased liver and kidney weights were noted at all doses. No treatment-related gross observations were recorded at necropsy. In the full study, groups of 12 male and 12 female rats were administered 0, 50, 150, or 500 mg DIBC/kg via oral gavage. Female rats were dosed once daily for approximately two weeks prior to breeding, continuing through breeding (two weeks), gestation (three weeks), and through postpartum day 4. Male rats were dosed beginning approximately two weeks prior to breeding and continuing through breeding (two weeks) for an exposure period of 33 days. A decrease in bodyweight weight and feed consumption was observed in females at the 500 mg/kg level. Liver weight changes were seen in the male rats at the middle and high doses; weights were increased by 17% and 28%, respectively. The weight of the livers in the high dose females was increased by 10%. The liver effects corresponded with very slight hypertrophy of centrilobular hepatocytes in males given 150 or 500 mg/kg and females given 500 mg/kg. However there were no effects on liver enzyme markers in the blood that would indicate evidence of liver toxicity. As such it appears likely that the observed liver enlargement and associated hypertrophy were a consequence of liver enzyme induction rather than target organ toxicity. Males given 500 mg/kg/day and females given 150 or 500 mg/kg/day had higher relative kidney weights that were interpreted to be treatment related, however there were no corresponding clinical pathologic or histopathologic alterations for the higher kidney weights that would indicate evidence of toxicity. There was no evidence of systemic toxicity in rats given 50 mg/kg/day. Additional treatment-related effects that were interpreted to be non-adverse consisted of transient salivation noted only around the time of dosing in the high dose males and females, slightly decreased urine pH in males at all dose levels, as well as increased serum total protein and cholesterol in males or females given 500 mg/kg/day. Olfactory and respiratory epithelium effects were seen at the 50, 150, and 500 mg/kg groups. These effects consisted of degeneration and/or inflammation, attributed to the gavage procedure. There were no neurological effects noted. In this study the NOEL was 50 mg/kg bw/day, however the effects observed at the mid and high dose in the liver and kidneys could be considered to be adaptive in nature rather than evidence of systemic toxicity. As such, based on the decreased bodyweight in the high dose, the NOAEL for this study is considered to be 150 mg/kg bw/day. Data on supporting substances

 

MIBK

Oral:

In a key study (Mulligan, 1986) groups of 30 male and 30 female Sprague-Dawley rats were administered MIBK by gavage in corn oil at daily dose levels of 0 (vehicle control), 50, 250, or 1000 mg/kg-day for 13 consecutive weeks and evaluated for exposure-related changes in body weight, food consumption, mortality, clinical signs, ophthalmological parameters, and terminal organ weights (heart, liver, spleen, brain, kidney, gonads, adrenals, thyroid, and parathyroid). The following evaluations were conducted in rats from each exposure level at interim (week 7) and final sacrifices: hematology, clinical chemistry, urinalysis, and comprehensive gross pathology. All tissue samples collected during gross necropsy in high-dose and control rats were evaluated for histopathology, and kidney samples were also histologically evaluated in mid-dose rats.

Reversible lethargy was observed in rats of both sexes receiving 1000 mg/kg-day (but not at lower dose levels) for a few hours following dosing and reportedly decreased in incidence and severity during the study. Males in the high-dose group showed a slight (9%) but significantly decreased mean body weight gain as compared to controls during the last 2 weeks of exposure, whereas female body weight gain was significantly increased during 5 of the last 6 weeks of exposure. Final bodyweights were not however different to control. Both male and female food consumption was significantly increased during the second half of the exposure period. The only potentially exposure-related hematological effects observed were slight but statistically significant increases in hemoglobin (+6%) and hematocrit (+8%) at terminal sacrifice in females administered 1000 mg/kg-day and a 15% decrease in lymphocyte count in high-dose males at terminal sacrifice. The lowest hepatic effect level that was observed in the oral exposure studies was 250 mg/kg-day for increased (+39%) serum glutamic-pyruvic transaminase (SGPT) in female rats at the terminal sacrifice. The following changes suggestive of adverse liver effects were observed at 1000 mg/kg-day at either interim and/or final sacrifice: increased SGPT (+73%, interim; +34%, terminal) in females as compared to controls, increased serum alkaline phosphatase (+84%, interim) in females, increased serum cholesterol in males (+30%, interim) and females (+59%, interim; +65%, final), increased terminal absolute (+34%, males; +39%, females) and relative (+42%, males; +38%, females) liver weights, decreased albumin/globulin ratio in males (-16%, interim), and minimally increased serum total protein in females (+9%, interim; +10%, terminal). The only renal effect occurring at 250 mg/kg-day was increased terminal absolute or relative kidney weights in males and females, ranging from 6 to 12% over controls. The following changes suggestive of adverse kidney effects were observed at 1000 mg/kg-day: increased terminal absolute and relative kidney weights (from 25 to 34% in males and from 20 to 22% in females) as compared to controls, increased blood-urea-nitrogen (BUN) in males (+37%, interim), increased serum potassium in males (+34%, terminal), decreased serum glucose in males (-27%, terminal), and a reported increase in urinary protein and ketones in males and females at terminal sacrifice (summary data were not provided). Histological examination of kidney tissues revealed an increased incidence of male rats with mild nephropathy (multifocally distributed swollen or hyperchromatic and flattened renal cortical tubular epithelial cells) at 1000 mg/kg-day (16/20) as compared to controls (4/20) but no increase in such lesions in females. Significantly increased relative adrenal weights in male (+29%) and female (+11%) rats and slightly increased relative testis weights (+9%) in males were also observed at 1000 mg/kg day. With the exception of the kidney, no exposure-related histopathologic lesions were evident in any tissue that was examined. The NOAEL was estimated to be 250 mg/kg bw/d, based on increases in relative kidney weights for male and females rats administered MIBK at doses of 250 mg/kg bw/d but without histological lesions. Effects at higher doses included kidney changes, hepatomegaly and alterations in clinical chemistry and urinalysis parameters. No treatment-related effects of any kind were observed at 50 mg/kg-day.

 

Inhalation:

In a whole body 2-year inhalation study in Fischer 344 rats, animals (50/sex/group) were exposed to MIBK at concentrations of 0 (control), 450 ppm, 900 ppm, or 1800 ppm for 6 hours per day, 5 days per week for 2 years (NTP, 2007; Stout et al., 2008). This GLP study was equivalent to OECD Test Guideline 451. Mortality was observed in all groups. However, survival was significantly decreased in males administered MIBK at 1800 ppm as compared to controls. Mean body weights also were decreased in males administered 900 ppm and 1800 ppm as compared to controls. The mean body weights and survival in treated females were similar to controls. The primary target of MIBK toxicity was the kidney. Briefly, chronic progressive nephropathy (CPN) similar to that which occurs in aged rats also was observed in all rats (including controls). There were treatment related significant increases in both the incidence (1800 ppm) and severity in all exposed groups. Kidney lesions that typically accompany CPN also were reported in males exposed to 900 ppm and 1800 ppm MIBK. The kidney lesions observed were suggestive of α2µ-globulin nephropathy (specific to male rat), a mechanism of xenobiotic-induced renal carcinogenesis for which there is no human counterpart. A NOAEC was not identified by the authors. Review of the study data suggests that a NOAEC of 450 ppm (1840 mg/m3) can be derived for neoplastic and non-neoplastic lesions, based on the non-neoplastic lesions observed in the kidneys at higher dose levels and the irrelevance to humans of the tumour types observed in the kidneys of male rats.

 

In a supporting 2-year inhalation study in B6C3F1 mice, animals (50/sex/group) were administered MIBK at concentrations of 0 (control), 450 ppm, 900 ppm, or 1800 ppm for 6 hours per day, 5 days per week for 2 years (NTP, 2007; Stout et al., 2008). This GLP study was equivalent to OECD Test Guideline 451. Survival of male and female mice was similar to controls. There were no clinical findings observed related to MIBK exposure. Mean body weights of male mice were generally similar to the controls throughout the study. Mean body weights of females exposed to 1800 ppm MIBK were 9-16% less than the controls. Eosinophilic foci in the liver were increased in all exposed groups of female mice, and the increases over the controls were significant in the 450 and 1800 ppm groups; this lesion was not significantly increased in exposed male mice. Hepatocellular adenomas and carcinomas were reported in males and females at various doses. Review of the study data suggests that a NOAEC of 450 ppm (1840 mg/m3) can be derived for neoplastic and non-neoplastic lesions, based on the reported neoplastic effects in the liver of female mice (multiple adenomas) at higher dose levels. Subsequent investigations (Geter et al., 2009) reported that MIBK-related hepatocellular findings in mice may be due to induction of cytochrome P450 enzymes following activation of the mouse constitutive androstane receptor (CAR) in a manner that is similar to Phenobarbital-like compounds. The authors of the study noted that a carcinogenic effect in mice that can be attributed to Phenobarbital-like activation of CAR is not relevant to humans.

 

In a supporting subchronic inhalation study in rats and mice, groups of 14 male and 14 female Fischer 344 rats and B6C3F1 mice were exposed to measured mean concentrations of 0, 50, 252, and 1002 ppm (0, 205, 1033, and 4106 mg/m3) MIBK for 6 hrs/day, 5 days/week, for 14 weeks and sacrificed following their final exposure day (Dodd and Eisler, 1983; Phillips et al., 1987). The following endpoints were evaluated: clinical signs, body weights, organ weights (kidneys, heart, liver, lungs, and testes), urinalysis, hematology, serum chemistry (including glucose and hepatic enzyme levels), complete gross pathology, targeted histopathology (nasal cavity, trachea, liver, kidneys, and lungs) in all animals and complete histopathology in control and high-exposure groups.

No effects of any kind were observed in rats or mice of either sex at 205 mg/m3. Terminal body weights were significantly increased in female rats at >=1033 mg/m3. Mouse hematology was unaffected at all exposure levels, but platelet numbers in male rats were significantly increased at 4106 mg/m3 by 13% over controls, and eosinophil number in female rats was significantly decreased at 4106 mg/m3 by 57% as compared to controls. Serum cholesterol in male rats was significantly increased at the 1033 and 4106 mg/m3 exposure levels by 23 and 35%, respectively, as compared to controls. Male rats and male mice showed a significant increase in absolute (+13%, rats; +7%, mice) and relative (+9%, rats; +11%, mice) liver weight at 4106 mg/m3; absolute, but not relative, liver weight was also slightly increased in male mice (+8%) at 1033 mg/m3. No histological lesions were observed in the liver and no changes were seen in serum liver enzymes and bilirubin in any exposure group; thus, the observed liver enlargement may have been an adaptive response to increased hepatic metabolic activity rather than a toxic effect.

Urine glucose was significantly increased in male rats at 1033 mg/m3 (+37%) and 4106 mg/m3 (+55%) and in female rats at 4106 mg/m3 (+26%). Significantly increased urine protein (+28%) was also observed in male rats at 4106 mg/m3. The only renal histological lesion observed was hyaline droplet formation in all male rats; the severity of the lesion generally increased with exposure level. The U.S. EPA has concluded that renal alpha2u-globulin hyaline droplet formation is unique to male rats and is probably not relevant to humans for the purposes of risk assessment. In conclusion, other than the male rat kidney effect, exposure of male and female rats and mice to MIBK at levels up to 1000 ppm for 14 weeks was without significant toxicological effect.

In a supporting sub-acute inhalation study, groups of six male and six female F344 rats and B6C3F1 mice were exposed for 6 hrs/day, 5 days/week, for 9 days to measured concentrations of 0, 101, 501, or 1996 ppm (0, 410, 2053, or 8178 mg/m3) MIBK (Dodd et al., 1982; Phillips et al 1987). Groups were evaluated for changes in clinical signs, body weight, organ weights (liver, lungs, kidneys, and testes), ophthalmology, gross pathology, and histopathology. The only exposure-related effects observed were periocular wetness in rats exposed to 8178 mg/m3, increased relative liver weights in male rats at 2053 and 8178 mg/m3 and in female rats and female mice at 8178 mg/m3, increased kidney weights in male rats and female mice at 8178 mg/m3, and hyaline droplet degeneration in kidneys of male rats exposed to 2053 or 8178 mg/m3, with epithelial regeneration of proximal convoluted tubules in the high-exposure group. No effects of any kind were observed in rats at 410 mg/m3 and in mice at 2053 mg/m3.

 

MIBC

Inhalation:

A non-GLP 6-week inhalation study was conducted in male and female Wistar rats with methyl isobutyl carbinol (MIBC) (Blair, 1982). Animals were exposed to concentrations of MIBC at 0 (control), 211, 825, or 3698 mg/m3for 6 hours per day for 5 days per week for 6 weeks. There were no adverse effects reported for the following parameters evaluated: mortality and clinical signs, hematology, gross pathology, and histopathology. The mean alkaline phosphatase value was increased in females exposed to 3698 mg/m3. Mean concentrations of ketone bodies in urine samples from all the exposed groups (except males exposed to 211 mg/m3) showed a significant increase above those of the control groups. The mean concentrations of protein were increased in all the exposed female groups and in the male group exposed to 3698 mg/m3. The mean specific gravities of both male and female groups exposed to 3698 mg/m3were increased compared to the control values as was the female group exposed to 825 mg/m3. The mean pH of urine from the male group exposed to 3698 mg/m3was reduced, and the mean glucose content of the urine of females exposed to the same concentration was increased compared to the control values. Finally, the mean urine volumes of females exposed to 211 and 3698 mg/m3were reduced compared to the control values. Although significantly increased kidney weights were noted in males exposed to 3698 mg/m3as compared to controls, there were no macroscopic or microscopic correlates associated with this finding. Although the authors did not define the NOAEC for this study, NOAEC can be considered to be the highest exposure concentration of 3698 mg/m3.

Justification for classification or non-classification

The overall data base supports the conclusion that in animal studies, liver and kidney are target organs for this set of chemicals. In the assessment of much of the data, effects on these organs have been considered adaptive given the absence of corroborating histopathological and clinical chemistry findings to indicate damage to organs. The exception is the effects on the male rat kidney. This is considered to be due to alpha 2u globulin associated nephropathy, although the quality of the evidence supporting that conclusion is limited, and currently subject to additional research. Although there are often reports of kidney enlargement in the females and in mice (to varying degrees) it is again felt that these findings are more adaptive in the absence of any other significant pathological findings. As such, in most cases, effects on the liver and kidney have been considered as non-adverse and not taken into consideration in the determination of the NOAEL.

 

There is no clear trend in toxicity across the group with all substances having effects on the liver and kidneys within a similar range of doses. Given the consistency in the findings and the similarity in dose response it is proposed to use the long term studies on MIBK to derive the DNELs for DIBK. An additional assessment factor of 2 is proposed to address any residual uncertainty associated with the use of a read across/weight of evidence approach for this endpoint.

 

With respect to the conversion of the NOAEC for MIBK into a NOAEC for DIBK, a molar conversion will be used.

With respect to the route-to-route extrapolation, the toxicokinetics information and physical chemical properties indicate that all category members are bioavailable via oral, inhalation and dermal routes. Comparing oral and inhalation studies (where they exist for the same substance) demonstrates a similar pattern of toxicity with the same target organs. As such it is not expected that extrapolating from an inhalation NOAEC to a dermal NOEL would result in misrepresentation of the hazards of that route of exposure. In fact, extrapolating from inhalation to dermal route is likely to represent a conservative assessment due to the slower rate of uptake via the dermal route compared to the inhalation route. i.e. the toxicokinetic differences between these routes would typically lead to a conclusion that dermal exposure would have a lower hazard potential relative to the oral and inhalation routes.

 

The Key study taken for the DNEL derivation will therefore be the 2 year inhalation study using MIBK.

The NOAEC from this study is 1840 mg/m3, this is 18.4mmol/m3.

18.4 mmol/m3 DIBC = 2650 mg/m3

In doing this conversion it is recognised that the converted NOAEC is greater than the highest attainable vapour concentration for DIBC (approx 1000mg/m3 based on a vapour pressure of 17 Pa). However this is not an issue for the derivation of the DNEL since the resulting DNEL would be expected to fall within a possible vapor concentration of DIBC.

 Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint: Reliability 1 repeated dose oral study (OECD 422 study design). Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint: Chronic toxicity/carcinogenicity study performed using an analogue (refer to endpoint summary and Section 13 of IUCLID) for justification of read across. NOAEC from study is converted based on a molar calculation to give the equivalent NOAEC of DIBC.