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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 based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
17 Jan - 20 May 2008
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:
2008
Report date:
2008

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
Ministerium für Umwelt und Naturschutz, Landwirtschaft und Verbraucherschutz des Landes Nordrhein-Westfalen, Düsseldorf, Germany

Test material

Constituent 1
Reference substance name:
97593-30-1 (C12)
IUPAC Name:
97593-30-1 (C12)
Details on test material:
- Name of test material (as cited in study report): only trade name given
- Chemical name: Acetylided glyceride-mixture (multiconstituent substance)
- Physical state: brownish liquid
- Analytical purity: taken as 100%
- Lot/batch No.: CH70920C
- Expiration date of the lot/batch: 20 Sep 2008
- Stability under test conditions: stability of the test substance in the administration vehicle was checked prior to the study start. This analytical investigation showed the test substance to be stable in the concentration range used beyond the period of use.
- Storage condition of test material: at room temperature

Test animals

Species:
rat
Strain:
other: Hsd Cpb:WU
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Borchen, Germany
- Age at study initiation: 5 weeks
- Weight at study initiation: 145-171 g (males), 134-153 g (females)
- Housing: in groups with 2 or 3 animals into Makrolon cages Type IV. For environmental enrichment wooden blocks were provided to each cage and renewed as necessary. During the acclimatization and experimental period animals were kept under controlled environmental conditions on low-dust wood granules. Cages and bedding were replaced weekly by new ones.
- Diet: fixed-formula standard diet (Provimi Kliba SA, Kaiseraugst, Switzerland), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 5
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 17 Jan 2008 To: 14 Feb 2008

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test item (test substance) was formulated with corn oil at the appropriate concentrations at room temperature and maximally used over the stability period of eight days.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The correct concentrations in the formulations given to the rats were determined twice by analytical examination during the study.
A gas chromatographic method for quantifying the test item in liquid formulations was developed. For analytical investigations, representative samples from the test item formulation covering the test item concentration range used in the study, were taken. These samples were diluted with ethanol into the calibration range and subsequently quantified by gas chromatography (GC) with FID-detection (flame ionization detection). Standard solutions of the authentic test item were used for calibration. Linearity, Precision, Specificity, Robustness and Accuracy of the analytical method were evaluated apart from this GLP-study and fulfil the predefined acceptance criteria. Additional system suitability tests in terms of specificity, precision and linearity indicated that qualified analytical procedures were followed during the study.
Analysis of blank samples (0 mg/mL) revealed no measurable traces of test item.
The content checks assure that during the study appropriate and equal mixture procedures were followed (see Table 1).
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected according to results obtained in a previous oral study performed in rats. In this study the test substance was administered to 3 males and 3 females per group for 2 weeks orally by gavage in daily doses of 0, 250, 500, 750 and 1000 mg/kg.
Survival was not affected by the treatment with the test substance. At clinical observations no treatment-related findings were recorded. Body weight development in both sexes was comparable to that of the respective control group up to 1000 mg/kg. The food and water intake was not affected by the treatment. Determination of organ weights (liver, kidneys, spleen) might indicate a slight increase at 1000 mg/kg in males.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: morbidity and mortality (twice daily, once on weekends and public holidays), general clinical examinations (once daily).

DETAILED CLINICAL OBSERVATIONS: Yes, including Open Field Observation (OFO)
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: daily

FOOD CONSUMPTION:
- Food consumption determined per groups and mean daily diet consumption calculated as g food/kg body weight/day: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 28
- Anaesthetic used for blood collection: Yes (CO2/O2 (from room air))
- Animals fasted: No
- How many animals: all
- Parameters examined: differential blood count, erythrocyte morphology, erythrocyte count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular volume, haemoglobin concentration, haematocrit, leucocyte count, reticulocyte count, thrombocyte count, thromboplastin time (Hepato-Quick).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 28
- Animals fasted: No
- How many animals: all
- Parameters examined: alanine aminotransferase, alkaline phosphatase, aspartate aminotransferase, albumin, cholesterol, creatinine, total protein, urea, glucose, potassium, sodium.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Day 21 (males), Day 22 (females)
- Dose groups that were examined: all
- Battery of functions tested:

Functional Observational Battery

The following observations/examinations were performed:
- home cage observation: posture, piloerection, gait abnormalities, involuntary motor movements, vocalization, others
- observations during handling: ease of removing, reaction to being handled, muscle tone, palpebral closure, lacrymation, nasal discharge, salivation and stains
- open field observations: piloerection, respiratory abnormalities, posture, involuntary motor movements, stereotypy, bizarre behaviour, gait abnormalities, vocalization, arousal, rearing, defecation, urination, others
- the following manipulative tests were additionally performed: approach response, touch response, auditory response, tail pinch response, righting reflex, grip strength, landing foot splays, body temperature, pupil size and pupil response.

Motor Activity ("Figure 8 Maze")
Motor activity (MA) and locomotor activity (LMA) were examined as activity for the entire 60-minute session (Summary Session MA and LMA) and activity during each 10-minute interval (Summary Interval MA and LMA). Motor activity was measured as the number of beam interruptions that occurred during the test session. Locomotor activity was measured by eliminating consecutive counts for a given beam. Thus, for locomotor activity, only one interruption of a given beam was counted until the animal relocated in the maze and interrupted one of the other beams. Habituation was evaluated as a decrement in activity during the test session.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, the necropsy was a systematic gross examination of each animal's general physical condition, body orifices, external and internal organs and tissues.
The following organs of the animals killed at the end of the treatment period were weighed before fixation: brain, heart, liver, spleen, kidneys (both), thymus, adrenal glands (both), epididymides and testes (both).
HISTOPATHOLOGY: Yes (see Table 2)
Statistics:
See "Any other information on material and methods incl. tables".

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: one female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation (non-adverse).
Mortality:
mortality observed, treatment-related
Description (incidence):
1000 mg/kg bw/day: one female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation (non-adverse).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
300 and 1000 mg/kg bw/day: mean body weight values of females in these groups were marginally to slightly lower than in the respective control group (non-adverse).
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:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: Foot splay landing slightly higher in females (non-adverse). In males, motor activity slightly increased over the 60-minute observation period as well as within the first two 10-minute intervals (non-adverse).
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Necropsy revealed only a few findings in control and treated animals. None of them had to be attributed to the treatment with the test substance (see also Details on results).
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
All findings in the examined organs were evenly distributed among the groups and are known as spontaneous findings in similar incidence and severity from other studies with rats of this source (see also Details on results).
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
During the treatment period there was no evidence of intercurrent morbidity and mortality related to dosing with the test substance.
No clinical findings could be observed in males up to 1000 mg/kg bw/day and in females up to 300 mg/kg bw/day.
Within the last two weeks of treatment, one 1000 mg/kg bw/day female showed increased motility (and loss of hair which is considered as incidental) during detailed clinical observation.

BODY WEIGHT AND WEIGHT GAIN
Body weight development was comparable to controls in treated males up to 1000 mg/kg bw/day and in treated females at 100 mg/kg bw/day (see Table 3).
In general, mean body weight values were marginally to slightly lower in females at 300 and 1000 mg/kg bw/day than in the respective control group. This finding is most presumably based on the fact that mean body weights of these both groups were already lower than controls before the first administration of the test substance (day 1: 6% lower than control).
Therefore, a toxicologically relevant effect on the body weight is not considered in both sexes up to 1000 mg/kg bw/day, which is supported by the fact that the body weight gain during the study was comparable with that of controls.

FOOD CONSUMPTION
Food intake per animal and per kg body weight of treated animals was comparable to that of controls up to 1000 mg/kg bw/day.

WATER CONSUMPTION
Water intake per animal and per kg body weight of all dose groups was not toxicologically relevantly changed compared to the respective controls up to 1000 mg/kg bw/day.

HAEMATOLOGY
Haematological investigation revealed no toxicologically relevant changes in red and white cell parameters as well as in blood coagulation in any dose group of male and female rats (see Table 4).
Mean erythrocytes, haemoglobin and haematocrit values of the 300 mg/kg bw/day males were only slightly but statistically significantly increased (by ca. 7, 6 and 7%, respectively) compared to control values.

CLINICAL CHEMISTRY
Determinations of enzyme activities in peripheral blood evidenced no toxicologically relevantly changes in males and females up to 1000 mg/kg bw/day.
Substrates in peripheral blood revealed few statistically significant changes in 300 mg/kg bw/day males: a ca. 5% increase in total protein and albumin concentration, respectively; without dose dependency, no toxicologically relevant changes were considered (see Table 5).
Determination of serum electrolyte concentrations showed no changes which were attributable to the treatment with the test substance.

NEUROBEHAVIOUR
- Functional Observation Battery (FOB):
Corresponding to the clinical observation, one 1000 mg/kg bw/day female showed increased motility during the FOB investigation on Day 22 of the study; however, the values of this animal quantified in the MA and LMA (see below) were not indicative of any treatment-induced effect.
Additionally in this rat, the number of rearing was relatively high; alopecia is not considered as treatment-related.
Foot splay landing revealed slightly higher mean values in 1000 mg/kg females compared to control. With increasing dosages the mean values were: 77 ± 5; 85 ± 10 ; 79 ± 11; 94 ± 16 mm. The difference to the control mean was not statistically significant and the value of 94 ± 16 mm was within the range of historical data determined in other studies (mean values: 83-98 mm) (see Table 6).

- Motor and Locomotor Activity (MA and LMA):
The activity determination over the entire 60-minute observation period as well as of the 10-minute intervals did not reveal any significant effect on locomotor activity in treated rats at any dose and on motor activity in males up to 300 mg/kg bw/day and in females up to 1000 mg/kg bw/day (see Table 7).
In males at 1000 mg/kg bw/day, motor activity appeared slightly but not statistically significantly increased over the entire 60-minute observation period as well as within the first two 10-minute intervals. The differences between 0 and 1000 mg/kg bw/day males were comparable to historical values measured in former studies.
The motility observed during detailed clinical observation and FOB (see above) of one female (1000 mg/kg bw/day) appeared higher than in control animals; the individual values of this animal quantified in the MA and LMA were not indicative of any treatment-induced effect.

ORGAN WEIGHTS
Absolute and relative organ weights were inconspicuous in any dose group; no toxicologically relevant changes were observed in both sexes. The only change noted was a ca. 15% decrease in the mean relative spleen weight in females dosed 100 mg/kg bw/day compared to control (200 vs. 236 mg/100 g bw, respectively).

GROSS PATHOLOGY
Necropsy revealed only a few findings. None of them had to be attributed to the treatment with the test substance (see Table 8).

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathology revealed no findings that had to be attributed to the treatment. All findings in the examined organs were evenly distributed among the groups and are known as spontaneous findings in similar incidence and severity from other studies with rats of this source (see Table 9).

HISTORICAL CONTROL DATA
Historical reference mean values for foot splay of control females and motor activity of control males are given Tables 6 and 7, respectively.

Effect levels

Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects; NOAEL corresponding to the highest dose tested

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 3. Group mean body weights and body weight gains.

 

Dose

(mg/kg bw/day)

Body weight Day 1 (g)

Body weight Day 29, prior to necropsy (g)

Body weight gain Days 1-29 (g)

Males

0

158

294

136

100

163

313

150

300

162

283

121

1000

156

291

135

Females

0

152

212

60

100

148

217

69

300

143++

205

62

1000

143+

201

58

 

+ p ≤ 0.05

++ p ≤ 0.01

 

 

Table 4. Haematology.

 

Dose (mg/kg bw/day)

ERY (10E12/L)

HB (g/L)

HCT (L/L)

MCV (fl)

MCH (pg)

MCHC (g/L ERY)

RETI (‰)

THRO (10E9/L)

HQUICK (sec)

Males Day 28

0

7.64

150

0.480

62.9

19.7

313

23

1251

41.7

100

7.56

153

0.490

64.9

20.2

311

24

1337

39.4

300

8.17++

159++

0.514++

62.9

19.5

309

19

1251

40.4

1000

7.74

154

0.490

63.3

19.9

314

21

1167

40.5

Females Day 28

0

7.88

148

0.466

59.1

18.8

318

17

1244

35.2

100

7.81

149

0.471

60.4

19.2

317

20

1336

35.4

300

7.86

150

0.476

60.6

19.1

314

21

1254

35.1

1000

7.76

147

0.466

60.1

18.9

315

22

1228

36.6

 

ERY = Erythrocytes; HB = Haemoglobin; HCT = Haematocrit; MCV = Mean corpuscular volume erythrocytes; MCH = Mean corpuscular haemoglobin; MCHC = Mean corpuscular haemoglobin concentration; RETI = Reticulocytes; THRO = Thrombocytes/Platelets; HQUICK = Hepato Quick.

++ p ≤ 0.01

 

 

Table 5. Clinical chemistry: Substrates.

 

Dose (mg/kg bw/day)

GLUCOSE (mmol/L)

CHOL (mmol/L)

CREA (mcmol/L)

UREA (mmol/L)

PROT (g/L)

ALBUMIN (g/L)

Males Day 28

0

5.70

1.59

56

7.70

65.7

34.5

100

5.76

1.66

57

7.87

66.3

34.6

300

5.14

1.76

57

7.51

69.2+

36.3+

1000

5.51

1.93

55

7.53

67.5

35.4

Females Day 28

0

5.40

1.65

56

7.16

68.5

37.3

100

5.16

1.65

59

7.54

68.4

37.2

300

5.17

1.75

56

7.44

68.2

36.8

1000

5.29

1.63

55

7.31

64.9

35.5

 

CHOL = Cholesterol; CREA = Creatinine; PROT = Protein

+ p ≤ 0.05

 

 

Table 6. Footsplay.

 

Dose (mg/kg bw/day)

Footsplay (mm ± SD)

Reference values (mm) / Date

Males Day 21

0

81 ± 10

-

100

87 ± 12

-

300

98 ± 9

-

1000

92 ± 9

-

Females Day 22

0

77 ± 5

98 ± 15 / 07 Apr 2008
91 ± 13 / 05 May 2008
83 ± 9 / 27 Feb 2007

100

85 ± 10

-

300

79 ± 11

-

1000

94 ± 16

-

 

 

Table 7. Motor activity (males).

 

Dose (mg/kg bw/day)

Interval 1

Interval 2

Interval 3

Interval 4

Interval 5

Interval 6

60 min period

Reference values Interval 1

Reference values 60 min period

Dates of reference values

Males Day 22

0

132 ± 26

94 ± 61

52 ± 24

39 ± 21

23 ± 12

8 ± 12

347 ± 98

172 ± 51
272 ± 104
209 ± 85
179 ± 36

394 ± 178
549 ± 215
431 ± 195
367 ± 91

20 Aug 2007
07 Apr 2008
20 May 2008
26 May 2008

100

110 ± 29

85 ± 24

42 ± 10

25 ± 14

16 ± 9

13 ± 11

282 ± 30

-

-

-

300

132 ± 27

86 ± 43

61 ± 39

28 ± 34

39 ± 38

25 ± 22

364 ± 150

-

-

-

1000

187 ± 61

127 ± 44

50 ± 12

28 ± 11

24 ± 21

26 ± 24

427 ± 96

-

-

-

 

 

Table 8. Number of animals with necropsy findings by organ/group/sex.

 

Organ / Finding

Dose group (mg/kg bw/day)

0

100

300

1000

Sex

m

f

m

f

m

f

m

f

Animals examined

5

5

5

5

5

5

5

5

Heart:
- coloured area(s)


-


-


1


-


-


-


-


-

Lungs:
- coloured area(s)


-


-


-


-


-


1


-


-

Liver:
-discolouration(s)
- distinct lobulation


-
1


-
-


-
-


1
1


1
1


-
-


-
-


-
2

Kidneys:
- retraction(s)
- surface change(s)


-
-


-
-


-
1


1
-


-
-


-
-


-
-


-
-

Spleen:
- surface change(s)


-


-


-


-


2


-


-


-

 

 

Table 9. Number of animals with microscopic findings by organ/group/sex.

 

Organ / Finding

Dose group (mg/kg bw/day)

0

100

300

1000

Sex

m

f

m

f

m

f

m

F

Animals examined

5

5

5

5

5

5

5

5

HEART

- Mononucl. Infiltrat.

-

2

1

-

-

-

2

3

- Hyperemia/Congestion

-

-

1

-

-

-

-

-

LUNGS

- Hemorrhage,(m-) focal

4

2

-

-

-

1

3

2

- Inflammat. Infiltr.

1

3

-

-

-

1

1

1

ESOPHAGUS

- Mononuc. Infiltrat.

1

-

-

-

-

-

-

-

LIVER

- Periport. Infiltrat.

-

2

-

-

-

-

-

1

- Kupffer Cell Focus/i

4

3

-

-

2

-

3

3

LIVER (ORO)

- Fat-pos. Reaction/PP

5

5

-

-

-

-

4

5

- Fat-pos. Reaction/SC

1

2

-

-

-

-

3

3

- Fat-pos. Reaction/CL

-

-

-

-

-

-

1

-

KIDNEYS

- Basophilic Tubules

3

2

1

-

-

-

2

3

- Cystic Tub. Dilation

-

1

-

-

-

-

-

-

- Mineralization/CMT

-

2

-

-

-

-

-

3

- Pelvic Dilation

-

-

-

-

-

-

-

1

- Pyelonephritis

-

-

-

1

-

-

-

-

- Trans. Cell Hyperpl.

-

-

-

1

-

-

-

-

URINARY BLADDER

- Foc. Simple Hyperpl.

1

-

-

-

-

-

1

-

- Mononucl. Infiltrat.

-

-

-

-

-

-

1

-

EPIDIDYMIDES

- Sperm Granuloma

1

-

-

-

-

-

-

-

PROSTATE

- Interst. Inflammat.

2

-

-

-

-

-

2

-

OVIDUCTS

- Debris/Oocyte Lumen

-

-

-

-

-

-

-

1

UTERUS

- Dilation/Horns

-

1

-

-

-

-

-

-

- Keratinization/Cerv.

-

1

-

-

-

-

-

2

- Mucification/Cervix

-

2

-

-

-

-

-

1

THYROID GLAND

- Foil. Cell Hypertr.

2

-

-

-

-

3

1

 

- Ectopic Thymus Tiss.

1

2

-

-

-

-

1

2

ADRENAL GLANDS

- Incr. Vacuolation

1

-

-

-

-

-

-

-

- Access. Cort. Nodule

-

-

-

-

-

-

-

1

MANDIB.LYMPH NODES

- Blood Resorption

2

1

-

-

-

-

2

1

STERNUM

- Increased Fat Marrow

-

-

-

-

-

-

1

1

Applicant's summary and conclusion