Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2014

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP and OECD testing guideline compliant study with well-characterized test material

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2014

Report date:

2014

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

EC Number:

270-128-1

EC Name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Cas Number:

68411-46-1

Molecular formula:

C16H19N - C28H43N (main constituents)

IUPAC Name:

Benzenamine, N-phenyl-, reaction products with 2,4,4-trimethylpentene

Details on test material:

- Name of test material (as cited in study report): Benzenamine, N-phenyl-,reaction products with 2,4,4-trimethylpentene
- Substance type: UVCB
- Physical state: Clear slightly yellow viscous liquid
- Analytical purity: 100% UVCB
- Purity test date: 2014 , study number 13L00223
- Lot/batch No.: 40401913D
- Expiration date of the lot/batch: 09 February 2016
- Stability under test conditions: stable
- Storage condition of test material: At room temperature in the dark

Test substance handling: Avoid temperatures <10°C, maximum temperature:30°C. In the case of crystallization, heat up the substance (temperature up to 30°C) until a clear solution is obtained
Specific Gravity / Density 0.975 g/cm3 (20°C)
pH 5.1-6.2 (1%(m), 20-25°C) (as suspension)
Stability at higher temperatures Maximum temperature: 30°C

Test animals

Species:

rat

Strain:

other: Crl:WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 10-12 weeks
- Weight at study initiation: 353 g (males), 210 g (females)
- Fasting period before study: none
- Housing: groups of 5 animals/sex/cage (premating males and females and postmating males), otherwise single cages
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 to 24°C
- Humidity (%): 40 to 70%,
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 13 March 2014 To: 1 May 2014

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) are be prepared daily within 6 hours prior to dosing.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Test item is insoluble in water, but soluble in corn oil
- Amount of vehicle (if gavage): 5 mL/kg body weight. Actual dose volumes were calculated according to the latest body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature was also determined (highest and lowest concentration).
The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110% of the target concentration. Homogeneity was demonstrated if the coefficient of variation was ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

28 days (males), ca 53 days (females)

Frequency of treatment:

once daily

Doses / concentrations

Remarks:

Doses / Concentrations:
25, 75 and 225 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10
5 animals/sex/group were selected for functional observations, locomotor activity, clinical pathology, organ weights and histopathology

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results of a 14-day range-finding study in rats

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily
- Cage side observations: mortality, clinical observations

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the first exposure and at the end of the study as part of the functional observation battery (FOB)

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights of male and female animals were recorded shortly before the start of
administration of the test item at randomization and at the start of the study (day 0). Males
were weighed weekly until sacrifice. Females were weighed once per week during the premating period. Mated females were
weighed on days 0, 7, 14 and 21 during presumed gestation and on day 0 and 4 of lactation.
Non-mated females were weighed once per week after the mating period. In addition, the
animals were weighed on their scheduled necropsy date in order to calculate the correct organ to body weight ratios.


FOOD CONSUMPTION:
Feed consumption was measured per cage over weekly intervals during the study, with exemption of the mating period, during which no feed consumption was registered.


HAEMATOLOGY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst under CO2/O2 anaesthesia. K3-EDTA was used as anticoagulant. In each sample the following determinations were carried out:
haemoglobin
packed cell volume
red blood cell count
reticulocytes
total white blood cell count
differential white blood cell counts (neutrophils, lymphocytes, eosinophils,
basophils, monocytes)
prothrombin time
thrombocyte count
mean corpuscular volume (MCV; calculated)
mean corpuscular haemoglobin (MCH; calculated)
mean corpuscular haemoglobin concentration (MCHC; calculated)
activated partial prothoplastin time (APTT)
red blood cell districution width (RDW)

CLINICAL CHEMISTRY
Prior to sacrifice, 5 animals/sex/group were fasted overnight (water was freely available) and blood was taken from the aorta during necropsy, whilst CO2/O2 anaesthesia. Blood was collected in heparinized plastic tubes and plasma was prepared by centrifugation. The following measurements were made in the plasma:
alkaline phosphatase activity (ALP),
aspartate aminotransferase activity (ASAT),
alanine aminotransferase activity (ALAT),
gamma glutamyl transferase activity (GGT),
total protein,
albumin,
ratio albumin to globulin (calculated),
urea,
creatinine,
glucose (fasting),
bilirubin (total),
cholesterol (total),
triglycerides,
phospholipids,
calcium (Ca),
sodium (Na),
potassium (K),
chloride (Cl),
inorganic phosphate (PO4),
bile acids

Thyroid hormone analyses:
Thyroid stimulating hormone (TSH) μIU/mL
total triiodothyronine (T3) ng/dL
total thyroxine (T4) μg/dL


NEURO-BEHAVIOURAL TESTING (FOB) AND SPONTANEOUS MOTOR ACTIVITY
During neuro-behavioural testing, the observer was unaware of the treatment of the animals. FOB and spontaneous motor activity were assessed in all study animals during the predose phase and in 5 animals/sex/group at the end of the study.

Sacrifice and pathology:

GROSS NECROPSY AND HISTOLOGY OF PARENTAL ANIMALS
All surviving male and female parent animals were sacrificed by exsanguination from the abdominal aorta whilst under CO2/O2 anaesthesia at necropsy and then examined grossly for pathological changes. Male animals were sacrificed after the mating period. Female animals were sacrificed at or shortly after day 4 of lactation. A necropsy was performed on animals that died intercurrently (if not precluded by autolysis) or that had to be killed because they were moribund. Prior to preservation of organs/tissues, the following organ weights were recorded: adrenals, brain, heart, kidneys, liver, ovaries, prostate, seminal vesicles, spleen, thymus, thyroid, uterus. Samples of the following tissues and organs of all parent animals were preserved in a neutral aqueous phosphate-buffered 4% solution of formaldehyde; except for the testes which were preserved in Bouin's fixative:

- ovaries (after counting the corpora lutea)
- uterus (after counting of the implantation sites)
- testes
- epididymides
- seminal vesicles
- prostate
- all gross lesions
In addition of 5 animals/sex/group following organs were preserved:
adrenals
bone marrow (femur)
brain (including sections of cerebrum, cerebellum, medulla/pons)
caecum
cervix
clitorial gland
colon
coagulation gland
duodenum
eyes
femur including joint
heart
ileum
jejunum (including Peyer's patches)
kidneys
liver
lungs
mesenterial and axillary lymph nodes
ovaries
peripheral nerve (sciatic or tibial)
pituitary gland
preputial gland
prostate
rectum
seminal vesicles (including coagulation gland)
skeletal muscle (thigh)
spinal cord (cervical, mid-thoracic and lumbar)
spleen
stomach*
thymus
thyroid (including parathyroid)
trachea
urinary bladder
uterus
vagina
* Non glandular (“forestomach”) and glandular (fundus, pylorus) parts were examined
microscopically.

Microscopic examination was performed on the collected organs of all animals of the control (group 1) and high dose group (group 4). If treatment-related changes were observed in the high-dose group, the evaluation of these tissues/organs was extended to the intermediate-dose groups (2 and 3).
In addition, reproductive organs of males that failed to sire (did not mate or mated females were not pregnant) and females that were non-mated or non-pregnant, of the low- and mid-dose groups, were microscopically examined. Furthermore, organs showing gross lesions of animals of all groups were microscopically examined.

Statistics:

If the variables could be assumed to follow a normal distribution, the Dunnett-test (Ref. 2; many-to one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

various liver parameters, see tables 1 and 2

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

liver, see tables 3 and 4

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

liver

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

liver

Histopathological findings: neoplastic:

not examined

Details on results:

Clinical biochemistry details
Compared to controls, animals at 225 mg/kg bw/day had relevant increases in alkaline phosphatase (ALP, both sexes), bilirubin (both sexes), glucose (males), cholesterol (females) and decreased albumin (both sexes), total protein (females) inorganic phosphate (both sexes) and total thyroxine (T4,
both sexes).
At 75 mg/kg bw/day, animals had increased ALP (both sexes, not significantly different for males), glucose (males, not statistically significant), total bilirubin (both sexes) and decreased albumin (both sexes) and total protein (females).
Calcium was lower for all treated animals of both sexes. T4 measurements were significantly lower for females of Groups 2 and 3 as well. The toxicological relevance of these effects is questionable, however, as no clear dose response relationship was noted for either parameter and no relevant effects were seen in the thyroid for females at the microscopic examination.
The statistically significant decrease in cholesterol seen for males at 75 mg/kg bw/day was not considered to be toxicologically relevant as it occurred in the absence of a dose-dependent distribution.
Thyroid stimulating hormone (TSH) was much higher than controls for all groups (both sexes, not always statistically significant). These data showed high variability with one or more individuals in each treated group showing extremely high values (Group 2, male no. 11, Group 3 male nos. 22, 25, and female no. 62 and Group 4 male no. 33). When recalculated excluding the outliers, group means remained higher than controls (males Group 2: 0.518, Group 3: 0.548, Group 4: 0.475; females Group 3: 0.386), though no dose-dependent distribution was apparent. In the absence of a clear relationship with total T3 or T4, and in the absence of adverse findings seen in the thyroids during the microscopic examination, no toxicological relevance was attributed to higher TSH values.

Macroscopic findings
At 225 mg/kg bw/day treatment related macroscopic findings were seen in the liver including accentuated lobular pattern of the liver (6 males, 2 females), pale discoloration (7 males, 1 female), and an enlarged liver (1 male). Taken together with relevant clinical biochemistry and microscopic findings, these were considered to be toxicologically relevant.
Pale discoloration of the liver was also seen for 2 males at 75 mg/kg bw/day, this was also considered to be treatment related but since microscopic evidence indicated this was an adaptive response, it was not considered to be adverse at this level.
One male at 25 mg/kg bw/day (no. 13) was noted with a liver that was enlarged and with black discoloration. At the microscopic examination, this animal was found with centrilobular vacuolation,
congestion and hepatocellular degeneration/necrosis. These were not considered toxicologically relevant as they occurred in the absence of other relevant findings and were seen for a single animal.
The macroscopic findings noted for the female that was euthanized after struggling with an extended parturition (no. 50) included pale discoloration of the whole body and 11 dead and autolytic pups were
found in the uterus.

Organ weights
Relative kidney weights were significantly higher for males at 225 mg/kg bw/day only. Absolute and relative liver weights were significantly higher for males of all treatment groups (approximately 20-54%
increase in relative weight for males). Liver weights were also higher for females at 75 and 225 mg/kg bw/day, but the difference from controls was only significantly different for females at 75 mg/kg
bw/day.

Histopathology
Test item-related microscopic findings were noted in the liver of both sexes starting at 75 mg/kg bw/day, the thyroid gland of the males in all treated groups, and in the kidney of females at 225 mg/kg bw/day.
Hepatocellular hypertrophy of the liver was noted in 6/6 males (6 minimal) and 1/5 females (1 minimal) treated at 75 mg/kg bw/day and in 8/8 males (2 minimal, 6 slight) and 6/6 females (2 minimal, 4 slight) at 225 mg/kg bw/day.
Hepatocellular vacuolation of zone 1 (periportal) and/or zone 2 (midzonal) of the liver was noted in 2/5 females (2 minimal) treated at 75 mg/kg bw/day and in 8/8 males (3 minimal, 5 slight) and 6/6 females
(3 minimal, 1 slight, 2 moderate) at 225 mg/kg bw/day. This finding consisted of microvesicular vacuolation of the midzonal hepatocytes (zone 2) in the males, whereas in the females the vacuolation ranged from microvesicular vacuolation of the periportal hepatocytes (zone 1) to combined micro-and macrovesicular vacuolation of periportal and midzonal hepatocytes (recorded as slight and moderate degrees) in the 225 mg/kg bw/day group.

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Test-item related effects on liver at 75 and 225 mg/kg bw

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%). The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%). Group 1 (control group) did not contain the test substance.

Table 1: Clinical chemistry values for males at the end of treatment
		control	25 mg/kg bw	75 mg/kg bw	225 mg/kg bw
ALAT	MEAN	55.3	61.1	57.3	63.6
U/L	ST.DEV	22.9	29.9	14.2	13.3
	N	5	5	5	5
ASAT	MEAN	78.8	84.6	86.1	84.0
U/L	ST.DEV	14.4	14.0	4.1	8.4
	N	5	5	5	5
ALP	MEAN	144	176	220	409 **
U/L	ST.DEV	52	43	32	64
	N	5	5	5	5
Total protein	MEAN	60.7	58.3	58.3	58.3
g/L	ST.DEV	1.5	2.0	3.0	2.2
	N	5	5	5	5
Albumin	MEAN	31.2	30.1	29.5 **	28.4 **
g/L	ST.DEV	0.5	0.9	0.8	0.8
	N	5	5	5	5
Total bilirubin	MEAN	2.1	2.5	3.4 *	5.6 **
umol/L	ST.DEV	0.2	0.3	1.0	1.0
	N	5	5	5	5
Urea	MEAN	5.4	5.1	5.1	5.8
mmol/L	ST.DEV	1.8	0.6	0.7	0.9
	N	5	5	5	5
Creatinine	MEAN	39.4	39.4	41.0	39.2
umol/L	ST.DEV	3.2	1.2	2.5	1.3
	N	5	5	5	5
Glucose	MEAN	8.59	10.47	11.08	11.27 *
mmol/L	ST.DEV	0.96	1.32	2.37	1.46
	N	5	5	5	5
Cholesterol	MEAN	1.94	1.81	1.44 *	1.55
mmol/L	ST.DEV	0.44	0.23	0.18	0.18
	N	5	5	5	5
Sodium	MEAN	142.2	141.6	141.9	141.3
mmol/L	ST.DEV	1.4	0.4	1.4	0.5
	N	5	5	5	5
Potassium	MEAN	4.31	4.10	4.24	4.32
mmol/L	ST.DEV	0.30	0.18	0.14	0.24
	N	5	5	5	5
Chloride	MEAN	104	105	105	105
mmol/L	ST.DEV	1	1	2	1
	N	5	5	5	5
Calcium	MEAN	2.55	2.44 *	2.38 **	2.45 *
mmol/L	ST.DEV	0.05	0.05	0.07	0.05
	N	5	5	5	5
Inorg.Phos	MEAN	2.08	1.88	1.90	1.79 *
mmol/L	ST.DEV	0.25	0.05	0.14	0.07
	N	5	5	5	5
TSH	MEAN	0.143	1.116	0.706	0.794
uIU/mL	ST.DEV	0.070	1.347	0.253	0.740
	N	5	5	5	5
Total T3	MEAN	85.1	74.3	77.9	73.6
ng/dL	ST.DEV	9.8	4.4	5.9	12.8
	N	5	5	5	5
Total T4	MEAN	4.28	3.97	3.79	3.04 *
ug/dL	ST.DEV	1.10	0.74	0.27	0.24
	N	5	5	5	5
Bile acids	MEAN	24.2	15.8	11.7*	10.7**
umol/ml	ST.DEV	10.3	4.3	4.2	2.2
	N	5	5	5	5

 */** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 2: Clinical chemistry values for females at the end of treatment
		control	25 mg/kg bw	75 mg/kg bw	225 mg/kg bw
END OF TREATMENT ALAT	MEAN	48.5	56.9	56.1	63.1
U/L	ST.DEV	12.0	17.9	16.3	19.5
	N	5	5	5	5
ASAT	MEAN	77.3	77.9	75.3	87.1
U/L	ST.DEV	11.3	7.1	7.9	14.4
	N	5	5	5	5
ALP	MEAN	99	99	206 *	292 **
U/L	ST.DEV	55	24	62	55
	N	5	5	5	5
Total protein	MEAN	61.5	60.4	55.8 **	57.0 *
g/L	ST.DEV	0.3	2.7	2.3	3.1
	N	5	5	5	5
Albumin	MEAN	31.6	29.8	26.5 **	27.3 **
g/L	ST.DEV	0.6	1.8	1.5	2.2
	N	5	5	5	5
Total bilirubin	MEAN	1.9	1.9	2.8 **	4.4 **
umol/L	ST.DEV	0.5	0.3	0.4	0.4
	N	5	5	5	5
Urea	MEAN	7.1	7.5	7.4	7.4
mmol/L	ST.DEV	1.3	0.9	1.3	0.9
	N	5	5	5	5
Creatinine	MEAN	43.4	44.0	45.4	41.9
umol/L	ST.DEV	2.0	1.5	2.0	3.2
	N	5	5	5	5
Glucose	MEAN	6.91	6.72	7.95	7.41
mmol/L	ST.DEV	1.19	0.93	0.33	0.88
	N	5	5	5	5
Cholesterol	MEAN	1.65	1.94	2.62	2.77 *
mmol/L	ST.DEV	0.21	0.31	0.94	0.76
	N	5	5	5	5
Sodium	MEAN	139.9	140.2	140.0	139.2
mmol/L	ST.DEV	1.6	2.2	1.1	2.5
	N	5	5	5	5
Potassium	MEAN	3.53	3.55	3.79	3.79
mmol/L	ST.DEV	0.21	0.18	0.18	0.21
	N	5	5	5	5
Chloride	MEAN	98	100	99	100
mmol/L	ST.DEV	1	1	1	2
	N	5	5	5	5
Calcium	MEAN	2.60	2.51 *	2.46 **	2.44 **
mmol/L	ST.DEV	0.06	0.03	0.05	0.02
	N	5	5	5	5
Inorg.Phos	MEAN	2.54	2.21	2.16	1.86 **
mmol/L	ST.DEV	0.29	0.16	0.29	0.16
	N	5	5	5	5
TSH	MEAN	0.146	0.490 *	0.488 *	0.224
uIU/mL	ST.DEV	0.037	0.233	0.282	0.075
	N	5	5	5	5
Total T3	MEAN	71.1	62.2	72.6	73.3
ng/dL	ST.DEV	8.0	6.0	8.0	9.0
	N	5	5	5	5
Total T4	MEAN	3.15	1.96 *	2.05 *	2.02 *
ug/dL	ST.DEV	0.44	0.49	0.58	0.71
	N	5	5	5	5
Bile Acids	MEAN	23.6	6.9**	15.5	12.9
umol/ml	ST.DEV	11.7	1.7	4.3	4.5
	N	5	5	5	5

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Table 3: Organ and body weight values for males at the end of treatment
		control	25 mg/kg bw	75 mg/kg bw	225 mg/kg bw
BODY W.	MEAN	388	385	384	376
(GRAM)	ST.DEV	28	25	20	20
	N	10	10	10	10
BRAIN	MEAN	2.11	2.10	2.17	2.13
(GRAM)	ST.DEV	0.08	0.07	0.08	0.08
	N	10	10	10	10
HEART	MEAN	1.026	1.026	1.044	1.047
(GRAM)	ST.DEV	0.087	0.069	0.058	0.062
	N	10	10	10	10
LIVER	MEAN	9.10	10.84 *	11.15 *	13.55 **
(GRAM)	ST.DEV	0.79	2.56	0.82	1.18
	N	10	10	10	10
THYROIDS	MEAN	0.016	0.016	0.018	0.017
(GRAM)	ST.DEV	0.004	0.003	0.003	0.004
	N	10	10	10	10
THYMUS	MEAN	0.321	0.324	0.313	0.315
(GRAM)	ST.DEV	0.082	0.065	0.048	0.046
	N	10	10	10	10
KIDNEYS	MEAN	2.36	2.46	2.42	2.52
(GRAM)	ST.DEV	0.25	0.22	0.21	0.14
	N	10	10	10	10
ADRENALS	MEAN	0.051	0.053	0.056	0.056
(GRAM)	ST.DEV	0.006	0.010	0.006	0.007
	N	10	10	10	10
SPLEEN	MEAN	0.620	0.616	0.658	0.608
(GRAM)	ST.DEV	0.066	0.074	0.110	0.079
	N	10	10	10	10
TESTES	MEAN	3.52	3.64	3.75	3.63
(GRAM)	ST.DEV	0.29	0.34	0.27	0.29
	N	10	10	10	10
PROSTATE	MEAN	0.695	0.761	0.616	0.642
(GRAM)	ST.DEV	0.100	0.153	0.072	0.155
	N	10	10	10	10
EPIDIDYMIDES	MEAN	1.158	1.182	1.180	1.147
(GRAM)	ST.DEV	0.066	0.092	0.079	0.122
	N	10	10	10	10
SEMINAL VES	MEAN	1.625	1.651	1.558	1.601
(GRAM)	ST.DEV	0.133	0.242	0.160	0.222
	N	10	10	10	10

*/** Dunnett-test based on pooled variance significant at 5% (*) or 1% (**) level

Applicant's summary and conclusion

Executive summary:

Hepatic toxicity was noted for animals of both sexes at 225 mg/kg bw/day. This was supported by changes in clinical pathology endpoints (increase of ALP, bilirubin, cholesterol and decrease of albumin, total protein and inorganic phosphate, among others), macroscopic abnormalities (accentuated lobular pattern, enlargement and discoloration), increased organ weights (relative weights approximately 50% and 19% higher for males and females, respectively) and microscopic findings including hepatocellular vacuolation and hypertrophy. Taken together, the findings at this dose level were considered to be adverse.

At 75 mg/kg bw/day, morphologic liver findings were regarded to be an adaptive, non-adverse response based on the minimal severity of the hypertrophy (correlated to pale discoloration) and low incidence and minimal degree of vacuolation in the females. However, when taken together withsimilar changes seen in clinical biochemistry parameters as high dose animals, and an approximate

relative liver weight increase of 24% and 17% for males and females, respectively, the findings were also considered toxicologically relevant at 75 mg/kg bw/day.