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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Jul 26, 2016 - Aug 16, 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
other: Justification for dose selection rationale

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2017
Report date:
2017

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)

Test material

Constituent 1
Chemical structure
Reference substance name:
4-trans-Propyl-4-trans-vinyl-[1,1-bicylohexyl]
EC Number:
601-409-2
Cas Number:
116020-44-1
Molecular formula:
C17H30
IUPAC Name:
4-trans-Propyl-4-trans-vinyl-[1,1-bicylohexyl]
Test material form:
solid: crystalline

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Species: Rat
Strain: Crl:WI (Han)
Breeder: Charles River, Germany
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 8-9 weeks
- Weight at study initiation: m: 234 (214 – 267) g, f: 166 (152 – 179) g
- Fasting period before study: no
- Housing: IV Makrolon® cages with means to hide
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2ºC
- Humidity (%): 40 – 70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12 h

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


VEHICLE
- Justification for use and choice of vehicle (if other than water): standard vehicle
- Concentration in vehicle: 5-100 mg/mL
- Amount of vehicle (if gavage): 1 mL
- Lot/batch no. (if required): -
- Purity: -
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
GC with FID detection

Mean recoveries
day 0: 84-93%
day 6: 80 - 102 %

Duration of treatment / exposure:
90 consecutive days
Frequency of treatment:
daily
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
control
Dose / conc.:
5 mg/kg bw/day (actual dose received)
Remarks:
LD
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Remarks:
MD
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
HD
No. of animals per sex per dose:
Control: main: 10 m, 10 f, recovers: 5 m, 5 f
LD: main: 10 m, 10 f
MD: main: 10 m, 10 f
HD: main: 10 m, 10 f, recovers: 5 m, 5 f
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:

A structurally similar compound (CAS 96624-41-8) was tested in a 28-day repeat-dose oral toxicity study (OECD 407) in HanRcc: WIST (SPF) rats at 30, 100 and 300 mg/kg/d in corn oil. At 300 mg/kg/d a slight decrease in body weight gain, slight to moderate effects on some clinical pathology parameters (e.g. reticulocytes, glucose) was observed. At 100 mg/kg/d some clinical pathology parameters were also affected, but less pronounced. Treatment-related histopathological findings at 300 mg/kg/d consisted of minimal centrilobular hepatocellular hypertrophy in 2 male animals. The histopathological and most clinical pathology findings proved to be reversible. Only some slight effects on clinical pathology parameters were
observed at the end of recovery. Therefore, 300 mg/kg/d was defined as the NOAEL (no adverse effect level) and 30 mg/kg/d as the NOEL (no observed effect level).

Another structurally similar compound (CAS: 129738-34-7) was tested in a 28-day repeat-dose oral toxicity study (OECD 407) in Crj: CD (SD) IGS (SPF) rats at 5, 20, 80 and 320 mg/kg/d in warm olive oil. At 320 mg/kg/d effects on some clinical pathology parameters (e.g. white blood cells, thromboplastin time, LDH). At 80 mg/kg/d some clinical pathology parameters were also affected, but less pronounced. Treatment-related histopathological findings at 320 mg/kg/d consisted of hyperthrophy of alveolar lining cells in the lung, centrilobular hepatocellular hypertrophy, focal myocarditis and hypertrophy of the paracortex in the mediastinal lymph nodes, increased mitosis of hepatocytes and germinal center development in the mediastinal lymph nodes, foamy cells and focal proliferation of atypical pneumocytes in the lung, atypical basophilic tubules and mineralization of the cortico-medullary junction in the kidney. The histopathological and clinical pathology findings proved to be only partial reversible. Therefore, 20 mg/kg/d was defined as the NOEL (no observed effect level).

Therefore, it was expected for the current study that the high dose level of 100 mg/kg/d over a time period of 90 days would cause mild to moderate systemic toxicity, the low dose level of 5 mg/kg/d was expected to cause no or mild systemic toxicity. The dose of 20 mg/kg/d was used to investigate dose-dependent effects.


- Rationale for animal assignment (if not random): random
- Post-exposure recovery period in satellite groups: yes
- Section schedule rationale (if not random): random

Examinations

Observations and examinations performed and frequency:
Observations/Measurements Time schedule No. of rats/sex/group
Appearance and behavior Daily 10 or 15
Mortality Daily 10 or 15
Detailed Clinical Observations Predose (day -1), weekly 10 or 15
Motor activity Day 77 10
Functional observational battery Day 77 10
Body weight Pre-dose,
there after weekly 10 or 15
Food consumption Weekly 10 or 15
Ophthalmological Investigations Day -1, 84 10 or 15
Hematology (including Coagulation) Week 13 10
Week 17 5
Clinical chemistry Week 13 10
Week 17 5
Urinalysis Week 13 10
Week 17 5
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
Statistics:
To compare the treatment groups with the control group, the following statistical procedures were applied separately for each sex and each measuring point. To take the number of dose groups into account all the test procedures used maintain a multiple significance level of 0.05.

Body weight and body temperature: Dunnett-test (2-sided)
Food consumption: Dunnett-test (2-sided)
Water consumption: Dunnett-test (2-sided)
Clinical pathology parameters (hematology including coagulation, clinical chemistry serum parameters, specific gravity and urine weight): Dunnett-test (2-sided)
Functional observational battery (numerical parameters): Kruskal-Wallis followed by Wilcoxon-test
Motor activity (total counts): Dunnett-test (2-sided)
Dunnett-test (2-sided): Dunnett-test (2-sided)

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
for details, see results and tablles below
Mortality:
no mortality observed
Description (incidence):
for details, see results and tablles below
Body weight and weight changes:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Haematological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Urinalysis findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
for details, see results and tablles below
Gross pathological findings:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
for details, see results and tablles below
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
for details, see results and tablles below
Details on results:
for details see "any other information on results"

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: non-neoplastic

Target system / organ toxicity

Key result
Critical effects observed:
no
Lowest effective dose / conc.:
100 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
liver

Any other information on results incl. tables

Clinical Signs

The following symptoms were observed during the study:


0 mg/kg/d
5 mg/kg/d
20 mg/kg/d
100 mg/kg/d
Males
Females
Males Females Males Females Males Females
No of animals
15
15
10
10
10
10
15
15
Symptom

Hair loss
1a
3a 3a -
1a 1a 1a 6a
Animal No
9
43-46
12, 16, 18
-
26
61
88
73-77, 79
Skin, wound scabbed,
- back
- head
1a
(d7 - 27)b
-
-
-
1a
(d39 - 65)b
-
1a
(d80 - 89)b
-
Animal No
9
-
-
-
26
-
88
-
Symptom

Skin, swelling soft
- hindleg, right
-
-
-
-
-
1a
(d20 - 21)b
-
-
Animal No -
-
-
-
-
61
-
-

a: No. of animals with this symptom;b: study days (d) when symptom was observed from earliest to latest observation

Several animals of all dose groups showed hair loss. These findings were without a dose-response relationship and also observed control animals. Therefore, these findings are considered incidental.
Single animals of the 100 and 20 mg/kg/d groups showed skin wounds. These findings were without a dose-response relationship and also observed in a control animal. Therefore, these findings are considered incidental.
One 20 mg/kg/d female showed a soft skin swelling at the right hindleg on days 20 and 21. This finding was observed for a very short time period and without a dose-response relationship. Therefore, this finding is considered incidental.

Detailed Clinical Observations
One 100 mg/kg/d female (animal No. 75) showed vocalization on days -1 (pre-dose), 7, 14, 21, 28, 35, 42, 49, 56, 63 and 70. This observation was already present pre-dose and without a dose-response relationship and therefore considered incidental.
Hair loss was observed in the control group (0 mg/kg/d) in 1 male and 2 females on days 7, 14, 21, 28 and 35, in 2 females on days 42, 49, 56, 63 and 70 and in 3 females on days 77, 84 and 90. At 5 mg/kg/d hair loss was seen in 1 male on days 7, 14, 21 and 35, in 2 males on days 42, 56, 63, 84 and 90 and in 3 males on days 49, 70 and 77. At 20 mg/kg/d hair loss was found in 1 female on days 35, 49, and 56. At 100 mg/kg/d hair loss was observed in 2 females on day 42, in 1 female on days 49, 56 and 63, in 1 male and 1 female on day 70, in 1 male and 4 females on days 77 and 84, in 1 male and 5 females on day 90 and in 1 male on days 98, 105, 112 and 118.
Hair loss is a common background finding in Wistar rats and was observed in control and dose group animals of both sexes without showing a dose-response relationship. Therefore, these findings were considered incidental.
A skin wound was seen in the control group (0 mg/kg/d) in 1 male on day 7. At 20 mg/kg/d skin wounds were observed in 1 male (animal No. 26) on days 42, 49, 56, and 63. At 100 mg/kg/d a skin wound was found in 1 male (animal No. 88) on day 84.
In the 20 and 100 mg/kg/d groups only single animals showed skin wounds. These findings were without a dose-response relationship and also observed in a control animal. Therefore, these findings are considered incidental.

Motor Activity
All motor activity parameters – number of counts, on-time, off-time, total distance, rearing, rearing time – did not show treatment-related differences of dose groups versus control.
 
Additional Motor Activity Parameters
Additional parameters of motor activity – off-time (minutes), on-time (minutes), rearing no., rearing time (minutes) – measured on day 77 did not show treatment-related differences of treatment groups versus control.

Functional Observational Battery (FOB)
In the functional observational battery (FOB) no treatment-related changes in autonomous, sensomotoric, neuromuscular, and central nervous system including body temperature measurements were noted.

Autonomous Nervous System
No significant effects on lacrimation, salivation, pupil response, piloerection, defecation (numberof fecal boluses, feces consistency), urination (number of urine pools, urine stain size), and palpebral closure were seen in any dose group on day 77.

Sensomotoric System
No statistically significant effects on approach response, click response, touch and tail pinch response were seen in any dose group on day 77.

Central Nervous System
No statistically significant effects on ease of removal and handling, arousal, fur appearance, raising behavior, or catalepsies were seen in any dose group on day 77.

Body Temperature
No statistically significant effects on ease of removal and handling, arousal, fur appearance, raising behavior, or catalepsies were seen in any dose group on day 77.

Body Weight (Gain)
Mean body weight (absolute values): day 1 - 118

Dose / [mg/kg/d]
Mean Body Weight / [g]
Day 1
Day 42
Day 90
Day 118

Males
Females
Males Females Males Females Males Females
0
234.4
166.1
366.0
215.2
437.9
236.6
457.6
241.4
5
234.4
166.7
359.6
219.1
425.0
241.4
-
-
20
235.8
166.0
363.2
219.7
429.3
241.8
-
-
100
232.2
165.6
354.4
214.3
422.0
236.2
451.8
242.8


Mean body weight (relative compared to control): Day 1 - 118

Dose / [mg/kg/d]
Mean Body Weight / [%]
Day 1
Day 42
Day 90
Day 118

Males
Females
Males Females Males Females Males Females
0
100.0
100.0 100.0 100.0 100.0 100.0 100.0 100.0
5
100.0 100.4 98.3
101.8 97.1
102.0 -
-
20
100.6 99.9
99.2
101.8
98.0
102.2
-
-
100
99.1
99.7
96.8
99.6
96.4
99.8
98.7
100.6


Mean body weight gain (absolute and relative compared to control): Day 1 - 118

Dose / [mg/kg/d]
Weight Gain / [g]
Weight Gain / [%]
Day 1-90
Day 1-118
Day 1-90
Day 1-118

Males
Females
Males Females Males Females Males Females
0
203.5
70.5
229.9
74.4
100.0
100.0
100.0 100.0
5
190.6
74.7
-
-
93.7
106.0
-
-
20
193.5
75.8
-
-
95.1
107.5
-
-
100
189.8
70.6
221.2
80.4
93.3
100.1
96.2
108.1


Body weight and body weight gain did not show any treatment-related changes at the end of the treatment and recovery period.

Food Consumption

Food consumption: Day 1-28

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 1-7
Day 7-14
Day 14-21
Day 21-28

Males
Females
Males Females Males Females Males Females
0
24.14
16.08
25.10
16.53
23.95
16.90
24.22
16.75
5
24.31
16.77
25.14
16.91
24.41
17.46
23.73
17.10
20
24.19
16.07
24.68
16.57
24.12
17.15
23.62
16.79
100
24.65
16.59
25.12
16.92
24.62
17.24
24.38
17.30


Food consupmption: Day 25-56

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 28-35
Day 35-42
Day 42-49
Day 49-56

Males
Females
Males Females Males Females Males Females
0
24.00
16.50
24.01
15.69
23.75
15.21
23.82
15.26
5
23.55
17.11
23.48
17.13**
22.79
16.43**
23.46
16.37
20
23.35
16.98
23.49
16.47**
22.96
15.99
23.20
16.24*
100
23.55
16.89
23.70
16.81**
23.29
16.34**
23.24
16.63**

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consupmption: Day 56-84

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 56-63
Day 63-70
Day 70-77
Day 77-84

Males
Females
Males Females Males Females Males Females
0
24.06
15.25
23.42
14.95
22.71
14.96
22.79
15.08
5
23.41
16.13
22.75
15.88
22.34
15.99*
22.50
15.54
20
23.36
15.61
22.61
15.17
22.51
15.42
22.46
15.37
100
23.46
16.37**
23.16
15.84
22.46
15.45
22.92
15.78

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consupmption: Day 84-112

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 84-90
Day 90-98
Day 98-105
Day 105-112

Males
Females
Males Females Males Females Males Females
0
22.80
14.79
23.28
15.18
24.41
15.57
24.09
15.74
5
22.43
15.80*
-
-
-
-
-
-
20
22.42
15.20
-
-
-
-
-
-
100
22.74
15.72*
23.05
15.03
24.54
15.61
24.84
15.38

**: Dunnett’s test 1% significance level; *: Dunnett’s test 5% significance level

Food consumption: Day 112-118

Dose / [mg/kg/d]
Food Consumption / [g/animal/d]
Day 112-118

Males
Females
0
24.80
15.73
5
-
-
20
-
-
100
25.14
15.61


Food consumption was slightly increased in females compared to control at 5 mg/kg in week 6-8 (day 35-56), week 11 (day 70-77) and week 13 (day 90-98), at 20 mg/kg in week 6 (day 35-42) and week 8 (day 49-56) and at 100 mg/kg in week 6-9 (day 35-63) and week 13 (day 90-98). These slight increases were without clear dose dependency and no corresponding effects on body weight were observed. Therefore, these changes were considered incidental.

Gross Pathology
Main kill

No treatment-related macroscopic organ lesions were found.

Recovery
No treatment-related macroscopic organ lesions were found.

Body and Organ Weights
Main kill

100 mg/kg
Determination of terminal body weights revealed no treatment-related changes in rats of both genders.
Determination of organ weights revealed in males a tendency to increased absolute liver weights (statistically not significant) and a slight increase of relative liver weights. In females, an increase of absolute and relative liver weights was found.

20 and 5 mg/kg
Determination of terminal body and organ weights revealed no treatment-related changes.The decrease of absolute and relative adrenal weights in females dosed with 20 mg/kg was considered to be arisen by chance.
Summary of discussed absolute and relative organ weights (mean values):


Test groups 1 - 4
Males
Females
Group
1
2
3
4
1
2
3
4
Dose / [mg/kg/d] 0
5
20
100
0
5
20
100
Number of animals
10
10
10
10
10
10
10
10
Body weight / [g]

410
395
398
383
216
217
220
215
Liver
abs. / [g]
10.2
9.9
10.2
10.3
5.6
5.8
5.9
6.7**
rel. / [%] 2.5
2.5
2.6
2.7*
2.6
2.7
2.7
3.1**
Adrenals
abs. / [g] 0.061
0.054
0.057
0.051
0.077
0.071
0.063**
0.069
rel. / [%] 0.015
0.014
0.014
0.013
0.036
0.032
0.029**
0.032

abs. = absolute weight
rel. = relative weight
g = gram
* - Test: Dunnett 5% significance level
** - Test: Dunnett 1% significance level

Number in bold are considered to represent treatment-related changes.


Recovery
Determination of terminal body weights and organ weights revealed no treatment-related changes.

Histopathology
Main kill

At histopathology, a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes was found in 1/10 (5mg/kg), 3/10 (20mg/kg) and 3/10 (100mg/kg) male rats and 0/10 (5mg/kg), 1/10 (20mg/kg) and 3/10 (100mg/kg) female rats. Incidence was a little bit higher in males than in females. The intracytoplasmic vacuolation of affected hepatocytes was characterized by the presence of multiple mid to large sized optically empty vacuoles. These vacuoles were distributed throughout the cytoplasm around the nucleus and enlarged the affected hepatocytes. Most intracytoplasmic vacuolated hepatocytes were located in periportal and midzonal areas of the liver lobules.

No further treatment-related changes were noted in the organs examined.

Immunohistochemistry results – Anti-Adipophilin

Immunohistochemistry for adipophilin (Anti-Adipophilin, Clone AP125, ProGen Kat.Nr. 610102 (LOT310281); Antibody dilution 0.5μg/ml) showed a prominent positive red immunostaining of the membranes surrounding the mid and large sized cytoplasmic vacuoles in midzonal/periportal
hepatocytes in treated animals.

Positive control: a slide from liver tissue of a Carbon tetrachloride treated rat (no. 69/A2002) and a slide from a rat (no. 182/16-IV013-N0) with known positive adipophilin staining.

Animal number
HE-stained slides
Antibody IHC staining Anti-Adipophilin, Clone AP125
69/A2002 = positive control (centrilobular)
-
+++
Red stained membranes of small and large hepatocellular intracytoplasmicvacuoles (centrilobular)
No. 182/16-IV013-N0 = positive control (periportal)
Vacuolation, hepatocellular, microvesicular, diffuse,marked, centrilobular, pronounced
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
-
Vacuolation, hepatocellular, microvesicular (negative for Anti-Adipophilin)
1 and 4 (control males)
No vacuolation, cytoplasmic hepatocytes present
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
31, 34, 35 (group 4 males)
Vacuolation, cytoplasmic, hepatocytes, focal/multifocal, minimal/mild
+++
Red stained membranes of mid to large sized hepatocellular intracytoplasmic vacuoles (midzonal, periportal)
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
71 (group 4 female)
No vacuolation, cytoplasmic hepatocytes present
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding
77, 79 (group 4 females)
Vacuolation, cytoplasmic, hepatocytes, multifocal, minimal and mild
+++
Red stained membranes of mid to large sized hepatocellular intracytoplasmic vacuoles (midzonal, periportal)
+
Red stained membranes of small hepatocellular intracytoplasmic vacuoles (periportal) = normal background finding


Immunohistochemistry for adipophilin showed a prominent positive immunostaining of the membranes surrounding the cytoplasmic vacuoles in hepatocytes. Adipophilin is a component of the surface membrane of lipid droplets (Obert at al., 2007). Enhanced expression of Adipophilin,
as noted here in treated animals, is indicative for increased lipid accumulation in the hepatocellular cytoplasm (also called “fatty change”). Most likely, hepatocytes of treated rats developed mid and large sized intracytoplasmic vacuoles as consequence of metabolic activation/adaption of
hepatocytes (with lipid accumulation) to the test item and the vehicle (100% corn oil).

Incidence summary table of discussed treatment-related findings in the liver:


Test groups 1 - 4, main kill
Males
Females
Group
1
2
3
4
1
2
3
4
Dose / [mg/kg/d] 0
5
20
100
0
5
20
100
Number of animals
10
10
10
10
10
10
10
10
Liver
Vacuolation, cytoplasmic; hepatocytes, multivocal
minimal
1

1



2
mild



2



1
Vacuolation, cytoplasmic; hepatocytes, vocal
minimal








mild


3
1


1




Recovery
100 mg/kg
At histopathology, a focal minimal intracytoplasmic vacuolation of hepatocytes was diagnosed in one male rat of the high dose group.
Incidence summary table of discussed treatment-related findings in the liver:


Test groups 1 - 4, main kill
Males
Females
Group
1
 
 
4
1
 
 
4
Dose / [mg/kg/d] 0


100
0


100
Number of animals
5


5
5


5
Liver
Vacuolation, cytoplasmic; hepatocytes, vocal
minimal







mild



1





All other findings noted were considered to be spontaneous and sporadic in nature.

Discussion Pathology

The test material was administered daily orally by gavage to Crl:WI (Han) rats for a period of 90 days. For the main kill, 10 rats per gender were dosed with 5, 20 or 100 mg/kg test material. 100% corn oil served as control. The treatment period was followed by four weeks of recovery. Reversibility of treatment-related changes was assessed in 5 rats per gender in the control group and 5 rats per gender in the high dose group. All rats survived until their scheduled necropsy date.
At gross pathology, no treatment-related changes were noted in the organs examined of main kill and recovery rats.
Determination of terminal body weights revealed no treatment-related changes in rats of both genders for main kill and recovery rats.
For main kill rats, at a dose level of 100 mg/kg test material, determination of organ weights revealed a tendency to increased absolute liver weights (statistically not significant) and a slight increase of relative liver weights in males and an increase of absolute and relative liver weights in females. No clear-cut histomorphological correlate to these weight changes was found in the liver of main kill rats. Maybe increased liver weights were triggered by metabolic activation/adaption of hepatocytes to the test item and the vehicle (100% corn oil).
At histopathology examination of main kill rats, a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes was found in some rats of both genders at a dose level of 100, 20 and 5 mg/kg. Males were affected more often than females.
Immunohistochemistry for adipophilin showed a prominent positive immunostaining of the membranes surrounding the cytoplasmic vacuoles in hepatocytes. Adipophilin is a component of the surface membrane of lipid droplets (Obert at al., 2007). Enhanced expression of Adipophilin,
as noted here in treated animals, is indicative for increased lipid droplet accumulation in the hepatocellular cytoplasm of main kill rats, often named “fatty change”. Most likely, hepatocytes of treated rats developed mid size and large intracytoplasmic vacuoles as consequence of metabolic
activation/adaption of hepatocytes (with lipid accumulation) to the test item and the vehicle (100% corn oil).
At histopathology examination of recovery rats, a nearly full recovery of the liver changes was noted in rats of the high dose group. One male still exhibited a focal minimal intracytoplasmic vacuolation of hepatocytes.
The oral administration of 5, 20 or 100 mg/kg of test material for 90 days was considered to be tolerated by the rats, as only minor treatment-related histomorphological liver changes were found in some main kill rats, that were considered non-adverse and rather a sign of metabolic
activation/adaption. Moreover, nearly full recovery was shown after 4 weeks without treatment.


Clinical pathology

Results Hematology and Coagulation

Week 13:

5 mg/kg: BASO (absolute) was increased in males.
20 mg/kg: PT R (%) was decreased and PT R (sec) increased in males. HGB was decreased in females.
100 mg/kg: PT R (%) was decreased in PT R (sec) increased in males. HGB, MCV and PTT were decreased in females.

Week 17:

100 mg/kg: HGB and MCHC were decreased in females.

Results Clinical Chemistry

Week 13:
5 mg/kg: TRIG was decreased in males.
20 mg/kg: CL was increased and TRIG decreased in males.
100 mg/kg: GLUC was increased in both sexes. In males, K and TRIG were decreased and BA and CL increased. In females, ASAT was decreased.

Week 17:
100 mg/kg: In females, GLUC was increased and ALB decreased. UREA was increased in males.

Results Urinalysis
Urinalysis did not show any treatment-related alterations in week 13 and 17.

Disussion Clinical Pathology
Regarding hematology, alterations were slight in degree, not consistent in both sexes and within internal reference ranges. They are not treatment-related. The slight but dose-dependent increase of prothrombin time (PT R (sec)) in 20 and 100 mg/kg males could be indicative for a prolongation of the plasmatic coagulation time. However, the alteration was minimal (< 10%) and not accompanied by further clinico-pathological, clinical or histopathological alterations and therefore considered not treatment-related.
Internal reference intervals given as 2.5 and 97.5 percentiles or *min and max:

Parameter
Unit
15 – 52 weeks


males
females
HGB
g/dL
-
13.6-17.0
MCV
fL
-
50.3-57.5
MCHC
g/dL
-
31.6 - 39.7
BASO
103/µL
0*-0.08*
-
PT% R
%
No data
No data
PT R
sec
No data
No data
PTT
sec
No data
No data



Alterations in clinical chemistry were low in degree, not present in both sexes and within or slightly below or above internal reference intervals. They were not treatment-related. Internal reference intervals given as 2.5 and 97.5 percentiles or *min and max:

Parameter
Unit
15 – 52 weeks


males
females
K
mmol/L
4.50-5.50
-
CL
mmol/L 104.6-108.6
-
GLUC
mmol/L 6.3*-7.9*
3.9-7.2
UREA
mmol/L 4.0-5.6
-
ALB
g/L
-
39.5-46.4
TRIG
mmol/L 0.32-0.98
-
BA
mmol/L 12.2-23.2
-
ASAT
U/L
-
62-99


-No alterations, reference intervals not presented.




Applicant's summary and conclusion

Conclusions:
Daily intravenous treatment with 5, 20 and 100 mg/kg the test material to Wistar (Han) rats was clinically tolerated over 90 days. The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 100 mg/kg/d.
Executive summary:

Objective

The purpose of this oral toxicity study was to assess the cumulative toxicity of the test material when administered daily to rats by gavage for a period of 90 days. The reversibility of treatment-related changes will be assessed after a 4-week treatment-free recovery period. This study should provide the basis for a toxicological risk assessment in man. The results of this study should indicate potential target organs and should identify chemicals with neurotoxic potential.

Study Design

The test material was administered orally by gavage, once daily, 7 times a week for 90 days to 3 groups of male and female Crl:WI (Han) rats at doses of 5, 20 or 100 mg/kg. A similarly constituted control group received the vehicle, 100% maize oil (corn oil), and served to generate contemporary control data.

The control and high dose groups consisted of 15 male and 15 female rats each. The low dose and mid dose groups consisted of 10 male and 10 female rats each. At the end of the treatment period, 20 (10 males and 10 females) rats per group were scheduled for necropsy. The remaining rats of groups 1 and 4 were scheduled for a 4-week recovery period. The rats were gang-housed under conventional conditions.

Survey of inlife investigations:

Observations/Measurements

Time schedule

Appearance and behavior

Daily

Detailed Clinical Observations

Predose (day -1), weekly

Mortality

Daily

Motor activity

Day 77

FOB

Day 77

Body Weight

Weekly

Food Consumption

Weekly

Ophthalmological Investigations

Day -1, 84 

Hematology (including Coagulation)

Week 13 + 17

Clinical chemistry

Week 13 + 17

Urinalysis

Week 13 + 17

At the end of the treatment or recovery period, all animals were subjected to a detailed necropsy. Organ weights were recorded and histopathological examinations performed on selected organs.

Results and Discussion

Formulation analysis revealed that the test material was homogeneously distributed in the vehicle and the anticipated test item concentrations were met in the formulations of all treatment groups at the beginning and at the end of usage. No test item was detected in the control formulations.

No mortality was observed, all animals survived until their scheduled sacrifice without showing any treatment-related clinical signs.

The detailed weekly clinical observations revealed not treatment-related findings.

In the functional observational battery (FOB) no treatment-related changes in autonomous, sensomotoric, neuromuscular, and central nervous system including body temperature

measurements were noted. All motor activity parameters – number of counts, on-time, off-time, total distance, rearing, rearing time – did not show treatment-related differences of dose groups versus control.

No treatment-related effects were observed on mean body weight, mean body weight gain and food consumption.

Ophthalmological examinations did not reveal any treatment-related effects.

In hematology including coagulation, clinical chemistry and urinalysis no treatment-related alterations were seen.

At gross pathology, no treatment-related changes were noted.

Terminal body weights at main (day 90) and recovery kill (day 118) necropsy showed no treatment-related changes. Determination of organ weights at main kill revealed a slight or at least a tendency towards an increase of absolute and relative liver weights in both sexes at 100 mg/kg/d. At recovery kill no treatment-related organ weight changes were seen.

Histopathology of the main kill animals revealed a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes in some rats of both sexes at all dose levels. The

incidence of these effects was more pronounced in males. Immunohistochemistry for adipophilin showed a prominent positive immunostaining of the membranes surrounding the cytoplasmic vacuoles in hepatocytes and therefore proved the presence of lipid storage vacuoles in the hepatocellular cytoplasm. At the end of the treatment-free recovery period the histopathological effects at 20 and 5 mg/kg/d showed full reversibility. And at 100 mg/kg/d the only histopathological finding consisted of a minimal, focal intracytoplasmic vacuolation of hepatocytes in one male.

Conclusions

Daily oral treatment with 5, 20 and 100 mg/kg the test material to Wistar (Han) rats was clinically tolerated over 90 days.

At main kill on day 90 histopathology of the main kill animals revealed a multifocal/focal minimal to mild intracytoplasmic vacuolation of hepatocytes due to lipid storage at all dose levels in both sexes. In addition, a slight increase of absolute (females only) and relative liver weights was seen in both sexes at 100 mg/kg/d. These findings are considered to be non-adverse.

At the end of the treatment-free recovery period the treatment-related lesions in the liver showed full reversibility in all dose groups, except for a minor finding in the liver of a single 100 mg/kg/d male.

The No Observed Adverse Effect Level (NOAEL) in Wistar (Han) rats was established at 100 mg/kg/d.