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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
17 Mar - 16 Jun 2006
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study. According to the ECHA guidance document "Practical guide 6: How to report read-across and categories (March 2010)", the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2006
Report Date:
2006

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Behörde für Wissenschaft und Gesundheit, Hamburg, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material (as cited in study report): propylheptyl caprylate, 2-propylheptyl octanoate
- Physical state: colourless liquid
- Analytical purity: approximately 84%
- Impurities (identity and concentrations): 4-methyl-2-propylhexyl octanoate and 5-methyl-2-propylhexyl octanoate in unknown concentrations
- Lot/batch No.: KLEE-ZL-1394
- Expiration date of the lot/batch: 2 Mar 2007
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: CD Crl:CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories Germany GmbH, Sulzfeld, Germany
- Age at study initiation: 27 days (males); 28 days (females)
- Weight at study initiation: 72.9-84.9 g (range, males); 70.2-80.9 g (range, females)
- Housing: the animals were kept individually in MAKROLON cages (type III, approximately W39 cm x D23 cm x H15 cm) with granulated textured wood (Granulat A2, J. Brandenburg, Goldenstedt, Germany) as bedding material. The cages were changed and cleaned once a week.
- Diet: Commercial ssniff® R/M-H V1530 (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 9 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 55 ± 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Mar 2006 To: 16 Jun 2006

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: soybean oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test substance solutions were freshly prepared every day. The amount of test substance was adjusted daily according the most recent body weight recorded.

VEHICLE
- Justification for use and choice of vehicle (if other than water): the test substance is soluble in soybean oil and practically insoluble in water
- Concentration in vehicle: 20, 60 and 200 mg/L
- Amount of vehicle (if gavage): 5 mL/kg bw/day
- Lot/batch no. (if required): 055K0055 (Sigma, Taufkirchen, Germany)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An analysis of the test substance in the solutions confirmed that the formulations used for the administration in the treatment groups were correctly prepared, homogenous and stable for at least 24 hours during storage at room temperature. The stability results were 95.4- 104.2% of the nominal value for all dose levels. The concentration was measured in samples taken on Day 1 immediately after preparation, 8 and 24 hours after preparation, and on Day 90 during dosing. Stability was analysed in samples taken on Day 1 immediately after preparation, and 8 and 24 hours after preparation. The homogeneity was analysed in samples taken on Day 1 at the start of administration, during administration and just before administration to the last animal per dose group.The results showed a range of 96.8-106.1% of nominal value for all dose groups, well within the specified range of 90-110%. The investigation of the validation parameters linearity, accuracy, method precision, stability of processed samples, specificity and sensitivity indicated that the method of analysis is suitable for the determination of the test substance concentration in soybean oil.
Duration of treatment / exposure:
90 days
Frequency of treatment:
Daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal by gavage
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: animals were checked for mortality twice daily and for clinical signs at least once daily
- Cage side observations included: skin/fur, eyes, mucous membranes, respiratory and circulatory systems, somatomotor activity and behaviour patterns

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first administration and once a week thereafter; 1, 2, 4, 8 and 24 hours after administration. In week 13 the observations were made prior to administration. Observations were made outside the home cage in a standard arena at the same time point, every time. Signs recorded included changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, autonomic activity (e.g. lacrimation, pilo-erection, pupil size, and unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, and stereotypes (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards).

BODY WEIGHT: Yes
- Time schedule for examinations: on Day 0 and weekly thereafter, on the same day every week

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes. The food consumption was measured weekly.

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: the drinking water consumption was monitored daily by visual evaluation. The water consumption was not measured per rat.

OPHTHALMOSCOPIC EXAMINATION: Yes. Prior to examination, mydriasis was produced after instillation of MYDRUM®11 eye drops into the conjunctival sacs. The adnexa oculi, conjunctiva, cornea, anterior chamber, iris (pupil dilated), lens, vitreous body and fundus were examined.
- Time schedule for examinations: prior to study start and at study termination in week 13
- Dose groups that were examined: control group and all treatment groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Anaesthetic used for blood collection: Yes, ether
- Animals fasted: Yes, overnight
- How many animals: control group and all treatment groups
- Parameters examined: haemoglobin, erythrocytes, leucocytes, reticulocytes, platelets, differential blood count (relative), differential blood count (absolute), haematocrit, thromboplastin time, activated partial thromboplastin time, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, plasma.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: control group and all treatment groups
- Parameters examined: albumin, total bilirubin, total cholesterol, creatinine, glucose, total protein, triglycerides, uric acid, urea, calcium potassium, chloride, sodium, alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, lactate dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: at study termination
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, overnight
- Parameters examined: volume, specific gravity, pH, protein, glucose, bilirubin, urobilinogen, ketones, haemoglobin (approximate value), nitrite, epithelial cells, leucocytes, erythrocytes, organisms, further constituents (i.e. sperm, casts), crystalluria, colour, turbidity

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In test week 13, approximately 1 - 2 hours after dosing and before any blood sampling for laboratory examinations
- Dose groups that were examined: control group and all treatment groups
- Battery of functions tested: grip strength; locomotor activity assessment; and sensory reactivity to stimuli of different types (e.g. auditory, visual and proprioceptive stimuli). The observational screening included: righting reflex, body temperature, salivation, startle response, respiration, mouth breathing, urination, convulsions, piloerection, diarrhoea, pupil size, pupil response, lacrimation, impaired gait, stereotypy, toe pinch, tail pinch, wire manoeuvre, hind leg splay, positional passivity, tremors, positive geotropism, limb rotation, auditory function.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. All superficial tissues were examined visually and by palpation; the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves; all subcutaneous tissues were examined; the condition of the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart; the abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation; the gastro-intestinal tract was examined as a whole and the stomach and caecum were incised and examined; the lungs were removed and all pleural surfaces examined; any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.
The weight of the adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testicle, thymus and uterus were recorded for all animals.
Blood smears were prepared from all animals for examination of pathological changes, to be examined only depending on necropsy findings and in agreement with the sponsor.

The following organs or parts of organs of all animals (including deceased or sacrificed animals) were fixed in 7% buffered formalin: adrenal glands, aorta abdominalis, bone marrow (os femoris), brain (cerebrum, cerebellum, medulla/pons), epididymis, eyes with optic nerve, gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, large (colon, rectum). intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), kidneys, ureters, liver, lungs (with mainstem bronchi and bronchioles [preserved by inflation with fixative and then immersion]), lymph node (cervical), lymph node (mesenteric), mammary gland, nerve (sciatic), oesophagus, ovaries, pancreas, pituitary, prostate, salivary glands (mandibular, parotid and sublingual gland), skin (left flank), spinal cord (3 levels: cervical, mid-thoracic, lumbar), spleen, stomach, testicle, thymus, thyroids (incl. parathyroids), tissue masses or tumours, (including regional lymph nodes), trachea (incl. larynx), urinary bladder, uterus (incl. cervix and oviducts), vagina, the eyes were preserved in Davidson's solution.

HISTOPATHOLOGY: Yes. The organs listed above under 'Gross pathology' of all animals in the control group and high-dose group were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. In addition, frozen sections of the heart, liver and one kidney were made and stained with scarlet R. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plan of section and in all cases where they were noted as grossly enlarged.
Other examinations:
Oestrus cycle: At the end of the study, vaginal lavages were taken daily from all female animals for periods of 2 weeks each (week 12 + 13). The stages of the oestrus cycles observed in each vaginal lavage were recorded.

Sperm parameters: Using the sperm in one testicle and epididymis from all control and treated animals, a sperm count was carried out, the sperm viability was determined and the sperm morphology (malformation percentage) was examined. The examinations were performed according to the method described by I. Chahoud and R. Franz (1993), and by S. Plassmann and H. Urwyler (2001).
Statistics:
STUDENT's t-test was used for all numerical functional tests / urinalysis (p ≤ 0.01). The following limit was used: p ≤ 0.01 ≙ t = 2.8784 for 18 degrees of freedom.
Multiple t-test based on 'DUNNETT, C. W. New tables for multiple comparisons with a control. Biometrics 482-491; Sep 1964' was used for body weight/ food consumption/ haematology/ clinical biochemistry/ organ weights (p ≤ 0.01). The following limit was used: p ≤ 0.01 ≙ t = 3.09 for 36 degrees of freedom.
Exact test of R.A. FISHER was used for histology (p ≤ 0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day, females: increased food intake week 9, 11 (non-adverse)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day, males: increased albumin level, blood urea nitrogen level (non-adverse)
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw/day, males: decreased pH value (non-adverse)
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased absolute and relative liver weight (non-adverse)
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
1/10 females in each of the low-, mid- and high dose groups, respectively, died during the laboratory examinations on the last treatment day after blood withdrawal. These deaths are considered to be caused by the ether narcosis and are therefore not treatment-related. No other mortality was noted during the study. The low-dose female that died during ether narcosis had piloerection on Day 72-78. This is considered to be an incidental occurrence. No other animals showed clinical signs.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant differences in body weight or body weight gain between the control group and the treatment groups.

FOOD CONSUMPTION
During week 9 and 11, females in the high-dose group showed a statistically significant increase in food consumption, compared to the control group during the same period. As the effect was temporary it is considered to be incidental.

WATER CONSUMPTION
The female in the low-dose group that died during ether narcosis had moderate to severe increased water consumption and diuresis from Day 40 until study termination. This effect is not considered to be treatment-related. No other effects on water consumption were noted.

OPHTHALMOSCOPIC EXAMINATION
No effects were noted during the opthamological examination.

HAEMATOLOGY
There were no statistically significant differences in the hematology parameters between the control group and the treatment groups.

CLINICAL CHEMISTRY
The albumin and blood urea nitrogen levels were significantly increased in the males in the high-dose group (see Table 1). As no effects were noted on these parameters in the females of the high-dose group and in the absence of related histopathological findings, these effects are considered to be treatment-related, but not toxicologically relevant.

URINALYSIS
A statistically significant decrease in pH value was noted in males in the mid-dose group and in males and females in the high-dose groups (see Table 2). As there were no other effects on urinary parameters or kidney histopathology, this is considered to be a treatment-related, but not toxicologically relevant effect. The specific gravity was statistically significant increased in mid-dose males, compared to the control group. As the effect was only observed in this group, it is not seen as treatment-related.

NEUROBEHAVIOUR
No treatment-related effects were observed during the neurobehavioral and observational tests.

ORGAN WEIGHTS
The absolute and relative liver weight in high-dose males and females was statistically significantly increased (see Table 3). This is probably an adaptive response to the increased metabolic load due to ingestion of the test substance. As no treatment-related effects were noted on liver enzyme levels or liver histopathology, this is not considered to be a toxicologically relevant effect. The increase in relative gonad weight in mid-dose females is seen as a incidental occurrence, as the effect was only noted at this dose level.

GROSS PATHOLOGY
No treatment-related findings were reported.

HISTOPATHOLOGY: NON-NEOPLASTIC
No treatment-related findings were reported.

OTHER FINDINGS
- Oestrus cycle: There were no statistically significant differences in the mean length of the oestrus cycles and the number of complete cycles between the females in the control group and the treatment groups.
- Sperm parameters: There were no statistically significant differences between the males in the control group and the treatment groups in the mean number and percentage of ultrasound-resistant spermatids, number of motile spermatozoa in the cauda epididymis, and in the percentage of spermatids with malformations.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No treatment-related effects 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: Clinical chemistry results

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Albumin (g/L)

30.94 ± 1.04

31.73 ± 0.91

31.74 ± 1.21

32.47 ± 0.99**

34.22 ± 1.10

33.68 ± 3.10

32.93 ± 1.84

33.89 ± 1.19

Blood urea nitrogen (mmol/L)

4.138 ± 0.58

4.260 ± 0.361

3.993 ± 0.430

4.859 ± 0.487**

4.956 ± 0.424

6.921 ± 5.989

4.897 ± 0.717

5.578 ± 1.045

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

 

Table 2: Urinary pH

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

pH

6.44 ± 0.22

6.21 ± 0.35

6.05 ± 0.25**

5.76 ± 0.05**

6.15 ± 0.28

6.18 ± 0.24

5.85 ± 0.18

5.78 ± 0.18**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Table 3: Liver weights

 

Group (mg/kg bw/day)

 

Males

Females

 

Control

100

300

1000

Control

100

300

1000

Absolute (g)

12.59 ± 1.74

13.02 ± 1.77

13.56 ± 2.03

16.39 ± 2.50**

7.11 ± 0.67

7.93 ± 0.82

8.12 ± 0.85

9.10 ± 0.89**

Relative (g/kg bw)

29.04 ± 2.60

30.06 ± 2.52

30.05 ± 2.04

36.52 ± 2.55**

30.22 ± 2.12

34.45 ± 7.31

33.38 ± 2.52

38.58 ± 2.33**

*Statistically significant (p < 0.05)

**Statistically significant (p < 0.01)

Applicant's summary and conclusion