9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From September 19, 2016 to February 17, 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

EC Number:

271-843-1

EC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Cas Number:

68609-93-8

Molecular formula:

A generic formula cannot be provided for this UVCB substance. The alkyl chain length of the sulfonated fatty acids range from C12-C22, however the major alkyl chain is C18.

IUPAC Name:

9-Octadecenoic acid (Z)-, sulfonated, potassium salts

Test material form:

solid

Details on test material:

Purity/Composition: UVCB (100%);
Appearance: orange-brown crystalline powder with lumps.

Test animals

Species:

rat

Strain:

Wistar

Remarks:

SPF-bred Wistar Han rats

Details on species / strain selection:

Rationale: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test system Rat: Crl:WI(Han), (outbred, SPF-Quality).
Source: Charles River Deutschland, Sulzfeld, Germany.
Total number of animals: 40 males, 40 females (females were nulliparous and nonpregnant).
Age at start of treatment: Approximately 6 weeks.
Identification: Earmark and tattoo.
Randomization: By computer-generated random algorithm according to body weight, with all animals within ± 20% of the sex mean.
Acclimatization period: At least 5 days before the start of treatment under laboratory conditions.
Health inspection: Upon receipt of the animals.

Conditions: Environmental controls for the animal room were a mean daily temperature range of 21 to 22°C, a mean daily relative humidity range of 44 to 63%, at least 10 air changes/hour, and a 12-hour light/12-hour dark cycle. The light/dark cycle was interrupted for study related activities.
Accommodation: Group housing of 5 animals per sex in Macrolon cages (MIV type, height 18 cm) with sterilized sawdust as bedding material (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG) and paper as cage-enrichment (Enviro-dri, Wm. Lilico & Son (Wonham Mill Ltd)). During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp).
Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten). During motor activity measurements, animals did not have access to food for a maximum of 1 hour and 43 minutes. Water: tap water. During motor activity measurements, animals did not have access to water for a maximum of 1 hour and 43 minutes.
Diet, water, bedding and cage enrichment evaluation for contaminants and/or nutrients was performed according to facility standard procedures. There were no findings that could interfere with the study.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

Method: Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing. A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Dose volume: 5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Frequency: Once daily, 7 days per week, approximately the same time each day with a maximum of 6 hours difference between the earliest and latest dose.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Sampling: Samples (0.5 mL) were taken using a pipette, and were weighed on an analytical balance at 4 decimals precision. During sampling, formulations were placed on a magnetic stirrer. Immediately after sampling (accuracy and homogeneity samples) or after 5 hours at room temperature under normal laboratory light conditions (stability samples), samples were stored on dry ice until receipt.

Analysis: Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 5 hours at room temperature under normal laboratory light conditions 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:

90 d

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.

Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals at least immediately after dosing. The grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. In the data tables, the scored grades as well as the percentage of animals affected were recorded.

Functional observations: during week 13 of treatment, functional observation tests were performed on the first 5 animals/sex/group. Tests were performed after dosing at no specific time point, but within a similar time period after dosing. The following tests were performed: hearing ability, pupillary reflex, static righting reflex, fore- and hind-limb grip strength, locomotor activity.

Body weights: weekly.
Food consumption: weekly.
Water consumption: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.
Ophthalmoscopic examination: following instillation of tropicamide solution both eyes were examined by means of an ophthalmoscope: at pretest: all animals (including spare animals), at week 13: groups 1 and 4 animals.

Blood samples were collected under anaesthesia using isoflurane between 7.00 and 10.30 a.m. at the end of the treatment. Animals were deprived of food overnight (for a maximum of 24 hours), but water was available.
Blood samples were drawn from the retro-orbital sinus and collected into tubes prepared with K3-EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into serum tubes for determination of bile acids.

The following parameters were determined: white blood cells, differential leucocyte count neutrophils, lymphocytes, monocytes, eosinophils, basophils, red blood cells, reticulocytes, red blood cell distribution width, haemoglobin, haematocrit, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelets.

Clotting Potential: prothrombin time, activated partial thromboplastin time.

Clinical Biochemistry: alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, total Protein, albumin, total bilirubin, urea, creatinine, glucose, cholesterol, sodium, potassium, chloride, calcium, inorganic phosphate.

Sacrifice and pathology:

Animals surviving to the scheduled day of necropsy and all moribund animals were deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy: adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, prostate, seminal vesicles including coagulating glands, spleen, testes, thymus, thyroid (including parathyroid), uterus (including cervix).
- Pathology examinations:
The numbers of corpora lutea and former implantation sites were recorded for all paired females. Samples of the following tissues and organs were collected and fixed in 10% buffered formalin for further histophatological examinations: ovaries, adrenal glands, pancreas, aorta, peyer's patches, brain (cerebellum, mid-brain, cortex), pituitary gland, caecum, preputial gland, cervix, prostate gland, clitoral gland rectum, colon, salivary glands (mandibular, sublingual), coagulation gland sciatic nerve, duodenum seminal vesicles, epididymide skeletal muscle, eyes, skin, female mammary gland area, spinal cord (cervical, midthoracic, lumbar), femur including joint, spleen, heart, sternum with bone marrow, ileum, stomach, jejunum, testes, epididymides, kidneys, thymus, lacrimal gland, exorbital, thyroid including parathyroid, larynx, tongue, liver, trachea, lung, urinary bladder lymph nodes, uterus, vagina, oesophagus, all gross lesions.

Histopathology
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Group 1 and 4 animals,
- all tissues from all animals of all dose groups which died spontaneously or were terminated in extremis,
- adrenal glands, pancreas, kidneys and mesenteric lymph nodes of all animals of Groups 2 and 3 (males and/or females) and spleen of all females of Groups 2 and 3, based on (possible) treatment-related changes in these organs in Group 4,
- all gross lesions.

All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

The following statistical methods were used to analyze the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-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 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. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In the surviving animals, diarrhoea was observed on single occasions in seven 1000 mg/kg animals towards the end of the treatment period. Swelling/gas distention of the abdomen was observed in three 1000 mg/kg and two 300 mg/kg animals, each for single short and transient bouts. Rales were also observed for single short and transient bouts in five 1000 mg/kg animals and two 100 mg/kg animals. No findings were noted during the arena observations in this study.
A dose-related increase in salivation seen after dosing among treated groups was not considered toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test substance rather than a sign of systemic toxicity.
Any other clinical signs noted during the treatment period occurred within the range of background findings.

Mortality:

mortality observed, treatment-related

Description (incidence):

There were two death males treated at 1000 mg/kg.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Slight (not statistically significant) trends towards a lower body weight and body weight gain were seen in 1000 mg/kg treated animals during a large proportion of the study.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

no effects observed

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A statistically significant increase in the incidence of focal corneal edema was observed in 100 mg/kg females at Week 13, however this finding was not considered treatment-related as it occurred in a non-dose related manner.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
- White blood cell count was increased in 1000 mg/kg females, lymphocyte levels were reduced in 1000 mg/kg males and 1000 mg/kg females, and neutrophil levels were increased in 300 mg/kg males and 1000 mg/kg males and females (males not statistically significant).
- Red blood cell distribution width (%RDW) was increased in 1000 mg/kg females, and haemoglobin levels, mean red blood cell volume (MCV), and mean cell haemoglobin concentration (MCH) were reduced in 1000 mg/kg males and females.
Any other statistically significant changes in haematology parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals:
- Alanine aminotransferase (ALAT) levels were significantly increased in both male and female 1000 mg/kg animals, and aspartate transaminase (ASAT) levels were increased in 1000 mg/kg females.
- Total protein and albumin levels were increased in 1000 mg/kg, and 100/300/1000 mg/kg males, respectively. Total bilirubin levels were increased in 100, 300 and 1000 mg/kg males.
- Urea levels were increased in 1000 mg/kg males and females, and creatinine levels were increased in 100 and 1000 mg/kg males and 1000 mg/kg females.
- Glucose and cholesterol levels both reduced in 1000 mg/kg males and females.
- Inorganic phosphate levels were increased in 1000 mg/kg males and females. Sodium levels were increased in 100 and 300 mg/kg males and females, however these findings occurred in the absence of a dose-related trend and therefore considered not toxicologically relevant.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Foregrip strength was reduced in 300 and 1000 mg/kg males and 1000 mg/kg females in a statistically significant manner. And a slight (not statistically significant) reduction in total movement and ambulatory behavior was observed in 1000 mg/kg females.
Further observations, hearing ability, pupillary reflex, static righting reflex and hindgrip strenght were normal in all examined animals. Motor activity was similar between treated and control males. And all groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related higher liver weights (relative to body weights) were noted in the 1000 mg/kg group females and increased kidney weights (absolute and/or relative to body weights) were noted in 1000 mg/kg group males and females. The increased kidney weight was related to the microscopic findings of tubular vacuolation with pigmented material.
Some organ weight differences were statistically significant when compared to the control group but were considered to be the result of a test substance-related effect on final body weight (including the liver weight change in males). There were no other test substance-related organ weight changes.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Test substance-related microscopic findings were noted, starting at 300 mg/kg in the kidneys and mesenteric lymph nodes of males and females, and at 1000 mg/kg in the adrenal glands of males and females, the pancreas of males, and the spleen of females.
Kidney findings included an increased incidence and severity of tubular vacuolation, with pigmented material in the cortex, which was present in males and females starting at a dose of 300 mg/kg. Tubular degeneration and an increased severity and/or incidence in tubular basophilia was observed at 1000 mg/kg.
In the mesenteric lymph node, an increased incidence and severity of macrophage foci was recorded, starting at 300 mg/kg in both sexes.
In the adrenal gland, vacuolation of the zona glomerulosa was recorded at an increased incidence and/or severity in both sexes at 1000 mg/kg.
In the pancreas, increased apoptosis of acinar cells was recorded in males treated at 1000 mg/kg. One female at 1000 mg/kg with minimal increased apoptosis was considered to be within background levels.
In the spleen, increased incidence and severity of extramedullary hematopoiesis and increased severity of pigmentation was recorded in females treated at 1000 mg/kg.
The remaining histological changes were considered to be incidental findings or within the range of background pathology encountered in rats of this age and strain. There were no test substance-related alteration in the prevalence, severity, or histological character of those incidental tissue alterations.

Histopathological findings: neoplastic:

not examined

Other effects:

effects observed, treatment-related

Description (incidence and severity):

Increased macrophages in male and female mesenteric lymph nodes starting at 300 mg/kg/day, vacuolation of the zona glomerulosa of male and female adrenal glands at1000 mg/kg/day and increased apoptosis in male pancreas at 1000 mg/kg/day were also considered to be non-adverse since these findings were slight in nature.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

mortality

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

System:

urinary

Organ:

kidney

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Any other information on results incl. tables

Results

(A) Body weight

Body weights (gms) - Male

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAY 1	MEAN	168	163	166	169
WEEK 1	ST.DEV	8.8	8.8	8.5	6.4
	N	10	10	10	10
DAY 8	MEAN	217	210	210	216
WEEK 2	ST.DEV	13.6	12.6	12.5	10.7
	N	10	10	10	10
DAY 15	MEAN	262	250	255	258
WEEK 3	ST.DEV	17.6	13.9	17.2	14.6
	N	10	10	10	10
DAY 22	MEAN	300	283	291	295
WEEK 4	ST.DEV	21.8	15.2	22.2	16.8
	N	10	10	10	10
DAY 29	MEAN	325	310	313	321
WEEK 5	ST.DEV	28.9	13.1	28.2	21.1
	N	10	10	10	10
DAY 36	MEAN	346	328	331	332
WEEK 6	ST.DEV	35.7	15.3	32.4	41.3
	N	10	10	10	10
DAY 43	MEAN	364	346	349	355
WEEK 7	ST.DEV	36.3	14.8	33.5	23.3
	N	10	10	10	9
DAY 50	MEAN	378	359	363	361
WEEK 8	ST.DEV	38.1	15.9	36.7	30.6
	N	10	10	10	9
DAY 57	MEAN	389	373	375	369
WEEK 9	ST.DEV	40.0	16.7	38.8	32.0
	N	10	10	10	9
DAY 64	MEAN	402	386	386	382
WEEK 10	ST.DEV	41.9	17.3	39.3	26.8
	N	10	10	10	9
DAY 71	MEAN	412	394	394	381
WEEK 11	ST.DEV	44.2	17.9	39.7	29.1
	N	10	10	10	9
DAY 78	MEAN	418	399	401	392
WEEK 12	ST.DEV	45.4	19.3	41.4	31.0
	N	10	10	10	8
DAY 85	MEAN	425	406	407	397
WEEK 13	ST.DEV	48.5	19.3	41.3	32.0
	N	10	10	10	8
DAY 91	MEAN	428	409	411	404
WEEK 13	ST.DEV	48.6	17.9	42.2	34.2
	N	10	10	10	8

Body weight gain (%) - Male

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0.0	0.0	0.0	0.0
	N	10	10	10	10
DAY 8	MEAN	29	28	26	28
WEEK 2	ST.DEV	1.9	3.3	3.1	5.6
	N	10	10	10	10
DAY 15	MEAN	56	53	53	53
WEEK 3	ST.DEV	3.9	5.4	5.5	9.6
	N	10	10	10	10
DAY 22	MEAN	78	74	75	75
WEEK 4	ST.DEV	6.4	9.9	8.9	11.2
	N	10	10	10	10
DAY 29	MEAN	93	90	88	90
WEEK 5	ST.DEV	10.9	10.9	11.9	14.6
	N	10	10	10	10
DAY 36	MEAN	105	102	99	96
WEEK 6	ST.DEV	14.4	13.5	14.7	24.6
	N	10	10	10	10
DAY 43	MEAN	116	113	110	110
WEEK 7	ST.DEV	14.7	15.3	14.5	16.8
	N	10	10	10	9
DAY 50	MEAN	124	121	118	113
WEEK 8	ST.DEV	15.8	16.9	16.1	20.8
	N	10	10	10	9
DAY 57	MEAN	130	129	125	118
WEEK 9	ST.DEV	16.5	18.2	17.3	21.1
	N	10	10	10	9
DAY 64	MEAN	139	137	132	126
WEEK 10	ST.DEV	17.7	19.2	18.0	18.3
	N	10	10	10	9
DAY 71	MEAN	145	142	136	125
WEEK 11	ST.DEV	18.6	20.1	18.2	16.5
	N	10	10	10	9
DAY 78	MEAN	148	146	141	131
WEEK 12	ST.DEV	18.5	21.0	18.8	19.5
	N	10	10	10	8
DAY 85	MEAN	152	150	145	133
WEEK 13	ST.DEV	20.8	20.5	19.8	20.1
	N	10	10	10	8
DAY 91	MEAN	154	151	147	137
WEEK 13	ST.DEV	20.9	19.4	20.4	21.9
	N	10	10	10	8

Body weight (gms) - Female

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAY 1	MEAN	132	133	133	134
WEEK 1	ST.DEV	6.4	7.4	8.3	7.4
	N	10	10	10	10
DAY 8	MEAN	152	156	153	157
WEEK 2	ST.DEV	9.3	10.6	11.8	10.0
	N	10	10	10	10
DAY 15	MEAN	175	174	174	180
WEEK 3	ST.DEV	9.8	13.0	13.6	12.9
	N	10	10	10	10
DAY 22	MEAN	189	188	190	196
WEEK 4	ST.DEV	8.4	14.1	14.8	9.7
	N	10	10	10	10
DAY 29	MEAN	202	199	200	203
WEEK 5	ST.DEV	10.1	13.9	17.4	11.2
	N	10	10	10	10
DAY 36	MEAN	208	210	208	207
WEEK 6	ST.DEV	11.7	17.4	19.3	17.9
	N	10	10	10	10
DAY 43	MEAN	218	217	216	219
WEEK 7	ST.DEV	10.6	19.4	18.1	17.1
	N	10	10	10	10
DAY 50	MEAN	224	222	222	221
WEEK 8	ST.DEV	8.7	17.6	18.8	14.7
	N	10	10	10	10
DAY 57	MEAN	229	227	228	225
WEEK 9	ST.DEV	10.6	18.5	20.2	14.8
	N	10	10	10	10
DAY 64	MEAN	232	235	231	228
WEEK 10	ST.DEV	12.9	22.9	21.1	15.6
	N	10	10	10	10
DAY 71	MEAN	237	239	235	233
WEEK 11	ST.DEV	12.1	23.1	19.2	17.8
	N	10	10	10	10
DAY 78	MEAN	241	242	239	235
WEEK 12	ST.DEV	10.6	23.2	19.6	16.7
	N	10	10	10	10
DAY 85	MEAN	243	244	241	233
WEEK 13	ST.DEV	11.4	22.1	21.5	19.7
	N	10	10	10	10
DAY 91	MEAN	245	249	242	235
WEEK 13	ST.DEV	12.9	24.9	21.2	16.5
	N	10	10	10	10

Body weight gain (%) - Female

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAY 1	MEAN	0	0	0	0
WEEK 1	ST.DEV	0.0	0.0	0.0	0.0
	N	10	10	10	10
DAY 8	MEAN	15	17	15	17
WEEK 2	ST.DEV	3.5	4.1	4.1	4.7
	N	10	10	10	10
DAY 15	MEAN	32	31	31	34
WEEK 3	ST.DEV	3.5	5.5	6.0	8.9
	N	10	10	10	10
DAY 22	MEAN	43	42	43	46
WEEK 4	ST.DEV	3.6	7.6	7.6	5.8
	N	10	10	10	10
DAY 29	MEAN	53	49	51	51
WEEK 5	ST.DEV	3.9	5.3	7.0	6.4
	N	10	10	10	10
DAY 36	MEAN	57	58	57	54
WEEK 6	ST.DEV	6.1	9.3	8.7	13.1
	N	10	10	10	10
DAY 43	MEAN	65	63	63	63
WEEK 7	ST.DEV	6.2	10.0	8.5	10.5
	N	10	10	10	10
DAY 50	MEAN	70	67	67	65
WEEK 8	ST.DEV	5.3	9.4	9.7	8.7
	N	10	10	10	10
DAY 57	MEAN	74	71	72	68
WEEK 9	ST.DEV	6.4	8.8	9.4	9.3
	N	10	10	10	10
DAY 64	MEAN	76	77	74	70
WEEK 10	ST.DEV	9.0	12.1	9.6	9.9
	N	10	10	10	10
DAY 71	MEAN	80	80	77	74
WEEK 11	ST.DEV	8.2	12.8	9.1	11.8
	N	10	10	10	10
DAY 78	MEAN	82	82	80	75
WEEK 12	ST.DEV	8.6	13.0	8.8	10.2
	N	10	10	10	10
DAY 85	MEAN	84	83	82	74
WEEK 13	ST.DEV	8.1	11.9	9.3	11.8
	N	10	10	10	10
DAY 91	MEAN	86	87	82	75
WEEK 13	ST.DEV	10.4	14.0	9.7	9.7
	N	10	10	10	10

(B) Food consumption

Food consumption (g/animal/day) - Males

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAYS 1-8	MEAN	22	21	21	23
WEEKS 1-2	ST.DEV	0.2	0.7	0.6	0.3
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	26	24	25	26
WEEKS 2-3	ST.DEV	0.4	0.6	0.2	0.1
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	25	24	25	27
WEEKS 3-4	ST.DEV	0.1	0.4	0.6	0.3
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	25	24	25	26
WEEKS 4-5	ST.DEV	0.5	0.4	1.0	0.0
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	25	24	24	25
WEEKS 5-6	ST.DEV	0.7	0.3	2.0	3.3
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	25	24	24	26
WEEKS 6-7	ST.DEV	0.1	0.0	1.5	0.1
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	24	23	23	26
WEEKS 7-8	ST.DEV	0.3	0.1	0.8	0.2
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	23	23	23	23
WEEKS 8-9	ST.DEV	0.4	0.7	1.2	2.2
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	23	23	22	25
WEEKS 9-10	ST.DEV	0.5	0.8	0.9	2.3
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	23	22	22	23
WEEKS 10-11	ST.DEV	0.2	1.6	1.3	3.3
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	22	21	22	22
WEEKS 11-12	ST.DEV	0.7	1.6	1.6	1.3
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	22	21	21	23
WEEKS 12-13	ST.DEV	0.5	0.8	1.0	1.4
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	23	22	21	25
WEEK 13	ST.DEV	0.4	1.4	1.3	0.5
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	24	23	23	25

Relative food consumption (g/kg bw/day) - Males

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAYS 1-8	MEAN	101	102	102	104
WEEKS 1-2	ST.DEV	0.3	0.5	3.1	0.1
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	98	98	98	102
WEEKS 2-3	ST.DEV	0.8	0.3	0.6	1.6
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	85	85	85	90
WEEKS 3-4	ST.DEV	1.6	0.6	0.1	0.9
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	77	77	79	82
WEEKS 4-5	ST.DEV	0.8	1.8	0.6	1.3
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	73	74	71	74
WEEKS 5-6	ST.DEV	0.2	1.7	3.4	6.1
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	70	70	69	75
WEEKS 6-7	ST.DEV	1.7	0.3	1.9	0.7
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	65	63	64	73
WEEKS 7-8	ST.DEV	1.0	0.2	0.1	3.8
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	60	62	62	62
WEEKS 8-9	ST.DEV	0.5	1.9	1.2	3.1
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	58	59	57	65
WEEKS 9-10	ST.DEV	0.1	1.7	0.2	7.1
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	56	57	56	61
WEEKS 10-11	ST.DEV	1.0	3.5	1.1	7.5
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	53	54	55	56
WEEKS 11-12	ST.DEV	0.1	3.7	1.5	5.2
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	52	52	51	57
WEEKS 12-13	ST.DEV	0.6	1.3	0.5	1.9
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	53	53	52	62
WEEK 13	ST.DEV	0.7	2.6	1.0	3.0
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	69	70	69	74

Food consumption (g/animal/day) - Females

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAYS 1-8	MEAN	15	15	15	16
WEEKS 1-2	ST.DEV	0.1	1.0	0.3	0.4
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	17	17	17	18
WEEKS 2-3	ST.DEV	0.3	1.3	0.1	0.2
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	16	16	16	16
WEEKS 3-4	ST.DEV	0.5	1.2	0.1	0.7
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	16	16	16	17
WEEKS 4-5	ST.DEV	0.4	1.3	0.0	0.1
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	16	17	17	16
WEEKS 5-6	ST.DEV	0.5	0.6	0.1	1.2
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	17	17	17	17
WEEKS 6-7	ST.DEV	0.4	0.7	0.0	0.9
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	16	17	16	16
WEEKS 7-8	ST.DEV	0.6	0.4	0.2	0.2
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	16	16	17	16
WEEKS 8-9	ST.DEV	0.5	0.2	0.2	0.8
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	16	16	16	15
WEEKS 9-10	ST.DEV	0.8	0.6	0.2	0.4
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	16	16	16	16
WEEKS 10-11	ST.DEV	1.0	0.3	0.3	0.3
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	15	16	16	15
WEEKS 11-12	ST.DEV	1.1	0.4	0.7	0.4
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	15	15	15	15
WEEKS 12-13	ST.DEV	1.1	0.1	0.5	0.3
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	16	15	15	14
WEEK 13	ST.DEV	1.5	0.0	0.4	0.3
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	16	16	16	16

Relative food consumption (g/kg bw/day) - Females

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
TREATMENT DAYS 1-8	MEAN	96	95	96	99
WEEKS 1-2	ST.DEV	0.9	4.4	1.0	0.7
	N (CAGE)	2	2	2	2
DAYS 8-15	MEAN	96	96	96	99
WEEKS 2-3	ST.DEV	0.7	5.1	0.1	4.0
	N (CAGE)	2	2	2	2
DAYS 15-22	MEAN	85	85	84	81
WEEKS 3-4	ST.DEV	3.0	3.6	0.3	5.7
	N (CAGE)	2	2	2	2
DAYS 22-29	MEAN	81	82	80	81
WEEKS 4-5	ST.DEV	2.3	3.9	0.3	1.4
	N (CAGE)	2	2	2	2
DAYS 29-36	MEAN	79	79	79	76
WEEKS 5-6	ST.DEV	1.9	1.2	0.3	2.1
	N (CAGE)	2	2	2	2
DAYS 36-43	MEAN	78	76	78	76
WEEKS 6-7	ST.DEV	2.0	1.0	0.5	0.1
	N (CAGE)	2	2	2	2
DAYS 43-50	MEAN	73	75	74	73
WEEKS 7-8	ST.DEV	3.2	0.6	0.4	4.5
	N (CAGE)	2	2	2	2
DAYS 50-57	MEAN	68	72	73	72
WEEKS 8-9	ST.DEV	2.4	1.1	1.1	0.4
	N (CAGE)	2	2	2	2
DAYS 57-64	MEAN	68	69	67	68
WEEKS 9-10	ST.DEV	3.0	0.0	1.0	1.5
	N (CAGE)	2	2	2	2
DAYS 64-71	MEAN	66	65	67	68
WEEKS 10-11	ST.DEV	3.8	1.7	0.7	5.3
	N (CAGE)	2	2	2	2
DAYS 71-78	MEAN	64	65	66	66
WEEKS 11-12	ST.DEV	4.1	1.4	3.0	1.5
	N (CAGE)	2	2	2	2
DAYS 78-85	MEAN	63	63	63	63
WEEKS 12-13	ST.DEV	3.5	2.0	2.1	1.1
	N (CAGE)	2	2	2	2
DAYS 85-91	MEAN	63	62	63	60
WEEK 13	ST.DEV	5.1	3.1	1.5	1.4
	N (CAGE)	2	2	2	2
MEAN OF MEANS OVER TREATMENT	MEAN	75	76	76	76

(C) Opthalmic observations

MALES
	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE-TEST No Findings	4/10	4/10	4/10	4/10
Corneal Edema	1/10	0/10	0/10	0/10
Focal Corneal Edema	3/10	1/10	0/10	0/10
Focal Corneal Opacity	5/10	6/10	6/10	6/10
Haemorrhage From Hyaloid Vessel	1/10	0/10	0/10	0/10
Pinpoint Corneal Opacities AT WEEK13 NoFindings	1/10   4/10	0/10	1/10	1/10   5/8
Focal Corneal Edema	1/10			1/8
Focal Corneal Opacity	4/10			3/8
Pinpoint Corneal Opacities	2/10			0/8
FEMALES
	GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
PRE-TEST No Findings	7/10	2/10	4/10	7/10
Focal Corneal Edema	0/10	6/10 #	2/10	0/10
Focal Corneal Opacity	2/10	7/10	4/10	3/10
Haemorrhage From Hyaloid Vessel	1/10	0/10	0/10	0/10
Pinpoint Corneal Opacities AT WEEK13 NoFindings	0/10   5/10	2/10	2/10	2/10   6/10
Focal Corneal Edema	1/10			1/10
Focal Corneal Opacity	5/10			4/10
Pinpoint Corneal Opacities	0/10			1/10

# / ## Fisher's Exact test significant at 5% (#) or 1% (##) level

(D) Haematology summary

Males

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT WBC	MEAN	7.1	6.9	6.5	7.4
10E9/L	ST.DEV	1.4	1.0	1.1	1.4
	N	10	10	10	8
Neutrophils	MEAN	14.6	18.6	20.2 ++	20.8
%WBC	ST.DEV	3.1	4.2	2.9	7.5
	N	10	10	10	8
Lymphocytes	MEAN	82.4	77.5	76.5 ++	75.7 ++
%WBC	ST.DEV	3.3	5.1	2.7	8.2
	N	10	10	10	8
Monocytes	MEAN	1.8	2.2	1.9	2.1
%WBC	ST.DEV	0.5	1.0	0.6	0.8
	N	10	10	10	8
Eosinophils	MEAN	1.1	1.6 +	1.3	1.3
%WBC	ST.DEV	0.3	0.4	0.7	0.6
	N	10	10	10	8
Basophils	MEAN	0.1	0.1	0.1	0.2
%WBC	ST.DEV	0.0	0.1	0.1	0.1
	N	10	10	10	8
Red blood cells	MEAN	9.01	9.20	9.02	9.21
10E12/L	ST.DEV	0.41	0.33	0.45	0.35
	N	10	10	10	8
Reticulocytes	MEAN	2.1	2.1	2.0	2.0
%RBC	ST.DEV	0.3	0.2	0.2	0.3
	N	10	10	10	8
RDW	MEAN	13.7	12.5	14.0	14.0
%	ST.DEV	3.6	0.5	3.3	0.8
	N	10	10	10	8
Haemoglobin	MEAN	10.0	10.1	9.9	9.5 *
mmol/L	ST.DEV	0.4	0.2	0.5	0.3
	N	10	10	10	8
Haematocrit	MEAN	0.481	0.490	0.486	0.469
L/L	ST.DEV	0.013	0.015	0.019	0.023
	N	10	10	10	8
MCV	MEAN	53.4	53.3	53.9	50.9 **
fL	ST.DEV	1.9	1.1	1.8	1.0
	N	10	10	10	8
MCH	MEAN	1.10	1.09	1.10	1.03 *
fmol	ST.DEV	0.06	0.03	0.07	0.02
	N	10	10	10	8
MCHC	MEAN	20.67	20.54	20.39	20.18
mmol/L	ST.DEV	0.48	0.35	0.63	0.41
	N	10	10	10	8
Platelets	MEAN	726	769	692	686
10E9/L	ST.DEV	121	121	83	63
	N	10	10	10	8
PT	MEAN	16.7	15.6 *	15.2 **	16.3
s	ST.DEV	0.6	0.5	0.6	1.4
	N	10	10	9	8
APTT	MEAN	19.2	20.7	18.9	18.9
s	ST.DEV	2.0	0.9	2.2	2.1
	N	10	10	9	8

+/++ Steel-test significant at 5% (+) or 1% (++) level

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

Females

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
WBC	MEAN	3.9	3.6	4.6	5.9 **
10E9/L	ST.DEV	0.9	0.8	0.8	1.3
	N	10	10	10	10
Neutrophils	MEAN	13.8	21.3	17.6	21.1 ++
%WBC	ST.DEV	2.4	13.8	8.9	4.9
	N	10	10	10	10
Lymphocytes	MEAN	82.4	73.1	78.9	75.7 ++
%WBC	ST.DEV	2.6	16.4	9.3	4.4
	N	10	10	10	10
Monocytes	MEAN	2.3	2.2	2.2	2.2
%WBC	ST.DEV	0.5	0.7	0.4	0.8
	N	10	10	10	10
Eosinophils	MEAN	1.4	3.3 +	1.2	0.9
%WBC	ST.DEV	0.6	3.2	0.5	0.2
	N	10	10	10	10
Basophils	MEAN	0.1	0.1	0.1	0.1
%WBC	ST.DEV	0.1	0.1	0.1	0.1
	N	10	10	10	10
Red blood cells	MEAN	8.19	8.12	8.17	8.37
10E12/L	ST.DEV	0.40	0.36	0.24	0.48
	N	10	10	10	10
Reticulocytes	MEAN	2.9	2.9	2.6	2.6
%RBC	ST.DEV	0.4	0.3	0.3	1.5
	N	10	10	10	10
RDW	MEAN	11.4	11.6	11.2	12.8 **
%	ST.DEV	0.6	0.4	0.4	1.0
	N	10	10	10	10
Haemoglobin	MEAN	9.3	9.1	9.3	9.0 *
mmol/L	ST.DEV	0.3	0.2	0.2	0.4
	N	10	10	10	10
Haematocrit	MEAN	0.455	0.452	0.456	0.444
L/L	ST.DEV	0.015	0.015	0.008	0.022
	N	10	10	10	10
MCV	MEAN	55.6	55.7	55.9	53.1 **
fL	ST.DEV	1.3	1.5	1.5	1.7
	N	10	10	10	10
MCH	MEAN	1.14	1.12	1.14	1.07 **
fmol	ST.DEV	0.04	0.03	0.03	0.04
	N	10	10	10	10
MCHC	MEAN	20.49	20.13	20.35	20.18
mmol/L	ST.DEV	0.31	0.28	0.37	0.41
	N	10	10	10	10
Platelets	MEAN	785	750	815	711
10E9/L	ST.DEV	100	96	95	175
	N	10	10	10	10
PT	MEAN	17.2	16.2	16.5	16.6
s	ST.DEV	1.4	0.8	1.1	1.6
	N	10	10	10	10
APTT	MEAN	19.4	19.2	20.0	18.5
s	ST.DEV	1.4	1.8	1.0	1.4
	N	10	10	10	10

+/++ Steel-test significant at 5% (+) or 1% (++) level

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

(E) Clinical biochemistry summary

Males

END OF TREATMENT		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
ALAT	MEAN	43.0	45.1	46.6	83.6 **
U/L	ST.DEV	7.1	13.5	11.8	17.2
	N	10	10	10	8
ASAT	MEAN	76.0	69.4	73.2	81.7
U/L	ST.DEV	7.8	6.8	9.0	5.8
	N	10	10	10	8
ALP	MEAN	126	132	124	147
U/L	ST.DEV	38	50	25	49
	N	10	10	10	8
Total protein	MEAN	65.7	67.9	67.9	69.7 **
g/L	ST.DEV	1.4	2.6	3.0	3.0
	N	10	10	10	8
Albumin	MEAN	32.0	33.3 *	33.6 **	34.5 **
g/L	ST.DEV	1.0	0.8	1.2	0.9
	N	10	10	10	8
Total bilirubin	MEAN	1.9	2.4 *	2.8 **	2.8 **
umol/L	ST.DEV	0.3	0.3	0.6	0.6
	N	10	10	10	8
Urea	MEAN	7.0	7.1	7.6	8.3
mmol/L	ST.DEV	1.0	0.9	1.5	1.4
	N	10	10	10	8
Creatinine	MEAN	41.5	45.2 **	42.2	45.8 **
umol/L	ST.DEV	2.9	2.6	1.7	2.5
	N	10	10	10	8
Glucose	MEAN	10.42	10.39	9.32	9.03 *
mmol/L	ST.DEV	1.10	1.16	1.04	1.27
	N	10	10	10	8
Cholesterol	MEAN	2.37	2.04	2.06	1.67 **
mmol/L	ST.DEV	0.42	0.34	0.35	0.17
	N	10	10	10	8
Bile Acids	MEAN	19.8	34.4 **	23.7	11.3
umol/L	ST.DEV	9.8	14.8	8.0	2.7
	N	10	10	10	8
Sodium	MEAN	138.8	141.0 **	142.6 **	138.7
mmol/L	ST.DEV	1.5	1.4	1.0	1.5
	N	10	10	10	8
Potassium	MEAN	3.88	3.79	3.90	3.96
mmol/L	ST.DEV	0.11	0.19	0.22	0.24
	N	10	10	10	8
Chloride	MEAN	101	101	103 *	102
mmol/L	ST.DEV	2	1	1	2
	N	10	10	10	8
Calcium	MEAN	2.55	2.56	2.56	2.56
mmol/L	ST.DEV	0.04	0.05	0.04	0.08
	N	10	10	10	8
Inorg.Phos	MEAN	1.49	1.39	1.60	1.99 **
mmol/L	ST.DEV	0.16	0.15	0.12	0.15
	N	10	10	10	8

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

Females

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
ALAT	MEAN	31.3	29.0	32.7	94.2 **
U/L	ST.DEV	7.9	6.1	5.2	24.1
	N	10	10	10	10
ASAT	MEAN	73.0	69.4	70.0	88.2 *
U/L	ST.DEV	11.0	12.7	15.5	11.9
	N	10	10	10	10
ALP	MEAN	43	45	47	53
U/L	ST.DEV	19	20	15	22
	N	10	10	10	10
Total protein	MEAN	68.4	67.2	69.9	70.6
g/L	ST.DEV	2.4	3.6	2.1	3.6
	N	10	10	10	10
Albumin	MEAN	35.4	35.0	36.2	35.5
g/L	ST.DEV	1.3	1.9	1.6	1.8
	N	10	10	10	10
Total bilirubin	MEAN	2.2	2.5	3.0	3.3 **
umol/L	ST.DEV	0.3	0.5	0.7	1.3
	N	10	10	10	10
Urea	MEAN	7.3	6.1	6.9	10.0 **
mmol/L	ST.DEV	1.1	0.9	0.9	1.5
	N	10	10	10	10
Creatinine	MEAN	45.1	45.8	46.6	52.0 **
umol/L	ST.DEV	2.3	5.3	2.5	4.8
	N	10	10	10	10
Glucose	MEAN	7.32	7.86	7.92	7.10
mmol/L	ST.DEV	0.78	1.16	0.85	0.94
	N	10	10	10	10
Cholesterol	MEAN	1.75	1.84	1.73	1.57
mmol/L	ST.DEV	0.23	0.53	0.29	0.35
	N	10	10	10	10
Bile Acids	MEAN	18.8	16.1	15.4	13.3
umol/L	ST.DEV	12.6	4.0	6.6	7.5
	N	10	10	10	10
Sodium	MEAN	140.0	141.5 **	141.8 **	139.8
mmol/L	ST.DEV	0.7	0.9	0.9	1.1
	N	10	10	10	10
Potassium	MEAN	3.52	3.45	3.45	3.67
mmol/L	ST.DEV	0.20	0.16	0.20	0.29
	N	10	10	10	10
Chloride	MEAN	102	102	102	102
mmol/L	ST.DEV	1	2	1	2
	N	10	10	10	10
Calcium	MEAN	2.58	2.56	2.56	2.56
mmol/L	ST.DEV	0.05	0.07	0.06	0.07
	N	10	10	10	10
Inorg.Phos	MEAN	1.37	1.30	1.35	1.79 **
mmol/L	ST.DEV	0.15	0.12	0.18	0.13
	N	10	10	10	10

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

(F) Organ weight and organ/body weight ratio summary

Males - organ weight

ORGAN WEIGHTS MALES	(GRAM) SUMMARY
		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT BODY W.	MEAN	408	388	388	367 *
(GRAM)	ST.DEV	44	18	38	29
	N	10	10	10	8
BRAIN	MEAN	2.07	2.05	2.03	2.06
(GRAM)	ST.DEV	0.06	0.06	0.07	0.06
	N	10	10	10	8
HEART	MEAN	1.055	0.978	0.992	0.945 *
(GRAM)	ST.DEV	0.122	0.089	0.079	0.061
	N	10	10	10	8
LIVER	MEAN	9.72	8.72	8.89	9.57
(GRAM)	ST.DEV	1.15	0.73	1.10	1.34
	N	10	10	10	8
THYROIDS	MEAN	0.017	0.016	0.016	0.015 *
(GRAM)	ST.DEV	0.002	0.003	0.003	0.001
	N	10	10	10	8
THYMUS	MEAN	0.317	0.319	0.288	0.233 *
(GRAM)	ST.DEV	0.053	0.069	0.065	0.025
	N	10	10	10	8
KIDNEYS	MEAN	2.40	2.24	2.32	2.57
(GRAM)	ST.DEV	0.22	0.14	0.23	0.20
	N	10	10	10	8
ADRENALS	MEAN	0.058	0.056	0.053	0.060
(GRAM)	ST.DEV	0.009	0.008	0.008	0.009
	N	10	10	10	8
SPLEEN	MEAN	0.581	0.557	0.551	0.615
(GRAM)	ST.DEV	0.080	0.076	0.081	0.120
	N	10	10	10	8
TESTES	MEAN	3.71	3.71	3.60	3.55
(GRAM)	ST.DEV	0.19	0.28	0.19	0.23
	N	10	10	10	8
PROSTATE GLAND	MEAN	0.870	0.779	0.844	0.865
(GRAM)	ST.DEV	0.158	0.132	0.192	0.123
	N	10	10	10	8
EPIDIDYMIDES	MEAN	1.279	1.303	1.243	1.226
(GRAM)	ST.DEV	0.128	0.284	0.099	0.181
	N	10	10	10	8
SEMINAL VESICLES	MEAN	1.338	1.338	1.344	1.321
(GRAM)	ST.DEV	0.219	0.294	0.209	0.179
	N	10	10	10	8

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

Males - organ/body weight ration (%) summary

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT BODY W.	MEAN	408	388	388	367 *
(GRAM)	ST.DEV	44	18	38	29
	N	10	10	10	8
BRAIN	MEAN	0.51	0.53	0.53	0.56
(%)	ST.DEV	0.05	0.02	0.05	0.05
	N	10	10	10	8
HEART	MEAN	0.260	0.252	0.256	0.258
(%)	ST.DEV	0.033	0.019	0.018	0.015
	N	10	10	10	8
LIVER	MEAN	2.38	2.25	2.29	2.60 **
(%)	ST.DEV	0.10	0.15	0.13	0.18
	N	10	10	10	8
THYROIDS	MEAN	0.004	0.004	0.004	0.004
(%)	ST.DEV	0.001	0.001	0.001	0.000
	N	10	10	10	8
THYMUS	MEAN	0.078	0.082	0.074	0.064
(%)	ST.DEV	0.014	0.017	0.014	0.006
	N	10	10	10	8
KIDNEYS	MEAN	0.59	0.58	0.60	0.70 **
(%)	ST.DEV	0.05	0.03	0.03	0.06
	N	10	10	10	8
ADRENALS	MEAN	0.014	0.014	0.014	0.016
(%)	ST.DEV	0.002	0.002	0.001	0.002
	N	10	10	10	8
SPLEEN	MEAN	0.143	0.144	0.142	0.168
(%)	ST.DEV	0.020	0.020	0.019	0.031
	N	10	10	10	8
TESTES	MEAN	0.92	0.96	0.94	0.97
(%)	ST.DEV	0.12	0.08	0.12	0.07
	N	10	10	10	8
PROSTATE GLAND	MEAN	0.214	0.201	0.217	0.235
(%)	ST.DEV	0.036	0.036	0.045	0.017
	N	10	10	10	8
EPIDIDYMIDES	MEAN	0.318	0.335	0.323	0.334
(%)	ST.DEV	0.056	0.067	0.040	0.049
	N	10	10	10	8
SEMINAL VESICLES	MEAN	0.330	0.345	0.351	0.360
(%)	ST.DEV	0.055	0.075	0.074	0.043
	N	10	10	10	8

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

Females - organ weight

ORGAN WEIGHTS FEMALES	(GRAM) SUMMARY
		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT BODY W.	MEAN	231	231	226	210 *
(GRAM)	ST.DEV	12	22	21	16
	N	10	10	10	10
BRAIN	MEAN	1.91	1.87	1.87	1.85
(GRAM)	ST.DEV	0.09	0.09	0.07	0.07
	N	10	10	10	10
HEART	MEAN	0.683	0.692	0.659	0.645
(GRAM)	ST.DEV	0.052	0.074	0.059	0.053
	N	10	10	10	10
LIVER	MEAN	5.64	5.76	5.36	6.27
(GRAM)	ST.DEV	0.37	0.94	0.42	0.51
	N	10	10	10	10
THYROIDS	MEAN	0.014	0.014	0.014	0.013
(GRAM)	ST.DEV	0.003	0.003	0.002	0.002
	N	10	10	10	10
THYMUS	MEAN	0.269	0.299	0.285	0.215
(GRAM)	ST.DEV	0.038	0.061	0.067	0.048
	N	10	10	10	10
KIDNEYS	MEAN	1.57	1.56	1.59	1.72 *
(GRAM)	ST.DEV	0.08	0.14	0.15	0.11
	N	10	10	10	10
ADRENALS	MEAN	0.068	0.063	0.066	0.069
(GRAM)	ST.DEV	0.011	0.011	0.008	0.010
	N	10	10	10	10
SPLEEN	MEAN	0.443	0.432	0.436	0.465
(GRAM)	ST.DEV	0.044	0.078	0.029	0.053
	N	10	10	10	10
OVARIES	MEAN	0.139	0.147	0.134	0.128
(GRAM)	ST.DEV	0.017	0.017	0.019	0.026
	N	10	10	10	10
UTERUS	MEAN	0.684	0.822	0.611	0.607
(GRAM)	ST.DEV	0.244	0.287	0.293	0.282
	N	10	10	10	10

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

Female - organ/body weight ratio (%) summary

		GROUP 1 CONTROL	GROUP 2 100 MG/KG	GROUP 3 300 MG/KG	GROUP 4 1000 MG/KG
END OF TREATMENT BODY W.	MEAN	231	231	226	210 *
(GRAM)	ST.DEV	12	22	21	16
	N	10	10	10	10
BRAIN	MEAN	0.83	0.82	0.83	0.88
(%)	ST.DEV	0.05	0.08	0.06	0.06
	N	10	10	10	10
HEART	MEAN	0.296	0.299	0.292	0.307
(%)	ST.DEV	0.016	0.015	0.009	0.016
	N	10	10	10	10
LIVER	MEAN	2.44	2.49	2.38	2.98 **
(%)	ST.DEV	0.12	0.34	0.10	0.16
	N	10	10	10	10
THYROIDS	MEAN	0.006	0.006	0.006	0.006
(%)	ST.DEV	0.001	0.001	0.001	0.001
	N	10	10	10	10
THYMUS	MEAN	0.117	0.129	0.125	0.102
(%)	ST.DEV	0.015	0.020	0.021	0.019
	N	10	10	10	10
KIDNEYS	MEAN	0.68	0.68	0.70	0.82 **
(%)	ST.DEV	0.04	0.07	0.05	0.07
	N	10	10	10	10
ADRENALS	MEAN	0.029	0.027	0.029	0.033
(%)	ST.DEV	0.005	0.005	0.004	0.003
	N	10	10	10	10
SPLEEN	MEAN	0.192	0.187	0.194	0.222 *
(%)	ST.DEV	0.018	0.031	0.016	0.029
	N	10	10	10	10
OVARIES	MEAN	0.060	0.064	0.060	0.061
(%)	ST.DEV	0.008	0.011	0.008	0.011
	N	10	10	10	10
UTERUS	MEAN	0.294	0.358	0.269	0.284
(%)	ST.DEV	0.094	0.132	0.120	0.111
	N	10	10	10	10

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

For more parameters and data tables, kindly refer to the attached background material section of the IUCLID.

Applicant's summary and conclusion

Conclusions:

Under the study conditions, based on the early mortality and adverse microscopic findings in the kidneys of 1000 mg/kg animals, a NOAEL for the test substance was established at 300 mg/kg bw/day.

Executive summary:

A study was conducted to determine repeated dose toxicity of the test substance when administered to rats according to OECD Guideline 408, EU Method B.26 and EPA OPPTS 870.3100. The test substance, formulated in water, was administered daily for at least 90 days by oral gavage to SPF-bred Wistar Han rats. One control group and three treated groups were tested, each consisting of 10 males and 10 females, at the dose levels of 0, 100, 300 and 100 mg/kg bw/day. Chemical analyses of formulations were conducted on three occasions during the study to assess accuracy and homogeneity. The following parameters were evaluated: mortality and clinical signs daily; functional observation tests in week 13; body weight and food consumption weekly; ophthalmoscopy at pretest and in week 13; estrous cycle determination during the last 3 weeks; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. Formulation analyses confirmed that formulations of test substance in water were prepared accurately and homogenously. Adverse test substance-related morphologic alterations were observed in the kidneys of males and females treated at 1000 mg/kg. These findings consisted of the accumulation of pigmented material in vacuolar structures in the cortical tubules. This pigmented material may well represent (parts of) test substance accumulated in the tubular organelles (lysosomes). At higher severity, the pigmented material most likely caused additional degeneration of the tubules and regenerative changes of the tubules, which was visible by the increased presence of basophilic tubules. The rather severe vacuolation with pigmented material, in combination with degeneration of tubules and increased tubular basophilia, is considered to be adverse. Furthermore, kidney weights were increased in 1000 mg/kg animals, an effect attributed to tubular vacuolation with pigmented material. The spontaneous death of a 1000 mg/kg male was also most likely attributed to the abovementioned test-substance related effects. At 300 mg/kg, in a few males and females, some small vacuoles containing a minimal amount of pigmented material were still present. This was not considered to be adverse based on severity and absence of the additional degenerative changes seen at 1000 mg/kg. Haematological alterations such as increased neutrophil levels in 300 mg/kg males, and 1000 mg/kg males and females, and increase in white blood cell counts in 1000 mg/kg females, and reductions of relative lymphocyte level  in 1000 mg/kg males and females, and 300 mg/kg males do not seem to have any clear microscopic correlate. Also no clear correlation could be determined for changes in clinical biochemistry parameters, including increased urea, creatinine and inorganic phosphate levels in 1000 mg/kg males and females. In addition increased levels of total protein (1000 mg/kg), albumin (300/1000 mg/kg), and bilirubin (100/300/1000 mg/kg) were noted in males. At 1000 mg/kg, increases were observed in the levels of liver enzymes alanine aminotransferase and aspartate transaminase, and in other liver-related parameters including cholesterol and glucose levels. Although these occurred in correlation with increased liver weight, no corroborative microscopic correlates were noted. Haemoglobin, mean red blood cell volume, and mean cell haemoglobin concentration were all reduced in 1000 mg/kg animals, and red blood cell distribution width was increased in 1000 mg/kg females. These changes were small in scale and may be related to the marginally higher incidence of haematopoiesis (slight) observed in the spleen of 1000 mg/kg animals. Based on these slight changes and the histopathological evaluation this was considered to be non-adverse. Other test substance-related findings as increased macrophages in male and female mesenteric lymph nodes starting at 300 mg/kg/day, vacuolation of the zona glomerulosa of male and female adrenal glands at1000 mg/kg/day and increased apoptosis in male pancreas at 1000 mg/kg/day were also considered to be non-adverse since these findings were slight in nature. Clinical signs including diarrhoea, swelling/gas distention of the abdomen and rales were incidentally observed in individual surviving animals, mainly in 1000 mg/kg animals. These signs were most likely related to the test substance, however their respective durations were transient, and had no clear clinical pathology or microscopic correlate. The reduced foregrip strength in 300 mg/kg males and 1000 mg/kg males/females, and reduced motor activity in 1000 mg/kg females occurred in the absence of any clear correlation of clinical signs or morphological changes and therefore considered not adverse. No toxicologically significant changes were noted in any of the remaining parameters investigated in this study (i.e. ophthalmoscopy, body weight, food consumption, functional observations, and macroscopic examination). Under the study conditions, based on the early mortality and adverse microscopic findings in the kidneys of 1000 mg/kg animals, a NOAEL for the test substance was established at 300 mg/kg bw/day (Beerens-Heijnen, 2017).