Hexanoic acid, 3,5,5-trimethyl-, 2,2,6,6-tetramethyl-1-[2-[(3,5,5-trimethyl-1-oxohexyl)oxy]ethyl]-4-piperidinyl ester

Endpoint summary

Description of key information

Subacute toxicity was evaluated in a combined repeated dose toxicity / screening for reprotoxicity study according OECD 422 (GLP). Male and female rats were exposed orally to 15, 50 and 150 mg/kg bw/d. No adverse effects were observed. The NOAEL is 150 mg/kg bw/d.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and ServiceGmbH, Sulzfeld, Germany
- Age at study initiation: 11-13 weeks
- Fasting period before study: no
- Housing: individually, following exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together until PND 4 (end of lactation).
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks:

suspension

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): daily
- Mixing appropriate amounts with (Type of food): corn oil
- Storage temperature of food: RT

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- according GLP
- stability of the test substance was proven before the start of the study
- method stability of test item in corn oil: UV/VIS spectroscopy

Duration of treatment / exposure:

The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males,
and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females.

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
15, 50, 150 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: range finder test 300 and 1000 mg/kg bw, histopathological findings at 300 and 1000 mg/kg bw
- Rationale for animal assignment (if not random): Randomization

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: before the start of the administration period in order to randomize the animals. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).

FOOD CONSUMPTION
- weekly
- Food consumption was not determined during the mating period (male and female F0 animals).
- Food consumption of the F0 females with evidence of sperm was determined on GD 0, 7, 14 and 20.
- Food consumption of F0 females, which gave birth to a litter, was determined for PND 4

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in the course of FOB
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of administration period
- Anaesthetic used for blood collection: Yes, anaesthetized using isoflurane (Isoba®, Essex GmbH, Munich, Germany)
- Animals fasted: Yes
- How many animals: 5/sex/dose

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of administration period
- Animals fasted: Yes
- How many animals: 5/sex/dose

URINALYSIS: Yes
- Time schedule for collection of urine: males: after mating, females: 1 day before end of administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: male: postnatal day 0, female: 10d after gestation
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
1. Adrenal glands
2. All gross lesions
3. Aorta
4. Bone marrow (femur)
5. Brain
6. Cecum
7. Cervix
8. Coagulating glands
9. Colon
10. Duodenum
11. Eyes with optic nerve
12. Esophagus
13. Extraorbital lacrimal gland
14. Epididymides (modified Davidson’s solution)
15. Femur with knee joint
16. Heart
17. Ileum
18. Jejunum (with Peyer’s patches)
19. Kidneys
20. Larynx
21. Liver
22. Lungs
23. Lymph nodes (axillary and mesenteric)
24. Mammary gland (male and female)
25. Nose (nasal cavity)
26. Ovaries (modified Davidson’s solution)
27. Oviducts
28. Pancreas
29. Parathyroid glands
30. Pharynx
31. Pituitary gland
32. Prostate gland
33. Rectum
34. Salivary glands (mandibular and sublingual)
35. Sciatic nerve
36. Seminal vesicles
37. Skeletal muscle
38. Spinal cord (cervical, thoracic and lumbar cord)
39. Spleen
40. Sternum with marrow
41. Stomach (forestomach and glandular stomach)
42. Target organs
43. Testes (modified Davidson’s solution)
44. Thymus
45. Thyroid glands
46. Trachea
47. Urinary bladder
48. Uterus
49. Vagina

HISTOPATHOLOGY: Yes, control and high dose group, gross lesions in all animals
1. All gross lesions
2. Adrenal glands
3. Bone marrow (femur)
4. Brain
5. Cecum
6. Cervix
7. Coagulating glands
8. Colon
9. Duodenum
10. Epididymides
11. Heart
12. Ileum
13. Jejunum
14. Kidneys
15. Liver
16. Lung
17. Lymph nodes (mesenteric and axillary lymph nodes)
18. Ovaries
19. Oviducts
20. Peyer’s patches
21. Prostate
22. Rectum
23. Sciatic nerve
24. Seminal vesicles
25. Spinal cord (cervical, thoracic and lumbar cords)
26. Spleen
27. Stomach (forestomach and glandular stomach)
28. Testes
29. Thymus
30. Thyroid glands
31. Trachea
32. Urinary bladder
33. Uterus
34. Vagina

Statistics:

Blood parameters:
For parameters with bidirectional changes:
Non-parametric one-way analysis using KRUSKAL-WALLIS test. If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose
group with the control group was performed using WILCOXON-test (twosided) for the hypothesis of equal medians
For parameters with unidirectional changes:
Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urinalysis parameters: WILCOXON-test (one-sided)

Food consumption: DUNNETT-test (twosided)

fertility indices: FISHER'S EXACT test

Proportions of affected pups per litter with necropsy observations: WILCOXON-test

Weight parameters: KRUSKAL-WALLIS test

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Ophthalmological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Behaviour (functional findings):

no effects observed

Gross pathological findings:

no effects observed

Details on results:

Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 during the entire study period. Salivation after treatment was seen sporadically in single animals of all substance treated groups. From the temporary, short appearance immediately after dosing it is likely, that this finding was induced by a bad taste of the test substance or local affection of the upper digestive tract. This finding was not considered to be an adverse and toxicologically relevant effect.

Concerning clinical pathology, no treatment-related, adverse effects were observed up to the highest tested dose of the compound. In males of test group 3 (150 mg/kg bw/d) total bilirubin levels were lower compared to controls. In absence of any signs of anemia this finding is most probably due to an increased conjugation rate of bilirubin as consequence of liver enzyme induction followed by an accelerated excretion of bilirubin via the bile. This effect is regarded as adaptive and nonadverse.

Regarding pathology, there were neither treatment-related organ weight changes, nor gross lesions.

In the kidneys of males, histopathology revealed a dose-dependent increased accumulation of eosinophilic droplets in the proximal convoluted tubules of test groups 1, 2 and 3. Eosinophilic droplets were consistent with α2u-globulin. α2u-globulin is a male and ratspecific poor soluble protein synthesised in the male rat liver, filtered by the glomerulus and reabsorbed in the S2 segment of the proximal tubules where it is slowly hydrolysed by lysosomal digestion. Reversible binding of the test-substance or their metabolites to α2uglobulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible in form of characteristic eosinophilic droplets. Although the increase of eosinophilic droplets is considered treatment-related, this finding does not represent a risk for humans since they do not synthesise this protein (Durham and Swenberg, 2013).
All other histopathological findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Key result

Dose descriptor:

NOAEL

Effect level:

150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed up tp the highest dose

Key result

Critical effects observed:

no

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

150 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Description of the OECD 422 guideline study

The test item was administered orally by gavage to groups of 10 male and 10 female Wistar rats (F0 animals) at dose levels of 0 mg/kg body weight/day (corn oil served as vehicle), 15 mg/kg bw/d, 50 mg/kg bw/d and 150 mg/kg bw/d. The duration of treatment covered a 2-week premating and a mating period in both sexes, approximately 1 week post-mating in males, and the entire gestation period as well as 4 days of lactation and 2 weeks thereafter in females. A detailed clinical observation was performed in all animals. Body weights and food consumption were determined in F0 animals. Clinicochemical and hematological examinations as well as urinalyses and FOB were performed in all animals towards the end of the administration period. All animals were assessed by gross pathology; weights of selected organs were recorded and a histopathological examination was performed.

 

Results of the OECD 422 guideline study

There were no test substance-related or spontaneous mortalities in any of the substance treated groups.

One female parental animal of the control was sacrificed moribund on premating day 11 due to gavage error and poor general condition (grade: severe), gasping and piloerection.

Three high-dose (150 mg/kg bw/d) and three mid-dose (50 mg/kg bw/d) male animals showed slight to moderate salivation after treatment on premating days 10, 12 and 13. Three high-dose, two mid-dose and two low-dose male animals showed slight to moderate salivation after treatment during several parts of the mating period. This transient salivation for a few minutes immediately after treatment was likely to be induced by the unpleasant taste of the test substance or by local irritation of the upper digestive tract. It is not considered to be a sign of systemic toxicity. One mid-dose female animal showed a palpable mass (palpable through skin, location: mammary line) during PND 11 - 13). This was assessed as an incidental finding. No other clinical signs or changes of general behavior, which may be attributed to the test substance, were detected in any male or female F0 generation parental animal during the entire study period.

Food consumption, mean body weight and mean body weight change of the male and female animals in all test substance-treated groups (test groups 1 - 3; 15, 50 and 150 mg/kg bw/d) was comparable to the concurrent control group during the entire study period.

Male and female animals of all dose groups did not show any abnormalities at detailed clinical observations. No test substance-related or spontaneous findings were observed during the functional observation battery. Also motor activity measurement did not reveal significant changes in comparison to the concurrent control group.

No treatment-related, adverse changes were observed among hematological, clinical chemistry and urinalysis parameters. In males of test group 3 total bilirubin levels were lower compared to controls and in males of test group 2 urine volume was increased and urine specific gravity was decreased.

All mean absolute and relative organ weight parameters showed no significant differences when compared to the control group 0. Likewise no treatment related gross lesions occurred in any of the test groups. In males of test group 3 the incidences of transitional epithelial cells and granulated and epithelial casts in the urine sediment were increased. Concurrently in an immunohistological staining α2u-Globulinurie was detected in the kidneys in males of group 3. Apart from this finding, no other treatment related histopathological changes were observed.

 Discussion of results

In males of test group 3 total bilirubin levels were lower compared to controls. In absence of any signs of anemia this finding is most probably due to an increased conjugation rate of bilirubin as consequence of liver enzyme induction followed by an accelerated excretion of bilirubin via the bile. This effect is regarded as adaptive and non-adverse.

In males of test group 2 urine volume was increased and urine specific gravity was decreased. These findings were not dose-dependent and therefore the effect is regarded as incidental and not treatment-related.

In males of test group 3 the incidences of transitional epithelial cells and granulated and epithelial casts in the urine sediment were increased. This correlates well with the histopathological findings of α2u-Globulinurie in the kidneys and is regarded as a rat specific finding not relevant for humans (Hard et al., 1993). Reversible binding of the test-substance or their metabolites to α2u-globulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible in form of characteristic eosinophilic droplets. Although the increase of eosinophilic droplets is considered treatment-related, this finding does not represent a risk for humans since they do not synthesize this protein (Durham and Swenberg, 2013).

Justification for classification or non-classification

Classification, Labeling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008.