Ethylenebis(oxyethylene) bis[3-(5-tert-butyl-4-hydroxy-m-tolyl)propionate]

Specific investigations: other studies

Link to relevant study record(s)

Reference

Reference 1

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

No OECD guideline available with which to compare the study design. Reasonable description of methods and results but individual data was not provided.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Induction of xenobiotic metabolizing enzymes in rat

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Species:

rat

Strain:

not specified

Sex:

male

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

As no stability issues were identified in the previous feeding studies, no analytics were included.

Duration of treatment / exposure:

2 weeks (experiment 1); 3 to 6 weeks (experiment 2)

Frequency of treatment:

daily

Post exposure period:

none (experiment 1); 1, 2, or 4 weeks (Reversibility was examined in 3 of the 9 groups in experiment 2)

Dose / conc.:

50 ppm

Dose / conc.:

150 ppm

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

No. of animals per sex per dose:

5 groups of 9 animals (experiment 1); 9 groups of 4 animals (experiment 2)

Control animals:

yes, plain diet

Details on results:

Administration of 50, 150, 500, and 1000 ppm to male rats for two weeks causes a dose-dependent increase in the liver weights. Biochemically, this hepatomegaly is accompanied by an induction of specific cytochrome P-450 isozymes, cytochrome P-450-associated monooxygenases, microsomal epoxide hydrolase, UDP-glucuronosyltransferase, cytosolic glutathione S-transferase and peroxisomal beta-oxidation of fatty acyl-CoA esters. Morphologically, the test compound causes a moderate proliferation of smooth endoplasmic reticulum membranes and a moderate proliferation of peroxisomes containing metrical inclusions. In addition, helically arranged fibrils within inner membrane spaces of mitochondria are observed.
Essentially the same biochemical and morphological effects are observed, when 1000 ppm are administered for more extended periods of time (three, four, or six weeks). Two or four weeks after cessation of a two-week treatment at the highest dose-level (1000 ppm), the investigated parameters returned to control levels with the exception of the morphological alterations in mitochondria, which persisted to some extent.

IN-LIFE PHASE:

No signs of toxicity were observed during the treatment period. Food consumption and body weight gains were similar in control and treated groups (data not shown).

FINAL BODY AND LIVER WEIGHTS:

Before killing, the body weights of the anesthesized animals were recorded. No differences were found between treated groups and their respective controls. Treatment with TK 12627 for two weeks caused a dose-dependent increase in the absolute liver weights (25% above control at the highest dose-level). The observed increases were statistically significant at 150, 500, and 1000 ppm. Slightly increased liver weights were also observed after a three or four week treatment (1000 ppm), whereas treatment for six weeks at the same dose-level was without an effect on this parameter. Within a two or four week recovery period, the absolute liver weights of treated animals (1000 ppm, two weeks) returned to control levels.

PROTEIN CONTENTS:

The protein contents in homogenates, 100 g supernatants and cytosolic fractions were not affected following treatment with TK 12627. Compared with the respective controls, the microsomal protein content was increased to a statistically significant extent following treatment with 500 or 1000 ppm for two weeks. Increased microsomal protein contents were also found after treatment with 1000 ppm for three, four or six weeks. Within a four week recovery period, microsomal protein contents of treated animals (1000 ppm, two weeks) returned to control levels.

ENZYME ACTIVITIES:

Following administration of the substance for two weeks, the content of microsomal cytochrome P-450 was enhanced in a dose-dependent manner at 500 and 1000 ppm (139% above control at the highest dose-level). Treatment with 1000 ppm of the test compound for three, four or six weeks caused also increased cytochrome P-450 levels.

Ethoxycoumarin O-de-ethylase and lauric acid 12-hydroxylase, two cytochrome P-450-associated monooxygenase activities, were found to be induced after treatment for two weeks at 500 and 1000 ppm. The observed activities at 1000 ppm were 153% (ethoxycoumarin O-de-ethylase) and 330% (lauric acid 12-hydroxylase) above the respective control values. The same parameters were also found to be induced after administration of 1000 ppm for up to six weeks. Slightly increased activities were found for lauric acid II-hydroxylase, another cytochrome P-450-dependent hydroxylase activity, after treatment with 1000 ppm for two or six weeks. However, the degree of induction for this parameter did not reach a level of statistical significance.

In selected animals (three control rats and three rats treated with 1000 ppm for two weeks), the microsomal oxidative metabolism of R-warfarin was investigated. With respect to untreated controls, the formation-rates of six metabolites were found to be increased to different extents in microsomal fractions from treated animals. The most prominent effects (about 350 % above control) were observed for hydroxylation at positions 7 and 8 and for 9,10-dehydration.

After a two-week treatment, microsomal epoxide hydrolase activity was induced to a statistically significant extent at 150, 500, and 1000 ppm. At the highest dose level, the activity was 336% above control. Treatment with 1000 ppm for three, four, or six weeks caused also a pronounced effect on this parameter.

Microsomal UDP-glucuronosyltransferase as well as cytosolic glutathione S-transferase were induced in a dose-dependent manner following treatment with the test compound. The observed increases at 1000 ppm were 104 and 115% above control for UDP-glucuronosyltransferase and glutathione S-transferase, respectively. The two transferase activities were also increased in animals treated with 1000 ppm for three, four, or six weeks.

A marginal but dose-dependent effect of the test compound was observed on the activity of fatty acyl-CoA oxidase (peroxisomal B-oxidation). Irrespective of the duration of treatment (two, three, four, or six weeks), this activity was increased by a factor of about 1.5 at the highest dose-level. Within one month after cessation of the treatment, the investigated biochemical parameters had essentially reverted to control levels.

IMMUNOBLOT ANALYSES:

Treatment with 500 or 1000 ppm for two weeks caused a clear induction in the content of peroxisome proliferator-inducible cytochrome P-450IVA isozymes. The same treatment caused also moderately increased contents of phenobarbitone-inducible cytochrome P-450 isozymes (P450IIB proteins). Administration of the test compound did not induce the microsomal levels of the major polycyclic aromatic hydrocarbon-inducible cytochrome P-450 isozymes.

MORPHOLOGICAL OBSERVATIONS:

The structure, relative number and distribution of organelles within hepatocytes of all control animals was similar to that described for untreated rats.

Rats treated for two weeks with 50 ppm showed no striking morphological changes of hepatocyte organelle structure. After treatment with 150 ppm, some minor alterations of mitochondrial structure were observed. These consisted of (a) the occurrence of a few unusually shaped organelles (i,e„ elongated, ring- and dumbell-like forms), and (b) the presence of a few dilated cristae mitochondriales per organelle which contained a fibrillar material. After administration of 500 and 1000 ppm of the test compound these mitochondrial changes became in a dose-related manner more prominent; in addition, the fibrillar material within dilated cristae was arranged in a helical fashion. At the highest dose-level (1000 ppm) the morphology of peroxisomes was also changed; their number appeared to be slightly augmented, their size was definitely increased, and they showed elongated or distorted shapes. The majority of peroxisomes contained matrical inclusion bodies, the so-called matrical plates. The smooth endoplasmic reticulum membranes responded to this treatment with a marginal proliferation.

Treatment of rats with 1000 ppm TK 12627 for three, four or six weeks accentuated the alterations in mitochondria already described for a two-week treatment at the same dose level. In addition, a moderate proliferation of smooth endoplasmic reticulum membranes was observed after administration for three or four weeks, whereas the effects on the peroxisomal compartment were comparable to those described for the two-week treatment period. After a six-week treatment with 1000 ppm, the morphological modifications of peroxisomes and smooth endoplasmic reticulum membranes were less prominent.

When the test compound was withdrawn for one week after a two-week treatment (1000 ppm), the morphological changes of mitochondria apparently persisted to a full extent. However, a proliferation of smooth endoplasmic reticulum membranes was no longer discernible and also the peroxisomal changes were less prominent, although metrical plates were still present in a minority of peroxisomes. After discontinuation of the treatment (1000 ppm, two weeks) for two and four weeks, the mitochondrial changes still persisted, although in an attenuated manner; the structure of peroxisomes, however, was completely normalized.

Table 1: Effects on final body weights and liver weights after a two-week treatment period
dose (ppm)	0	50	150	500	1000
body weight (g)	325 (11)	317 (31)	335 (20)	324 (23)	314 (14)
liver weight (g)	14.6 (0.8)	15.5 (2.3)	17.1 (2.4)*	18.0 (2.1)*	18.3 (1.3)**
n = 9, SD given in parentheses
Asterisks indicate results significantly different (two-sided Dunnett's test) from control : * p < 0.05, ** p < 0.01, *** p < 0.001 .

Reference 2

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline available with which to compare study design.

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Effects on serum thyroid hormone and serum thyroid-stimulating hormone (TSH) values in monkeys.

GLP compliance:

no

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once/day, at the same approximate daily time, 7 days a week

Post exposure period:

None

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

3 males/group

Control animals:

yes, concurrent vehicle

Details on study design:

Statistical analysis was performed using ANOVA with one grouping factor and one within factor.

Details on results:

All three groups showed some changes over time with the serum concentrations of total T4 and T3; T4 increasing slightly and T3 decreasing. Serum rT3 levels were very low and showed no trend but fluctuated more than serum T4 and T3 levels. Most importantly, there is no significant difference between the groups for any variable and over time, all three groups behaved in a similar manner. Over time there was a significant difference in T3 and T4 yet there is no difference (interaction) between the groups. No difference in rT3 and T4 values were observed between the groups. No difference in TSH levels was observed.

Reference 3

Endpoint:

endocrine system modulation

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

Report of well designed non-guideline study. No individual data on food consumption.

Qualifier:

no guideline available

Principles of method if other than guideline:

Investigation on the level of thyroid stimulating hormone (TSH) and thyroid hormones in serum of male rats after dietary administration of 1000 ppm for 3, 6, 13 or 20 days

GLP compliance:

no

Type of method:

in vivo

Species:

rat

Strain:

other: RAI

Sex:

male

Details on test animals or test system and environmental conditions:

Male adult rats (RAI, starting mean body weight 179 g) were kept in a room maintained at 22° +_ 1°C with a relative humidity of 55 + 5% and a 12 hour light/dark cycle. The animals were given food (Nafag 890, Nafag, Gossau, Switzerland) and drinking water ad libitum.

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

3, 6, 13, and 20 days

Frequency of treatment:

daily

Post exposure period:

none

Dose / conc.:

1 000 ppm

Remarks:

coresponding to ca 85 mg/kg/bw

No. of animals per sex per dose:

4

Control animals:

yes, plain diet

Details on study design:

At time of sacrifice, 3 - 6 mL of blood were collected from abdominal aorta. Levels of T4 and T3 were determined by an enzyme immunoassay, T3 uptake test was performed, and TSH levels were measuered by a radioimmuno assay.

Examinations:

T4 and T3 were determined by an enzyme immunoassay using a kit (Serono Diagnostics, Coinsins, Switzerland) according to the manufacturers instructions. Direct addition of the test item to the serum at concentrations up to 50 µg/ml did not interfere with the measurement of either T4 or T3.
A T3-uptake (T3U) test, which assessed the degree of saturation of serum binding proteins by the thyroid hormones T3 and T4, was performed using a Serono MAIA kit (Serono Diagnostics, Coinsins, Switzerland) according to the manufacturer's instructions.
This method is based upon the partition of a trace amount of radioiodinated T3between serum-binding proteins and a small amount of an immobilized anti T3-antibody. The amount of radioactivity bound or taken up by the immobilized anti T3-antibody (T3U value) is inversely related to the availability of unoccupied thyroid hormone-binding sites in the test serum. The product of the total serum T4 or T3 concentration and the T3U uptake value yields the Free T3 or T4 Index, respectively. This value generally correlates well with direct measurement of the free serum hormone concentration.


TSH levels were measured by a radioimmuno assay. TSH purified from rat and rabbit antiserum against rat TSH were obtained from Dr.A.F.Parlow (Pituitary Hormones and Antisera Center, California, USA) under the NIADDR Rat Pituitary Hormone Program. The radioimmunoassay was carried out according to the outline protocol suggested by Dr. Parlow.


Samples of liver, thyroid and pituitary gland from selected control and treated animals were fixed in formalin. The tissues were embedded in paraplast, sectioned at 3-5 microns, stained with hematoxylin and eosin and examined under the light microscope.

Selected tissue samples from control or treated animals were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4 (liver), or with 3% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4 (thyroid gland and pituitary gland), for 4 hr at 4 °C.
Postfixation was performed with 1% osmium tetroxide in 0.1 M cacodylate buffer, pH 7.4, for 2.5 hr in the cold. The tissue was dehydrated in a series of acetone solutions and embedded in Epon. From each tissue one block was selected per time point for thin sectioning. The sections were double-stained with uranyl acetate and lead citrate.

Positive control:

no

No signs of toxicity were observed during the treatment period. Administration for different time periods resulted in a significant and time-dependent increase in serum TSH levels up to 10 -fold the control value after 20 days, the longest time point tested. Treatment for 13 and 20 days resulted in slight to moderate hypertophy of the follicular epithelium and a pronounced depletion of colloid. After 3 or 6 days treatment, there was no substantial change in the histological appearance of the thyroid gland except for a minimal depletion of the thyroid colloid by 6 days. The severity of the lesion was comparable after 13 and 20 days of treatment. Due to the considerable morphological heterogeneity of follicles, it was impossible to describe unambigously treatment-related ultrastructural alterations with the restricted number of tissue samples available.

Histological examination of tissue from pituitary glands indicated the presence of cells containing cytoplasmic vacuoles, similar to those occuring in thyroidectomy cells following both 13 and 20 days treatment. The changes which occurred to a minimal extent were also characterized by electron microscopy. In control animals presumptive thyrotroph cells were characterized by an oval shape and often contained excentricalIy located nuclei. Their cytoplasm contained abundant amounts of rough endoplasmic reticulum displaying extensively dilated cisternae which harbored an electron-dense fuzzy material. In addition, a variable number of small secretory granules with a tendency to be more crowded in the cell periphery was present. After treatment of rats with the test substance the number of secretory granules in the thyrotrophs appeared to be reduced. Instead, similar granules appeared within the dilated cisternae of the rough endoplasmic reticulum.

After 3-days treatment the overall morphology of the hepatocytes resembled that of the controls. However, the presence of some enlarged peroxisomes was noted. In addition, a few peroxisomes contained matrical inclusions, so-called matrical plates. The structural alterations of peroxisomes described above were more prominent after treatment for 6 and 13 days. The total number of peroxisomes did not appear to be increased above that found in the controls, however enlarged peroxisomes with elongated and polygonal shapes were more prominent. Most of these contained abundant matrical plates. In some hepatocytes of rats treated for 13 days, the cisternae of rough endoplasmic reticulum tended to be dilated. Liver parenchymal cells of animals fed for 20 days displayed similar organelle changes, although to a lesser extent than at 13 days.

Total serum T4 levels in the treated group were significantly lower than those of the corresponding control group after three and six days of treatment. No difference was observed between the control and treated groups at 13 or 20 days.

When recorded before killing, very similar body weights were found in control and treated animals. The test compound caused an increase in the absolute liver weights at all time points. The most significant increases were observed after treatment for 6 and 13 days. At day 20, the thyroid gland weights recorded from three control animals were 39.7, 26.7 and 29.5 mg and from two treated animals 74.9 and 67.3 mg.

The serum free T4 levels expressed as FT4I values in the treated groups followed the same trend. However, with this parameter relatively large variations were observed between the control groups.

The total serum T3 levels and the free T3 concentration, expressed as FT3l, were found to be significantly decreased in the treated groups at all time points investigated. In treated animals serum levels of rT3 increased in a time-dependent manner, up to 3-fold the control values by 20 days treatment.

Reference 4

Endpoint:

biochemical or cellular interactions

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

From Mar. 19, 1997 to Oct. 10, 1997

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

No OECD guideline available with which to compare study design

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline available

Principles of method if other than guideline:

Hepatic enzyme induction in monkey

GLP compliance:

yes

Type of method:

in vivo

Endpoint addressed:

repeated dose toxicity: oral

Species:

monkey

Strain:

Macaca fascicularis

Sex:

male

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

4 weeks

Frequency of treatment:

once/day, at the same approximate daily time, 7 days a week

Post exposure period:

None.

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

3 males/group

Control animals:

yes, concurrent vehicle

Details on results:

No effects observed on content of liver protein or microsomal overall CYP. No effects on activity of EROD, Coumarin-7-hydrolase, lauric acid 11- and 12-hydroxylation, Cyanide-insensitive peroxisomal β-oxidation, UDP-glucuronosyltransferase, GST. Treatment had no effect on UDPGT activity towards thyroxine. Treatment resulted in a slight increase of hydroxylation at the 16β position of testosterone of control at the highest dose. Treatment had no effect on 2β-, 6α-, 6β-, 7α-, 15β- and 16α-hydroxylation and oxidation to androstenedione.

Final body weight measurements as well as absolute and relative liver weights were provided. Animal No. 7 treated with 1000 mg/kg was killed prematurely on day 16 of dosing due to poor clinical condition. No investigation of biochemical liver parameters was performed for this animal and therefore the high dose group comprised only two animals.   Protein contents: Treatment had no effect on the protein content of liver 100 g supernatant, microsomal or cytosolic fraction. The microsomal cytochrome P450 content:  Treatment had no effect on the microsomal cytochrome P450 content. Microsomal 7-ethoxyresorufin-O-dealkylase activity (EROD): Treatment had no effect on EROD activity, indicating that the test article is not a polycyclic aromatic hydrocarbon-type inducer of xenobiotic metabolising enzymes. Microsomal coumarin 7-hydrolase activity: Treatment had no effect on coumarin 7-hydroxylase activity, indicating that the test article is not a phenobarbital-type inducer of xenobiotic metabolising enzymes. Microsomal lauric acid 11- and 12-hydroxylation: Treatment had no effect on lauric acid 11- and 12-hydroxylation activities, suggesting that the test article does not behave as a peroxisome proliferator-type inducer of xenobiotic metabolising enzymes in the cynomolgus monkey. Regio- and stereoselective microsomal hydroxylation of testosterone: Treatment resulted in a slight increase of hydroxylation at the 16β position to 292% of control at the highest dose. Treatment had no effect on 2β-, 6α-, 6β-, 7α-, 15β- and 16α-hydroxylation and oxidation to androstenedione. Cyanide-insensitive peroxisomal β-oxidation: Cyanide-insensitive peroxisomal β-oxidation activity was not increased in the two treatment groups. At the lower dose a slightly lower activity was observed, which is regarded to have no biological significance. Microsomal UDP-glucuronosyltransferase (UDPGT) activity using 3-methyl-2-nitrophenol as substrate: Treatment had no effect on UDPGT activity towards 3-methyl-2-nitrophenol. Microsomal UDP-glucuronosyltransferase activity with thyroxine (T4) as substrate: Treatment had no effect on UDPGT activity towards thyroxine. Cytosolic glutathione S-transferase activity: Treatment had no effect on GST activity.

Description of key information

Rats were found to adapt to exposure at 1000 ppm (ca 85 mg/kg bw) with reversible liver enzyme induction predominantly of the phenobarbitone-type (CYP P450 2B), but also slightly of the peroxisomal enzymes (CYP P450 4A). This interferes with thyroid hormone metabolism and results in a strong increase in TSH and changes in thyroid hormone concentrations in serum. These changes are not observed in monkeys even at exposure to 1000 mg/kg bw for 28 days.

Additional information

As a result of the carcinogenicity study and the different responses of rats, dogs and monkeys, special investigations in regard to thyroid hormone status and induction of hepatic metabolism were performed with rats and cynomolgus monkeys. Homeostatic responses to low thyroid hormone concentrations result in a compensatory increase in the release of thyroid-stimulating hormone (TSH) from the pituitary gland, which in turn stimulates the thyroid gland to increase thyroid hormone synthesis and release. Persistent elevation of TSH levels leads to thyroid follicular-cell hypertrophy and hyperplasia, which if maintained (due to continuous exposure to the compound) can eventually lead to neoplasia. This neoplastic mode of action in rats is well accepted by the scientific community (IARC Monograph no 79. (2001)).

Two studies (Novartis, 1997, also published by Waechter F 1999) describe effects biochemical liver and thyroid hormone parameters in groups of monkeys (3 males/group), which were orally administered the test article by gavage for a period of 4 weeks at dosages of 0, 200, or 1000 mg/kg body weight/day, as reported in the study "Four-week Toxicity study by Oral Administration (Gavage) in Cynomolgus Monkeys" Study No. 14759 TSP. One animal treated with 1000 mg/kg was killed prematurely on day 16 of dosing due to poor clinical condition. No investigation of biochemical liver parameters was performed for this animal and therefore the high dose group comprised only two animals. Serum thyroxine (T4), T3, rT3, serum free T4 and thyroid stimulating hormone (TSH) levels were measured prior to treatment and on days 3, 7, 14, 16, and 28. All three groups showed some changes over time with the serum concentrations of total T4 and T3; T4 increasing slightly and T3 decreasing. Serum rT3 levels were very low and showed no trend but fluctuated more than serum T4 and T3 levels. Most importantly, there was no significant difference between the groups for any variable and over time, all three groups behaved in a similar manner. Over time there was a significant difference in T3 and T4 yet there is no difference (interaction) between the groups. No difference in rT3 and T4 values were observed between the groups. TSH levels were not affected. In addition, the crucial enzyme transforming T4 to T3, monodeiodinase type 1, was not affected by this treatment. This is reflected by the lack of increase in serum rT3 which would be, together with the fall in serum T3, a typical consequence of an inhibition of this enzyme.

For biochemical investigations, liver tissue (about 20 to 25 g) collected from the different lobes was taken from each monkey. Biochemical parameters as indicated below were determined in the appropriate subcellular liver fractions. Liver microsomal suspensions of all animals were subjected to SDS-polyacrylamide gel electrophoresis, and monoclonal antibodies (purified IgG fractions) against purified rat liver cytochrome P450 of families CYP1A, CYP2B, CYP3A, and CYP4A were used for immunoblot analysis.

Treatment had no effect on the protein content of liver 100xg supernatant, microsomal or cytosolic fraction. It had also no effect on the total microsomal cytochrome P450 content and on microsomal 7-ethoxyresorufin-O-dealkylase activity, indicating that the test article is not a polycyclic aromatic hydrocarbon-type inducer of xenobiotic metabolising enzymes. Treatment had no effect on microsomal coumarin 7-hydroxylase activity, indicating that the test article is not a phenobarbital-type inducer of xenobiotic metabolising enzymes. Treatment had no effect on microsomal lauric acid 11- and 12-hydroxylation activities, suggesting that the test article does not behave as a peroxisome proliferator-type inducer of xenobiotic metabolising enzymes in the cynomolgus monkey. Treatment resulted in a 2.9 fold increase of hydroxylation at the 16β-position of testosterone compared to control animals. Treatment had no effect on 2β-, 6α-, 6β-, 7α-, 15β- and 16α-hydroxylation of testosterone and oxidation to androstenedione. Cyanide-insensitive peroxisomal β-oxidation activity was not increased in the two treatment groups. Microsomal UDP-glucuronosyltransferase activity using either 3-methyl-2-nitrophenol or thyroxine as substrate was not affected. Treatment had no effect on glutathione S-transferase activity.

The effects of the substance on male rat thyroid hormone status, morphology of the thyroid gland, pituitary gland and liver were investigated in 1988 (Kelly 1988, published in Muakkassah-Kelly 1991). Thirty-two rats were randomly assigned to eight groups each consisting of 4 animals. The test compound was administered for 3, 6, 13, and 20 days to the animals of four groups added to the food at a dose of 1000 ppm. Animals of four control groups received food without the test compound for the same time periods. At time of sacrifice, 3 -6 mL of blood were collected from abdominal aorta. Levels of T4 and T3 were determined by an enzyme immunoassay, T3 uptake test was performed, and TSH levels were measured by a radioimmuno assay. No signs of toxicity were observed during the treatment period. Administration for different time periods resulted in a significant and time-dependent increase in serum TSH levels up to 10 -fold the control value after 20 days, the longest time point tested. Treatment for 13 and 20 days resulted in slight to moderate hypertrophy of the follicular epithelium and a pronounced depletion of colloid. After 3 or 6 days treatment, there was no substantial change in the histological appearance of the thyroid gland except for a minimal depletion of the thyroid colloid by 6 days. The severity of the lesion was comparable after 13 and 20 days of treatment. Due to the considerable morphological heterogeneity of follicles, it was impossible to describe unambigously treatment-related ultrastructural alterations with the restricted number of tissue samples available.

Histological examination of tissue from pituitary glands indicated the presence of cells containing cytoplasmic vacuoles, similar to those occuring in thyroidectomy cells following both 13 and 20 days treatment. The changes which occurred to a minimal extent were also characterized by electron microscopy. In control animals presumptive thyrotroph cells were characterized by an oval shape and often contained excentricalIy located nuclei. Their cytoplasm contained abundant amounts of rough endoplasmic reticulum displaying extensively dilated cisternae which harbored an electron-dense fuzzy material. In addition, a variable number of small secretory granules with a tendency to be more crowded in the cell periphery was present. After treatment of rats with the test substance the number of secretory granules in the thyrotrophs appeared to be reduced. Instead, similar granules appeared within the dilated cisternae of the rough endoplasmic reticulum. After 3-days treatment the overall morphology of the hepatocytes resembled that of the controls. However, the presence of some enlarged peroxisomes was noted. In addition, a few peroxisomes contained matrical inclusions, so-called matrical plates. The structural alterations of peroxisomes described above were more prominent after treatment for 6 and 13 days. The total number of peroxisomes did not appear to be increased above that found in the controls, however enlarged peroxisomes with elongated and polygonal shapes were more prominent. Most of these contained abundant matrical plates. In some hepatocytes of rats treated for 13 days, the cisternae of rough endoplasmic reticulum tended to be dilated. Liver parenchymal cells of animals fed for 20 days displayed similar organelle changes, although to a lesser extent than at 13 days. Total serum T4 levels in the treated group were significantly lower than those of the corresponding control group after three and six days of treatment. No difference was observed between the control and treated groups at 13 or 20 days. When recorded before killing, very similar body weights were found in control and treated animals. The test compound caused an increase in the absolute liver weights at all time points. The most significant increases were observed after treatment for 6 and 13 days. At day 20, the thyroid gland weights recorded from three control animals were 39.7, 26.7 and 29.5 mg and from two treated animals 74.9 and 67.3 mg. The serum free T4 levels expressed as FT4I values in the treated groups followed the same trend. However, with this parameter relatively large variations were observed between the control groups. The total serum T3 levels and the free T3 concentration, expressed as FT3I, were found to be significantly decreased in the treated groups at all time points investigated. In treated animals serum levels of rT3 increased in a time-dependent manner, up to 3-fold the control values by 20 days treatment.

Administration of 50, 150, 500, and 1000 ppm to male rats for two weeks causes a dose-dependent increase in the liver weights (Waechter 1990). Biochemically, this hepatomegaly is accompanied by an induction of specific cytochrome P-450 isozymes, cytochrome P-450-associated monooxygenases, microsomal epoxide hydrolase, UDP-glucuronosyltransferase, cytosolic glutathione S-transferase and peroxisomal beta-oxidation of fatty acyl-CoA esters. Morphologically, the test compound causes a moderate proliferation of smooth endoplasmic reticulum membranes and a moderate proliferation of peroxisomes containing metrical inclusions. In addition, helically arranged fibrils within inner membrane spaces of mitochondria are observed. Throughout this experiment, special emphasis was put on the characterization of effects on the microsomal cytochrome P-450 system. In this context, studies with monoclonal antibodies as well as monooxygenase activity measurements demonstrate that the substance shares properties of phenobarbitone-type inducers (increased levels of cytochrome P450 2B proteins, induction of ethoxycoumarin O-de-ethylase activity and the site-specific hydroxylation pattern observed with R-warfarin as substrate) and peroxisome proliferatortype inducers (increased levels of cytochrome P450 4A proteins and induction of lauric acid 12-hydroxylase activity). Essentially the same biochemical and morphological effects are observed, when 1000 ppm are administered for more extended periods of time (three, four, or six weeks). Two or four weeks after cessation of a two-week treatment at the highest dose-level (1000 ppm), the investigated parameters returned to control levels with the exception of the morphological alterations in mitochondria, which persisted to some extent.