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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
09 Mar - 12 May 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-guideline study with acceptable restrictions. No neurological examinations were performed, no clinical observations outside the home cage, the test substance was administered 5 days/week, the analytical purity of the test substance was not specified.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guidelineopen allclose all
Qualifier:
equivalent or similar to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
no neurological examinations performed, no clinical observations outside the home cage
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
no neurological examinations performed, no clinical observations outside the home cage
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): fatty acid ester (C8-10, C12-18-alkyl)
- Physical state: colourless liquid
- Analytical purity: 100 %
- Lot/batch No.: 00071226
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Wistar Bor:WISW (SPF) Cpb
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Winkelmann, Borchen, Germany
- Age at study initiation: approximately 4 weeks
- Weight at study initiation: 44-61 g (males), 46-60 g (females)
- Housing: 2-3 animals of the same sex in macrolon cages, type III with wood shavings (ARWI-Center, Essen, Germany)
- Diet: nitrosamine-poor pellets, Altromin 1324 DK (Fa. Altromin GmbH, Lage, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21-24
- Humidity (%): 41-55
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 Feb 1992 To: 9-10 Mar 1992 (main groups), 12 May 1992 (satellite groups)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: 0.5% carboxymethylcellulose and 0.25% cremophor in aqua dest. (CMCC)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: dosing solutions were prepared daily prior to administration. The application volumes were adopted weekly to the current body weights.

VEHICLE
- Concentration in vehicle: 1, 2 and 10% for the 100, 300 and 1000 mg/kg bw/day groups, respectively.
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
28 days (control and treatment group)
28 days and 33 days post-exposure observation period (satellite control and treatment group)
Frequency of treatment:
once daily, 5 days/week (23-24 doses in total)
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10 (main groups)
5 (satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose levels were selected in order to find the highest possible safety margin for the determination of the non-cumulative toxic dose level and the determination of toxic dose ranges.
- Rationale for selecting satellite groups: satellite control and high-dose groups were selected to determine the reversibility of potentially cumulative toxic effects
- Post-exposure recovery period in satellite groups: 33 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed for mortality and clinical signs twice daily (once daily during weekends and public holidays)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, the intake per cage per week was recorded and calculated as g/rat/week
- 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: Yes
- Time schedule for examinations: once weekly, per cage

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: one day prior to the sacrifice of the animals. Mydriaticum Roche R (Hoffmann-La Roche, Grenzach-Wyhlen) was instilled into the right eye, which was then examined using a Topcon-SL-5D slit lamp (Bon, D-Wahlstedt).
- Dose groups that were examined: control and 1000 mg/kg bw/day group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: No
- How many animals: all animals in the main groups
- Parameters examined: haematocrit, haemoglobin content, red blood cell count, white blood cell count, mean cell volume, thrombocyte cell count, differential blood count

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: No
- How many animals: all animals in the main groups
- Parameters examined: gamma-glutamyl transferase, aspartate-aminotransferase, alanine-aminotransferase, alkaline phosphatase, sodium, potassium, glucose, urea, total protein, calcium, creatinine, cholesterol, chloride, bilirubin

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The animals in the main groups and satellite groups were sacrificed using an overdose of ether and gross pathology was conducted with all animals. The eyeballs and the inner organs were removed and fixed in 10% neutral formalin solution (Formol-Fixx-Concentrate, Shandon). The weight of the brain, testes, heart, liver, spleen, adrenals, kidneys and thymus of all main and satellite groups was recorded.

HISTOPATHOLOGY: Yes. For the control and 1000 mg/kg bw/day groups, the following organs were histologically examined: aorta thoracia, eyes, colon, stomach, small intestine, cerebrum, urinary bladder, skin, heart, testes, hypophysis, cerebellum, liver, trachea, lung, axilliary lymph nodes, mesenteric lymph nodes, spleen, epididymides, adrenals, periphery nerve, kidneys, ovaries, pancreas, prostate, seminal vesicle, thyroid, salivary gland, oesophagus, sceletal muscle, thymus, uterus, forestomach, tongue.
In the satellite groups and the 500 mg/kg bw/day main group, the forestomach was examined as a target organ.
Statistics:
The inter-groups variations for body weight, clinical chemistry and hematology were determined using a t-test. The differences in organ weight were evaulated using a Steel-test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw/day: 1/10 females died at the end of the study during the collection of blood (not test substance-related)
Mortality:
mortality observed, treatment-related
Description (incidence):
100 mg/kg bw/day: 1/10 females died at the end of the study during the collection of blood (not test substance-related)
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased level leucoytes with segmented nuclei, males (non-adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased ALT level, males (non-adverse)
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/d: increased relative heart weight, males; 100 mg/kg bw/day: increased relative/absolute heart weight, males (non-adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: effects on the mucosa of the forestomach
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female administered 100 mg/kg bw/day died when a blood sample was drawn during anesthesia. There was no substance-related mortality. No clinical signs were observed during the study period.

BODY WEIGHT AND WEIGHT GAIN
There was no difference in body weight gain between the control and treatment groups.

FOOD CONSUMPTION
There was no statistically significant difference in food consumption between the control and treatment groups.

WATER CONSUMPTION
There was no statistically significant difference in water consumption between the control and treatment groups. The females in the 1000 mg/kg bw/day group showed an increase in water consumption in week 2, however, as this was due to a mechanical failure this is not considered to be a relevant finding (see Table 1).

OPHTHALMOSCOPIC EXAMINATION
There were no substance-related changes to the eyes of the examined animals.

HAEMATOLOGY
The males administered 1000 mg/kg bw/day had a significant increase in the level of leucocytes with segmented nuclei (see Table 2). As this effect was only observed in one sex and there were no other haematologic or histopathologic effects, this is not considered to be a treatment-related effect.

CLINICAL CHEMISTRY
In males administered 1000 mg/kg bw/day, the ALT-level was statistically significantly increased (see Table 3). This increase in a liver enzyme may be caused by an increase in the liver metabolism due to the test substance. However, no other effects were seen on clinical chemistry parameters or in the histopathological results and the effects are considered not to be of toxicological relevance. An increase in the protein- and calcium levels of males in the 100 mg/kg bw/day group is not considered to be substance-related as these increases were not observed at any other dose level and in one sex only.

ORGAN WEIGHTS
A slight decrease was observed in the relative and absolute heart weight of males in the 100 mg/kg bw/day group and in the relative heart weight in males in the 300 mg/kg bw/day group (see Table 4). As no effect was observed in the highest dose group, this is not deemed to be a treatment-related effect.

GROSS PATHOLOGY
1/10 females administered 1000 mg/kg bw/day had severe oedema in the forestomach and 1/10 had thickening of the forestomach mucosa. These findings were probably compound-related, however, humans do not have a forestomach and this effect is therefore not relevant to human exposure. No other effects were noted during necropsy.

HISTOPATHOLOGY: NON-NEOPLASTIC
4/10 males and 2/10 females in the 1000 mg/kg bw/day group had oedema in the mucosa of the forestomach, and the 2 females also had ulcerations (see Table 5). These findings were probably compound-related, however, as humans do not have a forestomach this effect is not relevant to human exposure. As none of the animals in the satellite group had effects on the forestomach, this is considered to be a reversible effect.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
other: No treatment-related effects were observed up to and including the highest dose level

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Water consumption as group mean value (mL/animal/day)

 

Group (mg/kg bw/day)

 

Males

Females

Week

Control

100

300

1000

Control

100

300

1000

1

209

196

205

197

156

154

159

155

2

209

215

217

192

145

152

158

195

3

229

215

209

207

138

141

147

149

4

217

208

203

211

141

143

145

149

 

 

Table 2: Haematological results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

leucocytes with segmented nuclei (%)

0.3

0.1

0.4

1.1*

0.7

0.7

0.7

0.7

*Statistically significant (p < 0.01)

 

Table 3: Clinical chemistry

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

ALT (U/L)

30

30

30

40*

23

23

21

27

Protein (g/L)

60

62**

61

60

63

63

63

63

Calcium (mmol/L)

2.3

2.4**

2.4

2.4

2.4

2.4

2.4

2.4

*Statistically significant (p < 0.01)

**Statistically significant (p < 0.05)

 

Table 4: Organ weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Heart, absolute (g)

0.84

0.75**

0.77

0.82

0.58

0.61

0.59

0.60

Heart, relative (%)

0.34

0.32**

0.31*

0.33

0.36

0.36

0.36

0.37

*Statistically significant (p < 0.01)

**Statistically significant (p < 0.05)

 

 

 

Table 5: Histopathological effects

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Edema in forestomach

0/10

-

-

4/10

0/10

-

-

2/10

Ulcer(s) in forestomach

0/10

-

-

0/10

0/10

-

-

2/10

 

 

 

 

 

 

 

 

 

Applicant's summary and conclusion