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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
17 Nov 1997 - 16 Mar 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP-Guideline study, tested with the source substance 146289-36-3. 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:
1998
Report Date:
1998

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
Ministerium für Raumordnung, Landwirtschaft und Umwelt des Landes Sachsen-Anhalt, Magdeburg, Germany
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Pentanoic acid, mixed esters with pentaerythritol, isopentanoic and isononanoic acid
- Physical state: colourless liquid
- Analytical purity: 100%
- Lot/batch No.: 6DP17
- Storage condition of test material: RT, protected from light
- Expiration date of the lot/batch: September 1999

Test animals

Species:
rat
Strain:
other: Wistar Crl:WI BR
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH, Sulzfeld, Germany
- Age at study initiation: 32-38 days
- Weight at study initiation: 148.5 g (males), 132-165 g (males), 136.7 g (females), 123-155 g (females)
- Housing: animals were housed individually or in groups of two animals in Makrolon Type 3 cages with soft wood bedding.
- Diet: pelleted standard diet (Altromin 1326), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6-8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-23
- Humidity (%): 30-60
- Air changes (per hr): air conditioned
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
other: distilled water containing 1% Tween 80
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Separate formulations were prepared for each dose level by mixing a weighed quantity of the test article with an appropriate volume of vehicle.

VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no.: S23350 739
- Amount of vehicle (if gavage): 10 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
HPLC analysis by means of the external standard method was applied. A greater deviation from the planned nominal concentration was determined only in one case (sample of the low dose group from Day 22). In the other cases the planned nominal concentrations, especially in the high dose group, were achieved.
Duration of treatment / exposure:
13 weeks (control and test groups)
13 weeks and 4 weeks post-exposure observation period (satellite control group and high-dose group)
Frequency of treatment:
once daily, 7 days/week
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg bw/day
Basis:
other: nominal
No. of animals per sex per dose:
10 (main study)
10 (satellite control and high-dose groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: 10 animals per sex from the high dose and the vehicle control group were used to investigate the reversibility of possible effects.
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behaviour
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling, occurrence of secretions and excretions
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

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

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: all (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: erythrocyte count, haemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombin time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to killing at the end of the study
- Animals fasted: Yes
- How many animals: all animals (including the satellite groups)
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli)/grip strength/motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: cranial, thoracic and abdominal cavities
HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, oesophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal chord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus
Statistics:
Group means and standard deviations were calculated for all numerical data. Analysis of variances (ANOVA) was carried out on all parameters, using treatment group as the factor in the analysis. Statistical significance was declared at the two-sided 5% level. Analysis of variance was carried out with different statistical methods:
body weights, food consumption: Welch t-test;
haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test;
differential leukocyte count: mean value, ranges and standard deviation

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
low- and mid-dose group: one animal died in each group, due to incorrect gavage, non adverse
Mortality:
mortality observed, treatment-related
Description (incidence):
low- and mid-dose group: one animal died in each group, due to incorrect gavage, non adverse
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):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
low- and high-dose group (female): increased erythrocyte count and decreased packed cell volume, non adverse; high-dose group (female): increase in fibrinogen concentration
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see Table 1 under "Any other information on results incl. tables".
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):
high-dose (males): significant increase in kidney weights, high-dose (females): significant increase in liver weights
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
mid- and high-dose (females): deposition of intracellular fat and fatty degeneration of hepatocytes in the liver
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood. Only a few clinical observations (e.g. ruffled fur, slight squatting position, slight nose bleeding) were made and considered to be substance independent and were made only for a short period of time. None of the animals showed any alterations of their general state of well-being and behaviour at any observation period.

BODY WEIGHT AND WEIGHT GAIN
Not affected by the test compound.

FOOD CONSUMPTION
Not dose-dependently influenced by the test compound.

OPHTHALMOSCOPIC EXAMINATION
No alterations were observed in any animal during the study period.

HAEMATOLOGY
Female animals in the low- and high dose group showed an increased erythrocyte count and decreased packed cell volume. These significant differences were in the normal ranges of values for the strain used.
The prothrombin time showed a dose-dependent increase in male animals, which was significant in the high-dose male group. At the end of the recovery period, no differences were observed in the satellite group. Due to a low prothrombin time in male controls it was assumed by the authors that the significance was not substance-dependent, but accidental. Increased fibrinogen concentration in females of the high-dose group was observed. The increase was not seen after the recovery period. The increase was therefore considered as caused by the administration of the test article (see Table 1 under "Any other information on results incl. tables").

CLINICAL CHEMISTRY
Activity of alkaline phosphatase was dose dependently increased in the male and female animals and was significantly increased in the animals of the high dose group (see Table 1 under "Any other information on results incl. tables"). After the recovery period normal activity was apparent. The activity of aspartate aminotransferase was dose dependently decreased in male animals of all groups. The values in the satellite groups of control and high dose animals were in the same range as the previous significant values of the male animals. Therefore and because the value of the control was relatively high, it was assumed that the significance was not substance-dependent, but accidental. Serum urea nitrogen was increased in female animals of the high dose group and in the mid- and high-dose males. The creatinine content was significantly increased in all male and the high dose group of female animals. The phosphorus content was significantly increased in all female animals and the sodium content was dose dependently decreased in male animals. These effects were not observed after the recovery period. The observed effects on the serum urea, creatinine and phosphorus content were considered to be substance-related.
Significant differences of the glucose and calcium levels were considered to be incidental due to small, not dose-dependent differences.
All other clinical-biochemical parameters investigated were unaffected.

NEUROBEHAVIOUR
No changes in grip strength, motor activity and sensory response were observed during the study period in any animal.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased in all male animals and statistically significant increased in the high dose group which was still present after the recovery period (see Table 2 under "Any other information on results incl. tables"). Absolute and relative liver weights were increased in both sexes and statistically significant increased in females of the high dose group. This increase was no longer apparent after the recovery period in females. The increase of the weights of the liver was considered to be in accordance with the lipopexia in the liver cells. Other significant differences (adrenals, brain, epididymides, heart, ovaries and thymus) seemed to be incidental.

GROSS PATHOLOGY
No substance-dependent changes were observed in the animals.

HISTOPATHOLOGY
Intracellular lipid droplets in hepatocytes of the female animals in the high- and mid-dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. The degree of severity was classified in 4/10 animals as low, in 4/10 as moderate and in 2/10 as severe in both groups.
There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals. No markedly degenerated hepatocytes or intracellular fat droplets were observed in the satellite groups.
Other histological findings were observed such as:
Stomach: oesophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach
Lungs: atelectactic and emphysemic areas
Thymus: partial substitution of the parenchyma by fibrinogenesis
Skin: benign fibrous proliferation
Sciatic nerve: thickening of the perineurium and thickening of the adventitia of the vessels.
No histological changes on ovaries, uterus, testes and epididymis were reported in any animal.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: increased activity of alkaline phosphatase, intracellular fat and fatty degeneration of hepatocytes and increased liver and kidney weights

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1. Results of haematology and clinical biochemistry tests.

Parameter

Results of statistical calculation

males in dose group [mg/kg bw/day]

females in dose group [mg/kg bw/day]

control

100

300

1000

control

100

300

1000

Erythrocyte count [106/µL]

8.57 ± 0.55

8.09 ± 0.7

8.52 ± 0.41

8.33 ± 0.57

7.52 ± 1.04

8.32 ± 0.29*

8.03 ± 0.53

8.31 ± 0.26*

Packed cell volume [%]

44 ± 2.4

42.2 ± 2.4

43.3 ± 2.4

42.4 ± 3.6

39.4 ± 5.6

43.1 ± 1.6*

41.9 ± 2.7

43.4 ± 1.8*

Fibrinogen [g/dL]

2.92 ± 0.25

3.07 ± 0.25

2.73 ± 0.47

2.83 ± 0.28

2.06 ± 0.34

2.11 ± 0.21

2.10 ± 0.4

2.86 ± 0.43*

Alkaline phosphatase [U/L]

145.0 ± 26.7

148.7 ± 50.6

167.4 ± 57.2

198.3 ± 51.0*

86.4 ± 15.3

96.3 ± 27.8

95.1 ± 17.8

141.7 ± 30.2*

Aspartate aminotransferase [U/L]

222.6 ± 32.6

168.2 ± 15.1*

147.4 ± 25.9*

144.9 ± 36.4*

151.0 ± 37.2

127.1 ± 20.3

140.6 ± 28.9

142.6 ± 16.7

Urea nitrogen [mg/dL]

20.9 ± 3.9

20.6 ± 1.8

24.6 ± 3.2*

25.6 ± 3.2*

19.2 ± 2.2

18.9 ± 3.3

20.9 ± 4.4

25.2 ± 3.7*

Glucose [mmol/L]

5.9 ± 0.9

6.0 ± 0.98

6.43 ± 1.11

6.26 ± 1.31

6.14 ± 0.75

5.99 ± 0.67

5.13 ± 0.73*

5.52 ± 0.76

Creatinine [µmol/L]

47.8 ± 4.5

53.3 ± 4.1*

58.0 ± 5.1*

58.0 ± 4.7*

51.1 ± 3.0

50.9 ± 6.2

53.5 ± 4.5

56.9 ± 3.2*

Sodium [mmol/L]

146.9 ± 3.9

144.9 ± 5.4

141.6 ± 3.0*

139.4 ± 3.1*

120.3 ± 1.8

123.4 ± 4.2

122.6 ± 3.4

121.7 ± 2.6

Potassium [mmol/L]

6.4 ± 0.5

5.93 ± 0.55

5.94 ± 0.75

5.41 ± 0.3*

5.47 ± 0.34

5.77 ± 0.68

5.28 ± 0.4

5.32 ± 0.45

Calcium [mmol/L]

9.8 ± 0.4

9.2 ± 0.3*

9.9 ± 0.3

10.0 ± 0.4

10.3 ± 0.2

10.4 ± 0.3

9.8 ± 0.2*

9.8 ± 0.2*

Phosphorus [mmol/L]

7.35 ± 0.54

7.1 ± 0.66

7.34 ± 0.55

7.79 ± 0.31

6.25 ± 0.55

6.98 ± 0.9*

7.12 ± 0.42*

7.48 ± 0.65*

*Dunnett test (two-sided 5% significance level)

Table 2. Absolut organ weights [mg].

Parameter

Results of statistical calculation

males in dose group [mg/kg bw/day]

females in dose group [mg/kg bw/day]

control

100

300

1000

control

100

300

1000

Adrenal left

33.2 ± 6.6

38.1 ± 5.3

38.8 ± 4.8

32.6 ± 4.5

36.8 ± 5.5

38.4 ± 6.9

38.0 ± 4.4

44.8 ± 7.1*

Kidney left

1626.8 ± 163.9

1823.

1 ± 197.5

1812.8 ± 229.9

2052.4 ± 289.9*

1041.5 ± 72.3

1057.1 ± 86.8

1038.4 ± 119.0

1103.0 ± 86.6

Kidney right

1721.4 ± 206.3

1885.5 ± 234.3

1875.4 ± 243.1

2138.7 ± 306.6*

1071.6 ± 86.1

1091.4 ± 104.2

1055.6 ± 121.0

1138.1 ± 104.2

Liver

21663 ± 2663

23150 ± 2881

23263 ± 2517

25997 ± 6899

9340 ± 1278

10562 ± 1286

10583 ± 1904

13337 ± 1855*

Testis (m) / Ovarium (f) right

1740.7 ± 92.3

1804 ± 172

1787.4 ± 96.2

1867.4 ± 177.6

64.2 ± 11.8

77.3 ± 9.2*

78.3 ± 9.5*

73.9 ± 11.5

Testis (m) / Ovarium (f) left

1702.1 ± 97

1757.4 ± 185.5

1784.6 ± 76.9

1824.1 ± 201.1

65.9 ± 13.5

70.2 + 9.8

65.4 ±9.5

70.9 ± 13.5

Epididymis left

574.6 ±124.1

575 ± 138.6

544.1 ± 91.9

521.3 ±47.5

-

-

-

-

Epididymis right

552.1 ± 58.1

548.2 ± 131.1

544.7 ± 94.1

479.5 ± 61.3

-

-

-

-

 

Parameter

Results of statistical calculation, satellite groups

males in dose group [mg/kg bw/day]

control

1000

Kidney left

1685.1 ± 191.5

2007.1 ± 270.2*

Kidney right

1740.3 ± 169.6

2016.1 ± 239.8*

*Dunnett test (two-sided 5% significance level)

Table 3. Relative organ weights [%].

Parameter

Results of statistical calculation

males in dose group [mg/kg bw/day]

females in dose group [mg/kg bw/day]

control

100

300

1000

control

100

300

1000

Adrenal left

0.0074 ± 0.0012

0.0080 ± 0.0009

0.0084 ± 0.0012

0.0068 ± 0.0012

0.0131 ± 0.0022

0.0136 ± 0.0019

0.0134 ± 0.0017

0.0150 ± 0.0022

Kidney left

0.363 ± 0.033

0.387 ± 0.080

0.392 ± 0.045

0.424 ± 0.047*

0.372 ± 0.034

0.376 ± 0.033

0.365 ± 0.029

0.369 ± 0.022

Kidney right

0.384 ± 0.036

0.398 ± 0.080

0.406 ± 0.050

0.442 ± 0.054

0.382 ± 0.033

0.389 ± 0.042

0.372 ± 0.037

0.380 ± 0.024

Liver

4.83 ± 0.39

4.86 ± 0.64

5.03 ± 0.44

5.30 ± 0.94

3.32 ± 0.38

3.75 ± 0.42

3.72 ± 0.59

4.44 ± 0.38*

Testis (m) / Ovarium (f) right

0.390 ± 0.038

0.380 ± 0.049

0.388 ± 0.033

0.389 ± 0.050

0.0228 ± 0.0037

0.0276 ± 0.0039*

0.0276 ± 0.0029*

0.0248 ± 0.0041

Testis (m) / Ovarium (f) left

0.382 ± 0.046

0.369 ± 0.047

0.387 ± 0.024

0.379 ± 0.047

0.0235 ± 0.0046

0.0250 ± 0.0036

0.0232 ± 0.0043

0.0237 ± 0.0038

Epididymis left

0.130 ± 0,032

0.120 ± 0.025

0.118 ± 0.017

0.108 ± 0.012

-

-

-

-

Epididymis right

0.124 ± 0.015

0.114 ± 0.021

0.118 ± 0.019

0.099 ± 0.009*

 

 

 

 

Parameter

Results of statistical calculation, satellite groups

males in dose group [mg/kg bw/day]

control

1000

Kidney left

0.333 ± 0.038

0.363 ± 0.037

Kidney right

0.345 ± 0.036

0.374 ± 0.034

*Dunnett test (two-sided 5% significance level)

Applicant's summary and conclusion

Conclusions:
The correlated deposition of intracellular fat and fatty degeneration of the hepatocytes, the increased weights of the liver in females, and the increased activity of alkaline phosphatase identified the liver as the target organ. The clinical-biochemical findings and the irreversible increase of the kidney weights in males indicated an effect on the kidney without any histological correlation, as well.
Except for the increased kidney weights in males, all changes were no longer apparent at the end of the treatment-free period.
Therefore, the results indicated a NOAEL of 300 mg/kg bw/day.