Hexanedioic acid, 1,6-bis(2-propylheptyl) ester

Endpoint summary

Description of key information

BASF SE (2015):
90-day feed study in rats, NOAEL 3000 ppm (approximately 209 mg/kg bw/day in male rats and approximately 244 mg/kg bw/day in female rats) 
NTP programme (81-768, 1982):
90-day feed study in mice, NOAEL 1600 ppm ( approximately 213 mg/kg bw/day, male mice and approximately 249 mg/kg bw/day, female mice) 
90-day feed study in rats, NOAEL 6300 ppm (approximately 623 mg/kg bw/day, male rats and approximately 829 mg/kg bw/day, female rats) 
103-weeks feed study in rats, NOAEL 12000 ppm (approximately 800 mg/kg bw/day, male rats and approximately 933 mg/kg bw/day, female rats) 
103-weeks feed study in mice, LOAEL 12000 ppm (approximately 1416 mg/kg bw/day, male mice and approximately 1368 mg/kg bw/day, female mice) 
Miyata et al. (2006)
28-day feed study in rats, NOAEL 200 mg/kg bw/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic 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:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

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 Services GmbH, Sulzfeld, Germany
- Age at study initiation: 42 ± 1 days
- Weight at study initiation:
- Housing: 5 animals per cage, polysulfonate cages, floor area about 2065 cm2, Dust-free wooden bedding
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24°C
- Humidity (%): 30-70%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light from 06.00-18.00 h, 12 hours dark from 18.00-06.00 h

IN-LIFE DATES: From: 31 Oct 2014 To: 03 Feb 2015

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency):
- Mixing appropriate amounts with (Type of food): ground Kliba maintenance diet mouse/rat “GLP”, meal, supplied by Provimi Kliba SA, Kaiseraugst, Switzerland
- Storage temperature of food: room temperature

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
3000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
12000 ppm (males); 15000 ppm (females)
Basis:
nominal in diet

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: 4 week dose finding study
- Rationale for animal assignment (if not random): random
- Section schedule rationale (if not random): random

Positive control:

not required

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least once a day


DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the start of the administration period and weekly thereafter

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations:Prior to the start of the administration period on day -1 and on study day 91
- Dose groups that were examined: all animals (day -1), control and high dose animals (day 91)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: towards the end of the administration period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.?] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: towards the end of the administration period
- Animals fasted: Yes
- How many animals: all
- Parameters checked in table [No.?] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: towards the end of the administration period
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [No.?] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: at the end of the administration period
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity / Home cage observations / Open field observations

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table)
HISTOPATHOLOGY: Yes (see table)

Organ weights: The following weights were determined in all animals sacrificed on schedule:

1. Anesthetized animals
2. Adrenal glands
3. Brain
4. Epididymides
5. Heart
6. Kidneys
7. Liver
8. Ovaries
9. Spleen
10. Testes
11. Thymus
12. Thyroid glands
13. Uterus with cervix

Organ/tissue fixation: The following organs or tissues were fixed in 4% neutral-buffered formaldehyde solution or in modified Davidson’s solution:

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

All gross lesions, liver and Thyroid glands were microscopically examined for all animals, all the other organs and tissues that have been fixed were examined for the control and the high dose group.

Statistics:

Body weight, body weight change: A comparison of each group with the control group was performed using DUNNETT's test (two-sided) for the hypothesis of equal means

Feces, rearing, grip strength forelimbs, grip strength hindlimbs, foot-splay test, motor activity: Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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 (two-sided) for the equal medians

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 (two-sided) 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) with Bonferroni-Holm adjustment for the hypothesis of equal medians

Urinalysis parameters (apart from pH, urine volume, specific gravity, color and turbidity: Pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians

Urine pH, volume, specific gravity, color and turbidity: 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 (two-sided) for the hypothesis of equal medians. Urine color and turbidity are not evaluated statistically.

Pathology (Weight parameters): Non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). 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 (two-sided) for the equal medians

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Test group 3: 12000 ppm in males and 15000 ppm in females
(875 mg/kg bw/d in males and 1361 mg/kg bw/d in females)

Clinical Examinations

• No treatment-related, adverse findings were observed.

Clinical Pathology

• Increased alkaline phosphatase (ALP) activities, urea and potassium levels were observed in male animals.
• Increased γ-glutamyltransferase (GGT) activities were observed in females.
• Decreased total protein and albumin levels were observed in females.
• Increased incidences of granulated and epithelial casts were observed in males.

Pathology

• Increased mean absolute and relative liver weights were observed in males and females (in combination with clinical pathology).
• Minimal diffuse hepatocellular hypertrophy was observed in 6 (out of 10) male and in 7 (out of 10) female animals (in combination with clinical pathology parameters).


Test group 2: 3000 ppm
(209 mg/kg bw/d in males and 244 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology and Pathology

• No treatment-related, adverse findings were observed.


Test group 1: 1000 ppm
(71 mg/kg bw/d in males and 85 mg/kg bw/d in females)

Clinical Examinations, Clinical Pathology and Pathology

• No treatment-related, adverse findings were observed.

Dose descriptor:

NOAEL

Effect level:

3 000 ppm

Based on:

other: test item in the diet

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

female

Critical effects observed:

not specified

Regarding clinical pathology, slightly increased alkaline phosphatase (ALP) activities in males of test group 3 (12000 ppm) and slightly increased γ-glutamyl transferase (GGT) activities in females of the same test group (15000 ppm) indicated a liver cell swelling because of liver enzyme induction. Higher urea levels in males of test group 3 (12000 ppm) and decreased total protein and albumin levels in females of the same test group (15000 ppm) were due to an increased protein metabolism in the liver.

 

Regarding pathology, target organs were the liver and the thyroid glands.

The absolute (♂ +16% / ♀ +26%) and relative (♂ +25% / ♀ +28%) liver weights were significantly increased in males and females of test group 3 (12000 [males] and 15000 [females] ppm).The increased liver weights correlated with a minimal diffuse hepatocellular hypertrophy that was observed in 6 (out of 10) male and in 7 (out of 10) female animals in this treatment group. These findings were considered to bea correlate for a microsomal enzyme induction in the liver. They were regarded to betreatment-related and adverse taking the changes in clinical pathology parameters into account.

The incidence of follicular hypertrophy/hyperplasia and of altered colloid in the thyroid glands was minimally increased in male animals of test group 3 (12000 ppm). These findings wereregarded to be secondary to an induced UDP-glucuronyl transferase activity in hepatocellular hypertrophy.The well-known phenomenon is characteristic for rodents (Curran et al., 1991) and leads to an accelerated degradation of T4 with a compensatory increase of TSH causing thyroid gland hypertrophy/hyperplasia of follicular cells. These findings were regarded to be treatment-related but not relevant for humans.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

209 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Additional information

90-days oral toxicity study

In a 90 days study, Wistar rats (each 10 animals/sex/dose) were treated with dietary concentrations of 0, 1000, 3000 and 12000 (males) or 15000ppm (females) Dipropylheptyladipat. Food consumption and body weight were determined weekly. The animals were examined for signs of toxicity or mortality at least once a day. Detailed clinical examinations in an open field were conducted prior to the start of the administration period and weekly thereafter.Beside this, a functional observational battery (FOB) as well as measurement of motor activity (MA) were carried out at the end of the administration period. Clinicochemical and hematological examinations as well as urinalyses were performed towards the end of the administration period. After the administration period, all rats were sacrificed and assessed by gross pathology. Organ weights were determined followed by histopathological examinations. Mid and high dose animals did not show any treatment-related adverse effects. High dose animals showed increased alkaline phosphatase (ALP) activities, urea and potassium levels (males), increased γ-glutamyltransferase (GGT) activities (females), decreased total protein and albumin levels (females) and an increased incidences of granulated and epithelial castswere observed (males). Pathology finings were increased mean absolute and relative liver weights observed in males and females (in combination with clinical pathology) and minimal diffuse hepatocellular hypertrophy observed in 6 (out of 10) male and in 7 (out of 10) female animals(in combination with clinical pathology parameters).

The slightly increased ALP activities and slightly increased (GGT) activities indicated a liver cell swelling because of liver enzyme induction. Higher urea levels and decreased total protein and albumin levels were due to an increased protein metabolism in the liver. Regarding pathology, target organs were the liver and the thyroid glands. The absolute (♂+16%/♀+26%) and relative (♂+25%/♀+28%) liver weights were significantly increased and correlated with a minimal diffuse hepatocellular hypertrophy. These findings were considered to be a correlate for a microsomal enzyme induction in the liver. They were regarded to be treatment-related and adverse taking the changes in clinical pathology parameters into account.

The minimally increased incidence of follicular hypertrophy/hyperplasia and of altered colloid in the thyroid glands of high dose male animals were regarded to be secondary to aninduced UDP-glucuronyl transferase activityinhepatocellular hypertrophy. The well-known phenomenon is characteristic for rodents (Curran et al., 1991) and leads to an accelerated degradation of T4 with a compensatory increase of TSH causing thyroid gland hypertrophy/hyperplasia of follicular cells. These findings were regarded to be treatment-related but not relevant for humans.

 

In addition, Diethylhexyladipat, a structural analogue or surrogate to Dipropylheptyladipat (for details please refer to the read across statement in chapter 13), has been evaluated for repeated dose toxicity in 14 -, 28- and 90 -days as well as 103-weeks oral feeding studies in rats and mice (NTP 1982, Miyata 2006).

14-days oral toxicity study

In a supporting 14-days study (NTP 1982), groups of 5 males and 5 females of each species were tested for 14 days with five concentrations (3100, 6300, 12500, 25000 and 50000 ppm) of the test substance in feed, followed by 1 day of observation with control diet. Groups of five males and five females of each species were maintained as untreated controls. Additionally, female rats and mice received 100000 ppm. However, feed consumption was reduced in these groups. All surviving animals were killed after 15 days. Survival, body weight and feed consumption were assessed. One female rat receiving 100,000 ppm died. Weight gain was depressed 25% or more in male rats fed 50,000 ppm and in females fed 25000 ppm or more. Feed consumption was reduced in rats fed 50,000 ppm or more. All female mice fed 100000 ppm died. Weight loss occurred among male mice fed 50000 ppm and females fed 25,000 ppm or more. In conclusion, the NOAEL was considered to be 25000 ppm for male rats (approximately 3424 mg/kg bw/day) and 12500 ppm for female rats (approximately 1919 mg/kg bw/day) considering mice the NOAEL was 25,000 ppm for male animals and 12500 ppm for female animals (approximately 3863 mg/kg bw/day, male mice and approximately 2257 mg/kg bw/day, female mice).

28-days oral toxicity study

In a supporting 28-days (Miyata 2006)oral toxicity study 10 male and female Sprague-Dawley rats received Diethylhexyladipat at dose levels of 40, 200 and 1000 mg/kg bw/day by gavage. Clinical signs, functional observation battery, body weight, food consumption, hematology, clinical biochemistry, hormone analysis, spermatology, estrous cycling, necropsy, organ weights and histopathology were assessed. Increased kidney weight without histopathological changes were observed in the males in the 200 mg/kg group, at 1000 mg/kg bw/day the increase in organ weight was seen in both males and females, accompanied by histopathological changes in the kidney. In addition, increased liver weight was also detected in the male and female rats in the 1000 mg/kg bw/day group, and this change appeared to be a toxic effect of Diethylhexyladipat. On this basis, the NOAEL was considered to be 200 mg/kg bw/day.

90-days oral toxicity study

In a supporting 90 days study (NTP 1982), F344 rats and B6C3F1 mice (each 10 animals/sex/dose) were treated with dietary concentrations of 1600, 3100, 6300, 12500 and 25,000 ppm. At the end of 91-days animals were killed and survival, feed consumption, body weight, gross pathology and histopathology analysis were assessed. Clinical observations were made twice daily and animals were weighed weekly. Weight gain depression was 10% or more for male mice fed 3100 ppm or more. Weight gain depression was 10% or more for females mice fed 6000 or 25000 ppm. One female rat receiving 1600 ppm died, but its death was not considered to be compound-related. Weight gain depression was 11% or more for male rats fed 12500 or 25000 ppm. No compound-related increased mortality, histopathological changes or reduction in feed consumption were observed. In conclusion, the NOAEL was considered to be 6300 ppm for male and female rats (approximately 623 mg/kg bw/day, male rats and approximately 829 mg/kg bw/day, female rats). For male and female mice the NOAEL was 1600 ppm (approximately 213 mg/kg bw/day male mice and approximately 249 mg/kg bw/day female mice).

103 weeks oral toxicity study

In a supporting 103 weeks study (NTP 1982) in which Diethylhexyladipat was administered to F344 rats and B6C3F1 mice (each 50 animals/sex/dose) at dietary levels of 12000 ppm and 25000 ppm body weight, clinical signs, survival, gross and histopathology were examined. The mean body weights of high-dose rats of either sex were lower than those of the controls throughout the study. For mice mean body weights of either sex were also lower than those of the corresponding controls throughout the study, and the decrease in weight gain was dose related. In female mice increased incidences of hepatocellular carcinomas developed, and in male mice hepatocellular adenomas occurred. The relevance of this finding to humans was considered to be low (see section 7.7). In conclusion, the NOAEL was considered to be 12000 ppm for male and female rats (approximately 600 mg/kg bw/day). For male and female mice the LOAEL was 12000 ppm (approximately 1715 mg/kg bw/day).

Reference

Curran, P.G. and Degroot, L. (1991). The effect of hepatic enzyme-inducing drugs on thyroid hormones and the thyroid gland.Endocrine Reviews12, 135-150

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The 90-day oral toxicity study is a reliable study and the administration period of 90-day is commonly used to determine the toxicity after repeated treatment.

Justification for classification or non-classification

Based on the results obtained from repeated dose testing Dipropylheptyladipat is not considered to be subject to classification and labelling for repeated dose toxicity according to Directive 67/548/EEC (DSD) and Regulation (EC) No 1272/2008 (CLP/GHS).