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Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
22 Apr - 17 Jun 2004
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP - Guideline study, tested with the source substance pentaerythritol tetra(2-ethylhexanoate). 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:
2005
Report Date:
2005

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
no
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): Pentaerythritol tetra(2-ethylhexanoate)- Physical state: colourless liquid- Analytical purity: 96.4%- Lot/batch No.: TOL-887- Stability under test conditions: verified by re-analysis- Storage condition of test material: at room temperature in the dark

Test animals

Species:
rat
Strain:
Crj: CD(SD)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS- Source: Charles River Laboratories Japan, Inc., Yokohama, Japan- Age at study initiation: 10 weeks- Weight at study initiation: males: 330.8 - 420.4 g (mean 374.0 g), females: 207.2 - 245.0 g (mean 228.3 g)- Fasting period before study: no fasting period- Housing: steel cage with wire floor- Diet: CE-2 from CLEA Japan, Inc., Tokyo, Japan, ad libitum- Water: tap water, ad libitum- Acclimation period: 2 weeksENVIRONMENTAL CONDITIONS- Temperature (°C): 23.0 - 24.5 - Humidity (%): 49.0 - 67.0- Air changes (per hr): 15- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:A 50% w/v solution was prepared by diluting test substance in vehicle. This solution was diluted serially with vehicle to prepare 5 and 15% w/v solutions. Gavage solution was prepared within 8 days before administration, since the stability was verified for 8 days. Prepared samples were stored at room temperature in the dark. VEHICLE- Justification for use and choice of vehicle: test substance is insoluble in water, but not in corn oil- Lot/batch no. (if required): V2P1825
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
0.5 mL of each prepared solution was mixed with hexane to be confirmed by GC. The resulting analytical concenrations were 107 - 110% of the nominal concentrations
Duration of treatment / exposure:
Males: 42 daysFemales: from Day 14 before mating to Day 4 of lactationFemales (satellite group): 42 daysRecovery period : - Males: 14 days- Females (satellite): 14 days
Frequency of treatment:
once daily
Doses / concentrations
Remarks:
Doses / Concentrations:100, 300, 1000 mg/kg bw/dayBasis:actual ingested
No. of animals per sex per dose:
Control: 7 males, 100 mg/kg: 12 males, 300 mg/kg: 12 males, 1000 mg/kg: 7 males;12 females per dose;control and 1000 mg/kg for satellite group; 5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose were based on the results of a preliminary test, where groups of male and female rats received doses of 100, 300 and 1000 mg/kg bw, respectively. No effects on general condition, body weight gain nor organ weights of liver, kidney or spleen was observed in any of the treated animals. Based on this, the maximum dose for the study was set to 1000 mg/kg bw, while the medium and low dose were set to 300 and 100 mg/kg bw, respectively.- Post-exposure recovery period in satellite groups: 14 days

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes- Time schedule: twice a day during treatment period and once a day during recovery periodDETAILED CLINICAL OBSERVATIONS: Yes - Time schedule: Males: Day 0, 7, 14, 21, 28, 35 and 42 of treatmentMales in satellite groups: Day 0, 7, 14, 21, 28, 35, 42 of treatment, Day 7 and 14 of recovery periodFemales: Day 0, 7, 14, 21, 28, 35, 42 of treatment and once from Day 0 of lactation to Day 4 of lactationFemales in satellite groups: Day 0, 7, 14, 21, 28, 35, 42 of treatment, Day 7 and 14 of recovery periodBODY WEIGHT: Yes- Time schedule for examinations: Males: Day 1, 7, 14, 21, 28, 35, 42 of administration and before sacrificeMales and females in satellite groups: Day 1, 7, 14, 21, 28, 35 , 42 of administration, Day 1, 7, 14 of recovery period and before sacrificeFemales: Day 1, 7, 14, 21 of treatment, Day 0, 7, 14, 20 of pregnancy, Day 0 and 4 of lactation, and before sacrificeFOOD CONSUMPTION:- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: YesHAEMATOLOGY: Yes - Time schedule for collection of blood: Day 43 (on day after last treatment) for males, Day 15 of recovery period for satellite animals of both sexes, Day 4 of lactation for dams, Day 26 of pregnancy for non delivered dams- Anesthetic used for blood collection: Yes (sodium pentobarbital)- Animals fasted: Yes (18 - 24 hours)- How many animals: 5 in each group- Parameters checked: erythrocytes count (RBC), hemoglobin, hematocrit, Mean Cell Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Con. (MCHC), leykocyte count (WBC), differential leukocyte count, platelet, Prothrombin time (PT), Activated partial thromboplastin time (APTT)CLINICAL CHEMISTRY: Yes- Time schedule for collection of blood: Day 43 (on day after last treatment) for males, Day 15 of recovery period for satellite animals of both sex, Day 4 of lactation for dams, Day 26 of pregnancy for non delivered dams- Anaesthetic used for blood collection: Yes (pentobarbital sodium)- Animals fasted: Yes (18 - 24 hours)- Parameters checked: total protein, albumin, A/G, blood urea nitrogen, creatinine, glucose, total cholesterol, triglyceride, Alkaline phosphatase (ALP), Alanine transaminase (ALT (GPT)), Aspartate transaminase (AST (GOT)), γ-GTP, total bilirubin, inorganic phosphorous, calcium, Na, K, Cl URINALYSIS: NoNEUROBEHAVIOURAL EXAMINATION: Yes - Time schedule for examinations: at the end of dosing period and at the end of recovery period- Dose groups that were examined: all group- Battery of functions tested: pupilary reflex, eyelid reflex, visual placing, withdrawal reflex, Payer's reaction, startle reaction, righting reflex
Sacrifice and pathology:
Termination: - Males: day 43 of treatment and day 15 of recovery- Females: day 5 of lactation- Females (satellite): day 15 of recovery- Offspring: 4 days after birthGROSS PATHOLOGY: YesHISTOPATHOLOGY: Yes brain, the pituitary gland, spinal cord, heart, lung and bronchus, liver, kidney, thymus, spleen, adrenal gland, thyroid gland, mandibular lymph node, stomach, duodenum, jejunum, ileum, cecum, colon, rectum, testis, epididymis, prostate, seminal vesicles including coagulation glands, ovary, uterus, vagina, mesenteric lymph node, sciatic nerve, urinary bladder, bone marrow of femur
Other examinations:
Estrus cycle, reproductive performance, observation of pups
Statistics:
Fisher test, Mann-Whitney-U-Test, Student's t-test, Aspin-Welch test, Bartlett test, Kruskal-Wallis test, Dunnett test

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
incidental effects which were not treatment-related (non adverse)
Mortality:
mortality observed, treatment-related
Description (incidence):
incidental effects which were not treatment-related (non adverse)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
100 mg/kg bw: reduced body weight in males (non adverse)
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: significantly more food consumption during days 29 - 30 by satellite group (non adverse)
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
300 mg/kg bw: increased hematocrit and hemoglobin in females (non adverse)
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw: increase of blood urea nitrogen in females at the end of recovery period (non adverse)
Urinalysis findings:
not examined
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: increase in relative liver weight in females (non adverse)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
one tumor in the femur (non adverse)
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
all changes were found distributed over all groups, thus they were regarded as spontaneous incidences (non adverse)
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
one tumor in the femur (non adverse)
Details on results:
CLINICAL SIGNS AND MORTALITYNo mortality was observed in all groups. An emaciation was observed at day 40 of treatment (Day 1 of lactation) in the female control group, but this was recovered after one day. Loss of the upper incisor was observed at day 25 - 32 in a female of the 300 mg/kg group but this was considered due to physical shock and were not affected by administration of the test substance. BODY WEIGHT AND WEIGHT GAINMales of the 100 mg/kg group showed body weight loss compared to the control group, but this was not statistically significant. There was no difference in female groups and satellite groups compared with control groups. FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)Females of the satellite 1000 mg/kg group showed significantly more food consumption during days 29 - 30 compared to the control group. However, this change was considered as not compound-related as no difference in other groups was found, both within the treatment period as well as in the recovery period. HAEMATOLOGYAt the end of administration, hematocrit and hemoglobin were significantly increased in females of the 100 mg/kg group. Prothrombin time was prolonged in females of the 300 mg/kg group. These changes were not considered as compound-related changes, since a dose dependency was not observed. No other significant differences were observed. CLINICAL CHEMISTRYAt the end of dosing period, significant differences between all treatment groups and the control groups were not found. Only concentration of blood urea nitrogen was increased in females of the 1000 mg/kg group at the end of recovery period. This change was not considered as compound-related change, since no corresponding abnormalities were observed in the kidney. NEUROBEHAVIOURNo abnormalities were observed.ORGAN WEIGHTSAt the end of dosing period, a significantly increase of absolute weight of brain was found in females (300 mg/kg), but the change of relative weight was not significant. No significant differences in males between treatment group and the control group were observed. At the end of recovery period, a significant increase of relative liver weight in females of the 1000 mg/kg group was noted, while no differences in males were found. The change was not considered as biologically significant. The statistical significance may be due to narrow variations of the organ and body weights at all (relative liver weight for control group: 2.69±0.08 g(%); high dose group: 2.83±0.09). This was supported by the fact that no effect on absolute organ weights was observed. In addition no findings were observed after gross pathology and histopathology examination. GROSS PATHOLOGYAt the end of dosing period, no abnormalities were observed. At the end of recovery period, a tumor in right hind femur was observed in one male of the control group. HISTOPATHOLOGY: NON-NEOPLASTICAt the end of dosing period, localized atrophy of the seminiferous tubules were observed in three males of the 100 mg/kg group and in one male of the 300 mg/kg group. Three of these animals had cell debris in lumen in epidedymis. Livers in males of the 1000 mg/kg and control groups exhibited fatty change in hepatocyte and microgranuloma. In all control group animals and three animals of 1000 mg/kg, eosinophilic bodies were found in cortex of the kidney, while slightly basophilic tubules were found in kidney cortex of two animals in the control group and in four animals of the 1000 mg/kg group. Extramedullary hematopoiesis and deposit of brown pigment was found in the spleen of all animals of the control and the 1000 mg/kg group. In addition, focal degeneration/fibrosis in the myocardium, accumulation of focal foam cell in the adveolus and mineralization of the arterial wall in lung, as well as lymphocytes and neutrophil cellular infiltration in prostate were observed both in control groups and 1000 mg/kg groups. However, these change were not significantly different but accidental.In one female of the 1000 mg/kg group, follicular cyst, increased arterial follicle and decreased corpus luteum was found in ovary at the end of dosing period. In liver, fatty change in periportal hepatocytes and microgranuloma was observed in all animals of control and 1000 mg/kg groups, respectively. Basophilic tubule in the cortex of the kidney was observed in one animal at 1000 mg/ kg and slight mineralization was found in one control animal and one animal dosed with 1000 mg/kg, respectively. In the spleen, extramedullary hematopoiesis and brown deposit were found in all animals of the control and the 1000 mg/kg group. In addition, focal degeneration/fibrosis in myocardium, mineralization on arterial wall of lung and atrophy in thymus were also found in both control and 1000 mg/kg. However, these change were not significantly different but accidental.At the end of recovery period, intramembranous ossification and periosteum proliferation were found in one male in control. These histopathological findings are due to fracture of the right femur. In one female of 1000 mg/kg, follicular cyst was found.

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 adverse effects were observed in males and females of all dose groups

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Body weights of males (g):

 

Dosage (mg/kg bw)

Days of treatment

0

100

300

1000

1

374.6 ± 24.0

373.6 ± 18.0

374.1 ± 26.2

373.7 ± 21.3

7

409.6 ± 29.0

405.4 ± 23.9

407.0 ± 31.9

404.4 ± 26.6

14

441.4 ± 32.0

431.4 ± 27.4

439.4 ± 38.2

437.5 ± 31.9

21

463.1 ± 34.7

450.4 ± 30.5

467.1 ± 44.7

461.1 ± 27.9

28

489.7± 37.1

474.8 ± 34.5

495.7 ± 53.5

493.4 ± 32.1

35

513.2 ± 40.9

493.6 ± 39.3

519.3 ± 58.2

514.5 ± 35.4

42

534.4 ± 46.4

511.8 ± 41.7

544.3 ± 62.4

540.3 ± 37.8

Days of recovery

 

 

 

 

1

545.3 ± 55.9

 

 

544.0 ± 34.9

7

562.3 ± 59.9

 

 

570.0 ± 30.7

14

580.4 ± 57.1

 

 

594.3 ± 31.6

Body weights of females (g):

 

Dosage (mg/kg bw)

Days of treatment

0

100

300

1000

1

228.4 ± 8.4

228.4 ± 5.4

227.7 ± 10.1

227.9 ± 9.8

7

237.6 ± 9.4

240.3 ± 7.9

237.6 ± 11.2

238.7 ± 12.9

14

247.5 ± 13.2

252.2 ± 11.1

249.3 ± 13.5

246.2 ± 14.9

Days of pregnancy

 

 

 

 

0

256.2 ± 12.2

260.3 ± 7.6

261.6 ± 17.5

259.0 ± 20.6

7

295.0 ± 16.1

298.5 ± 12.1

296.0 ± 23.7

299.3 ± 19.3

14

331.4 ± 17.4

334.5 ± 13.0

334.7 ± 26.4

332.8 ± 20.2

20

404.5 ± 24.0

406.8 ± 19.1

411.2 ± 31.0

401.9 ± 23.3

Days of lactation

 

 

 

 

0

292.1 ± 27.3

300.1 ± 24.7

297.6 ± 36.9

282.6 ± 29.1

4

327.9 ± 20.5

323.1 ± 14.8

325.0 ± 27.0

311.3 ± 22.6

Body weights of females, satellite group (g):

 

Dosage (mg/kg bw)

Days of treatment

0

1000

1

227.9 ± 5.9

230.8 ± 5.0

7

241.9 ± 4.5

242.5 ± 6.2

14

254.4 ± 3.6

253.8 ± 9.1

21

261.0 ± 11.5

261.8 ± 9.5

28

274.7 ± 12.0

273.4 ± 12.9

35

282.9 ± 13.3

278.4 ± 10.6

42

287.5 ± 11.2

286.9 ± 14.5

Days of recovery

 

 

1

290.1 ± 11.6

284.7 ± 17.4

7

297.2 ± 13.0

293.4 ± 17.0

14

307.1 ± 12.2

305.1 ± 20.3

Absolute organ weights of males at the end of dosing period (g):

 

Dosage (mg/kg bw)

 

0

100

300

1000

Number of animals

5

5

5

5

Terminal body weight

494.9 ± 36.3

466.3 ± 33.8

521.9 ± 61.8

489.6 ± 38.4

Brain

2.00 ± 0.03

1.98 ± 0.06

2.04 ± 0.10

2.06 ± 0.11

Thymus / mg

342.7 ± 99.4

330.9 ± 69.1

344.3 ± 79.6

350.3 ± 36.6

Heart

1.42 ± 0.10

1.43 ± 0.22

1.49 ± 0.11

1.51 ± 0.11

Liver

13.94 ± 1.18

12.64 ± 1.45

14.75 ± 2.39

13.90 ± 1.91

Kidneys

3.12 ± 0.19

3.23 ± 0.31

3.35 ± 0.12

3.36 ± 0.27

Spleen

0.77 ± 0.19

0.69 ± 0.06

0.85 ± 0.10

0.87 ± 0.10

Adrenal gland / mg

58.9 ± 3.6

55.7 ± 2.7

64.4 ± 14.7

64.6 ± 13.4

Testes

3.23 ± 0.15

3.24 ± 0.25

3.44 ± 0.47

3.33 ± 0.47

Epididymides

1.19 ± 0.10

1.18 ± 0.12

1.28 ± 0.08

1.26 ± 0.14

Absolute organ weights of females at the end of dosing period (g):

 

Dosage (mg/kg bw)

 

0

100

300

1000

Number of animals

5

5

5

5

Terminal body weight

282.8 ± 23.5

295.2 ± 14.9

297.1 ± 24.8

279.5 ± 17.4

Brain

1.81 ± 0.06

1.87 ± 0.07

1.93 ± 0.06**

1.84 ± 0.04

Thymus / mg

182.8 ± 122.0

201.1 ± 44.5

205.3 ± 94.8

198.9 ± 70.1

Heart

0.91 ± 0.15

0.93 ± 0.08

0.99 ± 0.18

0.91 ± 0.05

Liver

9.75 ± 1.20

10.84 ± 1.46

10.30 ± 0.94

9.95 ± 1.55

Kidneys

1.94 ± 0.29

1.99 ± 0.23

2.03 ± 0.20

1.88 ± 0.21

Spleen

0.59 ± 0.10

0.62 ± 0.02

0.69 ± 0.16

0.60 ± 0.13

Adrenal gland /mg

65.9 ± 7.0

76.1 ± 14.6

68.4 ± 7.8

75.0 ± 6.4

**: significantly different from control, p<0.05

Absolute organ weights of male and females (satellite group) at the end of the recovery period (g)

 

Dosage (mg/kg bw)

 

Males

Females

 

0

1000

0

1000

Number of animals

5

5

5

5

Terminal body weight

539.1 ± 54.2

548.2 ± 30.6

281.6 ± 0.10

280.5 ± 18.4

Brain 

2.06 ± 0.06

2.02 ± 0.08

1.89 ± .0.10

1.90 ± 0.04

Thymus / mg 

226.7 ± 23.7

269.8 ± 620.7

245.4 ± 43.9

261.9 ± 50.7

Heart 

1.49 ± 0.10

1.50 ± 0.15

0.93 ± 0.12

0.91 ± 0.05

Liver 

14.80 ± 2.06

16.21 ± 2.32

7.58 ± 0.44

7.95 ± 0.64

Kidneys 

3.45 ± 0.11

3.49 ± 0.33

1.92 ± 0.16

1.97 ± 0.25

Spleen 

0.73 ± 0.18

0.88 ± 0.14

0.48 ± 0.03

0.52 ± 0.05

Adrenal gland 

/ mg

61.5 ± 11.8

67.9 ± 10.2

66.8 ± 6.4

65.6 ± 3.1

Testes 

3.74 ± 0.33

3.41 ± 0.12

 

 

Epididymides 

1.32 ± 0.07

1.30 ± 0.01

 

 

Applicant's summary and conclusion