4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

9 May 2019 to 9 August 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

3 October 2008

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

EC Number:

253-781-7

EC Name:

4,4'-[(isopropylidene)bis(p-phenyleneoxy)]diphthalic dianhydride

Cas Number:

38103-06-9

Molecular formula:

C31H20O8

IUPAC Name:

5,5'-[propane-2,2-diylbis(4,1-phenyleneoxy)]bis(2-benzofuran-1,3-dione)

Test material form:

solid: flakes

Details on test material:

- Appearance: yellow flake
- Storage conditions: room temperature

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

The rat was selected because it is a standard species for use in toxicology studies. Rats are also being used as a rodent species per current EPA and OECD testing guidelines.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at first dose: 8 weeks
- Weight at first dose: 228.4 - 248.7 g (males); 183.0 - 203.0 (females)
- Housing: Animals were housed (two to three per cage within the same sex and group) in polycarbonate cages suspended on stainless steel racks. Non-plastic environmental enrichment was provided.
- Bedding: Certified hardwood bedding
- Diet: Certified Laboratory Diet 2018 (pellets), ad libitum
- Water: Filtered water, ad libitum
- Acclimation period: Animals were acclimated to laboratory conditions for seven days prior to the first dose.

DETAILS OF FOOD AND WATER QUALITY: The feed was analysed by the manufacturer for concentrations of specified heavy metals, aflatoxin, chlorinated hydrocarbons, and organophosphates. The water is routinely analysed for contaminants and specific microbes. The bedding was analysed by the manufacturer for acceptable levels of heavy metals, aflatoxins, bacteria, yeasts, moulds, and organophosphates prior to certification. No contaminants were known to be present.

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 26 °C
- Humidity: 30 to 70 %
- Air changes: Minimum of 10 air changes per hour
- Photoperiod: 12-hour light/12-hour dark

IN-LIFE DATES
From: 09 May 2019
To: 14 June 2019 (main study) or 28 June 2019 (recovery)

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

The oral route of administration was selected because it was used in a previous repeat dose reproductive screening study.

Vehicle:

corn oil

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: Formulations for Groups 2 (2 mg/mL), 3 (60 mg/mL), and 4 (200 mg/mL) were prepared five times by first triturating the required amount of test material. The required amount of corn oil was added, and the formulation mixed until visually uniform. Group 4 formulations were then homogenised at 9000 rpm for 5 minutes due to difficulties in sample aspiration and delivery. Formulations for dosing (including vehicle/control substance) were assigned a ten-day shelf life and stored in a refrigerator (5 ± 3 °C) and protected from light until used for dosing. With the exception of formulations prepared on 19-22 May 2019, prepared formulations were stirred overnight prior to use.

The neat control substance was considered 100 % pure for formulation purposes. The test material was corrected for purity (96.71 %).

- DOSE ADMINISTRATION: The formulations were brought to room temperature and stirred for at least 30 minutes prior to and during dosing. The animals were dosed once daily via oral gavage at a volume of 5.0 mL/kg for at least 28 consecutive days. Dosing volumes were based on the animals’ most recent body weights.
The first day of dosing was designated as SD 1 for each animal.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analysis of the dose formulations was performed. Additionally, samples were analysed for test material concentration and homogeneity.

Stability data demonstrate that the formulated test material is stable for up to 24 hours at room temperature (2.0 – 250 mg/mL), up to 10 days (2.0 mg/mL) or up to 34 days (250 mg/mL) when stored refrigerated (5 ± 3 °C).
Dose concentration analysis showed that the test material was properly formulated (92.8 to 116.4 % of the target concentrations) and homogenous (%RSD = 2.6 to 7.1 %). No test material was detected in the control formulation.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Group 1

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Group 2

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Group 3

Doses / concentrations 4

Dose / conc.:

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

Remarks:

Group 4

No. of animals per sex per dose:

Five main animals/sex/group and five recovery animals/sex/group in Groups 1 and 4.

One male (Group 2) was replaced due to unscheduled death on SD 2.

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on the results from previous toxicology studies performed by the Sponsor. Briefly, a repeat dose reproductive screening study was previously conducted at dose levels up to 1000 mg/kg/day for up to 63 days without dose-limiting toxicity.
- Rationale for animal assignment: Animals were initially accepted into the randomisation pool based upon pre-study body weights and physical examinations. They were assigned to study groups using computer-generated random numbers such that the mean body weight for each group was not statistically different (p < 0.05) from the control mean. Males and females were randomised separately.
- Post-exposure recovery period in recovery groups: 14 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day. Cage side observations included observation for mortality, moribundity, general health, and signs of toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to initiation of dosing, weekly thereafter, and prior to necropsy. Physical examinations included evaluation of skin and fur characteristics, eye and mucous membranes, respiratory, circulatory, autonomic, and central nervous systems, and somatomotor and behaviour patterns.

BODY WEIGHT: Yes
- Time schedule for examinations: prior to initiation of dosing, weekly thereafter (approximately the same time), a day prior to necropsy (unfasted), and prior to necropsy (fasted).

FOOD CONSUMPTION: Yes
- Time schedule: weekly. Food consumption values resulting from wet feed, spilled feed, and/or technical error were excluded from totals, summary data, and statistical analyses. Total food consumption values were presented only when data was present for all measured intervals. Total food consumption = g/animal

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: SD 29 (main phase animals); SD 43 (recovery phase animals)
- Blood collection: Cardiac puncture under 70 % CO2/30 % O2 anaesthesia. At least 0.5 mL was collected into K2 EDTA tubes for haematology samples; at least 1.8 mL was collected into sodium citrate tubes for coagulation samples.
- Animals fasted: Yes (overnight (with water available) prior to sample collection)
- How many animals: All surviving animals
- Parameters (haematology) checked included: white blood cells, red blood cells, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean platelet volume, platelets, red cell distribution width, absolute neutrophils, absolute lymphocytes, absolute monocytes, absolute eosinophils, absolute basophils, absolute reticulocytes and % reticulocytes.
- Parameters (coagulation) checked included: prothrombin time, activated partial thromboplastin time and fibrinogen.
Haematology parameters were measured using a Siemens Advia 120 Haematology analyser and coagulation parameters were measured using the Instrumentation Laboratory ACL Elite Pro Coagulation Analyser.
When the white blood cell differential or reticulocyte count could not be calculated by the Advia 120 due to mechanical or sample limitations, a manual count was performed. If platelet clumps were observed on the haematology smear, the platelet count and mean platelet volume were reported as clumped.
Blood smears for cellular morphology were prepared for each animal and stained using a Modified Wright-Giemsa stain. The slide was evaluated when the data results from the automated haematology evaluation for that particular animal were outside the system standards. Morphology findings (if any) were categorised as follows: 1+ = minimal; 2+ = mild; 3+ = moderate; 4+ = marked.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: SD 29 (main phase animals); SD 43 (recovery phase animals)
- Blood collection: Cardiac puncture under 70 % CO2/30 % O2 anaesthesia. At least 1 mL was collected into serum separator tubes.
- Animals fasted: Yes (overnight (with water available) prior to sample collection)
- How many animals: All surviving animals
- Parameters checked included: albumin, alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, blood urea nitrogen, calcium, cholesterol, creatine kinase, chloride, creatinine, gamma glutamyltransferase, glucose, potassium, sodium, phosphorus, total bile acid, total bilirubin, total protein, triglycerides.
Globulin (GLOB, g/dL) was calculated as the difference between total protein and albumin. The albumin to globulin ratio (A/G) was obtained by dividing the albumin value by the globulin value.
Clinical chemistry parameters were measured using a Siemens Dimension Xpand chemistry analyser

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: SD 27 or 28 (Function Observation Battery); SD 22 (Locomotor Activity)
Battery of functions tested:
- Activity arousal: hand held (handling reactivity, vocalisation), home cage (posture), open field (posture, rearings)
- Autonomic: hand held (exophthalmos, lacrimation, palpebral closure, piloerection, pupillary status, salivation), elicited responses (pupillary response), open field (defecation, urination)
- Neuromuscular: hand held (fur appearance), home cage (activity, unusual behaviour, gait), elicited responses (righting reflex), open field (activity, gait, unusual behaviour, grooms), forelimb grip strength, hindlimb grip strength, hindlimb splay
- Physiological: hand held (respiration), body weight, body temperature
- Sensory: elicited responses (approach response, auditory response, tail pinch response, pinna response)
For neurotoxicity observations (FOB, grip strength, and locomotor activity), animals were transported to the testing room and acclimated to white noise for at least 10 minutes prior to testing. For locomotor activity, animals were placed into an activity chamber for 60 minutes. The Kinder Scientific Motor Monitor II recorded the total number of occurrences of basic movement, fine movement, and rearing during the 60-minute testing timeframe. Data were tabulated in 10-minute intervals. Body temperatures were collected by scanning the implanted microchip transponder.

> OTHER:
VAGINAL LAVAGE: Prior to necropsy (only animals scheduled for necropsy)

HORMONE ANALYSIS
- Time schedule for collection of blood: SD 29 or 43
- Blood collection: Cardiac puncture under 70 % CO2/30 % O2 anaesthesia. At least 3 mL was collected into serum separator tubes.
- Animals fasted: Yes
- How many animals: Only animals scheduled for necropsy
- Parameters checked included: T3, T4 and TSH
Samples were processed to obtain serum, divided into two aliquots, and stored at -75 ± 15 °C for possible future analysis. Analysis was not required and samples were discarded prior to finalisation of the report.

Sacrifice and pathology:

TERMINAL PROCEDURES: On SD 29 for main phase animals and SD 43 for recovery animals, all surviving animals were euthanised by carbon dioxide inhalation followed by exsanguination prior to necropsy.

GROSS PATHOLOGY: Yes
- Animals were necropsied as soon as possible after the time of death or discovery (found dead). Animals were necropsied, bone marrow smears were prepared, required organs were weighed, and tissues were collected and preserved. No organ weights were collected and no bone marrow smears were prepared from animals found dead.
- Gross necropsy included examination of the external surface of the body, all orifices, and the cranial, thoracic, and abdominal cavities and their contents. Organ weights were collected as soon as possible after dissection and paired organs were weighed together. The thyroid gland (with parathyroid gland) was weighed after fixation. Tissues were preserved in 10 % neutral buffered formalin (NBF) with the exception of the eyes (and associated ocular tissue) and testes (with epididymides), which were preserved in modified Davidson’s fixative and subsequently transferred to 10 % NBF. Tissues from animals found dead were preserved in 10 % NBF only. Two bone marrow smears were prepared from the left femur and the slides were air-dried, fixed in methanol, and stored at room temperature for possible future evaluation. No analysis of the bone marrow smears was deemed necessary; therefore, the unstained slides were discarded prior to report finalisation.
- The following organs were collected/weighed as follows: thyroid with parathyroids (weighed after fixation), prostate with seminal vesicles and coagulating glands, and uterus with cervix. Organ weight data were presented as terminal body weights (TBW, g), absolute weights (Abs, g), organ-to-body weight ratios (/BW, %), and organ-to-brain weight ratios (/BR).

HISTOPATHOLOGY: Yes
Tissues were embedded in paraffin, sectioned, stained with haematoxylin and eosin, and examined by a board-certified veterinary pathologist. The uterus was identified as a potential target tissue and tissues from all female rats in Groups 2 and 3, and recovery phase females in Groups 1 and 4 were evaluated by the veterinary pathologist. Additionally, the vagina and ovaries from these animals were evaluated to facilitate interpretation of possible changes in the uterus.

Statistics:

See "Any other information on materials and methods incl. tables" for information

Results and discussion

Results of examinations

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test material did not affect clinical observations. No abnormalities were observed during cage side observations. Incidental findings during physical examinations included abrasions (shoulder or eye), alopecia, red discharge from the nose, and abnormal respiration (soft or loud). These observations were not considered test material-related due to lack of dose response, low incidence, or lack of persistence. The abnormal respiration findings were likely due to the viscous nature of corn oil vehicle, which can result in aspiration and/or reflux during gavage (Damsh et al, 2011).

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Treatment with the test material did not affect mortality. Animal 29523 (2m; 10 mg/kg bw/day) was found dead on SD 2. This early death was likely caused by trauma caused during gavage procedure as red discolouration of all lung lobes were noted at necropsy. This death was therefore not considered to be test material-related. All other animals survived to scheduled termination.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test material did not affect body weights or body weight changes. No significant differences were observed in mean body weights throughout the study. Males at 10 mg/kg bw/day and 1000 mg/kg bw/day had significantly lower mean body weight change from SD 8-15 compared to the mean of the concurrent control. However, no significant differences were noted in mean absolute body weight change from SD 1-28 (main phase), 29-42 (recovery phase), or from SD 1-42 for either sex.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Treatment with the test material did not affect food consumption. Statistically significant differences were observed but were not considered to be test material-related since no significant differences were observed in mean total food consumption for either sex from SD 1-28 (main phase), 29-42 (recovery phase), or 1-42. The majority (with the exception of Group 2 males) of the statistically significant differences were an increase in food consumption compared to the controls. Additionally, there was no corresponding impact on body weights or body weight gains.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

There were no test material-related alterations in haematology or coagulation parameters.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- Study Day 29: Minimal dose-dependent increased triglyceride concentration in females dosed at 300 mg/kg bw/day (+35.9 %; not statistically significant) and 1000 mg/kg bw/day (+69.1 %; statistically significant) occurred without correlating macroscopic or microscopic findings. The alterations were not considered adverse.
- Study Day 43: Minimal decreased blood urea nitrogen concentration in males dosed at 1000 mg/kg bw/day (-26.3 %; statistically significant) and minimal increased cholesterol in females dosed at 1000 mg/kg bw/day (+29.3 %; statistically significant) occurred without correlating macroscopic or microscopic findings. The alterations were of equivocal relationship to test material administration and were not considered to be adverse.
- Any other difference noted in clinical chemistry parameters on SD 29 and SD 43, regardless of statistical significance, were not considered to be test material-related because the changes were negligible in magnitude, were without a dose-response, and/or they were within the historical range provided.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

> Functional Observational Battery and Grip Strength
Treatment with the test material did not affect the functional observational battery, however, some abnormalities were noted, as follows:
Abnormal observations were noted in three males and one female at 0 mg/kg bw/day, one female at 300 mg/kg bw/day, and four males and two females at 1000 mg/kg bw/day. These findings were considered incidental as abnormal observations were noted in the control group, there was no observed dose-response, and the observations were in varying domains (neuromuscular and sensory) with no distinct pattern of disruption.
Treatment with the test material affected forelimb grip strength. Mean forelimb grip strength was statistically significantly decreased in treated groups compared to the control in both sexes. There was no effect on hindlimb grip strength in either sex. Mean landing splay of males was also significantly reduced in all treated groups compared to the control. Although increased landing splay has been correlated to distal axonopathy, there is no clear interpretation of the potential toxicity that would result in decreased landing foot splay or the translatability to humans (Ross, 2000). The functional behavioural effects on grip strength and landing foot splay were considered test substance-related, however, without corresponding pathological changes, these were not considered adverse (Moser, 2000 and Ross, 2000).
Mean number of rearings during the open field test was lower in males at 300 mg/kg bw/day and significantly lower in males at 1000 mg/kg bw/day compared to the control. Females at 10 mg/kg bw/day had significantly higher mean rearings during the open field test compared to the control. The effects on rearing were not replicated during locomotor activity measurements, which have been shown to be more sensitive as they are conducted for a longer duration and are automated (Moser, 2000). Therefore, these findings were considered incidental and not test material-related.
Mean number of urine pools was significantly higher in males at 10 mg/kg bw/day, but all other groups had zero urine pools, thus the significant difference was incidental. No notable differences were observed in the other quantitative measurements (hindlimb grip strength, number of grooms, and number of faecal boli). There was a statistically significant increase in body temperature at 10 and 300 mg/kg bw/day in male and females, however the increase was minimal (below 40 °C) and did not occur at the highest dose level, thus was not considered test material-related.

> Locomotor Activity
Treatment with the test material did not affect locomotor activity. Mean number of basic movements and rearings decreased over the six ten-minute intervals, showing normal habituation in all groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

> Terminal Necropsy (SD 29): Minimal decreased terminal body weight (-5.2 %; not statistically significant) and minimal decreased absolute and relative thymus weight (not statistically significant) were noted for males dosed at 1000 mg/kg bw/day. These alterations were without correlating macroscopic or microscopic findings or correlating changes in lymphocyte counts (haematology data). They were of equivocal relationship to test material administration and were not considered adverse.
> Recovery Necropsy (SD 43): There were no test material-related alterations in organ weights on SD 43.

Gross pathological findings:

no effects observed

Description (incidence and severity):

There were no test material-related macroscopic findings; all macroscopic observations on SD 29 and SD 43 were consistent with spontaneous background findings in rats.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

> Terminal Necropsy (SD 29): A possible test material-related microscopic finding was identified in the uterus on SD 29.
Minimal squamous metaplasia of uterine glands was identified in one animal dosed at 300 mg/kg bw/day and one animal dosed at 1000 mg/kg bw/day. Squamous metaplasia of the uterine epithelium may develop spontaneously in the rat uterus in response to increased endogenous oestradiol or may develop after administration of oestrogenic compounds. Spontaneous uterine squamous metaplasia is unusual in rats that are less than 5-6 months of age. However, given the minimal extent of the alteration, without evidence of a dose-response, a causal relationship to administration of the test material is uncertain. The alteration was considered non-adverse at the minimal severity level.
Mild or moderate ovarian corpus luteum hypertrophy accompanied by mild or moderate vaginal mucification were identified in one female given vehicle alone, two females dosed at 10 mg/kg bw/day, and one female dosed at 300 mg/kg bw/day. Mild hypertrophy/hyperplasia of mammary gland ducts and alveoli accompanied the ovarian and vaginal changes in the vehicle control animal (mammary gland was not examined in the animals given 10 or 300 mg/kg bw/day). This constellation of microscopic findings in the female rat reproductive tract indicates disruption of the oestrous cycle. A cause was not identified, but the presence of these findings in the vehicle control animal indicates that they were not related to administration of the test material.
Minimal to moderate degeneration of skeletal muscle in the oesophagus of vehicle control animals and animals dosed at 1000 mg/kg bw/day was considered secondary to minor trauma during the gavage procedure.
Pharynx was not present in the tissues provided for several animals. There were no microscopic findings in the pharynx of animals in which the pharynx was present, and the absence of pharynx was not considered to have had a negative impact on interpretation of the study results.

> Recovery Necropsy (SD 43)
There were no test material-related microscopic findings in the uterus, vagina or ovary on SD 43.
Mild vaginal mucification was identified in one vehicle control female on SD 43.
All other microscopic findings on SD 29 and SD 43 were unrelated to treatment with the test material because they occurred only sporadically and without evidence of dose-response, they were of low incidence and severity and/or they were observed with comparable incidence and severity in vehicle control animals, and/or they were considered common incidental findings in rats.

Histopathological findings: neoplastic:

not specified

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Remarks on result:

other: no adverse effects noted

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

Table 1: Selected Mean Organ Weight Differences at Terminal Necropsy

	Dose Group (mg/kg/day)
	Males				Females
	0	10	300	1000	0	10	300	1000
Thymus Absolute Weight	-	-5.8	-8.9	-20.0	-	-6.5	+6.0	+5.3
Thymus/Body Weight	-	-5.1	-9.7	-15.4	-	-8.0	+3.6	+7.0
Thymus/Brain Weight	-	-13.0	-12.1	-21.4	-	+3.5	+1.6	+11.5

 (% difference) = Percent difference between group means relative to control group;

- = Not applicable;

+/- = increase/decrease

Applicant's summary and conclusion

Conclusions:

Under the conditions of the study, the no-observed adverse-effect level (NOAEL) for the test material was considered to be 1000 mg/kg bw/day for male and female rats when dosed via oral gavage for 28 days.

Executive summary:

The potential for the test material to cause repeated dose toxicity was investigated in a study which was conducted in accordance with the standardised guideline OECD 407 and under GLP conditions.

Groups of male and female Sprague-Dawley rats were administered test material daily at 0 (vehicle control), 10, 300 and 1000 mg/kg bw/day, for a period of 28 days, by oral gavage and the persistence or reversibility of any toxic effects were determined over a 14-day recovery period.

Parameters evaluated during the study included mortality, physical examinations, cage side observations, body weights, body weight changes, food consumption, locomotor activity, functional observational battery, clinical pathology (clinical chemistry, haematology, and coagulation), gross pathology findings, absolute and relative organ weights, and histopathology findings.

No test material related effects were observed for mortality, body weights, body weight changes, cage side and clinical observations, and food consumption. During the last week of dosing, there were findings noted during the functional observation battery. Treatment with test material at all dose levels resulted in statistically significantly decreased forelimb grip strength in both sexes. Additionally, mean landing splay of males was also significantly reduced in all treated groups compared to the control. The functional behavioural effects on grip strength and landing foot splay were considered test substance related; however, without corresponding pathological changes, these were not considered adverse.

Minimal dose-dependent increased triglyceride concentration in females dosed at ≥ 300 mg/kg bw/day occurred without correlating macroscopic or microscopic findings. This change was considered of equivocal relationship to test material administration and was not adverse. At the end of recovery, minimal decreased blood urea nitrogen concentration in males dosed at 1000 mg/kg bw/day and minimal increased cholesterol in females dosed at 1000 mg/kg bw/day occurred without correlating macroscopic or microscopic findings. These alterations were also of equivocal relationship to test material administration and were not adverse. Minimal decreased terminal body weight and minimal decreased thymus weight in males dosed at 1000 mg/kg bw/day occurred without correlating macroscopic or microscopic findings or correlating changes in peripheral lymphocyte counts in animals at terminal necropsy and was not adverse. Minimal squamous metaplasia in the uterine glands of two females (one given 300 mg/kg bw/day and one given 1000 mg/kg bw/day) was of equivocal relationship to test material administration and was not adverse.

In conclusion, the no-observed adverse-effect level (NOAEL) for the test material was considered to be 1000 mg/kg bw/day for males and females rats when dosed via oral gavage for 28 days.