Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
November 2017 - June 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2018
Report date:
2018

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
SMILES Notation: O=C(O)C(C)(CC)CC(C)CCC

Test animals

Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley selected as this is the strain anticipated for use in any future developmental or reproductive toxicity studies.
Sex:
male/female

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
Thirteen weeks
Frequency of treatment:
Continuous daily exposure
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Dose / conc.:
100 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
700 mg/kg bw/day (nominal)
Remarks:
At study initiation the highest dose targeted was 1000 mg/kg/d, but was reduced to 700 mg/kg/d in the third week of the study due to weight loss in female rats and substantially decreased body weight gain in males observed in week 2.
No. of animals per sex per dose:
10-12
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
All animals were observed for mortality and general condition at least twice daily. Each study animal was removed from its cage and examined twice pretest and once weekly (once every 7 days) during the study period for general condition, skin and fur, eyes, nose, oral cavity, abdomen and external genitalia as well as an evaluation of respiration. Non-fasted body weights for study animals were recorded at least twice pretest and weekly (once every 7 days) during the study period. Fasted body weights were be obtained on the day(s) of the scheduled necropsy interval(s).

Food consumption was measured (weighed) weekly (for 6 days) for all study animals during the week prior to treatment initiation and throughout the study. Water consumption was no measured. Ophthalmology exams were performed pretest (all animals) and at the end of dosing (all surviving).

In week 12 of the study a functional observational battery including assessments in the home cage, open field, and reflex testing was conducted. The time of testing was balanced across treatment groups. All observations were performed by trained observers who were unaware of the animals’ treatment. After the functional observational battery, motor activity (horizontal and vertical) was monitored using an automated Motor Monitor (Kinder Scientific Company, LLC). Sessions were 60 minutes in length; each session was divided into 12 five minute intervals. Motor activity for each animal was individually started at the end of its functional observational battery assessment. The time of testing was balanced across treatment groups.

Terminal hematology included red blood cell count, hematocrit, hemoglobin, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, red cell distribution width, white blood cell count (total and differential), platelet count, and reticulocyte count. Blood coagulation measures included prothrombin time, activated partial thromboplastin time. Blood clinical chemisty included alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase, urea nitrogen, creatinine, glucose, total cholesterol, triglycerides, total protein, albumin, globulin, albumin/globulin ratio, bilirubin (total), sodium, potassium, chloride, calcium, and phosphorus.

Organs indicated from all surviving study animals at their scheduled necropsies were weighed, recorded and organ/body and organ/brain weight ratios calculated. Organs were not weighed for unscheduled decedents during the course of the study. Prior to weighing, all organs were carefully dissected and properly trimmed to remove fat and other contiguous tissues in a uniform manner. Organs were weighed as soon as possible after dissection to avoid drying. Paired organs were weighed together.
Sacrifice and pathology:
A complete macroscopic examination will be performed on all study animals, including all unscheduled decedents; all abnormal observations were recorded. The necropsy consists of an external examination as well as a detailed internal examination. With the exception of eye and testes, tissues from study animals were preserved using the following preservatives 10% neutral buffered formalin (lungs were infused with formalin to ensure fixation). Eye and testes were initially placed in modified Davidson's solution, then subsequently transfered to 10% neutral buffered formalin. Tissues for all animals in the control and high-dose groups will be processed, sectioned at approximately 5 microns, and examined microscopically. Hematoxylin and eosin staining will be used for histopathology.

Screening for alpha 2u-globulin nephropathy: A preliminary diagnosis will made on hematoxylin and eosin stained slides of the kidney and/or based on elevated kidney weights at the Terminal Necropsy. The sections will be stained (by amendment and at additional cost) with Mallory Heidenhain stain, if the following lesions are seen with H&E: angular hyalin inclusions in the proximal convoluted tubule, tubular degeneration and regeneration, and/or globular casts at the corticomedullary junction.
Statistics:
For all parameters, significant differences between control and test item-treated groups were expressed at the 5% (p<0.05) or the 1% (p<0.01) level.
Test item-treated groups will be compared to the control group using the following procedures:
Continuous Parameters
For comparisons involving more than two groups, if Bartlett's test for variance homogeneity was applied. If the F1 approximate test for monotonicity of dose-response is not significant at the 1% level, Williams' test for a monotonic trend was applied, otherwise Dunnett's test was performed. If Bartlett's test is significant at the 1% level then logarithmic and square-root transformations were applied. If Bartlett's test was still significant, then non-parametric methods was applied to mean ranks. If the H1 approximate test for monotonicity is not significant at the 1% level, Shirley's test for a monotonic trend was appliedotherwise Steel's test was performed.

Discrete Parameters
For comparisons involving more than two groups, if the Jonckheere-Terpstra test is significant at the 5% level, then the direction of the trend is established and one-tailed step-down testing in this direction was performed, otherwise the Kruskal-Wallis test was applied. If the Kruskal-Wallis test is significant at the 5% level, the test-item treated groups was compared to the control using exact Wilcoxon rank sum tests, otherwise, no further comparisons was made.

Binary Parameters
For comparisons involving more than two groups if the Cochran-Armitage test is significant at the 5% level, then the direction of the trend is established and one-tailed step-down testing in this direction was performed, otherwise then the chi-sqaured test was applied. If the chi-squared test is significant at the 5% level, the test-item treated groups will be compared to the control using Fisher’s Exact tests (Fisher, 1973), otherwise, no further comparisons were made.

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Description (incidence):
No unschedule deaths occured during the study.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
At study initiation female rats exposed to 1000 mg/kg/d showed significantly decreased body weight gain during the first week (gains averaged 23 g compared with 41 g in controls), and subsequent weight loss in the second week (gains of -1 g compared with 8 g in controls). Male rats also showed significant decreases in weight gain (average of 104 g compared with 118 g in controls) and week two (average of 19 g compared with 35 g in controls). Based on concerns for animal welfare the decision was taken to reduce the highest tested dose to 700 mg/kg/d at that point.

At terminal sacrifice body weight gain was significantly decreased in female rats at all doses. Control females averaged 108 grams of weight gain over 90 days of exposure, while neodecanoic acid exposed groups averaged weight gains of 87, 93, and 81 grams per rat in the 100, 300, or 700 mg/kg/day groups, respectively.

Terminal body weights were significantly decreased in female rats exposed to 700 mg/kg/d neodecanoic acid (average 265 g, P < 0.05) compared to controls (average 292 g).

Body weight gain and terminal body weight were not significantly affected by neodecanoic acid exposure for male rats.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Among female rats group mean food consumption on a g/animal basis was significantly decreased in most weeks for the 700 mg/kg/d exposed group, and occasionally for some weeks in rats exposed to 300 mg/kg/d. However, when food consumption was considered on a g/kg/d basis there was no significant effect of neodecanoic acid on food consumption.

Among male rats group mean food consumption on a g/animal basis was significantly decreased in some weeks, but without any consistent time or dose pattern. No statistically significant differences were found when food consumption was considered on a g/kg/d basis.
Food efficiency:
no effects observed
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):
In males only: Lymphocyte counts increased for all doses (1.25 to 1.58× control), but not significantly (p ≤ 0.001) until 1000/700 mg/kg/day. This corresponded with significantly increased (p ≤ 0.05 at 300 mg/kg/day and p ≤ 0.001 at 1000/700 mg/kg/day) white blood cell counts (1.20 to 1.51× control). Basophil counts significantly increased (p ≤ 0.001) in the 1000/700 mg/kg/day group compared to control (from 0.02 ± 0.009 to 0.05 ± 0.009 x10^3/uL).

In females only: At 1000/700 mg/kg/day, decreases in hemoglobin (0.94× control) and hematocrit (0.94× control);

In both males and females: red cell distribution width increased (1.08 to 1.17× control); and increases in blood urea nitrogen (BUN; 1.38× control; males only) and creatinine (1.33× control; males only) were considered test item related.

All of these changes were considered non-adverse due to their relatively small magnitude.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
In males only: At ≥ 300 mg/kg/day were total cholesterol increased (1.13 to 1.51× control; at 300 mg/kg/day). Consistent with kidney effects confirmed by histopatholgy as alpha 2u nephropathy, blood urea nitrogen (1.38× control) and creatinine (1.33× control) were significantly increased.

All of these changes were considered non-adverse due to their relatively small magnitude. Increased blood urea nitrogen and creatinine correlated with the histologic changes in the kidney of male rats. All other differences between control and treated mean values, including those that were statistically significant, were considered not test item related as they lacked a dose relationship and/or there was general overlap between individual control and treated values.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Among female rats there were significant increases in liver weight on an absolute basis and brain-weight relative basis at 700 mg/kg/d, and relative basis at 300 mg/kg/d and above. Adrenal gland weights were significantly decreased on an absolute basis at all doses, and relative to brain weight at 700 mg/kg/d, but not relative to body weight. Heart weights were unaffected by neodecanoic acid on a brain weight-relative and absolute basis, but significantly increased at all doses on a body weight-relative basis. However, the increase in heart weight did not appear dose responsive (0.379%, 0.353%, and 0.359% relative to body weight in the 100, 300, and 700 mg/kg/d groups compared with 0.331% in controls).

Among male rats there were significant increases in liver weight on an absolute, body weight-relative, and brain weight-relative basis at 300 mg/kg/d and above. Adrenal gland weights were significantly increased on an absolute basis at 100 and 700 mg/kg/d (non-significant, but marginally, increased at 300 mg/kg/d), relative to body weight only at 700 mg/kg/d, and relative to brain weight at all doses. Heart weights were marginally increased, but not significantly, by neodecanoic acid on an absolute and brain weight-relative basis, but significantly increased at 700 mg/kg/d on a body weight-relative basis. Prostate and seminal vesicle weights were significantly decreased in rats exposed to 700 mg/kg/d neodecanoic acid on an absolute and brain weight-relative basis, but not body-weight relative. Testes weights appeared unaffected. The most marked organ weight changes occurred in the kidney of male rats, with increased weights observed at all doses on an absolute and relative (both brain and body weight) basis, and increasing in a pattern consistent with a dose response. Alpha-2u nephropathy was expected in male rats exposed to neodecanoic acid, and pending histopathology confirmation, appears plausible.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Among female rats there were no remarkable gross pathology findings that occurred more than sporadically as single findings without any clear dose response pattern.

Among male rats the majority exposed to 700 mg/kg/d were reported to have some kind of finding for the kidney (abnormal color, enlarged, or masses), and a single rat exposed to 300 mg/kg/d was reported to have a dilated pelvis (a finding not reported for any rats at the 700 mg/kg/d exposure). Other gross pathology findings were sporadic and not clearly treatment related.
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Generalized hepatocellular hypertrophy was present in males and females at 1000/700 mg/kg/day. The enlarged hepatocytes had increased brightly eosinophilic granular cytoplasm, most notable in males with marked hypertrophy.

In male rats only: Increased hyaline droplets in the proximal tubular epithelium, tubular degeneration and regeneration and/or granular casts in all male rat kidneys at
≥100 mg/kg/day. The hyaline droplets were eosinophilic on hematoxylin and eosin staining and bright red with Mallory's Heidenhain stain, which enhanced visualization of the angular hyaline inclusions. Small scattered hyaline droplets are normally found in the proximal tubules of male rats. The hyaline droplets were larger and more numerous in males treated with the test article compared with controls, with a dose effect on incidence and severity. These findings were consistent with test item-induced alpha 2u-globulin nephropathy.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
A single 1000/700 mg/kg/day exposed male rat developed a renal tumor (4064S1) characterized as "malignant nephroblastoma, unilateral, incidental". This neoplasm is not uncommon in rats of this age and was considered spontaneous and not test item-related
Other effects:
not specified

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
>= 700 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
organ weights and organ / body weight ratios
Remarks on result:
not determinable due to adverse toxic effects at highest dose / concentration tested

Target system / organ toxicity

Critical effects observed:
yes
Lowest effective dose / conc.:
100 mg/kg bw/day (nominal)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
no

Any other information on results incl. tables

Liver weights were signficantly increased in a treatment-related manner in both male and female rats. Histopathology indicates liver hypertrophy, not hyperplasia, suggestive of an adaptive increase in metabolic capacity to increase clearance. Combined seminal vesicle and prostate weight were signficantly decreased on an absolute and brain weight-relative basis, but not body weight-relative. No histopathology difference was observed between the control and 1000/700 mg/kg/day group, suggesting the effect was not treatment related.

In male rats only kidney weights, gross pathology, and histopathology (including immunohistochemistry confirmation) indicate the occurrence of alpha2u globulin nephropathy. While it is an adverse effect for the male rat, it is well established as not relevant to humans.

Group/sex

2M

3M

4M

2F

3F

4F

Dose (mg/kg/day)

100

300

1000/700

100

300

1000/700

Kidney

 

 

 

 

 

 

    Absolute weight (%)

19

19

39b

-

-

-

    vs. body weight (%)

12

19

49

-

-

-

    vs. brain weight (%)

16

19

39

-

-

-

 

 

 

 

 

 

 

Liver

 

 

 

 

 

 

    Absolute weight (%)

-

12

34

-

-

28

    vs. body weight (%)

-

12

44

-

12

40

    vs. brain weight (%)

-

13

34

-

-

28

a Statistically significant difference between mean values for test item-treated and control groups.

bGroup 4 n=9 due to renal tumor in 1/10.

-= not test item-related.

Applicant's summary and conclusion

Conclusions:
Test article exposure resulted in liver hypertrophy, suggestive of adaptive increase in metabolism to increase test article clearance (non-adverse effect), and alpha 2u globulin nepropathy in male rats only (not human relevant effect). The results can be considered conclusive but not sufficient for classification. The human relevant NOAEL for the study is 700 mg/kg/day.
Executive summary:

Male and female Sprague-Dawley rats (n >= 10 per group) were exposed to neodecanoic acid by diet with target exposures of 0, 100, 300, or 1000/700 mg/kg/d for 13 weeks. The highest dose at study initiation targeted 1000 mg/kg/d, but were reduced to 700 mg/kg/d due to body weight losses following the second week of exposure. Body weight gain was significantly reduced in female rats in all exposure groups, but terminal body weights were significantly different from controls only for the 1000/700 mg/kg/d exposure group. Body weight gain and terminal body weight were not significantly affected in male rats. Liver weights were significantly increased in both sexes at target exposures of 300 mg/kg/d and greater. Histopathology indicated 1000/700 mg/kg/day exposure resulted in generalized liver hypertrophy, not hyperplasia.

In male rats only there were significant increases in kidney weight in all neodecanoic acid exposed groups, with weights increasing with exposure. Gross pathology findings in male rats were also consistent with a treatment-related effect. Histopathology findings were consistent with alpha2u globulin nephropathy, an effect not relevant to humans.

Combined prostate and seminal vesicle weights were decreased in male rats exposed to 1000/700 mg/kg/d neodecanoic acid on an absolute and brain weight-relative basis, but not body weight-relative. This was not correlated with any histopathology findings, thus the effects do not appear treatment-related.

In conclusion, dietary dosing of Neodecanoic Acid to rats for 90 days was generally well tolerated at dose levels up to 700 mg/kg/day based on clinical signs, body weights and food consumption. However, increased kidney weights in males at ≥ 100 mg/kg/day (consistent with an effect not relevant to humans, alpha 2u-globulin nephropathy in males at 1000/700 mg/kg/day) and increased liver weights in males and females at ≥ 300 mg/kg/day (consistent with generalized hepatocellular hypertrophy in both sexes at 1000/700 mg/kg/day) were seen. Thus, the NOEL (no observed effect level) was not identified but the NOAEL (no observed adverse effect level) was identified as the 700 mg/kg/day dose level.