[No public or meaningful name is available]

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20/10/2008-20/07/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline and in compliance with GLP

Cross-reference

Reason / purpose for cross-reference:

reference to other study

Remarks:

Preliminary study

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2008

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Dose Range-Finding study.

Remarks:

Preliminary study used as dose range-finding experiment for main study (OECD 408).

Guideline:

other: dose range-finding study

Principles of method if other than guideline:

- Principle of test: Dose range-finding study performed in the Wistar rat (repeated dose toxicity study)
- Short description of test conditions: In a dose range-finding toxicity study, test substance was administered daily by oral gavage to SPF-bred Wistar rats (5 animals/sex/dose) at dose levels of 100, 300 or 1000 mg/kg bw/day for a period of 14 days. A control group received a similar dose volume (5 mL/kg bw/day) of the vehicle, PEG 300.
- Parameters analysed / observed: Clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. At the end of the dosing period, all animals were killed, necropsied and examined post mortem. 

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

No data

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

PEG 300

Details on oral exposure:

DOSE VOLUME: 5 mL/kg bw/day

Analytical verification of doses or concentrations:

no

Duration of treatment / exposure:

14 days

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

300 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:

5

Control animals:

yes, concurrent vehicle

Positive control:

Not applicable

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: No

CLINICAL CHEMISTRY: No

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
At the end of the dosing period, all animals were killed, necropsied and examined post mortem.

Statistics:

No data

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related clinical signs of toxicological relevance were noted at any dose level.
- Slight salivation was noted immediately after administration in males and females treated with 300 mg/kg bw/day and 1000 mg/kg bw/day, although slightly less frequently in females treated with 300 mg/kg bw/day. Slight salivation was noted sporadically in males treated with 100 mg/kg bw/day and once in females. Insofar as this finding was noted immediately after treatment and not during later observation (ca 1 and 3 hours after treatment), it was considered to be related to the taste of the dose formulation, rather than as a indication of toxicity.
- During daily observations, moderate hair loss on the dorsal part of the neck and scabs on the lower mandible were noted in one male (no 11) treated with 300 mg/kg bw/day. These were considered to be typical findings resulting from internecine conflict and were unrelated to the test item. Dyspnea was recorded on one single occasion in one male (no 19) treated with 1000 mg/kg bw/day. The females of all groups were unaffected.

Mortality:

no mortality observed

Description (incidence):

All animals survived until scheduled necropsy.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

- The mean body weights of the test item-treated males and females compared favorably with those of the respective controls.
- The males treated with 1000 mg/kg bw/day gained slightly less weight than did the control males (+26.9% vs. 29.1%). These differences were likely to be the result of the slight (and transient) reduction of mean daily food consumption. The females were considered to be unaffected.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

- Slightly reduced mean daily body weights were noted during days 1-3 and 3-7 in males and females treated with 1000 mg/kg bw/day. During days 7-14 of treatment, the mean daily food consumption of these animals compared favorably with those of the respective control group.
- At 100 mg/kg bw/day and 300 mg/kg bw/day, the mean daily food consumption of the males and females compared favorably with those of the controls.

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

- Elevated mean absolute and relative liver weights were noted in males treated with 100 mg/kg bw/day, 300 mg/kg bw/day and 1000 mg/kg bw/day. Frequently these differences attained statistical significance (p<0.05 or p<0.01). In females, statistically significant elevations in mean absolute and relative liver weights were noted at 300 mg/kg bw/day (p<0.05) and 1000 mg/kg bw/day (p<0.01), when compared with the controls. These differences were considered to be related to the test item, but most likely due to metabolic adaptation.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- No test item-related macroscopical changes were noted at any dose levels.
- A number of typical background changes were noted in two males at 300 mg/kg bw/day (local alopecia and thymic foci). All other treated males (and all females) were unaffected.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

other: no NOAEL identified

Remarks on result:

other: the aim of this study was to determined the dose levels for a sub-chronic repeated dose toxicity study (OECD 408)

Key result

Critical effects observed:

no

None

Conclusions:

Based on these results of this 14-day dose range-finding study, dose levels of 30, 100 or 300 mg/kg bw/day were chosen for the main study (OECD 408).

Executive summary:

In a dose range-finding toxicity study, test substance was administered daily by oral gavage to SPF-bred Wistar rats (5 animals/sex/dose) at dose levels of 100, 300 or 1000 mg/kg bw/day for a period of 14 days. A control group received a similar dose volume (5 mL/kg bw/day) of the vehicle, PEG 300. Clinical signs, food consumption and body weights were recorded periodically during the acclimatization and treatment periods. At the end of the dosing period, all animals were killed, necropsied and examined post mortem. 

 

All animals survived until scheduled necropsy. No clinical signs of toxicological relevance were noted at any dose level. Initial, but slight reductions in mean daily food consumption were noted at the highest dose of 1000 mg/kg bw/day, but these differences were transient and no longer evident during the second week of treatment. Slightly lower mean daily body weight gain values were noted in males at 1000 mg/kg bw/day, whereas females were considered to be unaffected. Elevated mean absolute and relative liver weights were noted in males treated with 100 mg/kg bw/day and in both sexes at 300 and 1000 mg/kg bw/day. These differences were considered to be related to the test item. Although such changes are often due to metabolic adaptation, these tissues were not examined for morphological changes and therefore, any associated microscopic alterations were not evaluated. There were no macroscopical findings of toxicological relevance.

 

Based on these results of this 14-day dose range-finding study, dose levels of 30, 100 or 300 mg/kg bw/day were chosen for the main study (OECD 408).

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2009

Report date:

2009

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Molecular formula:

R-O-(CH2CH2O)nCH2CO-NH-CH2CH2OH

IUPAC Name:

Fatty alcohols C13-15 (odd numbered, linear and branched), reaction products with ethylene oxide, sodium chloroacetate and ethanolamine

Test material form:

liquid

Details on test material:

- Physical state: liquid
- Stability under test conditions: under storage conditions
- Storage condition of test material: at room temperature (20±5°C), light protected
- Expiry date: 10-APR-2009
- Stability of Test Item Dilution: Stable for 7 days in water

Test animals

Species:

rat

Strain:

Wistar

Remarks:

HanRcc: WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd (Switzerland)

- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding

Number of Animals:
Group 1: 15 males and 15 females
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 15 males and 15 females

Reserve animals:
Group 10: 2 males and 2 females

Age (at Delivery): Ca. 7 weeks

Body Weight Range (at Acclimatization):
Males: 176 g to 190 g (mean 184 g)
Females: 136 g to 156 g (mean 147 g)

- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no.44/08) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland),ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum

- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed batch was analyzed for contaminants.
Water: Bacteriological assay, chemical and contaminant analyses were made.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3ºC
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

A control group was treated similarly with the vehicle, PEG 300, only
Dose Volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On treatment day 1, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration were taken during week 4, 8 and 13 after commencement of gosing to confirm homogeneity and concentration. The analysis was performed using HPLC coupled to a UV detector and quantified with area under the peak. The test item was used as analytical standard.

Duration of treatment / exposure:

91/92 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

actual ingested

Doses / concentrations 2

Dose / conc.:

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

Remarks:

actual ingested

Doses / concentrations 3

Dose / conc.:

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

Remarks:

actual ingested

No. of animals per sex per dose:

10 animals/sex/dose for the following groups : 0, 30, 100 and 300 mg/kg body weight/day
Additional satellite group of 5 animals/sex/dose for the following groups: 0 and 300 mg/kg body weight/day

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (Harlan Laboratories study C02788).
In this dose range-finding toxicity study, AA 15 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 or 1000 mg/kg body weight for a period of 14 days. The study comprised five animals per group and sex which were sacrificed after 14 days of treatment.
No clinical signs of toxicological relevance were noted at any dose level.
Initial, but slight reductions in mean daily food consumption were noted at the highest dose of 1000 mg/kg/day, but these differences were transient and no longer evident during the second week of treatment. Slightly lower mean daily body weight gain values were noted in males at 1000 mg/kg/day, whereas females were considered to be unaffected.
Elevated mean absolute and relative liver weights were noted in males treated with 100 mg/kg/day and in both sexes at 300 mg/kg/day and 1000 mg/kg/day. These differences were considered to be related to the test item. Although such changes are often due to metabolic adaptation, these tissues were not examined for morphological changes and therefore, any associated microscopic alterations were not evaluated.
There were no macroscopical findings of toxicological relevance.
Based on the results of this 14-day dose range-finding study, dose levels of 30, 100 or 300 mg/kg body weight/day would be considered appropriate for a subsequent study with AA 15.


Dose Levels in Main study:
Group 1: 0 mg/kg/day
Group 2: 30 mg/kg/day
Group 3: 100 mg/kg/day
Group 4: 300 mg/kg/day

Dose Volume: 5 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6 mg/mL/day
Group 3: 20 mg/mL/day
Group 4: 60 mg/mL/day

Duration of Treatment Period: 91/92 days
Duration of Recovery Period: 28 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration as well as twice daily on days 1 to 3, once daily from on day to the end of the treatment period , and once daily during days 1 to 28 (recovery period).

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once during the acclimatization period and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals (satellite A and B) during acclimatization, during week 13 of treatment in animals of the control and high dose groups, and during week 17 in rats of satellite B.
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Anaesthetic used for blood collection: Yes : light isoflurane anesthesia
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals:
- Parameters checked in Table 7.5.1/1 were examined.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals:
- Parameters checked in Table 7.5.1/2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 7.5.1/3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.
- Dose groups that were examined: 0, 30, 100, 300 mg/kg bw/day
- Battery of functions tested: grip strength / locomotor activity. During week 13, relevant parameters from a modified Irwin screen test were evaluated in all animals.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 7.5.1/4)

HISTOPATHOLOGY: Yes : Slides of all organs and tissues (as defined in the table 7.5.1/4) which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (hearts (males only), livers and thyroids) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, organ weights and ratios as well macroscopic findings: the Dunnett-test, the steel-test and fisher's exact-test

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects seen during daily observations included initial salivation at the highest dose level. This finding was noted more sporadically as the treatment period progressed.
Pale feces was noted in both sexes from day 47 of treatment until the end of the treatment period and for one day of the recovery period. In females treated with 300 mg/kg/day, salivation was noted during weeks 2-6 in some animals, and then intermittently during several of the remaining treatment weeks.
At 100 mg/kg/day and 30 mg/kg/day, a variety of clinical signs (such as breathing noises, salivation, localized scabs) were noted sporadically in some animals. These findings were considered to be typical background clinical signs and considered to be of no toxicological significance, as indicated by the very sporadic occurrence and/or small number of affected animals.

CONCLUSION: No adverse effects

Mortality:

no mortality observed

Description (incidence):

One male (nº41) treated with 300 mg/kg/day was found dead on day 71 of treatment. This was considered to be a possible dosing error. All other animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A test item-related reduction in the mean body weights was noted during the treatment period in males treated with 300 mh/kg/day. These differences were largely reversible after the recovery period. In females, the differences to the control group during the treatment period were negligible and considered to be due to biological variation.
No effects were noted in the groups treated with 30 mg/kg/day or 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated rats was generally similar to that of the controls.

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted after 13 weeks of treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related changes of toxicological relevance were noted at any dose level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes in the clinical biochemistry parameters were characterized by alterations in the lipid metabolism parameters in males and females treated at 300 mgkg/day, as well as elevated activity of aspartate aminotransferase in males at 300 mg/kg/day.
The latter changes were considered to be related to metabolic adaptation. None of the remaining minor difference to the control values were considered to be of toxicological relevance.

CONCLUSION: No adverse effects

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in the urinalysis parameters at any dose level.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery
During the Functional Obsevational Battery performed after 13 weeks of treatment, slightly to moderately decreased activity was recorded in males treated with 100 mg/kg/day and 300 mg/kg/day. No other differences were noted.

Grip Strength
At 300 mg/kg/day, the mean hind limb grip strength was significantly reduced (p<0.01) in females when compared with that of the controls. Insofar as the forelimb grip strength of these females was unaffected, this was considered to be incidental. The mean fore- and hindlimb grip strength values of the males and females were considered to be unaffected at all dose levels.
No differences were ascertained at 30 mg/kg/day or 100 mg/kg/day.

Locomotor Activity
Minor differences in the mean locomotor activity were considered to be incidental.

CONCLUSION: No adverse effects

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, elevated mean absolute liver weights were noted in males and females when compared with controls.
In females, the mean absolute heart weight was elevated, along with mean brain-to-body weight, mean heart-to-brain weight, mean kidney-to-body weight and mean uterus-to-body weight ratios.
Females treated with 300 mg/kg/day also showed elevated liver-to-brain weight ratio at the end of the treatment period.

CONCLUSION: No adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related macroscopical changes were noted at any dose level.
The item-related microscopical inflammatory and degenerative changes were noted in the hearts of males treated with 100 and 300 mg/kg/day and centrilobular hypertrophy of the hepatocytes was recorded in the livers of males and females treated with 300 mg/kg/day. After the 28-day recovery period, the heart and liver changes reverted to normal.

CONCLUSION: No adverse effects

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Effect levels

1

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose level

2

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Treatment-related effects detected at 300 mg/kg bw/day were not considered to represent an adverse health effect.

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

Based on the results of this study, 30 mg/kg body weight/day of the substance was established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the substance as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

In a repeated dose toxicity study performed according to the OECD test guideline No. 408 and to the EU Method B.26 and in compliance with GLP, test material was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 30, 100 and 300 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after the end of the treatment period. Additional 5 rats per sex and group were used at 0 and 300 mg/kg. These animals were treated for 91/92 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Oral administration of test substance to Wistar rats at doses of 30, 100 and 300 mg/kg/day for 91/92 days resulted in no deaths from systemic toxicity, no relevant differences in mean fore- or hindlimb grip strength or locomotor activity, no ophthalmoscopic changes, no differences in mean daily food consumption, no changes of toxicological relevance in hematology or urinalysis and no macroscopical changes indicative of systemic findings. The single male (no 41) treated with 300 mg/kg/day which died on day 71 of treatment was considered to result from a dosing error that caused lung edema, hemorrhage and alveolar necrosis.

Test item-related findings were generally restricted to initial salivation at 100 mg/kg/day and 300 mg/kg/day (considered to be related to a bitter taste of the test item), pale feces at 300 mg/kg/day from day 47 of treatment onwards, decreased activity in both sexes at 300 mg/kg/day and in males at 100 mg/kg/day, reduced mean body weight development in males at 300 mg/kg/day, changes in clinical biochemistry (metabolic adaptation characterized by alterations in the lipid metabolism parameters in males and females treated at 300 mg/kg/day, as well as elevated activity of aspartate aminotransferase in males at 300 mg/kg/day), increased absolute and/or relative liver weights in males and females at 300 mg/kg/day and males at 100 mg/kg/day.

At the end of the treatment period, inflammatory and degenerative changes were seen in the heart of males treated with 100 mg/kg/day and 300 mg/kg/day. These heart changes reversed after a 28-day recovery period. Furthermore, centrilobular hypertrophy of the hepatocytes was seen in males and females treated with 300 mg/kg/day that reversed after a 28-day recovery period. This hypertrophy most likely reflected an induction of the mixed function oxidase (microsomal enzymes) that is responsible for the metabolism of the xenobiotics. The hypertrophy of the follicular epithelium of the thyroid gland was secondary to the hepatocytic hypertrophy. It is well documented, particularly in rats, that an increased turnover of T4 (due to the increased liver metabolism) is associated to an activation of the thyroid gland through the increased secretion of TSH (thyroid stimulating hormone) by the pituitary gland. The exposure to xenobiotics can induce the mixed function oxidase (MFO) in the liver with an increase in the microsome content resulting in an increase of the liver weight and a corresponding hepatocyte hypertrophy (predominantly centrilobular). In general, when the animals undergo a recovery period, the MFO activation disappears with a return to the normal morphology of the liver. The hypertrophy of the follicular epithelium of the thyroid gland of males and females at 300 mg/kg/day was very likely the result of the liver change. It is well documented that the induction of the mixed function oxidase in the liver determines an increased metabolism of the thyroid hormones (T4). As a result, the low level of T4 induces a feedback mechanism that leads to increased synthesis of TSH by the pituitary gland (pituitary-thyroid axis). This pathogenic mechanism is particularly observed in rats, the most sensitive species to the metabolism of thyroxine. Humans are far less sensitive to such mechanism.

Based on the results of this study, 30 mg/kg body weight/day of the substance was established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the substance as the no-observed-adverse-effect-level (NOAEL).