Bis(2-ethylhexyl) adipate

Endpoint summary

Description of key information

28-day, oral, gavage, rat: NOAEL = 200mg/kg (Miyata 2006, according to draft of the enhanced OECD 407 guideline)
2-year, oral, diet, rat: NOAEL = 600mg/kg (NTP, 1982, comparable to OECD 451)
  Within this study, additional range finding studies in rats and mice were performed (2-weeks, 90-days)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

April 1977- May 1979

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

other: OECD 451 (Carcinogenicity study)

Deviations:

yes

Remarks:

Urinalysis, haematological and clinical biochemistry measurements were not reported

Principles of method if other than guideline:

The aim of this study was to detect any carcinogenic potential of DEHA. Additionally clinical signs, body weights, survival, gross pathology also detecting in detail nonneoplastic effects in the tissue, and histopathology were performed.

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NCI Frederick cancer Research Center, Maryland
- Age at study initiation: 3 weeks
- Housing: 5 per cage
- Diet :e.g. ad libitum
- Water :e.g. ad libitum
- Acclimatisation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-31
- Humidity (%): 10-88
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

Dietary preparation:
Test diets were prepared by mixing the chemical with an aliquot of powdered Wayne® Lab Blox animal feed (Allied Mills, Chicago, IL), placing the mixture in a Patterson-Kelly twin-shell intensifier bar V-blender with the remainder of the feed, and mixing for 10 minutes . Test diets were sealed in labelled plastic bags and stored at 4°C for no longer than 14 days.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The amounts of di(2-ethylhexyl)adipate in selected batches of feed were measured by vapor-phase chromatography of 50-ml methanol extracts of 2-g samples. At each dietary concentration, the mean of the analytical concentration was usually within +/-10 % of the theoretical.

Duration of treatment / exposure:

103 weeks

Frequency of treatment:

Diet was available ad libitum for 103 weeks

Remarks:

Doses / Concentrations:
12000, 25000 ppm (600, 1250 mg/kg bw)
Basis:
nominal in diet

No. of animals per sex per dose:

50 rats

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: based on performed subchronic pre-study (14-week study)

Positive control:

No positive control used.

Observations and examinations performed and frequency:

Body weight: yes, recorded every 4 weeks.
Clinical observations: twice daily.
In addition, survival was recorded.

Sacrifice and pathology:

Necropsy: CO2 inhalation.

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Gross and microscopic examinations were performed on major tissues, major organs, and all gross lesions from killed animals and from animals found dead. Tissues were preserved in 10 % neutral buffered formalin, embedded in paraffin, sectioned, and stained with hematoxylin and eosin . The following tissues were examined microscopically: skin, lungs and bronchi, trachea , bone and bone marrow, spleen, lymph nodes, heart, salivary gland, liver, pancreas, stomach, small intestine, large intestine, kidneys, urinary bladder, pituitary, adrenal, thyroid, parathyroid, mammary gland, prostate and seminal vesicles or uterus, testis or ovary, brain, thymus, larynx, and esophagus. Necropsies were performed on all animals found dead unless precluded in whole or in part by autolysis or cannibalization. Thus, the number of animals from which particular organs or tissues were examined microscopically varies and does not necessarily represent the number of animals that were placed on study in each group.

Other examinations:

No other examinations were performed.

Statistics:

Probabilities of survival were estimated by the product-limit procedure of Kaplan and Meier (1958). Animals were statistically censored as of the time that they died of other than natural causes or were found to be missing; animals dying from natural causes were not statistically censored .
Statistical analyses for a possible dose-related effect on survival used the method of Cox (1972) for testing two groups for equality and Tarone's (1975) extension of Cox's methods for testing for a dose-related trend .
One-tailed P values have been reported for all tests except the departure from linearity test, which is reported only when its two-tailed P value is less than 0.05.
The incidence of neoplastic or non-neoplastic lesions has been given as the ratio of the number of animals bearing such lesions at a specific anatomic site (numerator) to the number of animals in which that site is examined (denominator).
The one-tailed Fisher exact test (Cox, 1970) was used to compare the tumor incidence of a control group with that of a group of dosed animals at each dose level.
The Cochran-Armitage test for linear trend in proportions, with continuity correction (Armitage, 1971), was also used. Life table methods were used to analyze the incidence of tumors. Curves of the proportions surviving without an observed tumor were computed as in Saffiotti et al . (1972).

Details on results:

CLINICAL SIGNS AND MORTALITY: No compound- related clinical signs were observed.

BODY WEIGHT AND WEIGHT GAIN: Mean body weights of high-dose rats of either sex were lower than those of the controls throughout the study.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): no data

FOOD EFFICIENCY: no data

HAEMATOLOGY: not examined

CLINICAL CHEMISTRY: not examined

URINALYSIS: not examined

NEUROBEHAVIOUR: not examined

ORGAN WEIGHTS: not examined

GROSS PATHOLOGY: no compound-related effects occurred.

HISTOPATHOLOGY: NON-NEOPLASTIC: No compound-related lesions were observed.

HISTOPATHOLOGY: NEOPLASTIC (if applicable): Tumors that were noted were those seen routinely in this strain of rat, and they occurred in comparable numbers in control and dosed rats. There were no compound-related effects.

HISTORICAL CONTROL DATA (if applicable): no data

Dose descriptor:

NOAEL

Effect level:

12 000 ppm

Sex:

male/female

Basis for effect level:

other: Mean body weights of high-dose rats of either sex were lower than those of the controls throughout the study.

Critical effects observed:

not specified

Endpoint conclusion

Dose descriptor:

NOAEL

600 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

NOAEL based on peroxisome proliferation

Additional information

Diethylhexyl adipate (DEHA) has been evaluated in two published repeated dose studies performed comparable to current guidelines.

In a 2 -year chronic study, 50 rats per sex and dose have been exposed via the diet to app. 600 and 1250mg/kg b.w. of the test substance (NTP 1982). Doses were selected based on two range-finding studies in which rats and mice were treated for 2 and 14 weeks. All studies were reviewed within the National toxicology program. Weight loss was observed in rats and mice after 14 -days at about 1000 mg/kg and 2000 mg/kg, respectively. After 90 -days, body weight gain was reduced in male rats at and above 1250mg/kg and in mice at and above app. 400 mg/kg. Thus, 1250mg/kg was selected as high dose in the chronic study. No analysis of haematology, clinical chemistry, urine, or neurobehavior was performed. No differences in organ weights were detected, and detailed histopathological examination revealed no substance related effects, including no indications of a cancerogenic potential, up to the highest dose tested. Mean body weights of high dose rats of both sexes were lower than those of control animals, so that the systemic NOAEL was set to 600mg/kg.

Additionally, as discussed in the section for carcinogenicity, 50 B6C3F1 mice per group and sex were exposed via diet for 2 years to app. 1715 and 3570 mg/kg DEHA following the same protocol as the rat study (NTP 1982). No systemic toxicity was observed up to 3570mg/kg.

In a published 28 -day study, 10 rats per sex and group were orally exposed via gavage to 40, 200, and 1000mg/kg DEHA in olive oil (Miyata 2006). No effects on body weight, clinical signs, haematologic parameters were observed. No analysis of urine, neurobehavior, and clinical chemistry blood parameters was performed. Kidney weights were increased in mid dose males and high dose males and females. No histopathological changes were seen in the kidneys of mid dose males, and since additionally no difference in kidney weight was observed for mid dose females, this change is considered as not adverse. In high dose males, eosinophilic bodies and hyaline droplets were detected in the kidneys. Furthermore, liver weights were increased in high dose animals, as well as adrenal weights in high dose females. Test substance administration had no effect on sperm parameters or hormone analysis. 4 high dose females showed increased ovarian follicle atresia and two of these had a prolonged estrous cycle (4 and 10 days, until sacrifice). But because these effects were observed with only 10 animals after 4 weeks and could not be confirmed by two studies using higher animal numbers, which were treated with comparable doses for significantly longer time periods (chronic exposure (NTP 1982), one-generation-study (CEFIC 1988, included in the section on reproductive toxicity)), it is concluded, that DEHA does not adversely effect fertility. Based on the effects on liver, kidney, and adrenals at 1000mg/kg, the NOAEL of the 28 -day repeated dose study was set at 200 mg/kg.

Similar results were obtained in an GLP compliant 28 day repeated dose study with a 14 day recovery period (Xu 2019). However in this study DEHA was administered intravenous, which does not represent a suitable application route for purposes under REACH.

Four groups of rats (15/sex/group) each received either vehicle or DEHA in vehicle (100, 200, or 450 mg/ kg/day). Criteria for evaluation included clinical observations, body weight, food consumption, clinical pathology (hematology, serum chemistry, coagulation, urinalyses), gross (necropsy) evaluation, organ weight and histopathological evaluation. There were no DEHA-related changes in all the endpoints evaluated at 100 or 200 mg/kg/day. There were no test article-related changes in clinical pathology or gross necropsy observation at 450 mg/kg/day. At the high-dose, DEHA-related adverse findings included clinical observations and decreased body weight gain and food consumption. All these findings were completely reversible within a 14-day recovery period. In conclusion the 200 mg/kg/day dose is considered to be the No-Observed-Adverse-Effect Level (NOAEL).

Justification for classification or non-classification

According to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not needed for repeated dose toxicity, as the LOAEL in the repeated dose toxicity tests are above the cut-off values with limited effects.