Bis(2-(2-butoxyethoxy)ethyl) adipate

Endpoint summary

Description of key information

Subchronic toxicity (OECD 408, GLP) oral:

NOAEL (rat): 300 and 100 mg/kg bw/day in males and females, respectively

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

25 May - 05 November 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

adopted 21 September 1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Health and Youth Care Inspectorate, Ministry of Health, Welfare and Sport, Utrecht, The Netherlands

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: 138 to 176 g (males), 118 to 147 g (females)
- Housing: on arrival and following randomization, 5 animals of the same sex and same dosing group in polycarbonate cages (Makrolon type IV, height 18 cm) containing appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) equipped with water bottles. Animals were separated during designated procedures/activities. During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cageenrichment, bedding material, food and water.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum. During motor activity measurements, animals did not have access to food for a maximum of 2 h.
- Water: municipal tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: analysis were performed

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 46 - 62
- Air changes (per hr): 10 or more
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 29 May 2018 To: 29 August 2018

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Remarks:

400

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Test item dosing formulations (w/w) were homogenized to visually acceptable levels at appropriate concentrations to meet dose level requirements. The dosing formulations were prepared daily as a solution and dosed within 5 h after adding vehicle to the test item. Test item dosing formulations were kept at room temperature until dosing. The dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the vehicle (1.125) and test item (1.1 at 25 °C). No correction was made for the purity/composition of the test item.

VEHICLE
- Justification for use and choice of vehicle: Trial preparations were performed at the test facility to select the suitable vehicle and to establish a suitable formulation procedure. Polyethylene glycol 400 (Merck, Darmstadt, Germany) was identified as suitable vehicle.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dose formulation samples were collected for analysis in week 1, 6 and 13 for homogeneity. For homogeneity analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was ± 10%. In addition, stability was tested in samples collected in week 1. Stability of the test item in the vehicle was determined over 5 h at room temperature under normal laboratory light conditions (lowest and highest concentration). Duplicate sets of each sample (approximately 500 mg, accurately weighed) were sent to the analytical laboratory. Stability results were considered acceptable if the sample analysis results were within or equal to ± 10% of the concentration determined by the initial analysis of each formulation. For concentration analysis, duplicate sets of samples (approximately 500 mg, accurately weighed) for each sampling time point were sent to the analytical laboratory.Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration. Analysis showed that concentrations analyzed were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).

Duration of treatment / exposure:

91 (males) or 92 (females) days

Frequency of treatment:

daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The oral route of exposure was selected because this is a possible route of human exposure during manufacture, handling or use of the test item. The dose levels were selected based on the results of the range finding prenatal developmental study (Test Facility Study No. 20148926) and in an attempt to produce graded responses to the test item.
- Fasting period before blood sampling for clinical biochemistry:

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once daily, beginning on day 1 of treatment

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly, beginning on day 1 of treatment until day of necropsy

BODY WEIGHT: Yes
- Time schedule for examinations: day - 5 and then weekly (before dosing), starting on day 1

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: No
- Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during pre-treatment and at the end of the dosing period in Week 13
- Dose groups that were examined: during pre-treatment in all animals, and at the end of the dosing in vehicle and 1000 mg/kg bw/day dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.1 in the "Any other information and methods incl. tables" section were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 in the "Any other information and methods incl. tables" section were examined.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 13
- Dose groups that were examined: 5 animals per dose group, all dose groups
- Battery of functions tested: grip strength / motor activity / other: hearing ability

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 3 in the "Any other information and methods incl. tables" section.)

HISTOPATHOLOGY: Yes (see table 3 in the "Any other information and methods incl. tables" section.)

Other examinations:

Estrous Cycle Determination
Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal lavage.
- Time schedule for examinations: week 11 (Day 71) up to and including week 14 (Day 92)
- Dose groups that were examined: females of all dose groups

Sperm analysis
For all surviving males, the following assessments were performed:
1. From all males, sperm samples were taken from the proximal part of the vas deferens (right) at necropsy:
- Sperm motility was assessed from all samples.
- Sperm smears for morphological evaluation were fixed from all samples. Abnormal forms of sperm from a differential count of 200 spermatozoa (if possible) per animal were recorded. Evaluation was performed for all males of the control and high-dose group.
2. One testis and one epididymis (left) from all males were removed, placed in labeled bags, and kept in the freezer at ≤-15°C. After thawing the epididymis was weighed, homogenized and evaluated for sperm numbers. Evaluation was performed for males of the control and high dose group.

Thyroid homone analysis:
- Time schedule for collection of blood: end of treatment
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked in table No.2 in the "Any other information and methods incl. tables" section were examined.

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels. Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed when possible, but excluded semi-quantitative data, and any group with less than 3 observations.

Variables for inferential analysis: body Weight and body weight gain, food consumption, organ weights, organ weight relative to body weight
Parametric/non parametric tests used: Levene’s test was used to assess the homogeneity of group variances. Datasets with at least 3 groups were compared using an overall one-way ANOVA F test when Levene’s test was not significant or the Kruskal-Wallis test when group variances were not homogeneous. When the overall F test or Kruskal-Wallis test was significant, then the above pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. Datasets with 2 groups (the designated control group and 1 other group) were compared using a t-test if Levene’s test was not significant or Wilcoxon Rank-Sum test if it was.

The following pairwise comparisons were made: Low dose group (2) vs. Vehicle dose group (1), Mid dose group (3) vs. Vehicle dose group (1), High dose group (4) vs. Vehicle dose group (1)

Incidence: An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

100, 300 and 1000 mg/kg bw/day: salivation and ploughing was noted in all animals. For salivation a dose-related trend in severity (up to severe on several occasions in high-dose rats) and time of onset (from day 8 in low-dose rats and from day 1 or 2 at the higher dose levels) was observed. The salivation was considered to be a physiological response and/or behavioural sign of discomfort in reaction to the bolus administration of the test item rather than a sign of systemic toxicity considering its severity (mostly slight or moderate) and the time of occurrence (i.e. within half an hour after dosing).

All other clinical signs noted occurred incidentally and represented normal background findings. At the incidence observed, these findings were considered not to be treatment-related. (Please refer to table 4 in the "Any other information on results incl. tables" section.)

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1000 mg/kg bw/day: two females died unscheduled. One female was found dead in the morning of study day 58. Prior to death, this animal showed no clinical signs of conditional decline and body weight development was normal. Possibly, its death could be related to the gavage procedure, however, this could not be confirmed by macroscopic or microscopic findings, due to cannibalism. The death was considered likely to be procedure-related rather than test-item related. Another female died during blood sampling on the scheduled day of necropsy. This death was considered to be related to the blood sampling procedure under anesthesia and was therefore regarded to be unrelated to treatment with the test item.

300 mg/kg bw/day: one females was euthanized for humane reasons on day 54 of the study, based on deep breathing and weight loss (9% between days 50-54). At macroscopic examination, red foci (multifocal) were noted in the glandular stomach. Microscopic examination showed marked mixed cell inflammation in the bronchus and moderate neutrophilic infiltration in the lumen of the larynx. These microscopic findings and the sudden onset of the moribundity suggested that the conditional decline was related to the gavage procedure rather than being test-item related

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: males gained slightly less weight than controls in most weeks of the study (reaching statistical significance in weeks 4 and 9 only). The slight non statistically significant differences in body weights gradually increased up to a mean value of 8 % lower for the high dose animals at the end of the treatment period (non-adverse, due to slight nature of the effect). (Please refer to table 5 in the "Any other information on results incl. tables" section.) Body weights and body weight gain of females showed no treatment-related changes.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: males consumed slightly less food (about 6-9%) than controls on several time periods during the study (weeks 1-2 and the second half of the 13-week treatment period) (non-adverse, due to slight nature of the effect). (Please refer to table 6 in the "Any other information on results incl. tables" section.) Food consumption of treated females was similar to that of controls over the study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Vehicle, 100, 300 and 1000 mg/kg bw/day: cornea opacity, cornea edema and cornea, opacity multifocal, pinpoint ocurred at similar incidences in all dose groups or only in single animals and were thus considered incidental, non-treatment-related and non-adverse. (Please refer to table 7 in the "Any other information on results incl. tables" section.) Ophthalmic examination of control and high-dose rats in the last week of the treatment period showed no test-item related ocular changes. The incidences of the findings did not indicate a relation with treatment and both the nature and incidence of the findings occurred within the normal range for rats of this age and strain.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
lower number of red blood cells in males (-10%) and females (-13%) (statistically significant),
lower mean corpuscular haemoglobin concentration (MCHC) in males (-9%) and females (-5%) (statistically significant),
lower red blood cell distribution width (RDW) in males (-7%)(statistically significant),
higher mean corpuscular haemoglobin (MCH) in females (+7%) (statistically significant),
higher mean corpuscular volume (MCV) in males and females (both +13%)(statistically significant), lower haemoglobin concentration in males (-9%) and females (-6%) (not statistically significant),
higher number of reticulocytes in males (+37%) and females (+30%) (not statistically significant)

All changes were regarded as non-adverse because of the minor magnitude of the change.

100 and 300 mg/kg bw/day: decreased prothrombin time in males (statistically significant). This finding was considered non-treatment-related and non-adverse due to the lack of a dose-related response.

100 mg/kg bw/day: higher MCHC in females (statistically significant). This isolated finding was considered non-treatment-related and non adverse due to the lack of a dose-related response.

(Please refer to table 8 in the "Any other information on results incl. tables" section.)

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day:
higher alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in males (+51% and +39%, respectively) (statistically significant),
higher alkaline phosphatase (ALP) in males (+54%) and females (+46%) (statistically significant),
higher bile acids in males (2.1-fold) (statistically significant). A higher mean bile acids concentration was also noted in females, but the difference from controls (1.9 fold) was not statistically significant and could be explained by the high concentration in a single animal,
lower cholesterol in males (-32%) and females (-28%) (statistically significant),
lower fasting glucose in males (-22%), (statistically significant),
higher creatinine in males (+7%)(statistically significant),
higher potassium in females (+10%).(statistically significant)
lower calcium in females (-3%) (statistically significant)

300 and 1000 mg/kg bw/day:
higher chloride in males (+2%) and (+3%) at the mid an high dose, respectively (statistically significant),
higher potassium in males (statistically significant, non-treatment-related, missing dose-related response)

100 and 300 mg/kg bw/day: higher sodium levels in males and females (statistically significant, non-treatment-related, missing dose-related response)

100 mg/kg bw/day: higher chloride in females (statistically significant, non-treatment-related, missing dose-related response)

Decreases noted in cholesterol (both sexes) and fasting glucose (males only) may also be related to the effect of
the test item on the liver. Slightly higher creatinine, chloride and potassium, and slightly lower calcium, mostly at 1000 mg/kg/day were not associated with adverse anatomic pathology alterations and therefore regarded as non-adverse.

(Please refer to table 9 in the "Any other information on results incl. tables" section.)

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: higher liver weights (absolute and relative to body weight) were noted in females (statistically significant, treatment-related, adverse), slight increase in relative kidney weights in both sexes (statistically significant, but not considered treatment-related,result of variations in terminal body weights), lower mean uterus weights (absolute and relative to body weight) in females (about -40%, not statistically significant) were considered to reflect normal physiological variation (non-treatment-related)

300 and 1000 mg/kg bw/day: increased heart weights in in mid dose males and high dose females (statistically significant, but not considered treatment-related, result of variations in terminal body weights). In the absence of a dose-related trend, the lower relative heart weight in males was regarded a chance finding.

(Please refer to table 10, 11 and 12 in the "Any other information on results incl. tables" section.)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All macroscopic findings were within the range of background gross observations encountered in rats of this age and strain and were therefore considered non-treatment-related. (Please refer to table 13 in the "Any other information on results incl. tables" section.)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: centrilobular hypertrophy at minimal to mild degree and periportal hepatocellular vacuolation at minimal degree in the liver (males), increased severity of pigment accumulation in the spleen (both sexes) and increased incidence and severity of diffuse vacuolation in the zona glomerulosa of the adrenal gland (both sexes) (treatment-related, non-adverse). Non-adversity was based on the nature of the finding, the low severity and the absence of any additional degenerative or proliferative changes.

300 and 1000 mg/kg bw/day: in mid dose females and high dose males and females adverse microscopic changes were observed in the liver and consisted of an increased incidence and severity (up to mild degree) of centrilobular mononuclear cell infiltrates accompanied by single cell necrosis as well as increased liver weighs (by about 20%) (treatment-related, adverse).

100 and 300 mg/kg bw/day: centrilobular mononuclear cell infiltration with single cell necrosis was observed in males of the low and mid dose group and in females of the low dose group (treatment-related, non-adverse). Non-adversity was based on the low severity/incidence of the lesions in these dose groups.

All other microscopic findings were within the range of background pathology encountered in rats of this age and strain. There was no test substance-related alteration in the prevalence, severity or histologic character of those incidental tissue alterations. (Please refer to table 14 in the "Any other information on results incl. tables" section.)

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

Neurobehavioural examination - functional tests (effects observed non-treatment-related):
1000 mg/kg bw/day: one male and one female showed low values for total movements and ambulations (below the normal ranges). Habituation profiles of these animals were normal and their lower motor activity was not associated with corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Moreover, all other high-dose animals showed normal motor activity. Therefore, the lower activity recorded for these two animals was not attributed to treatment.

Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In addition, forelimb and hind limb grip strength showed no treatment-related changes. (Please refer to table 15 and 16 in the "Any other information on results incl. tables" section.)

Estrous cycle determination (effects observed non-treatment-related):
1000 mg/kg bw/day: one female had irregular cycles of, subsequently, 6 and 2 days. Since irregular cycles were noted in only one female and adjacent cycles were normal, this finding was considered non-treatment-related.

Sperm analysis (effects observed non-treatment-related):
1000 mg/kg bw/day: epididymal sperm count was 35% higher compared to the control mean. Since the difference was not statistically significant and was atrributed to low counts in two control males, this finding was not considered treatment-related. (Please refer to table 17 in the "Any other information on results incl. tables" section.)

Thyroid homone analysis:
1000 mg/kg bw/day: T4 levels were lower compared to the control levels (-14% and -22% in males and females, respectively) Since statistical significance was not achieved and T4 levels in almost all high dose animals remained in the concurrent control range, these intergroup differences were considered non-treatment-related. (Please refer to table 9 in the "Any other information on results incl. tables" section.)

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no adverse effect observed at this dose level

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effect observed at this dose level

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

histopathology: non-neoplastic

Key result

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 4: Summary of clinical observations

Dose groups (mg/kg bw/day)	Male				Female
Observations From Day 1 to 93	Vehicle	100	300	1000	Vehicle	100	300	1000
eyeball, enlarged
number of animals affected	1	0	0	0	0	0	0	0
number of times recorded	2	0	0	0	0	0	0	0
% of affected animals	10	0	0	0	0	0	0	0
first to last seen	79 - 86	-	-	-	-	-	-	-
skin, lesion
number of animals affected	2	2	2	1	0	0	0	1
number of times recorded	4	3	10	5	0	0	0	2
% of affected animals	20	20	20	10	0	0	0	10
first to last seen	9 - 44	9 - 16	9 - 51	9 - 37	-	-	-	23 - 30
breathing, deep
number of animals affected	0	0	0	0	0	0	1	0
number of times recorded	0	0	0	0	0	0	1	0
% of affected animals	0	0	0	0	0	0	10	0
first to last seen	-	-	-	-	-	-	54 - 54	-
salivation
number of animals affected	1	10	10	10	2	10	10	10
number of times recorded	1	839	900	826	2	848	872	879
% of affected animals	10	100	100	100	20	100	100	100
first to last seen	62 - 62	8 - 91	2 - 91	1 - 91	54 - 62	7 - 92	1 - 92	1 - 92
skin, scab
number of animals affected	0	1	0	0	0	0	0	0
number of times recorded	0	2	0	0	0	0	0	0
% of affected animals	0	10	0	0	0	0	0	0
first to last seen	-	6 - 7	-	-	-	-	-	-
ploughing
number of animals affected	0	10	10	10	0	10	10	10
number of times recorded	0	839	840	763	0	845	811	815
% of affected animals	0	100	100	100	0	100	100	100
first to last seen	-	8 - 91	8 - 91	8 - 91	-	8 - 92	8 - 92	8 - 92

 

Table 5: Summary of body weight gains (g)

Males		Days - Change
Dose group (mg/kg bw/day)		-5-1	1-8	8-15	15-22	22-29	29-36	36-43
Vehicle	Mean	34.30	45.30	43.10	32.80	27.70	20.30	13.50
	SD	3.47	4.19	5.93	5.25	4.11	3.27	4.77
	N	10	10	10	10	10	10	10
100	Mean	35.00	46.70	45.80	36.80	23.40	21.20	11.50
	SD	2.62	3.77	5.67	4.54	5.42	4.32	2.80
	N	10	10	10	10	10	10	10
300	Mean	36.20	48.90	42.40	36.70	26.50	21.30	17.50
	SD	3.12	4.38	5.78	5.89	2.72	4.22	5.02
	N	10	10	10	10	10	10	10
1000	Mean	32.20	41.50	42.67	29.89	20.33b	16.22	14.33
	SD	6.51	6.31	3.91	3.10	4.69	5.29	7.84
	N	10	10	9	9	9	9	9
Males		Days - Change
Dose group		43-50	50-57	57-64	64-71	71-78	78-85	85-91
Vehicle	Mean	11.90	10.00	9.20	12.80	9.90	6.50	4.40
	SD	3.57	5.58	4.71	4.85	3.41	5.23	3.63
	N	10	10	10	10	10	10	10
100	Mean	11.60	10.00	11.00	10.30	8.10	5.10	10.20
	SD	5.27	3.56	2.26	4.35	2.60	3.87	8.77
	N	10	10	10	10	10	10	10
300	Mean	11.60	13.00	11.00	10.00	10.20	6.90	5.20
	SD	3.66	4.88	4.45	3.43	3.05	3.03	3.26
	N	10	10	10	10	10	10	10
1000	Mean	10.11	9.56	4.44a	8.22	7.78	4.11	2.78
	SD	5.25	5.92	3.54	5.36	3.67	3.62	5.70
	N	9	9	9	9	9	9	9
Females		Days - Change
Dose group		-5-1	1-8	8-15	15-22	22-29	29-36	36-43
Vehicle	Mean	19.20	21.00	20.40	14.00	10.50	8.60	4.70
	SD	3.58	4.92	4.62	3.65	4.84	4.30	3.86
	N	10	10	10	10	10	10	10
100	Mean	18.40	23.20	18.60	15.10	11.20	11.30	2.80
	SD	2.41	3.74	3.27	5.40	4.18	3.02	5.83
	N	10	10	10	10	10	10	10
300	Mean	15.80	23.20	17.10	14.20	8.30	8.90	5.50
	SD	2.70	5.98	4.89	3.08	3.40	5.67	5.21
	N	10	10	10	10	10	10	10
1000	Mean	19.70	23.00	18.60	14.80	7.90	10.10	6.40
	SD	3.74	4.62	3.44	4.83	2.88	1.91	2.37
	N	10	10	10	10	10	10	10
Females		Days - Change
Dose group		43-50	50-54	50-57	57-64	64-71	71-78	78-85	85-91
Vehicle	Mean	4.10	--	5.10	3.60	5.50	4.10	3.50	3.10
	SD	5.38	--	3.03	3.37	3.72	3.90	4.72	3.25
	N	10	--	10	10	10	10	10	10
100	Mean	6.20	--	5.40	4.40	4.40	4.00	1.40	3.40
	SD	7.63	--	5.02	4.60	4.12	6.51	3.86	3.63
	N	10	--	10	10	10	10	10	10
300	Mean	4.40	-22 .00z	6.44	4.67	6.33	2.00	3.44	2.22
	SD	7.52	--	5.77	5.34	2.78	4.30	4.48	3.93
	N	10	1	9	9	9	9	9	9
1000	Mean	5.30	--	4.70	1.89	4.89	2.11	3.00	-0 .67
	SD	5.31	--	3.47	4.40	3.14	5.93	2.74	4.42
	N	10	--	10	9	9	9	9	9

Significantly different from control group 1 value :a=p≤0.05,b=p≤0.01 (Dunnett)

Group excluded from statistical analysis (N<3): z

 

Table 6 Summary of food consumption (g/animal/day)

Males		Days (From – To)
Dose group (mg/kg bw/day)		1-8	8-15	15-22	22-29	29-36	36-43	43-50
Vehicle	Mean	21.85y	24.45y	25.25y	25.30y	25.45y	24.60y	24.40y
	SD	0.07	0.21	0.21	0.28	0.21	0.57	0.42
	N	2	2	2	2	2	2	2
100	Mean	21.15	23.70	25.00	25.40	24.65	23.35	23.10
	SD	0.49	0.14	0.14	0.14	0.07	0.49	1.41
	N	2	2	2	2	2	2	2
	% Diff G1	-3 .20	-3 .07	-0 .99	0.40	-3 .14	-5 .08	-5 .33
300	Mean	21.30	23.25	24.65	25.10	24.95	24.40	24.25
	SD	1.27	1.06	0.92	0.71	1.34	1.98	1.91
	N	2	2	2	2	2	2	2
	% Diff G1	-2 .52	-4 .91	-2 .38	-0 .79	-1 .96	-0 .81	-0 .61
1000	Mean	20.10	22.70	24.45	24.65	24.45	23.75	22.70
	SD	--	--	0.49	1.06	0.35	0.21	0.00
	N	1	1	2	2	2	2	2
	% Diff G1	-8 .01	-7 .16	-3 .17	-2 .57	-3 .93	-3 .46	-6 .97
Males		Days (From – To)
Dose group		50-57	57-64	64-71	71-78	78-85	85-91
Vehicle	Mean	23.80y	23.40y	23.75y	23.50y	22.70y	23.15y
	SD	0.85	0.85	0.49	0.99	1.27	0.78
	N	2	2	2	2	2	2
100	Mean	23.65	22.40	22.70	22.55	21.80	22.45
	SD	0.35	0.28	0.28	0.07	0.14	0.49
	N	2	2	2	2	2	2
		-0 .63	-4 .27	-4 .42	-4 .04	-3 .96	-3 .02
300	Mean	23.70	22.90	23.50	23.10	22.75	22.95
	SD	2.26	2.55	2.26	2.26	1.63	2.33
	N	2	2	2	2	2	2
		-0 .42	-2 .14	-1 .05	-1 .70	0.22	-0 .86
1000	Mean	23.15	22.05	22.20	22.10	21.45	22.55
	SD	0.35	0.21	0.28	0.14	0.35	1.20
	N	2	2	2	2	2	2
		-2 .73	-5 .77	-6 .53	-5 .96	-5 .51	-2 .59
Females		Days (From – To)
Dose group		1-8	8-15	15-22	22-29	29-36	36-43	43-50
Vehicle	Mean	15.60y	16.30y	16.35y	16.45y	16.20y	15.80y	16.60y
	SD	0.42	0.57	0.21	0.07	0.14	0.14	0.14
	N	2	2	2	2	2	2	2
100	Mean	15.40	16.05	16.30	16.55	16.50	16.25	16.75
	SD	0.14	0.35	0.28	0.07	0.57	0.92	0.78
	N	2	2	2	2	2	2	2
		-1 .28	-1 .53	-0 .31	0.61	1.85	2.85	0.90
300	Mean	15.35	16.05	16.40	16.60	16.60	16.50	17.15
	SD	0.07	0.21	0.14	0.14	0.28	0.14	0.07
	N	2	2	2	2	2	2	2
		-1 .60	-1 .53	0.31	0.91	2.47	4.43	3.31
1000	Mean	15.55	16.25	16.55	16.70	17.00	17.05	16.50
	SD	0.35	0.35	0.21	1.13	0.28	0.35	1.13
	N	2	2	2	2	2	2	2
		-0 .32	-0 .31	1.22	1.52	4.94	7.91	-0 .60
Females		Days (From – To)
Dose group		50-57	57-64	64-71	71-78	78-85	85-91
Vehicle	Mean	16.40y	15.75y	16.20y	16.25y	15.85y	16.10y
	SD	0.28	0.35	0.28	0.49	0.78	0.57
	N	2	2	2	2	2	2
100	Mean	16.20	16.00	16.15	16.70	15.55	16.15
	SD	0.71	0.99	1.06	0.85	0.78	0.78
	N	2	2	2	2	2	2
		-1 .22	1.59	-0 .31	2.77	-1 .89	0.31
300	Mean	16.55	16.50	16.50	16.65	16.15	16.70
	SD	0.78	0.00	0.57	0.21	0.21	0.57
	N	2	2	2	2	2	2
		0.91	4.76	1.85	2.46	1.89	3.73
1000	Mean	16.65	15.35	16.10	15.95	15.70	16.25
	SD	0.35	1.34	0.00	0.07	0.57	0.78
	N	2	2	2	2	2	2
		1.52	-2 .54	-0 .62	-1 .85	-0 .95	0.93

All Groups excluded from statistical analysis (N<3 in Control): y

 

Table 7: Summary of ophthalmoscopy findings

	Males				Females
Findings	Vehicle	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day	Vehicle	100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
Cornea, opacity	6	1		3	1	1	1	1
Cornea edema	2	2	2	1	1	1	1
Cornea, opacity multifocal, pinpoint			2	1

 

Table 8: Summary of haematology and coagulation values

End Of Treatment	Dose groups (mg/kg bw/day)	Control	100	300	1000
Males
WBC	mean	6.6	7.1	6.1	6.2
10E9/L	st.dev	1.7	1.2	0.9	0.9
	n	10	10	10	9
Neutrophils	mean	0.9	1.2	1.0	1.1
10E9/L	st.dev	0.4	0.2	0.1	0.4
	n	10	10	10	9
Lymphocytes	mean	5.5	5.7	4.9	4.9
10E9/L	st.dev	1.4	1.1	0.9	0.9
	n	10	10	10	9
Monocytes	mean	0.1	0.1	0.1	0.2
10E9/L	st.dev	0.0	0.1	0.0	0.1
	n	10	10	10	9
Eosinophils	mean	0.1	0.1	0.1	0.1
10E9/L	st.dev	0.0	0.0	0.0	0.0
	n	10	10	10	9
Basophils	mean	0.0	0.0	0.0	0.0
10E9/L	st.dev	0.0	0.0	0.0	0.0
	n	10	10	10	9
Red blood cells	mean	9.41	9.23	9.38	8.45 **
10E12/L	st.dev	0.27	0.28	0.31	0.47
	n	10	10	10	9
Reticulocytes	mean	178.8	199.5	172.2	245.0
10E9/L	st.dev	21.9	19.5	19.3	64.3
	n	10	10	10	9
RDW	mean	12.3	12.9	12.2	11.4 *
%	st.dev	0.5	0.9	0.6	0.7
	n	10	10	10	9
Haemoglobin	mean	10.2	10.1	10.0	9.3 **
mmol/L	st.dev	0.3	0.3	0.3	0.4
	n	10	10	10	9
Haematocrit	mean	0.510	0.499	0.496	0.514
L/L	st.dev	0.015	0.010	0.008	0.025
	n	10	10	10	9
MCV	mean	54.2	54.1	52.9	61.0 **
fL	st.dev	1.7	1.2	1.2	3.7
	n	10	10	10	9
MCH	mean	1.08	1.09	1.07	1.10
fmol	st.dev	0.04	0.04	0.02	0.03
	n	10	10	10	9
MCHC	mean	19.95	20.18	20.17	18.14 **
mmol/L	st.dev	0.25	0.43	0.32	0.61
	n	10	10	10	9
Platelets	mean	684	740	717	675
10E9/L	st.dev	109	43	68	73
	n	10	10	10	9
PT	mean	17.8	17.0 **	17.2 *	18.0
s	st.dev	0.5	0.4	0.5	0.3
	n	9	8	8	6
APTT	mean	20.8	20.1	20.8	20.1
s	st.dev	1.6	2.1	1.6	0.9
	n	10	9	10	9
Females
WBC	mean	3.9	3.8	3.9	4.6
10E9/L	st.dev	0.9	1.5	0.9	1.3
	n	9	10	9	8
Neutrophils	mean	0.4	0.7	0.6	0.6
10E9/L	st.dev	0.2	0.3	0.2	0.2
	n	9	10	9	8
Lymphocytes	mean	3.3	2.9	3.1	3.8
10E9/L	st.dev	0.8	1.5	0.7	1.1
	n	9	10	9	8
Monocytes	mean	0.1	0.1	0.1	0.1
10E9/L	st.dev	0.0	0.0	0.1	0.1
	n	9	10	9	8
Eosinophils	mean	0.1	0.1	0.1	0.1
10E9/L	st.dev	0.0	0.0	0.0	0.0
	n	9	10	9	8
Basophils	mean	0.0	0.0	0.0	0.0
10E9/L	st.dev	0.0	0.0	0.0	0.0
	n	9	10	9	8
Red blood cells	mean	8.29	8.14	8.35	7.21 **
10E12/L	st.dev	0.59	0.56	0.11	0.50
	n	10	10	9	8
Reticulocytes	mean	213.7	205.4	218.7	277.8
10E9/L	st.dev	35.2	22.2	33.9	60.0
	n	10	10	9	8
RDW	mean	11.0	11.0	11.1	11.0
%	st.dev	0.3	0.6	0.5	0.5
	n	10	10	9	8
Haemoglobin	mean	9.3	9.4	9.5	8.7
mmol/L	st.dev	0.7	0.7	0.2	0.7
	n	10	10	9	8
Haematocrit	mean	0.461	0.451	0.462	0.454
L/L	st.dev	0.035	0.033	0.012	0.032
	n	10	10	9	8
MCV	mean	55.7	55.5	55.4	63.0 **
fL	st.dev	1.2	1.3	1.0	2.2
	n	10	10	9	8
MCH	mean	1.13	1.15	1.14	1.21 **
fmol	st.dev	0.03	0.04	0.02	0.02
	n	10	10	9	8
MCHC	mean	20.25	20.72 *	20.66	19.16 **
mmol/L	st.dev	0.34	0.30	0.35	0.54
	n	10	10	9	8
Platelets	mean	779	750	864	744
10E9/L	st.dev	119	91	138	93
	n	10	10	9	8
PT	mean	17.4	17.2	18.7	18.5
s	st.dev	1.2	0.6	2.4	2.2
	n	10	10	9	8
APTT	mean	20.7	20.7	21.0	19.6
s	st.dev	1.6	1.1	1.8	1.8
	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

 

Table 9: Summary of clinical chemistry values

End Of Treatment	Dose groups (mg/kg bw/day)	Control	100	300	1000
Males
ALAT	mean	39.6	44.2	45.1	59.9 **
U/L	st.dev	5.0	7.0	5.9	20.1
	n	10	10	10	9
ASAT	mean	69.1	77.2	81.3	96.1 **
U/L	st.dev	9.6	9.8	9.9	19.9
	n	10	10	10	9
ALP	mean	102	98	98	157 **
U/L	st.dev	22	20	19	47
	n	10	10	10	9
Total protein	mean	65.8	66.6	66.2	64.4
g/L	st.dev	1.3	3.3	1.7	1.9
	n	10	10	10	9
Albumin	mean	33.3	33.7	33.6	33.8
g/L	st.dev	0.6	1.3	0.5	1.1
	n	10	10	10	9
Total bilirubin	mean	2.6	2.6	2.8	2.7
umol/L	st.dev	0.2	0.3	0.3	0.2
	n	10	10	10	9
Urea	mean	7.0	7.4	7.5	7.5
mmol/L	st.dev	0.6	0.9	0.9	0.3
	n	10	10	10	9
Creatinine	mean	34.5	34.5	34.5	36.9 **
umol/L	st.dev	1.1	1.5	1.6	1.8
	n	10	10	10	9
Glucose	mean	10.44	10.41	10.05	8.14 **
mmol/L	st.dev	1.70	1.56	1.30	1.13
	n	10	10	10	9
Cholesterol	mean	1.95	1.86	1.78	1.33 **
mmol/L	st.dev	0.22	0.19	0.28	0.33
	n	10	10	10	9
Bile Acids	mean	25.2	25.6	17.3	53.4 **
umol/L	st.dev	13.4	19.3	8.9	24.3
	n	10	10	10	9
Sodium	mean	141.8	143.3 **	143.6 **	142.7
mmol/L	st.dev	1.0	1.1	0.9	1.2
	n	10	10	10	9
Potassium	mean	3.74	3.83	4.02 *	3.82
mmol/L	st.dev	0.13	0.23	0.13	0.34
	n	10	10	10	9
Chloride	mean	102	103	104 **	105 **
mmol/L	st.dev	1	1	1	2
	n	10	10	10	9
Calcium	mean	2.63	2.66	2.68	2.63
mmol/L	st.dev	0.03	0.05	0.05	0.06
	n	10	10	10	9
Inorg.Phos	mean	1.69	1.63	1.75	1.78
mmol/L	st.dev	0.21	0.13	0.09	0.16
	n	10	10	10	9
Total T4	mean	3.61	3.97	4.04	3.09
ug/dL	st.dev	0.59	0.63	1.04	0.39
	n	10	10	10	9
females
ALAT	mean	36.6	38.7	37.3	41.6
U/L	st.dev	7.9	11.2	9.4	9.1
	n	10	10	9	9
ASAT	mean	73.5	83.8	72.8	71.9
U/L	st.dev	8.8	27.1	5.1	8.3
	n	10	10	9	9
ALP	mean	41	53	39	60 *
U/L	st.dev	13	13	7	26
	n	10	10	9	9
Total protein	mean	68.4	66.7	69.6	65.9
g/L	st.dev	2.8	1.8	3.3	2.2
	n	10	10	9	9
Albumin	mean	36.7	35.9	37.8	36.2
g/L	st.dev	1.5	1.3	1.5	1.1
	n	10	10	9	9
Total bilirubin	mean	3.0	3.0	3.0	3.2
umol/L	st.dev	0.4	0.3	0.3	0.4
	n	10	10	9	9
Urea	mean	7.6	7.4	7.7	8.1
mmol/L	st.dev	0.6	0.6	1.2	1.1
	n	10	10	9	9
Creatinine	mean	40.6	39.2	38.5	39.0
umol/L	st.dev	2.0	1.7	3.3	1.6
	n	10	10	9	9
Glucose	mean	7.12	6.74	6.74	7.42
mmol/L	st.dev	1.15	0.63	1.13	1.28
	n	10	10	9	9
Cholesterol	mean	1.95	1.72	1.70	1.40 *
mmol/L	st.dev	0.49	0.48	0.44	0.27
	n	10	10	9	9
Bile Acids	mean	19.7	20.2	19.1	36.6
umol/L	st.dev	8.3	12.2	12.5	42.9
	n	10	10	9	9
Sodium	mean	141.3	143.0 **	144.2 **	142.1
mmol/L	st.dev	1.0	0.7	0.8	1.1
	n	10	10	9	9
Potassium	mean	3.31	3.47	3.33	3.63 *
mmol/L	st.dev	0.30	0.20	0.10	0.25
	n	10	10	9	9
Chloride	mean	105	106 **	107 **	106 *
mmol/L	st.dev	1	1	1	1
	n	10	10	9	9
Calcium	mean	2.69	2.64	2.69	2.61 **
mmol/L	st.dev	0.07	0.05	0.04	0.06
	n	10	10	9	9
Inorg.Phos	mean	1.46	1.52	1.59	1.56
mmol/L	st.dev	0.22	0.09	0.19	0.14
	n	10	10	8	9
Total T4	mean	2.39	2.54	2.38	1.86
ug/dL	st.dev	0.79	0.69	0.85	0.52
	n	10	10	9	9

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

 

Table 10: Summary of absolute organ weights

Males
Group (mg/kg bw/day)		Body weight	Brain	Epididymis	Gland Adrenal	Gland Prostate	Gland Sem Ves	Gland Thyroid
		g	g	g	g	g	g	g
control	Mean	375.1	2.0836	1.1901	0.05781	0.8821	1.2902	0.01566
	SD	36.4	0.1148	0.1098	0.00720	0.2420	0.1764	0.00231
	N	10	10	10	10	10	10	10
100	Mean	374.7	2.0428	1.2244	0.05831	0.8008	1.4507	0.01603
	SD	36.8	0.0851	0.0970	0.00938	0.2282	0.2786	0.00411
	N	10	10	10	10	10	10	10
	%Diff G1	-0 .1	-1 .9581	2.8821	0.86490	-9 .2166	12.4399	2.36271
300	Mean	387.8	2.0560	1.2114	0.05979	0.8479	1.2558	0.01675
	SD	33.6	0.0735	0.1281	0.00682	0.2522	0.2168	0.00255
	N	10	10	10	10	10	10	10
	%Diff G1	3.4	-1 .3246	1.7898	3.42501	-3 .8771	-2 .6663	6.96041
1000	Mean	344.7	2.0511	1.1616	0.05176	0.6903	1.1749	0.01590
	SD	27.4	0.0681	0.0580	0.00805	0.1408	0.2073	0.00287
	N	9	9	9	9	9	9	9
	%Diff G1	-8 .1	-1 .5593	-2 .3985	-10 .47301	-21 .7398	-8 .9375	1.53257
Males
		Heart	Kidney	Liver	Spleen	Testis	Thymus
control	Mean	g	g	g	g	g	g
	SD	1.0080	2.4660	9.3318	0.5655	3.6530	0.2906
	N	0.1234	0.3757	1.5440	0.1083	0.3178	0.0637
100	Mean	10	10	10	10	10	10
	SD	0.9990	2.6050	9.4615	0.5563	3.7483	0.2642
	N	0.0947	0.2145	1.0497	0.0484	0.3845	0.0722
	%Diff G1	10	10	10	10	10	10
300	Mean	-0 .8929	5.6367	1.3899	-1 .6269	2.6088	-9 .0847
	SD	0.9664	2.5880	9.6277	0.5626	3.7919	0.2858
	N	0.0628	0.2555	0.8865	0.0876	0.3279	0.0575
	%Diff G1	10	10	10	10	10	10
1000	Mean	-4 .1270	4.9473	3.1709	-0 .5128	3.8024	-1 .6518
	SD	0.9701	2.5402	9.2477	0.5166	3.7129	0.2332
	N	0.0927	0.1934	0.7626	0.0630	0.2191	0.0423
	%Diff G1	9	9	9	9	9	9
		-3 .7588	3.0098	-0 .9016	-8 .6551	1.6394	-19 .7446
Females
Group (mg/kg bw/day)		Body weight	Brain	Gland Adrenal	Gland Thyroid	Heart	Kidney	Liver
control	Mean	g	g	g	g	g	g	g
	SD	223.8	1.9236	0.06453	0.01339	0.6761	1.5852	5.7994
	N	12.8	0.0888	0.01075	0.00190	0.0379	0.1127	0.7506
100	Mean	10	10	10	10	10	10	10
	SD	223.5	1.8803	0.06921	0.01389	0.6916	1.6120	5.6842
	N	15.5	0.0542	0.00910	0.00195	0.0600	0.1250	0.5325
	%Diff G1	10	10	10	10	10	10	10
300	Mean	-0 .1	-2 .2510	7.25244	3.73413	2.2926	1.6906	-1 .9864
	SD	213.4	1.8793	0.06820	0.01299	0.6957	1.5870	5.8367
	N	18.3	0.1129	0.00810	0.00247	0.0607	0.1268	0.8101
	%Diff G1	9	9	9	9	9	9	9
1000	Mean	-4 .6	-2 .3012	5.68728	-2 .99560	2.8940	0.1136	0.6426
	SD	218.1	1.9086	0.05995	0.01453	0.7194	1.7080	6.8550b
	N	22.6	0.0454	0.01010	0.00292	0.0815	0.1649	0.6071
	%Diff G1	8	8	8	7	8	8	8
Females
		Ovary	Spleen	Thymus	Uterus
control	Mean	g	g	g	g
	SD	0.1350	0.3937	0.2846	0.7898
	N	0.0186	0.0425	0.0475	0.4280
100	Mean	10	10	10	10
	SD	0.1513	0.3997	0.2863	0.8129
	N	0.0191	0.0294	0.0306	0.3222
	%Diff G1	10	10	10	10
300	Mean	12.0741	1.5240	0.5973	2.9248
	SD	0.1328	0.4026	0.2352a	0.7933
	N	0.0199	0.0545	0.0213	0.3912
	%Diff G1	9	9	9	9
1000	Mean	-1 .6461	2.2493	-17 .3499	0.4474
	SD	0.1365	0.4288	0.2893	0.4728
	N	0.0251	0.0471	0.0619	0.1671
	%Diff G1	8	8	8	8
		1.1111	8.9027	1.6339	-40 .1431

Significantly different from control group 1 value : a=p≤0.05,b=p≤0.01 (Dunn)

 

Table 11: Summary of organ weights relative to body weight

Males
Group (mg/kg bw/day)		Brain	Epididymis	Gland Adrenal	Gland Prostate	Gland Sem Ves	Gland Thyroid	Heart
		%	%	%	%	%	%	%
control	Mean	0.55840	0.31813	0.0155	0.23473	0.34545	0.0042	0.26838
	SD	0.03954	0.02148	0.0019	0.05825	0.04674	0.0005	0.01434
	N	10	10	10	10	10	10	10
100	Mean	0.54873	0.32851	0.0156	0.21599	0.38493	0.0043	0.26699
	SD	0.04436	0.03037	0.0020	0.06566	0.05075	0.0009	0.01329
	N	10	10	10	10	10	10	10
	%Diff G1	-1 .73141	3.26065	0.6025	-7 .98467	11.42982	1.9021	-0 .51709
300	Mean	0.53272	0.31351	0.0154	0.21703	0.32337	0.0043	0.24992a
	SD	0.03520	0.03331	0.0015	0.05592	0.04923	0.0006	0.01376
	N	10	10	10	10	10	10	10
	%Diff G1	-4 .59967	-1 .45401	-0 .2193	-7 .54035	-6 .39119	3.6075	-6 .87522
1000	Mean	0.59775	0.33879	0.0151	0.19946	0.34277	0.0046	0.28179
	SD	0.04170	0.03066	0.0025	0.03098	0.06930	0.0008	0.01982
	N	9	9	9	9	9	9	9
	%Diff G1	7.04767	6.49182	-2 .5076	-15 .02842	-0 .77639	10.8140	4.99732
Males
		Kidney	Liver	Spleen	Testis	Thymus
control	Mean	%	%	%	%	%	%	%
	SD	0.65756	2.47895	0.15014	0.97666	0.07756
	N	0.07344	0.23420	0.01989	0.06736	0.01581
100	Mean	10	10	10	10	10
	SD	0.69842	2.52911	0.14915	1.00536	0.07043
	N	0.05872	0.18531	0.01297	0.11174	0.01783
	%Diff G1	10	10	10	10	10
300	Mean	6.21360	2.02360	-0 .66122	2.93860	-9 .18779
	SD	0.66741	2.48456	0.14471	0.98160	0.07360
	N	0.03279	0.13234	0.01562	0.08770	0.01235
	%Diff G1	10	10	10	10	10
1000	Mean	1.49844	0.22631	-3 .61257	0.50572	-5 .10703
	SD	0.73750a	2.68451	0.15068	1.08346	0.06791
	N	0.02286	0.11401	0.02146	0.10781	0.01348
	%Diff G1	9	9	9	9	9
		12.15681	8.29233	0.36042	10.93501	-12 .44638
Females
Group (mg/kg bw/day)		Brain	Gland Adrenal	Gland Thyroid	Heart	Kidney	Liver	Ovary
control	Mean	%	%	%	%	%	%	%
		0.86159	0.0289	0.0060	0.30240	0.70923	2.59247	0.06058
	SD	0.05597	0.0048	0.0009	0.01375	0.05009	0.32206	0.00943
	N	10	10	10	10	10	10	10
100	Mean	0.84435	0.0310	0.0062	0.30964	0.72227	2.54130	0.06786
	SD	0.05406	0.0036	0.0009	0.02016	0.04813	0.11461	0.00910
	N	10	10	10	10	10	10	10
	%Diff G1	-2 .00053	7.3580	4.0112	2.39290	1.83943	-1 .97381	12.03147
300	Mean	0.88446	0.0320	0.0061	0.32681a	0.74485	2.72978	0.06200
	SD	0.06995	0.0030	0.0012	0.02617	0.03862	0.24753	0.00539
	N	9	9	9	9	9	9	9
	%Diff G1	2.65433	10.8381	2.1268	8.07136	5.02358	5.29637	2.35777
1000	Mean	0.88217	0.0275	0.0066	0.32993a	0.78421b	3.15242 b	0.06242
	SD	0.08159	0.0037	0.0014	0.02071	0.04232	0.21928	0.00805
	N	8	8	7	8	8	8	8
	%Diff G1	2.38831	-4 .8216	9.3446	9.10242	10.57337	21.59899	3.04074
Females
		Spleen	Thymus	Uterus
control	Mean	%	%	%
		0.17605	0.12699	0.35102
	SD	0.01824	0.01865	0.18264
	N	10	10	10
100	Mean	0.17904	0.12851	0.36804
	SD	0.01031	0.01480	0.15613
	N	10	10	10
	%Diff G1	1.70044	1.19996	4.84611
300	Mean	0.18829	0.11036	0.36826
	SD	0.01537	0.00751	0.17956
	N	9	9	9
	%Diff G1	6.95674	-13 .09221	4.90872
1000	Mean	0.19804	0.13413	0.21903
	SD	0.02761	0.03454	0.08351
	N	8	8	8
	%Diff G1	12.49191	5.62264	-37 .60137

Significantly different from control group 1 value :a=p≤0.05,b=p≤0.01 (Dunn)

 

Table 12: Mean percent liver weight differences from control groups

	Males			Females
Dose Level (Mg/Kg bw/day):	100	300	1000	100	300	1000
Liver
Absolute	1	3	-1	-2	1	18**
Relative To Body Weight	2	0	8	-2	5	22**
**: P<0.01

 

Table 13: Summary of gross pathology findings

	Males					Female
Dose group (mg/kg bw/day)	Vehicle	100	300	1000		Vehicle	100	300	1000
Number of animals	10	10	10	9		10	10	9	8
Adipose Tissue
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Bone, Femur
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Bone, Sternum
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Bone Marrow, Femur
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Bone Marrow, Sternum
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Brain
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Cervix
Submitted	-	-	-	-		10	10	9	8
No Visible Lesions	-	-	-	-		10	10	9	8
Epididymis
Submitted	10	10	10	9		-	-	-	-
No Visible Lesions	10	10	10	9		-	-	-	-
Esophagus
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	10	10	10	9		10	10	9	8
Eye
Submitted	10	10	10	9		10	10	9	8
No Visible Lesions	9	10	10	9		10	10	9	8
Enlargement	1	0	0		0	0	0	0	0
Galt
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Adrenal
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Clitoral
Submitted No Visible Lesions Focus GLAND, COAGULATING	-	-	-		-	10	10	9	8
No Visible Lesions	-	-	-		-	8	10	9	8
Focus	-	-	-		-	2	0	1	0
Gland; Coagulating
Submitted	10	10	10		9	-	-	-	-
No Visible Lesions	10	10	10		8	-	-	-	-
Abnormal Appearance	0	0	0		1	-	-	-	-
Gland, Harderian
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Lacrimal
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Mammary
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Parathyroid
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Pituitary
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Preputial
Submitted	10	10	10		9	-	-	-	-
No Visible Lesions	10	10	10		8	-	-	-	-
Small	0	0	0		1	-	-	-	-
Gland, Prostate
Submitted	10	10	10		9	-	-	-	-
No Visible Lesions	10	10	10		9	-	-	-	-
Gland, Salivary, Mandibular
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Salivary, Sublingual
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Gland, Seminal Vesicle
Submitted	10	10	10		9	-	-	-	-
No Visible Lesions	10	10	10		9	-	-	-	-
Gland, Thyroid
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Heart
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Kidney
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	9	10		9	10	10	9	8
Dilatation	0	1	0		0	0	0	0	0
Large Intestine, Cecum
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Large Intestine, Colon
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Large Intestine, Rectum
Submitted	10	10	0		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Larynx
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Liver
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	9	10	9		9	10	10	8	8
Hernia	1	0	0		0	0	0	0	0
Discoloration	0	0	1		0	0	0	1	0
Abnormal Consistency	0	0	1		0	0	0	0	0
Small	0	0	0		0	0	0	1	0
Lung
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Lymph Node Iliac
Submitted	-	-	-		-	1	0	0	0
No Visible Lesions	-	-	-		-	-	-	-	-
Lymph Node, Mandibular
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	9	9	9	7
Discoloration	0	0	0		0	1	1	0	1
Lymph Node, Mesenteric
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Muscle, Diaphgram
Submitted	1	0	0		0	-	-	-	-
No Visible Lesions	1	-	-		-	-	-	-	-
Muscle, Skeletal
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Nasopharynx
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Nerve, Optic
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Nerve, Sciatic
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Ovary
Submitted	-	-	-		-	10	10	9	8
No Visible Lesions	-	-	-		-	10	10	9	8
Pancreas
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Skin
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Small Intestine,
Duodenum
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Small Intestine, Ileum
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Small Intestine,
Jejunum
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Spinal Cord
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Spleen
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Stomach
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	9	10	9		9	9	7	9	6
Focus	1	0	1		0	0	1	0	1
Irregular Surface	0	0	0		0	1	2	0	1
Testis
Submitted	10	10	10		9	-	-	-	-
No Visible Lesions	10	10	10		9	-	-	-	-
Thymus
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	9		9	10	8	9	8
Focus	0	0	1		0	0	1	0	0
Discoloration	0	0	0		0	0	1	0	0
Tongue
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Trachea
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Urinary Bladder
Submitted	10	10	10		9	10	10	9	8
No Visible Lesions	10	10	10		9	10	10	9	8
Uterus
Submitted	-	-	-		-	10	10	9	8
No Visible Lesions	-	-	-		-	5	4	4	7
Fluid Accumulation	-	-	-		-	5	6	5	1
Vagina
Submitted	-	-	-		-	10	10	9	8
No Visible Lesions	-	-	-		-	10	10	9	8

 

Table 14: Summary test item-related microscopic findings – scheduled euthanasia animals

	Males				Females
Dose level (mg/kg):	Vehicle	100	300	1000	Vehicle	100	300	1000
LIVER a	10	10	10	9	10	10	9	8
Centrilobular infiltrate, mononuclear
Minimal	-	2	4	3	-	1	3	2
Mild	-	-	2	4	-	-	2	5
Centrilobular single cell necrosis
Minimal	-	-	2	2	-	1	1	3
Mild	-	-	-	3	-	-	1	2
Hypertrophy, centrilobular
Minimal	-	-	-	3	-	-	-	-
Mild	-	-	-	3	-	-	-	-
Vacuolation, periportal
Minimal	-	-	-	4	-	-	-	1
SPLEEN a	10	10	10	9	10	10	9	8
Pigment
Minimal	7	7	8	-	3	2	5	1
Mild	3	1	2	4	7	8	4	3
Moderate	-	-	-	5	-	-	-	4
ADRENAL GLAND a	10	10	10	9	10	10	9	8
Vacuolation, zona glomerulosa
Minimal	1	2	1	5	-	-	-	2
Mild	-	-	-	3	-	-	-	3

a = Number of tissues examined from each group.

 

Table 15: Summary of functional tests

Males	Dose (mg/kg bw/day)	group 1 control	group 2 100	group 3 300	group 4 1000
At Week 13 Hearing	median	0	0	0	0
Score 0/1	n	5	5	5	5
Pupil L	median	0	0	0	0
Score 0/1	n	5	5	5	5
Pupil R	median	0	0	0	0
Score 0/1	n	5	5	5	5
Static R	median	0	0	0	0
Score 0/1	n	5	5	5	5
Grip Fore	mean	1411	1387	1420	1281
Gram	st.dev	166	165	252	129
	n	5	5	5	5
Grip Hind	mean	634	688	706	593
Gram	st.dev	124	72	83	40
	n	5	5	5	5
Females	Dose (mg/kg bw/day)	group 1 control	group 2 100	group 3 300	group 4 1000
At Week 13 Hearing	median	0	0	0	0
Score 0/1	n	5	5	5	5
Pupil L	median	0	0	0	0
Score 0/1	n	5	5	5	5
Pupil R	median	0	0	0	0
Score 0/1	n	5	5	5	5
Static R	median	0	0	0	0
Score 0/1	n	5	5	5	5
Grip Fore	mean	1101	1193	1060	1248
Gram	st.dev	72	141	148	79
	n	5	5	5	5
Grip Hind	mean	569	502	550	483
Gram	st.dev	78	112	60	55
	n	5	5	5	5

 

Table 16: Motor activity test summary

Males	Dose (mg/kg bw/day)	group 1 control	group 2 100	group 3 300	group 4 1000
Total Movements	mean1	2411	2421	2897	2710
	st.dev	616	828	868	1227
	n	5	5	5	5
Ambulations	mean1	435	424	494	613
	st.dev	235	221	169	353
	n	5	5	5	5
Females	Dose (mg/kg bw/day)	group 1 control	group 2 100	group 3 300	group 4 1000
Total Movements	mean1	3630	3216	3656	3024
	st.dev	908	829	684	1157
	n	5	5	5	5
Ambulations	mean1	950	705	956	840
	st.dev	219	183	125	352
	n	5	5	5	5

 

Table 17: Summary of sperm analysis

Dose Group (mg/kg bw/day)		Vehicel	100	300	1000
Necropsy Nr of cells exam	mean	270	284	240	331
	st.dev	58	98	35	113
	n	10	10	10	9
Motile sperm	mean	76	78	72	81
%	st.dev	11	5	14	6
	n	10	10	10	9
Progressiv.sperm	mean	10	15	16	15
%	st.dev	7	5	7	9
	n	10	10	10	9
Spermcount epi	mean	454.4	---	---	500.9
10e6/Gram	st.dev	107.1	---	---	135.8
	n	8	0	0	9
Normal morph	mean	163	---	---	170
No of cells	st.dev	14	---	---	10
	n	10	0	0	9
Detached head	mean	1	---	---	1
No of cells	st.dev	1	---	---	2
	n	10	0	0	9
Abnormal head	mean	0	---	---	0
No of cells	st.dev	0	---	---	0
	n	10	0	0	9
Coiled tail	mean	25	---	---	24
No of cells	st.dev	9	---	---	11
	n	10	0	0	9
Other tail	mean	10	---	---	4
No of cells	st.dev	8	---	---	3
	n	10	0	0	9
Abnormal neck	mean	1	---	---	0
No of cells	st.dev	1	---	---	0
	n	10	0	0	9
Combined	mean	0	---	---	0
No of cells	st.dev	0	---	---	0
	n	10	0	0	9

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises an adequate, reliable (Klimisch score 1) and consistent study, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

liver

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

- Repeated dose toxicity: oral

Repeated dose toxicity following the oral route was tested in a GLP-compliant 90-Day gavagestudy inWistar rats (Crl: WI(Han)) according to OECD 408 (Charles River Laboratories 2019). In this study, 10 rats per sex and dose level were administered the test substance, dissolved in polyethylene glycol 400 at 100, 300 and 1000 mg/kg bw/day. Control animals received polyethylene glycol 400, only. Chemical analyses of formulations were conducted during the study to assess accuracy and homogeneity (weeks 1, 6 and 13). Stability of the test item in the vehicle was assessed in week 1. Analysis showed that analysed concentrations were in agreement with target concentrations (i.e. mean accuracies between 90% and 110%) and that no test item was detected in the vehicle formulation. Furthermore, the dose formulations were homogeneous (i.e. coefficient of variation ≤ 10%) and stable when stored at room temperature under normal laboratory light conditions for at least 5 h (i.e. differences between mean concentrations before and after storage ≤ 10%).

Three unscheduled deaths (one mid-dose female and two high-dose females) were observed during the study period, but none was considered treatment-related. Unscheduled deaths were most likely related to the oral gavage or blood sampling procedure. Salivation and ploughing was noted in all treated animals. For salivation a dose-related trend in severity (up to severe on several occasions in high-dose rats) and time of onset (from day 8 in low-dose rats and from day 1 or 2 at the higher dose levels) was observed. The salivation was considered to be a physiological response and/or behavioural sign of discomfort in reaction to the bolus administration of the test item rather than a sign of systemic toxicity considering its severity (mostly slight or moderate) and the time of occurrence (i.e. within half an hour after dosing). All other clinical signs noted occurred incidentally and represented normal background findings and were thus not considered treatment-related. Males at 1000 mg/kg/day showed slight and therefore non-adverse, reduced body weight gain and food consumption. There were no treatment-related adverse effects observed on body weight or food consumption in females.

 

Adverse microscopic changes were observed in the liver at 300 mg/kg/day (females only) and 1000 mg/kg/day (both sexes) and consisted of an increased incidence and severity (up to mild degree) of centrilobular mononuclear cell infiltrates accompanied by single cell necrosis as well as increased liver weighs (by about 20%) in females treated at 1000 mg/kg only.

 

Non-adverse, treatment-related centrilobular mononuclear cell infiltration with single cell necrosis was observed in males at 100 and 300 mg/kg/day and in females at 100 mg/kg/day. Non-adversity was based on the low severity/incidence of the lesions in these dose groups.

 

Other non-adverse microscopic changes at the highest dose level were centrilobular hypertrophy and hepatocellular vacuolation in the liver (males), increased severity of pigment in the spleen (both sexes), and increased incidence and severity of diffuse vacuolation in the zona glomerulosa of the adrenal gland (both sexes). Due to the low severity and the absence of any additional degenerative or proliferative changes, these findings were not considered to be adverse.

 

Treatment-related higher liver weights (absolute and relative to body weight) were noted in females and were considered adverse, due to histopathology correlates.

Other organ weight findings at high dose, slight increase in relative kidney weights in both sexes and lower mean uterus weights (absolute and relative to body weight, not statistically significant) in females were considered to reflect normal physiological variation and were thus not considered treatment-related. In addition, increased heart weights in in mid dose males and high dose females were also not considered treatment related, due to the absence of a dose-related trend and regarded a chance finding.

 

Clinical chemistry showed changes in several liver-related parameters at 1000 mg/kg/day, mostly in males, namely: increases in alanine aminotransferase (ALAT), aspartate aminotransferase (ASAT), alkaline phosphatase (ALP) and bile acids. Decreases noted in cholesterol (both sexes) and fasting glucose (males only) may also be related to the effect of the test item on the liver. The other clinical chemistry findings (slightly higher creatinine, chloride and potassium, and slightly lower calcium, mostly at 1000 mg/kg/day) were not associated with adverse anatomic pathology alterations and therefore regarded as non-adverse.

Treatment-related changes in red blood cell parameters were noted at 1000 mg/kg/day, generally in both sexes, and consisted of decreases in the number of red blood cells, haemoglobin, MCHC and RDW, and increases in MCV, MCH and reticulocytes. These changeswere regarded as non-adversebecause of the minor magnitude of the change.

All opthalmology findings occurred at similar incidences in all dose groups or only in single animals and were thus considered incidental, non-treatment-related and non-adverse.

There were no indications of possible reproductive toxicity as evidenced by the absence of treatment-related changes in the length and regularity of the estrous cycle, sperm parameters (motility, concentration, morphology), and weight and morphology of male and female reproductive organs.

 

Thyroid hormone analysis revealed lower T4 levels in the high dose group compared to the control levels (-14% and -22% in males and females, respectively). Since statistical significance was not achieved and T4 levels in almost all high dose animals remained in the concurrent control range, these intergroup differences were considered non-treatment-related.

 

Neurobehavioural examination revealed low values for total movements and ambulations in one male and one female (below the normal ranges). However, habituation profiles of these animals were normal and their lower motor activity was not associated with corroborative clinical signs or changes in other measures in the neuromuscular domain (including gait, air righting reflex and grip strength). Moreover, all other high-dose animals showed normal motor activity. Therefore, the lower activity recorded for these two animals was not attributed to treatment. Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. In addition, forelimb and hind limb grip strength showed no treatment-related changes.

 

Based on effects observed on the liver at 300 mg/kg bw/day in females and 1000 mg kg bw/day in both sexes, the LOAEL/NOAEL for female rats was determined at 300 mg/kg bw/day and 100 mg/kg bw/day and for male rats at 1000 mg/kg bw/day and 300 mg/kg bw/day.

Justification for classification or non-classification

Adverse effects, in form of liver toxicity were observed in the sub-chronic rat study with oral test substance administration at an dose of 1000 and 300 mg/kg bw/day and above in males and females, respectively. The dose at which adverse effects were observed does exceed the current valide cut-off level for classification in category 2 of 10 < c ≤ 100 mg/kg bw/day (oral route) (Annex I: 3.9.2.9.7.) and thus, the data requirements for classification are not met according to Regulation (EC) No. 1272/2008 (CLP).

The available data on oral repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008 and are therefore conclusive but not sufficient for classification.