Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

01 Dec 1992 to 31 Mar 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1993

Report date:

1993

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)

Version / remarks:

May 1981

Test guideline 2

Qualifier:

according to guideline

Guideline:

EPA OPP 82-1 (90-Day Oral Toxicity)

Version / remarks:

Nov 1982

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: Notification of Director General, Agricultural Production Bureau, Ministry of Agriculture, Forestry and Fisheries, Japan, 59 Noh San No. 4200

Version / remarks:

Jan 1985

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

EC Number:

276-696-7

EC Name:

Ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Cas Number:

72490-01-8

Molecular formula:

C17 H19 N O4

IUPAC Name:

ethyl [2-(4-phenoxyphenoxy)ethyl]carbamate

Test animals

Species:

rat

Strain:

other: Tif: RAIf (SPF)

Remarks:

hybrids of RII/l x RII/2 (Sprague-Dawley derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 4 weeks at delivery.
- Weight at study initiation: 99.7 - 119.4 g and 90.6 - 110.0 g for males and females, respectively.
- Housing: groups of 5 in macrolon cages type 4 with wire mesh tops and standardised granulated soft wood bedding Individually in cages (size 3) with wood chips.
- Diet: ad libitum (admixed to pelleted food).
- Water: Tap water, ad libitum.
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 10
- Air changes (per hr): 16 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE dates: From 01 Dec 1992 To 31 Mar 1993

Administration / exposure

Route of administration:

oral: feed

Vehicle:

acetone

Remarks:

(removed during preparation)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet: at about monthly intervals
- Mixing appropriate amounts with: The test substance was dissolved in acetone (130 g in 500 mL acetone). A premix was made for each group using aliquots of this solution added to fixed amount of Bolus alba and diet with the addition of a further amount of acetone to equalise the quantity of acetone used for each group. After removal of the acetone by vacuum at 22 ± 2 °C, the premix for each group was mixed with further fixed quantities of diet to yield diets containing the appropriate concentration of test substance. Diet was stored in stainless steel containers at room temperature in a separate area.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Control analyses of the test article content were undertaken with diet used for treatment weeks 1 - 4 and 10 - 14, respectively.

Duration of treatment / exposure:

13 weeks (satellite group: 4 weeks recovery)

Frequency of treatment:

Continuously

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

30 ppm

Remarks:

Dietary concentration; equivalent to 2.17 and 2.29 mg/kg bw/day for males and females, respectively.

Doses / concentrations 2

Dose / conc.:

150 ppm

Remarks:

Dietary concentration; equivalent to 9.71 and 10.1 mg/kg bw/day for males and females, respectively.

Doses / concentrations 3

Dose / conc.:

750 ppm

Remarks:

Dietary concentration; equivalent to 45.1 and 49.6 mg/kg bw/day for males and females, respectively.

Doses / concentrations 4

Dose / conc.:

3 000 ppm

Remarks:

Dietary concentration; equivalent to 199 and 203 mg/kg bw/day for males and females, respectively.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Post-exposure recovery period in satellite groups: The first experimental group was composed of 10 animals per sex and group for evaluation of toxicity, including laboratory investigations. A second experimental group contained 10 animals per sex and group for reversibility evaluation after 4 weeks of recovery, including laboratory investigations

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS:
- All animals were checked daily (a.m. and p.m. on working days, a.m. on weekends and holidays), in order to record mortalities, and to allow dead or moribund animals to be submitted to necropsy as soon as possible.
- In-life observations: In order to detect changes in state of health or behaviour, or any reaction to treatment, examination was carried out daily, and observations were recorded at least weekly.

BODY WEIGHT
- The weight of all animals was recorded individually at weekly (midweek) weighing sessions. The first weights were recorded during the acclimatation period.

FOOD CONSUMPTION
- The food consumption was recorded weekly (cage-wise) and was calculated for periods of one week. The calculation was based on the weight of the offered diet at the beginning of a Weighing period and its difference to the re-weighed amount after several days. The individual food consumption values were calculated from the food consumption per cage and the number of animals present.

FOOD CONSUMPTION, UTILISATION AND ACHIEVED DOSE:
- The food consumption ratios were calculated as mean of individual ratios according to the following formula:
(Weekly food consumption (g) / midweek body weight (g) ) x (1000/7)
Unit: g food/kg body weight per day.
- The test substance intake was calculated according to the following formula (rounded to 3 meaningful digits):
(food consumption ratio x nominal dose (ppm))/1000
Unit: mg test article/kg body weight per day
An overall mean value (MEAN1) was calculated based on the nominal content of the test article in the food. Additionally, this mean value was corrected for the analytically determined test article content in the food (MEAN2)

WATER CONSUMPTION:
- The water consumption was recorded weekly (cage-wise). The water consumption was calculated for periods of one week. The calculation was based on the weight of the offered water at the beginning of a weighing period and its difference to the re-weighed amount after one day. The individual water consumption was calculated from the water consumption per cage and the number of animals present

OPHTHALMOSCOPY EXAMINATION:
- Animals of the highest dose group and of the control group were examined prior to dosing (day -4), towards the end (day 87) of the treatment period and towards the end of the recovery period (day 115). Examination included observation of eye appearance and of periocular region, and detection of pupillary reflex using an ophthalmoscope.

HAEMATOLOGY, CLINICAL CHEMISTRY AND URINALYSIS:
- Laboratory investigations (haematology, blood chemistry, urine analysis) were carried out on all animals of each dose group at the end of the treatment period, and additionally at the end of the recovery period on animals of the control and high dose group kept for reversibility evaluation. To reduce the biological variability due to circadian rhythms, blood sampling was performed in the morning. Food was withheld overnight prior to blood removal. Ether anaesthesia was used to restrain the animals. Blood was withdrawn from the orbital sinus using glass capillary tubes. Blood samples from each animal with the respective anticoagulant (EDTA for performing the complete blood count, 3.8% Sodium citrate for coagulation testing, Heparin for blood chemistry and methaemoglobin measurements) were collected into individual vials.‘
Urine for analysis was collected overnight. The individual animals were housed in special metabolism cages. Food and water was withheld during the time of urine collection.
- Chemical urine components, pH and relative density were estimated by an automated urine analyser Clinitek Auto 2000 (Ames) using solid-phase reagent systems combined with reflectance spectroscopy
(chemical constituents and pH), and applying the falling drop method (relative density).

Sacrifice and pathology:

MACROSCOPIC EXAMINATION:
At the end of the treatment period all controls and treated animals of experimental group I, and at the end of the recovery period, all animals of experimental group II, were under ether anaesthesia and subjected to detailed necropsy. At necropsy the following weights were recorded from all animals: body (exsanguinated), brain, heart, liver, kidneys, adrenals, thymus, ovaries/testes, spleen, thyroid gland.
The following organs and tissues were preserved in neutral buffered 4 % formalin:
skin; mammary area; spleen; mesenteric lymph node; axillary lymph node; sternum with bone marrow; femur with joint; skeletal muscle; trachea; lung; heart; aorta; submandibular salivary gland, both; liver; pancreas; oesophagus; stomach; small intestine; large intestine; kidney, both; urinary bladder; prostate; seminal vesicle; testis, both; epididymis, both.; uterus; vagina; ovary, both
pituitary gland; adrenal gland, both; thyroid with parathyroid gland; thymus; peripheral nerve; brain; spinal cord; eye with optic nerve, both; orbital gland, both; extraorbital lacrimal gland, both; Zymbal gland, both; muzzle; tongue; any gross lesions, masses.

MICROSCOPICAL EXAMINATION
After the fixation, organ samples listed below were taken from all animals of the control and all treated groups (experimental group I) embedded in paraplast, sectioned at 3-5 microns, stained with haematoxylin and eosin, and subjected to a microscopical examination: spleen; mesenteric lymph node; axillary lymph node; sternum with bone marrow; femur with joint; trachea; lung; heart; aorta; submandibular salivary gland, both; liver; pancreas; esophagus; stomach; small intestine; large intestine; kidney, both; urinary bladder; testis, both; epididymis, both; uterus; vagina; ovary, both; pituitary gland; adrenal gland, both; thyroid with parathyroid gland; thymus; peripheral nerve; brain; any gross lesions, masses.
In addition, since the examination of the experimental group I revealed treatment-related changes in the liver and thyroid gland, these organs were processed and examined also in animals of experimental group II.
Where practicable, gross lesions were identified by a capital letter, e.g. A, B, C, etc. at necropsy. At the subsequent histopathological evaluation the diagnosis or diagnoses corresponding to the macroscopically identified lesions were given the same alphabetical label in order to correlate the microscopical findings with the changes seen at necropsy. The histopathological lesions observed were graded as to degree of severity according to the following criteria: Grade "+" : Minimal (slight). Includes histopathological change that is a noticeable but not prominent feature of the tissue. Grade "++“ : Moderate. Includes histopathological change that is a prominent but not dominant feature of the tissue. Grade "+++" : Marked. Includes histopathological change that is a dominant feature of the tissue. In the "Summary table of microscopical findings", the incidence "0" was listed when no finding was reported, i.e., the organ/tissue was free of changes or not selected for microscopical examination according to the study protocol. For technical reason, the term "no changes observed" was used only for the topographical site "organs and tissues as a whole", referring to organs and tissues selected for microscopical examination according to the study protocol.

Statistics:

For each time point and parameter an univariate statistical analysis was performed. Nonparametric methods <1> were applied, to allow for non normal as well as normal data distribution. Each treated group was compared to the control group by Lepage's two-sample test <2> and tested for increasing or decreasing trends from control up to the respective dose group by Jonckheere's test for ordered alternatives <3>. The Lepage test is a combination of Wilcoxon and Ansari—Bradley statistics, i.e. a combined test for location and dispersion. The Lepage test has a good power against the more general alternative that the distributions differ not only in location but also in dispersion. The Jonckheere test is sensitive to mono— tone dose—related effects. Two—sided asymptotic p—values are reported in the "statistics" tables. Flags for significant differences between groups (*) or trends over groups (+ or -) are given in the “means" tables according to the specified significance level. Statistical tests and flags used are indicated in the header or each table. Statistical significance does not necessarily imply biological relevance. Hence, the responsible scientist may not comment on statistically significant values lying within the physiological range and on the other hand may comment on values, which differ substantially from the expected normal values although this difference was not statistically significant.

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

The low incidence of clinical signs recorded in treated rats was not attributed to treatment, as these signs are of spontaneous nature in untreated rats of this age and colony.

Mortality:

no mortality observed

Description (incidence):

All rats survived the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The individual body weight values collected during the study. Lower body weight gains were recorded for males and females of group 5 (3000 ppm) with mean body weights approximately 5% and 10 % lower, respectively, than control values by the end of the treatment period. By the end of the recovery period, mean body weights for group 5 males were similar to control values, whereas lower mean body weights continued to be recorded for group 5 females. Body weights of other treated groups were considered not to have been influenced by treatment.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Differences between food consumption ratios for treated and control groups were of insufficient magnitude to be of toxicological relevance.
During the treatment period, lower food intakes (below 9 % the control value) were recorded for females of group 5 (3000 ppm). Food intakes for other treated female groups as well as for all treated male groups were not influenced by treatment. Slight variations in food intakes of high dose males during the recovery period were not considered associated with treatment.
Mean test substance intake values were calculated based on the food consumption ratios. They were corrected for actual amount of test substance in the diet according to the results of chemical analysis (analytical concentration as of nominal concentration). The calculated mean daily intake of test substance, based on the nominal concentrations in the diet was 2.09, 10.6, 50.5 and 214 mg/kg body weight in males, and 2.21, 11.1, 55.5 and 219 mg/kg body weight in females. Corrected for the analytically determined test substance concentrations in the diet, the mean daily intake of test substance was 2.17, 9.71, 45.1 and 199 mg/kg body weight in males, and 2.29, 10.1, 49.6 and 203 mg/kg body weight in females.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

The individual water consumption was calculated from the water consumption per cage and the number of animals present. The water intake values recorded were within the expected range and gave no indication of a treatment-related influence.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

Examinations included inspection of the surroundings of the eyes, of sclera, cornea, iris and adaptation of the pupil to the ophthalmoscopic light beam. Animals of the control and highest treatment level groups were examined during the acclimatation period (day -4), towards the and (day 87) of the application period, and towards the end of the recovery period (day 115). At each examination, no changes were detected in any of the animals.

Haematological findings:

no effects observed

Description (incidence and severity):

The treatment had no influence on the haematological profile. All the minor intergroup differences achieving a level of statistical significance reflect the biological variation of the parameters, and were therefore considered not to be treatment related.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

At treatment end, plasma cholesterol levels were slightly increased in males and females of groups 4 and 5 (750 and 3000 ppm). High dose males (3000 ppm) additionally had minimally lower triglyceride levels, increased activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, whereas high dose females had only minimally increased alkaline phosphatase activities. Furthermore, both males and females of the top dose group (3000 ppm) showed higher albumin to globulin ratios which in males resulted mainly from lower globulin levels and in females from higher albumin levels. All these alterations were reversible within the recovery period. Decreased bilirubin levels as recorded for high dose animals and for females of group 4 are without any toxicological relevance. All the other intergroup differences attaining a level of statistical significance reflect the physiological variation of the parameters and were therefore considered not treatment-related.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

Microhaematuria or haemoglobinuria observed in one male and one female of the high dose group (3000 ppm) at treatment end were considered to be incidental findings without any toxicological relevance.

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At the end of the treatment period reduced carcass weight in males (-7 % when compared with the control group) and in females (-11 % were noted. Organ weight analysis revealed higher liver weights and liver to body weight ratios in males (+ 26 and +35 %) and in females (+ 35 and + 52 %) of group 5 (3000 ppm). The absolute thyroid weight was increased in females (+29 %) and the thyroid to body weight ratio was increased in males (+ 8 %) and in females (+ 46 %)
In group 4 (750 ppm) the absolute and relative mean liver weights were increased in males (+ 6 and +7 %) and in females (+ 17 and + 17 %). Values for other absolute or relative organ weights were not disturbed by treatment. At the end of the recovery period, mean weights for liver and thyroid were similar to the control values.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The liver of 2/10 female rats of group 4 and of 4/10 males and 7/10 females of group 5 (750 and 3000 ppm, experimental group I) was enlarged. This finding correlated microscopically with a hypertrophy of the hepatocytes which was considered treatment-related. A mass in the liver observed in one male of group 5 (3000 ppm, experimental group II) correlated microscopically with a developmental malformation and it was regarded as incidental. All other findings occurred in comparable numbers in all experimental groups and were similar to those occurring spontaneously in our colony of rats. Thus, no experimental relevance was attributed to these findings.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following histopathological differences observed between control and treated rats were considered treatment-related: In the liver, a hypertrophy of the hepatocytes was seen in 8/10 female rats of group 4 and in-all males and females of group 5 (750 and 3000 ppm, experimental group I). This change, mainly confined to the centrilobular region of the liver, was minimal in females of group 4 and minimal to moderate in males and females of group 5. This cellular hypertrophy was most likely an adaptive response to changes in metabolism resulting from the administration of the test article. A hypertrophy of the follicular epithelium of the thyroid gland, associated with numerous resorption vacuoles in the cytoplasm of follicular cells and paler and more vacuolated colloid, was detected in 7/10 females of group 4 and in 3/10 males and in all females of group 5 (750 and 3000 ppm, experimental group I). This finding was minimal in females of group 4 and in males of group 5 and minimal to moderate in females of group 5. The thyroid gland changes were likely the result of a glandular activation secondary to the increased metabolism of the liver leading to an accelerated break-down of thyroid hormones. Within 4 weeks of recovery, both the liver and the thyroid gland findings were fully reversible. Additionally, a variety of other changes was found in this study. They commonly occur in our colony of rats, and, neither their incidences nor their distribution and morphological appearance gave any indication of a treatment-related association.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

150 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

histopathology: non-neoplastic

organ weights and organ / body weight ratios

serum/plasma biochemistry

Remarks on result:

other:

Remarks:

Dietary equivalent to 9.7 and 10.1 mg/kg bw/day in males and females, respectively

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

VERIFICATION OF TEST DIET

- Stability: Prior to the beginning of the study, food samples containing the test article at concentrations of 0, 10, 100, 1000 and 20000 ppm were dispatched for determination of content, homogeneity and stability. The results indicated stability of the test article in pelleted food for 5 weeks when stored at room temperature.

- Homogeneity: Homogeneity of the test article distribution in pelleted food was demonstrated in pre-test food preparations. The results showed a homogeneous distribution of the test article in the diet

- Concentrations: The analytical results showed that the contents of test substance in the diet were in agreement with the nominal concentrations. The analyses revealed concentrations of 89 – 105 % of the nominal values, resulting in mean concentrations of 104, 92, 89 and for dose groups 2, 3, 4, and 5, respectively.

Table 1 Results after 13 weeks

Dose (mg/kg food)	0	0	30	30	150	150	750	750	3000	3000	dr
	m	f	m	f	m	f	m	f	m	f
Mortality	none
Clinical signs	no treatment-related findings
Body weight									d	dc
Food consumption										d
Water consumption	no treatment-related findings
Ophthalmoscopy	no treatment-related findings
Haematology
white blood cells									ic
haemoglobin										dc
neutrophils										dc
lymphocytes										ic
monocytes										dc
large unstained cells										dc
Clin. Chemistry
glucose										ic
urea									ic
creatinine									dc
total bilirubin								dc	dc	dc
total protein										ic
albumin									ic	ic
globulin									dc
A/G ratio									ic	ic
cholesterol							ic	ic	ic	ic	m/f
triglycerides									dc
potassium									dc
phosphate									ic
ASAT									ic
ALAT									ic
alkaline phosphatase									ic	ic
Urine analysis	no treatment-related findings
Organ weights
liver								i^a, ic^r	ic^a,r	ic^a,r	m/f
thyroid										i^a, ic^r
Pathology
Macroscopy
liver large	0/10	0/10	1/10	0/10	0/10	0/10	0/10	2/10	4/10	7/10	f
Microscopy
liver: centrilobular hypertrophy	0/10	0/10	0/10	0/10	0/10	0/10	0/10	8/10	10/10	10/10	f
thyroid: follicular hypertrophy	0/10	0/10	0/10	0/10	0/10	0/10	0/10	7/10	3/10	10/10

dr = dose related

dc/ic = statistically significantly decreased/increased compared to the controls

d/i = decreased/increased, but not statistically significantly compared to the controls

a/r = absolute/relative

Table 2 Results after 4-week recovery period

Dose (mg/kg food)	0	0	30	30	150	150	750	750	3000	3000	dr
	m	f	m	f	m	f	m	f	m	f
Mortality	none
Clinical signs	no treatment-related findings
Body weight										d
Food consumption	no treatment-related findings
Water consumption	no treatment-related findings
Ophthalmoscopy	no treatment-related findings
Haematology	no treatment-related findings
Clin. Chemistry	no treatment-related findings
Urine analysis	no treatment-related findings
Organ weights	no treatment-related findings
Pathology
Macroscopy
Liver, mass	0/10	0/10	0/10	0/10	0/10	0/10	0/10	0/10	1/10	0/10
Microscopy	no treatment-related findings

dr dose related

dc/ic statistically significantly decreased/increased compared to the controls

d/i decreased/increased, but not statistically significantly compared to the controls

a/r absolute/relative

Applicant's summary and conclusion

Conclusions:

The study results indicate that the liver and thyroid are potential targets of test substance in rats. The NOAEL is set at 150 mg/kg food (dietary equivalent to 9.71 mg/kg bw for males and 10.1 mg/kg bw for females) based on effects on the liver and thyroid.

Executive summary:

In an OECD TG 408 study in compliance with GLP, the test substance was administered in the diet for 3 months at dietary levels of 0, 30, 150, 750, and 3000 ppm (= mg/kg food) to a total of 140 Tif: RAIf (SPF) albino rats. In each dose group 10 animals per sex and group sacrificed at the end of the treatment period; 10 animals per sex in the control and high dose groups were kept for a 4-week recovery period before sacrifice. Corrected for the analytically determined test article concentrations in the diet, the mean daily intake of test substance was 2.17, 9.71, 45.1, and 199 mg/kg body weight in males, and 2.29, 10.1, 49.6, and 203 mg/kg body weight in females.

Treatment did not affect the appearance or behaviour of the rats. All rats survived the study. During the treatment period the mean body weight gain in males and females of group 5 (3000 ppm) was minimally to slightly lower than in the control group. The effect was reversible in males but not in females over the 4-week recovery period. A reduced food intake by females of group 5 (3000 ppm) became similar to that of the controls during the recovery period.

Food consumption ratios were similar between treated and control groups and no effects on water intake was observed. No evidence of ocular toxicity was detected. The treatment had no influence on the haematological profile. At the end of the treatment, plasma cholesterol levels were slightly increased in males and females of groups 4 and 5 (750, 3000 ppm). In addition, high dose males (3000 ppm) had minimally lower triglyceride levels, increased activities of alanine aminotransferase, aspartate aminotransferase and alkaline phosphatase, whereas high dose females had only minimally increased alkaline phosphatase activities. Furthermore, both males and females of the top dose group (3000 ppm) showed higher albumin to globulin ratios which in males resulted mainly from lower globulin levels and in females from higher albumin levels. All these alterations were reversible within the recovery period. Treatment had no influence on urine parameters. In the high dose group (3000 ppm), reduced carcass weights, higher absolute and relative mean liver weights, and higher relative thyroid weights in both sexes and higher absolute thyroid weights in females were observed at treatment end. Normal values were recorded at the end of the recovery period. Macroscopically, enlarged livers were seen in 2/10 female rats of group 4 and in 4/10 males and 7/10 females of group 5 (750 and 3000 ppm) at the end of the treatment period. Minimal to moderate hypertrophy of the hepatocytes was observed in females of group 4 and in males and females of group 5. Hypertrophy of the follicular epithelium of the thyroid gland in females of group 4 and in both sexes of group 5 was considered of secondary nature. Within 4 weeks of recovery, both the liver and the thyroid gland changes were fully reversible.

Under the conditions of this test, treatment with test substance resulted in reduced food intake and depressed body weight gain in high dose animals. Liver changes at the higher dose levels were substantiated by increased weight, hepatocellular hypertrophy and changes to plasma levels for proteins, cholesterol, and liver enzymes. Hypertrophy of thyroid follicular epithelium was considered of secondary nature. This slight liver toxicity accompanied adaptive changes of a functional nature, indicative of metabolic load or hormonal adjustment, were completely reversible upon cessation of treatment. It can be inferred from the observations during the above study, that a "no observable effect level" for test substance when offered to rats continuously in their food over a period of 3 months is 150 ppm, corresponding to a mean daily intake of 9.7 mg/kg body weight in males and 10.1 mg/kg body weight in females.

Hence, the study results indicate that the liver and thyroid are potential targets of test substance in rats. The NOAEL is set at 150 mg/kg food (equal to 9.71 mg/kg bw for males and 10.1 mg/kg bw for females) based on effects on the liver and thyroid.