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Administrative data

Endpoint:
specific investigations: other studies
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
3 or 14 days
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
The study was performed according to standard operating procedures but not conducted in accordance with GLPs in that no formal Quality Assurance audit occurred during the course of the study. Study chemical is tertiary butyl alcohol, the primary metabolite of tertiary butyl acetate.

Data source

Reference
Reference Type:
publication
Title:
Unnamed
Year:
2009

Materials and methods

Test guideline
Qualifier:
no guideline available
Deviations:
not specified
Principles of method if other than guideline:
Tertiary butyl alcohol was administered continuously via the drinking water to groups of 15 female B6C3F1 mice for 3 or 14 days at concentrations of 0, 2 and 20 mg/mL. A positive control group of 15 female mice received 80 mg/kg bw/day of phenobarbital by oral gavage once per day. Before necropsy, a blood sample was taken from the aorta for thyroid hormone analysis (TSH, T3 and T4) by radioimmunoassay. At necropsy, liver and brain were weighed. The thyroid and a portion of the liver were fixed in 10% buffered formalin and examined microscopically. Small pieces of liver from each animal were used for gene expression analyses by quantitative Polymerase Chain Reaction (qPCR) and the remaining portions of the liver were homogenized for microsomal preparations to determine cytochrome P-450 specific isoenzyme profiles.
GLP compliance:
no
Type of method:
in vivo
Endpoint addressed:
not applicable

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Details on test material:
Tertiary butyl alcohol:
-Source: Lyondell Chemical Company (Houston, TX)
-Batch number: HL80417001
-Physical description: clear, colorless liquid
-Purity: 99.58%
-Storage: air-tight, light-resistant container at room temperature

Test animals

Species:
mouse
Strain:
B6C3F1
Sex:
female
Details on test animals and environmental conditions:
Test animals:
-Source: Harlan Laboratories, Gannat, France
-Age at start of treatment: 7-10 weeks
-Acclimation period: 7 days
-Randomization: Animals were allocated to groups using a randomization procedure that ensured a similar body weight distribution among groups that was within 20% of the mean body weight.
-Body weight: 19.7 to 23.5 g for females in the 3-day exposure group and 18.7 to 22.1 g for females in the 14-day exposure group
-Identification: stainless steel ear tag bearing a unique four-digit animal number.
-Housing: mice were housed individually in suspended, stainless steel, wire-mesh cages
-Feed: Certified rodent pelleted and irradiated diet A04C-10 (Scientific Animal Food and Engineering, Augy, France) ad libitum
-Water: filtered and softened water from local municipality ad libitum

Environmental conditions:
-Temperature: 20 °C- 24 °C
-Humidity: 40-70%
-Lighting: 12-hour light, 12-hour dark cycles (7 am - 7 pm)
-Ventilation: 10-15 air changes/hour

In-life study dates:
-Experimental starting date: August 27, 2008
-Final sacrifice dates:
September 8-9, 2008 (3-Day exposure)
September 18-19, 2008 (14-Day exposure)

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Tertiary butyl alcohol was administered continuously via the drinking water to groups of 15 female B6C3F1 mice for either 3 or 14 days at concentrations of 0, 2 and 20 mg/mL. A positive control group of 15 female mice received 80 mg/kg body weight/day of phenobarbital by oral gavage once per day for 3 or 14 days. Solutions were stored at +4 °C when not in use.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Tertiary butyl alcohol:
Analysis was performed at Défitraces Laboratory (Brindas, France), where solutions of the test substances were verified at each dose concentration. The stability of the solutions was determined in a separate study at concentrations of 0.2 and 2% m/v. and obtained from an analytical quantification. Solutions were analyzed by gas chromatography with FID.

-Phenobarbital:
Analysis was performed at Bayer CropScience. The homogeneity in a 0.5% aqueous suspension of methylcellulose was verified to demonstrate adequate formulation procedures and was used as a measure of the dose concentrations. The stability of phenobarbital in aqueous methylcellulose at a concentration of 8 g/L was demonstrated in a previous study. Solutions were analyzed by HPLC.
Duration of treatment / exposure:
3 or 14 days
Frequency of treatment:
Continuous 24 hour exposure
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
20 mg/mL tertiary butyl alcohol
Basis:
nominal in water
Remarks:
Doses / Concentrations:
80 mg/kg bw/day phenobarbital
Basis:
actual ingested
No. of animals per sex per dose:
15 female mice/group
Control animals:
yes, concurrent vehicle

Examinations

Examinations:
TEST SUBSTANCE INTAKE: Yes
-The mean achieved dosage intake in mg/kg bw/day for each period and for Weeks 1 to 2 was calculated using the formula:
Test substance intake (mg/kg bw/day) = [Dose level (mg/mL) x Group mean water consumption (g/day) x 1000]/ [Group mean body weight (g) at the end of the water consumption period]

HEMATOLOGY:
-At necropsy, animals were anesthetized with Isoflurane; blood samples were obtained from all surviving animals via the abdominal aorta; blood was collected on lithium heparin for hormone level evaluation; plasma samples were prepared and kept frozen at -80 °C until analysis.

HORMONE MEASUREMENT: Yes
-Analysis performed at Ecole Nationale Vétérinaire de Lyon (ENVL), Laboratoire d’endocrinologie clinique et toxicologique, France under GLP.
-Samples analyzed for TSH, T4 and T3 hormone levels with specific radio-immunoassay kits (supplied by Amersham for TSH and by Diasorin for T3 and T4).

SACRIFICE AND PATHOLOGY: Yes
-On study Days 5 or 16, all surviving animals from their respective groups were sacrificed; animals were not fasted overnight prior to sacrifice.
-Brain and liver were weighed fresh.
-A piece of median and left lobes of the liver of 5 animals selected randomly in each group and the thyroid gland of all animals were saved in neutral buffered 10% formalin and embedded in paraffin wax for histopathological examination. Histological sections were stained with hematoxylin and eosin and histopathological examinations were performed on all prepared tissues from all animals selected.
-One high-dose animal from the 3-day exposure was found dead and necropsied; necropsy included examination of all major organs, tissues and body cavities. Macroscopic abnormalities were recorded but not sampled.

HEPATOTOXICITY TESTING: The remaining portions of the liver of one animal/ group also used for microscopic examination and the entire liver of two other animals were pooled by group and homogenized for microsomal preparations. Total cytochrome P-450 content and specific cytochrome P-450 isoenzyme profile were determined.
-Total cytochrome P-450 content was determined by spectrophotometry using a reduced CO differential spectrum; a single quantification was performed for each pool.
-Specific cytochrome P-450 enzymatic activities were evaluated by spectrofluorimetry using benzoxyresorufin (BROD), ethoxyresorufin (EROD), pentoxyresorufin (PROD) and by HPLC using fluorimetric detection following derivatization by 4-(bromomethyl)-7 methoxycoumarin of 12-hydroxy-lauric acid (lauric acid used as substrate). Ethoxyresorufin is a highly selective substrate for isoform 1A, the isoform 2B metabolizes preferentially the O-dealkylation of pentoxyresorufin, while the benzoxyresorufin O-debenzylation is mainly metabolized by the isoform 3A. Cytochrome P-450 dependent dealkylation of resorufin derivatives was followed over a period of 2, 5 or 7 minutes at 37 °C. Samples were prepared to follow the hydroxylation of lauric acid by the isoform 4A over a period of 10 minutes at 37 °C. Two replicates of each incubation mixture were collected. One replicate was analyzed; the other one was stored frozen.

TOTAL RNA PURIFICATION:
-Total cytoplasmic RNA was isolated from the liver of individual control and treated mice using RNeasy Mini kits (Qiagen). Total RNAs were quantified by a spectrophotometer Nano Drop (Agilent Technologies). RNA quality controls were performed based on the ribosomal RNA electrophoretic profiles using a Bioanalyser (Agilent Technologies).

QUANTITATIVE PCR:
- Five μg of total RNA was used for Reverse transcription (RT) using a High Capacity cDNA Archive kit (Applied Biosystems). The assay was performed in duplicate using Taqman assays (Assay on demand, Applied Biosystems), 1/50 diluted first strand cDNA, AmpliTaq Gold® PCR Master Mix on an ABI prism 7900 HT machine (Applied Biosystems). For each gene transcript measured, a negative control condition was included in which H2O MQ was used as template instead of first strand cDNA.

The following gene families were analyzed:
- Cytochrome P450 (isoforms Cyp1a1, Cyp2b9, Cyp2b10, Cyp3a11)
- Sulfotransferase (isoforms Sulta1, Sulta2, Sultn)
- UDP Glucuronosyltransferase (isoforms Ugt1a1, Ugt2b1, Ugt2b5)
- Beta-2 microglobulin (isoform B2m). This was selected as reference gene for the quantitative calculations of transcripts.

The relative quantity (RQ) value of each test transcript was calculated using the following formula:

ΔΔCt = (Cttest – CtB2m) treated - (Cttest - CtB2m) control
RQ = 2-ΔΔCt, where Ct is the threshold cycle at which PCR amplification started to be significantly different from the background signal. Each RQ value obtained for a given gene was normalized by dividing by the RQ value obtained for the control animal.

STATISTICAL ANALYSES:
Statistical tests were conducted using SAS programs (Version 8.2). Mean and standard deviation was calculated for the following parameters: body weight and body weight gain parameters, food and water consumption, organ weight parameters, hormonal parameters, total cytochrome P450 content, hepatic enzymatic activities, quantity of gene transcripts.

Data were further analyzed using the F test, t-test (2-sided), Bartlett test, ANOVA, Kruskal-Wallis test, and/or Mann-Whitney test (2-sided) as appropriate.
Positive control:
Phenobarbital:
-Name: Phenobarbital
-Batch number: 06100228
-Physical description: white powder
-Purity: 99.6%
-Storage: air-tight, light-resistant container at room temperature

Results and discussion

Details on results:
MORTALITY: No treatment-related deaths.


Tertiary butyl alcohol– At 20 mg/mL, consumption was reduced 78% (p≤0.01) during the study in animals exposed for 3 days and 65% (p≤0.01) in animals exposed for 14 days. At 2 mg/mL, no effect in animals exposed for 3 days and a reduction of 8% (week 1) and 12% (week 2) that did not reach statistical significance in animals exposed for 14 days.

TEST SUBSTANCE INTAKE:
The test substance intake for animals exposed to tertiary butyl alcohol at dose concentrations of 0, 2 and 20 mg/mL equated approximately to 0, 344 and 818 mg/kg body weight/day for the 3-day exposure groups and 0, 418 and 1616 mg/kg body weight/day for the 14-day exposure groups.

HORMONE ANALYSIS:
Phenobarbital - After 3-days of exposure, animals had significantly decreased (p≤0.01) T3 (12%) and T4 (34%) concentrations compared to the control; mean TSH concentrations were slightly increased by 12% (not statistically significant). After 14 days of exposure, animals had significantly decreased (p≤0.01) T3 (21%) and T4 (48%) concentrations compared to the control; TSH levels were comparable to controls. See tables in section below for exact values.

Tertiary butyl alcohol – After 3 days of exposure, mean T3 and mean T4 concentrations for both treatment groups were comparable to the control; at 20 mg/mL, mean TSH concentrations were increased by 18% (not statistically significant) compared to the control. After 14 days of exposure, animals had a significant mean decrease in T3 concentrations (12% and 13% at 2 mg/mL and 20 mg/mL, respectively) compared to control and a significant mean decrease in T4 concentrations (15% and 22% at 2 mg/mL and 20 mg/mL, respectively); mean TSH concentrations for both treatment groups were comparable to the control values. See tables in section below for exact values.

GROSS NECROPSY:
Animals exposed to phenobarbital or tertiary butyl alcohol (2 or 20 mg/mL) for 14 days had treatment-related findings consisting of enlarged liver in 13/15 and 3/15 animals, respectively.

TERMINAL BODY WEIGHTS AND ORGAN WEIGHTS:
Animals exposed to phenobarbital (3 or 14-day exposure) had a statistically significant increase in mean absolute and relative liver weights compared to control.

HISTOPATHOLOGY:
Phenobarbital – Treatment-related changes in the liver consisted of minimal to slight diffuse centrilobular hepatocellular hypertrophy in 3- and 14-day exposure groups.

Tertiary butyl alcohol – Treatment-related changes in the liver consisting of minimal to slight diffuse centrilobular hepatocellular hypertrophy were observed in mice exposed to 20 mg/mL tertiary butyl alcohol for 14-days.

TOTAL P-450 CONTENT:
Phenobarbital - Significant increase of the total cytochrome P450 content by approximately 130% when compared to the controls after 3 or 14 days of treatment.

Tertiary butyl alcohol - No relevant change in total cytochrome P450 content at either dose level after 3 days of exposure; statistically significant slight increase (+56%) in total cytochrome P-450 observed at 20 mg/mL after 14-days of exposure; no changes at 2 mg/mL.


ENZYMATIC ACTIVITIES:
Phenobarbital - 3 days of treatment:
- marked increase in PROD activity (+678%, p≤0.05) and in BROD activity (6976%, p≤0.05) when compared to the control values.
- EROD appeared to be very slightly increased (p≤0.05) but this change was considered not to be toxicologically relevant.
- LAH was unaffected.

Phenobarbital - 14 days of treatment:
- marked increase in PROD activity (+710%, p≤0.01) and in BROD activity (+7075%, p≤0.01) when compared to the control values.
- EROD appeared to be slightly increased (p≤0.01) but this change was considered not to be toxicologically relevant.
- LAH was unaffected.

Tertiary butyl alcohol -3 days of treatment:
- no change in enzymatic activities EROD, PROD, BROD and LAH at either dose level.

Tertiary butyl alcohol - 14 days of treatment:
- slight increase (+109%, p≤0.01) in PROD activity at 20 mg/mL only; no change at 2 mg/mL.
- moderate increase (+1129%, p≤0.01) in BROD activity at 20 mg/mL; BROD was very slightly increased (+105%, p≤0.01) at 2 mg/mL but this was considered not to be related to the treatment.
- EROD and LAH were unaffected by treatment.

Overall, these data indicate that tertiary butyl alcohol at 20mg/mL has the ability to induce total cytochrome P-450, BROD and PROD activities in female mice following 14-day exposure.

Q-PCR ANALYSIS:
Phenobarbital -3 days of treatment:
-Cyp1a1 gene transcripts were down-regulated (-37%, p≤0.01)
-Cyp2b10 and Cyp3a11 gene transcripts were up-regulated (+2013%, p≤0.01 and +187%, p≤0.01; respectively) in the liver of phenobarbital treated animals when compared with controls.
-The transcripts of isoforms of sulfotransferases and UDP glucoronosyltransferases were statistically significantly up-regulated (ranging from +46% to +295%, p≤0.01 and from +83% to +110%, p≤0.01; respectively) in the liver of phenobarbital treated animals with the exception of Ugt2b5.

Phenobarbital -14 days of treatment:
-Cyp1a1 gene transcripts were down-regulated (-25%, p≤0.05)
-Cyp2b10 and Cyp3a11 gene transcripts were up-regulated (+3201%, p≤0.01 and +113%, p≤0.01; respectively) when compared with controls.
- The transcripts of isoforms of sulfotransferases and UDP glucoronosyltransferases were statistically significantly up-regulated (ranging from +58% to +825%, p≤0.01 and from +29% to +83%, p≤0.01; respectively) with the exception of Ugt2b5.

Tertiary butyl alcohol - 3 days of treatment:
No significant change was observed at either dose level.

Tertiary butyl alcohol - 14 days of treatment:
20 mg/mL:
-Cyp2b9 and Cyp2b10 gene transcripts were up-regulated (+21%, p≤0.01 and +2085%, p≤0.01; respectively) in the liver of tertiary butyl alcohol treated animals when compared with controls.
-The transcripts of sulfotransferase Sult 1a1 were statistically significantly up-regulated (+28%, p≤0.01) in the liver of tertiary butyl alcohol treated animals when compared with controls.

2 mg/mL:
-Cyp2b10 gene transcripts were up-regulated (+68%, p≤0.01) when compared with controls.
-The transcripts of sulfotransferase Sult 1a1 were statistically significantly up-regulated (+20%, p≤0.05) when compared with controls.

Any other information on results incl. tables

Mean hormone data ± SD at 3 days of treatment
Hormone Control Phenobarbital 2 mg/mL Tertiary butyl alcohol 20 mg/mL Tertiary butyl alcohol
T3 (nmol/L) 2.17 ± 0.24 1.90 ± 0.21S 2.02 ± 0.27 2.14 ±0.39
T4 (nmol/L) 43.4 ± 8.0 28.7 ± 6.9S 44.9 ± 6.5 44.0 ± 7.5
TSH (ng/mL) 2.00 ± 0.54 2.23 ± 0.37 1.95 ± 0.49 2.35 ± 0.60
 
 
Mean hormone data ± SD at 14 days of treatment
Hormone Control Phenobarbital 2 mg/mL Tertiary butyl alcohol 20 mg/mL Tertiary butyl alcohol
T3 (nmol/L) 2.53 ± 0.22 2.01 ± 0.20S 2.22 ± 0.19S 2.20 ± 0.19S
T4 (nmol/L) 59.7 ± 7.9 31.0 ± 5.0S 50.8 ± 9.2S 46.7 ± 9.7S
TSH (ng/mL) 2.21 ± 0.44 2.14 ± 0.28 2.33 ± 0.75 2.17 ± 0.54

S = Statistically significant compared to control.

  

Analytical Analysis:

  

Phenobarbital

Homogeneity and concentration checks of phenobarbital were within 97% and 99% of the nominal concentration and were therefore considered to be acceptable.

  

Tertiary butyl alcohol

Concentration checks of tertiary butyl alcohol were within 104% and 105% of the nominal concentration and were therefore considered to be acceptable. Tertiary butyl alcohol was found to be stable in solution at both dose levels under the conditions of storage and usage of the test compound solution on the study.

Applicant's summary and conclusion

Conclusions:
Overall, tertiary butyl alcohol appears to be a weak liver enzyme inducer in female B6C3F1 mice (weaker than phenobarbital) exposed for 14 days. The induction was specific for the cyp2b10 enzyme, with a minor increase in Cyp2b9. Tertiary butyl alcohol also induced the Phase II enzyme sulfotransferase 1a1, which is involved in thyroid hormone homeostasis.

Inclusion of this study on tertiary butyl alcohol in the overall evaluation of tertiary butyl acetate is relevant since tertiary butyl acetate is rapidly metabolized in vivo and the primary metabolite is tertiary butyl alcohol.
Executive summary:

In a study to examine the effects of tertiary butyl alcohol on hepatotoxicity and thyroid hormones, tertiary butyl alcohol was administered continuously via the drinking water to groups of 15 female B6C3F1 mice for either 3 or 14 days at concentrations of 0, 2 and 20 mg/mL. A positive control group of 15 female mice received 80 mg/kg body weight/day of phenobarbital by oral gavage once per day. There were no treatment-related deaths or adverse clinical signs in tertiary butyl alcohol treated animals at either dose level, and body weights, body weight gains, and food consumption were comparable to controls. At 20 mg/mL tertiary butyl alcohol, mean water consumption was significantly reduced for 3 or 14 days of exposure. After a 3-day exposure, there was a non-statistically significant increase in TSH concentration at the high-dose level only but no effect on mean T3 or T4 levels at either dose level. There was a statistically significant decrease in T3 and T4 concentrations in animals exposed to tertiary butyl alcohol for 14 days at both dose concentrations. No thyroid pathology was reported in the study. Following 14 days of exposure, enlarged livers were found in a few animals in both exposure groups and minimal to slight diffuse centrilobular hepatocellular hypertrophy was observed in the 20 mg/mL group only. Following 3 days of exposure to tertiary butyl alcohol, there were no significant changes in mean total cytochrome P-450 content, no change in enzymatic activities EROD, PROD, BROD or LAH, and no change in gene transcripts using Quantitative PCR analysis at either dose level. Following 14 days of treatment, there was a slight increase in total cytochrome P-450, a slight increase in PROD activity, and a moderate increase in BROD activity at 20 mg/mL; there were no effects observed on total P-450 content or enzymatic activities in the 2 mg/mL dose group. QPCR analyses of transcripts of genes revealed a slight up-regulation of Cyp2b9 transcripts, a marked up-regulation of Cyp2b10 transcripts, and a slight up-regulation of sulfotransferase Sult 1a1 transcripts for animals exposed to 20 mg/mL for 14 days; in the lower dose group, there was a slight up-regulation of Cyp2b10 and sulfotransferase Sult 1a1 transcripts.