Bis(2-ethylhexyl) adipate

Repeated dose toxicity: other routes

Administrative data

Endpoint:

short-term repeated dose toxicity: other route

Type of information:

experimental study

Adequacy of study:

other information

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2019

Materials and methods

Test guideline

Qualifier:

no guideline followed

Principles of method if other than guideline:

The present study shows the toxicity effects after intravenous administration of DEHA for 28 consecutive days and the reversibility of the effects following a 14-day recovery period. The study was conducted under GLP conditions. 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.

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Bis(2-ethylhexyl) adipate

EC Number:

203-090-1

EC Name:

Bis(2-ethylhexyl) adipate

Cas Number:

103-23-1

Molecular formula:

C22H42O4

IUPAC Name:

bis(2-ethylhexyl) adipate

Specific details on test material used for the study:

DEHA was produced by Polynt S.P.A., Italy (Product commercial name: DIPLAST D/MG, Lot number: 3502216356).

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on species / strain selection:

Sprague-Dawley rats (Rattus norvegicus)/Crl:CD® [SD] VAF/Plus®/SPF) were used in the study. The animals were 6–7 weeks at dosing initiation and body weights ranging from 214 to 258 g for males and 165–198 g for females. All rats were obtained from BioLASCO Taiwan Co., Ltd and were acclimated for at least 11 days prior to dosing.

Sex:

male/female

Details on test animals or test system and environmental conditions:

The rats were group housed (up to 3 animals of the same sex and same dosing group together) in solid-bottom plastic cages elevated off the floor with corncob bedding. The rooms were on a 12-h light/dark cycle with controlled room conditions.

Administration / exposure

Route of administration:

intravenous

Vehicle:

other: 10% lipid emulsion, same formulation as a marketed product intended for intravenous administration

Details on exposure:

Sixty (60) male and 60 female rats were assigned to 4 groups of 15/sex/group, and the last 5 rats/sex in each group were allocated for recovery. DEHA in 10% lipid emulsion was administered intravenously via tail vein once daily at a dose volume of 5 mL/kg as a bolus dosing. DEHA dose levels were 100, 200, and 450 mg/kg/day. The control group was administered the vehicle only.

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

15

Control animals:

yes, concurrent no treatment

Examinations

Observations and examinations performed and frequency:

Assessments were made of morbidity/mortality (twice daily), daily clinical observations, weekly body weight and food consumption, clinical pathology (hematology, coagulation, serum chemistry, and urinalyses), organ weights, and macroscopic and microscopic examinations. Urine samples were collected prior to necropsy and blood samples for clinical pathology were collected at necropsy. Parameters evaluated included: Haematology (haemoglobin concentration, haematocrit,
platelet count, red blood cell count, white blood cell count with WBC differential, mean corpuscular volume, mean platelet volume), Coagulation (prothrombin time, activated partial thromboplastin time, fibrinogen), Clinical chemistry (albumin, ALP, ALT, AST, calcium, chloride, cholesterol, creatinine, GGT, glucose, inorganic
phosphorus, potassium, sodium, total bilirubin, total protein, triglycerides, urea, creatine kinase), Urinalysis (appearance, bilirubin, glucose, ketones, occult blood, protein, specific gravity, pH, volume, urobilinogen, and sediment microscopic examination).

Sacrifice and pathology:

All animals in the study were subjected to a full, detailed gross necropsy. The following organs were weighed: adrenal glands, brain, epididymis, heart, kidney, liver, ovaries, pituitary gland, prostate gland, spleen, testes, thymus, thyroid glands with parathyroid gland(s), and uterus. Tissues collected at necropsy included the adrenal glands (bilateral), aorta, bone and bone marrow (sternum), bone (femur, including stifle joint), brain, epididymis (bilateral), esophagus, eye (with optic nerve) (bilateral), fallopian tubes (bilateral), Harderian glands (bilateral), heart, kidneys (bilateral), large intestine (cecum, colon, rectum), small
intestine (duodenum, jejunum, ileum), liver, lungs with mainstem bronchi, lymph nodes (mandibular and mesenteric), mammary glands (inguinal, female only), sciatic nerve (bilateral), ovaries (bilateral), pancreas, pituitary gland, prostate gland, salivary glands (mandibular, bilateral), seminal vesicles (bilateral), skeletal muscle (biceps femoris), skin (inguinal), spinal cord (cervical, thoracic, lumbar), spleen, stomach, testes (bilateral), thymus, thyroid (with parathyroid) (bilateral), trachea, urinary bladder, uterus (including cervix), vagina, injection sites and gross lesions. The above tissues from the control and high dose groups (including recovery animals) were removed from fixative, processed, and embedded in paraffin, sectioned, stained with hematoxylin and eosin, and examined by light microscopy. For the lower dose groups from dosing and recovery phases, slides were prepared and examined microscopically only for 1) gross lesions, 2) target organ, i.e., liver. The slides were peer reviewed by Dr. Steven Brunnert (DVM, DACVP, DACLAM, consultant pathologist at WuXi App Tec). The study pathologist and peer review pathologist came to a mutual agreement on the data interpretation.

Statistics:

Statistical analyses were performed on body weights, body weight changes, food consumption, hematology, coagulation, clinical chemistry, urinalysis (quantitative data), and organ weights. Males and females were analyzed separately. Whenever there were more than two groups, the homogeneity of the group variances was evaluated using the Levene's test at the 0.05 significance level. If differences between group variances were not found to be significant (p > 0.05), then a parametric one way analysis of variance (ANOVA) was performed. When significant differences among the means were indicated by ANOVA test (p≤0.05), the Dunnett's test was used to perform the group mean comparisons between the control group and each treated group. When Levene's test indicates heterogeneous group variances (p≤0.05) and the data set contains just positive values, log transformation was performed. If transformed data still failed test for homogeneity of variance (p≤0.05) or where the data contained zero and/or negative values, then the non parametric Kruskal Wallis test was used to compare all considered groups. When the Kruskal Wallis test was significant (p≤0.05), the Dunnett's test on ranks was used to perform the pairwise group comparisons of interest. Each pairwise group comparisons of interest was conducted via a two-sided test at the 5% significance level. Significant results were reported as either p≤0.001, p≤0.01, or p≤0.05, where p represents the observed probability.

Results and discussion

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Any other information on results incl. tables

There were no test article-related clinical signs from male or female rats when dosed at ≤200 mg/kg/day DEHA during the in-life phase.

At 450 mg/kg/day, test article-related clinical signs included prostration, salivation, abnormal gait, activity decreased, entire body atonia, abnormal stool, impaired righting reflex, and/or ataxia, piloerection in males and/or females during dosing phase. Motor activity related signs were not observed prior to next administration. Aforementioned clinical signs were no longer observed during recovery phase.

There were no test article-related body weight gain changes in male rats at ≤200 mg/kg/day DEHA or female rats at ≤450 mg/kg/day DEHA during the in-life phase. At 450 mg/kg/day in males during the dosing phase, and when

compared with the concurrent controls, test article-related lower bodyweight means were noted. In general, the percent difference (from control group) increased as the study progressed, and ranged from −7.06% to −11.73% during the dosing phase. The lower mean bodyweight correlated with reduced food consumption, this change was

considered to be test article related. During recovery phase, although lower absolute mean body weight was observed in male rats administered 450 mg/kg/day DEHA when compared to the concurrent controls, the decrease was attributed to the initial lower bodyweight at the start of the recovery phase, actually, higher body weight gain was observed when compared to the concurrent controls indicating recovery.

There were no test article-related food consumption changes in male or female rats at ≤200 mg/kg/day DEHA during the in-life phase. At 450 mg/kg/day in males during dosing phase, and when compared with the concurrent controls, reduced group mean food consumption was noted, the percent difference (from control group means) ranged

−14.1% to −20.4% (differences narrowed as the study progressed), while in females, reduced (−16.9%) mean food consumption was mainly noted during 1st week, the food consumption was comparable with pretest/concurrent control group means thereafter. Reduced group mean food consumption during dosing phase was considered as test article-related. The decreases in food consumption were no longer observed during recovery phase.

There were no test article-related changes in hematology, coagulation, serum chemistry, or urinalysis parameters noted during the course of the study. There were no test article-related macroscopic changes in any treatment groups at the end of dosing or recovery phase. Pelvis dilation in the left kidney was noted in one male at 450 mg/kg/day but was not considered test article-related as it was considered common background or spontaneous findings consistent with the age and strain of this species (McInnes EF, 2012).

There were no DEHA-related organ weight alterations noted except for the weight of the liver and thymus in the high-dose group at the end of the dosing phase. Increased liver weights were noted in females at 450 mg/kg/day, which correlated microscopically with centrilobular hepatocellular hypertrophy. Decreased thymus weights without microscopic correlation were noted in males and females at 450 mg/kg/ day; this change was considered most likely to be the result from stress secondary to multiple clinical signs (decreased activity, abnormal gait, and/or prostration) (Everds et al., 2013) observed at dosing phase. Test article-related microscopic changes noted in the liver in females at 450 mg/kg/day at the end of the dosing phase were minimal centrilobular hepatocellular hypertrophy (8/10 females), which correlated with liver weight increases. This change was completely reversible following a 14-day recovery period. This finding was not observed in the males in the same dose group or in any sex in the two lower dose groups.

Inflammatory cell infiltration at the injection site(s) was noted in all groups at the end of dosing phase, this finding represents injection site trauma and was not an effect of the test article. This observation was completely reversible following a 14-day recovery period. All other microscopic changes were not considered test article-related as they were considered common background or spontaneous findings consistent with the age and strain of this species.

Applicant's summary and conclusion

Executive summary:

In the present study, DEHA administered by intravenous injection to Sprague Dawley rats once daily at dosages of 100, 200, and 450 mg/kg/day for up to 28 consecutive days resulted in no test article-related effects for hematology, coagulation, clinical chemistry, urinalysis, or gross necropsy observations. Test article-related clinical signs in males and/or females at 450 mg/kg/day group during dosing phase were observed during treatment but reversible when treatment was terminated. Decreased body weight gains; that is, the bodyweights were lower at all measured timepoints during treatment period in males at 450 mg/kg/day, when compared to the concurrent control were associated with a decreased food consumption. Food consumption reduction was reversible and increase body weight gain was seen after treatment was terminated. Test article-related organ weight changes were an increase in liver weight in females and decrease in the thymus

weight (most likely to be resulted from stress) in males and females at 450 mg/kg/day. The liver weight changes were associated with hypertrophy in the centrilobular hepatocytes, this change was completely reversible with a 14-day recovery phase. The minimal centrilobular hepatocellular hypertrophy is considered non-adverse change (Hall AP et al., 2012; Kerlin et al., 2016).