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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
14/05/2018 - 04/01/2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Deviations:
not applicable
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
not applicable
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Reference substance name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
EC Number:
701-008-3
Molecular formula:
C27H34O6
IUPAC Name:
Reaction mass of benzyl 1-(isobutyryloxy)-2,2,4-trimethylpentan-3-yl phthalate and benzyl 3-(isobutyryloxy)-2,2,4-trimethylpentyl phthalate
Specific details on test material used for the study:
TEST MATERIAL NAME: Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate, a Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate
CAS Number: 16883-83-3
EC Number: 701-008-3
Molecular weight: 454.4
Trading name: Santicizer® 278
Batch number: 2984
Appearance: clear oily liquid
Molecular weight: 454.4 g/mol
Received on: 02 March 2018
Storage Temperature: 15 25°C protected from light
Purity: 98.51%
Retest date: 10 April 2019

The test article information and certificate of analysis provided by the Sponsor are considered an adequate description of the characterisation, purity and stability of the test article. Determinations of stability and characteristics of the test article were the responsibility of the Sponsor.

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
The rat was selected because it is a readily available rodent species acceptable to the regulatory authorities and is recommended for toxicity studies due to the historical control data available at Covance Harrogate.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, Margate, United Kingdom
- Age at study initiation: At the start of dosing, animals were 5 to 7 weeks old.
- Weight at study initiation: Males weighed between 157.6g and 222.1g, and females weighed between 136.7g and 173.8g.
- Housing: Animals were housed in groups of up to three by sex. During neurobehavioral assessments, animals remained in their home cage, except when placed in the testing apparatus. Bedding was provided on a weekly basis to each cage by use of clean Aspen wood chips (Datesand Ltd, Manchester, United Kingdom). Each batch of bedding was analysed for specific constituents and contaminants.
- Diet (e.g. ad libitum): Animals had ad libitum access to 5LF2 EU Rodent Diet (International Product Supplied Ltd., London, United Kingdom). Each batch of diet was analysed for specific constituents and contaminants.
- Water (e.g. ad libitum): Main supply water was provided ad libitum via water bottles. The water is periodically analysed for specific contaminants.
- Acclimation period: Animals were acclimated for 12 to 14 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 3°C
- Humidity (%): 30% to 70%
- Air changes (per hr): The room was air-conditioned to provide a minimum of 15 air changes/hour.
- Photoperiod (hrs dark / hrs light): Fluorescent lighting was controlled automatically to give a cycle of 12 hours of light and 12 hours of dark, except when otherwise dictated by experimental procedures.

IN-LIFE DATES: From: 14/05/2018 To: 23/10/2018

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations were prepared weekly and were frozen (<10°C) until required. The test article was formulated as a suspension in corn oil following Dispensary SOPs and the formulation method (Method 8381235_O_01D), as maintained in the study data.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial preparations performed at the Test Facility to select a suitable vehicle and to establish a suitable formulation procedure.
- Concentration in vehicle: Group 1 (Control): 0 mg/mL; Group 2: 25 mg/mL; Group 3: 75 mg/mL; Group 4: 250 mg/mL.
- Amount of vehicle (if gavage): 4 mL/Kg
- Lot/batch no. (if required): Not specified
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by using a validated analytical procedure.

Concentration Analysis:
Formulations prepared for use on the first and last day of dosing were analysed to determine the achieved concentration. Triplicate samples were removed from the middle of the test article formulations and analysed. A single sample was taken from the middle of vehicle formulations and was analysed.

Stability and Homogeneity Analysis:

Test article formulations at 1 mg/mL concentration have been shown to be stable for 6 hours at room temperature (15 to 25°C) and at least 12 days in frozen storage (<-10°C) and were homogenous (Covance Study 8381238).

Test article formulations at a concentration of 1000 mg/mL have been shown to be stable for up to 12 days at room temperature (15 to 25°C) and in frozen storage (<-10°C) and were homogenous (Covance Study 8381238).
Duration of treatment / exposure:
90 days, 7 days a week
Frequency of treatment:
Once daily, oral gavage, 7 days a week
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Control
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Low Dose
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Medium Dose
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
High Dose
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: A dose level of 1000 mg/kg/day was selected as the high-dose level based on the absence of significant clinical observations or effects on body weight and only minor reductions in food consumption at this dose level during the dose range-finding phase. An intermediate dose level of 300 mg/kg/day represented a dose level approximately 3-fold lower than the high dose (i.e. half-log spacing), and a low dose level of 100 mg/kg/day represented a dose level 3-fold lower than the intermediate dose (i.e. half- log spacing).
- Rationale for animal assignment (if not random): Animals were assigned to dose groups upon arrival using a total randomisation procedure. Animals were individually identified by electronic implant. During the FOB assessments, applicable animals were identified by tail marks. Following the first full weighing, group mean body weights and standard deviations were calculated and inspected to ensure no unacceptable differences occurred between groups for main study animals only. Animal R0203 (Group 3 main study phase male) was replaced with a spare male following the body weight review. Cages were positioned on batteries to avoid potential for cross contamination and to enable exposure of each cage/battery to similar environmental conditions, except during FOB assessments.
- Fasting period before blood sampling for clinical biochemistry: All samples were collected after animals were fasted overnight.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

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/animal/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

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: Not specified

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Pre-dose and Week 12
- Dose groups that were examined: All animals pre-dose and Groups 1 and 2 (Week 12)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All animals

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Week 13
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All animals

URINALYSIS: No
- Time schedule for collection of urine:
- Metabolism cages used for collection of urine: No
- Animals fasted: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Week 12
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other:

IMMUNOLOGY: Not specified

OESTROUS CYCLES: Yes
- Time schedule or examinations: Week 13
- Dose groups that were examined: All groups
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, all animals were subjected to a full external and internal examination.

ORGAN WEIGHTS: Yes (see Table 1)

HISTOPATHOLOGY: Samples of selected tissues (see Table 1) were removed from all animals and preserved. Tissues from all control and high dose group animals were examined microscopically. Thyroid, liver, and gross lesions were also examined from low and mid dose groups for possible treatment-related findings.
Statistics:
Please see 'Any other information on materials and methods incl. tables' for information on Statistics.

Results and discussion

Results of examinations

Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
Salivation was observed after dosing for all test article-treated groups, and the incidence increased dose-proportionally. This finding was also observed in two control females. Paddling behaviour was observed for a few animals administered 300 or 1000 mg/kg/day, and a dose response was observed. These findings were considered attributable to an unpalatable or unpleasant tasting test article formulation; as such, this possible test article-related finding was considered not to represent systemic toxicity, as it was also observed in two control females. Post-dose mouth rubbing was observed for all groups, including control, which suggested that this finding was in response to the vehicle and did not represent systemic toxicity.
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No adverse effect on body weight change was noted for test article-treated animals, compared with controls. Statistical analysis of the data did not reveal any significant intergroup differences.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No adverse effect on food consumption was noted for test article-treated animals, compared with controls. Any statistically significant changes noted were isolated considered to have arisen incidentally and were unrelated to test article toxicity, as the overall differences in food consumption over the duration of the study phase were comparable across groups, including controls.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
There were no ocular changes observed at the end of dosing.
Haematological findings:
no effects observed
Description (incidence and severity):
No toxicologically important haematology changes were noted for test article-treated animals, compared with controls. All minor intergroup differences observed in haematology parameters including those which were statistically significant, were attributed to normal biological variation and were considered not to be test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant increases in cholesterol levels were evident for males administered 300 or 1000 mg/kg/day (P < 0.05), although all values were within the historical control ranges (1.1 to 2.7 mmol/L). Cholesterol levels were also elevated for females administered 1000 mg/kg/day (P < 0.01), compared with controls, with all values from controls and animals administered 1000 mg/kg/day within the historical control ranges (0.9 to 2.5 mmol/L). These increases may be associated with the liver and thyroid gland changes noted.

Creatinine levels for males administered 1000 mg/kg/day were significantly lower than controls (P < 0.01), with two values for males administered 1000 mg/kg/day lower than the historical control ranges (19 to 39 µmol/L). Blood urea nitrogen (BUN) and urea levels were also significantly lower for males administered 1000 mg/kg/day, compared with controls (P < 0.01), with urea levels for five males that were outside of the historical control ranges (4.2 to 9.2 mmol/L [no background data available for BUN]). No such effects were noted in females administered 1000 mg/kg/day.

All other minor intergroup differences observed in clinical chemistry parameters including those which were statistically significant, were attributed to normal biological variation and were considered not to be test article related as they were small in magnitude, not dose dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate. Alanine aminotransferase activity was significantly lower for females administered 300 or 1000 mg/kg/day, compared with controls (P < 0.001), with one value for animals administered 1000 mg/kg/day lower than the expected ranges (18 to 67 IU/L).

Alkaline phosphatase activity was also significantly lower for females administered 300 or 1000 mg/kg/day, compared with controls (P < 0.05), with one or two low values observed for controls and animals administered 300 or 1000 mg/kg/day,
compared with the historical control ranges for this parameter (19 to 124 IU/L). Nevertheless, a reduction in activity of these enzymes was considered of no toxicological significance.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No indication of neurotoxicity was evident following the functional observation battery assessments. Weekly open field arena observations did not reveal any toxicologically significant effects of the test article. The observations noted were either isolated, with no dose response, or occurred in all groups, including controls; as such, these observations were considered not related to test article toxicity. Any intergroup differences were considered to have arisen incidentally as isolated occurrences that did not show a dose response.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Group mean liver:brain weight ratios, liver:body weight ratios, and unadjusted liver weights were higher for males administered 100 mg/kg/day and both sexes administered 300 or 1000 mg/kg/day, compared with concurrent controls. Group mean thyroid/parathyroid:brain weight ratios, thyroid/parathyroid:body weight ratios, and unadjusted thyroid/parathyroid weights were higher for males administered 300 or 1000 mg/kg/day and females administered 100 or 1000 mg/kg/day, compared with concurrent controls. All other organ weight and organ weight ratio changes, including those which were statistically significant, were attributed to normal biological variation and were considered not test article related as they were small in magnitude, not
dose-dependent, inconsistent between sexes, due to normal inter-animal variability, and/or lacked a microscopic correlate.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Large liver was recorded for six males administered 1000 mg/kg/day, which correlated with findings noted microscopically. No other macroscopic findings considered test article-related were recorded.

Other tissues were macroscopically unremarkable, or the findings recorded were generally consistent with the usual pattern of findings in rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Hypertrophy of centrilobular hepatocytes was recorded for the liver of most males and some females administered 100 mg/kg/day, all males and some females administered 300 mg/kg/day, and both sexes administered 1000 mg/kg/day. It was characterized by an increase in hepatocyte size, cell rounding, and diffusely pale eosinophilic cytoplasm, with centrilobular distribution, and it generally correlated with increased liver weights and large liver observed macroscopically in males.

Follicular cell hypertrophy in the thyroid gland was recorded for some animals administered 300 mg/kg/day and all males and most females administered 1000 mg/kg/day. It was characterized by an increase in the size of cells and cytoplasmic basophilia in the follicular epithelium, and it generally correlated with increased thyroid/parathyroid weights.

No other microscopic findings considered test article-related were recorded. Microscopic findings in other tissues were generally infrequent, of a minor nature, and consistent with the usual pattern of findings in rats of this strain and age.
Histopathological findings: neoplastic:
not examined

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: No adverse effects

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Dose Formulation Analyses

The mean % nominal concentration should be between 85% to 115% and with a relative standard deviation (RSD) 10.0%. Results were within these criteria. The test article was not detected in the control samples.

Applicant's summary and conclusion

Conclusions:
The test material Santicizer 278 adminstered at 100, 300, or 1000 mg/kg/day, for up to 90 consecutive days resulted in test article‑related changes at all dose levels, which generally consisted of changes in the liver and thyroid gland, however, these were considered to be adaptive changes and not adverse. Therefore, the no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day.
Executive summary:

The objective of the study was to determine the toxicity of the test article, Benzyl 3‑isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate a Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan-3-yl benzene‑1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate, following daily oral (gavage) administration to the rat for 90 days. Four groups of 10 male and 10 female Crl:WI(Han) strain rats were assigned to the main study phase. Animals were administered 0 (vehicle), 100, 300, or 1000 mg/kg/day of the test article for up to 90 consecutive days, by oral gavage at a dose volume of 4 mL/kg. The vehicle was corn oil.

Assessment of toxicity was based on mortality, clinical and postdose observations, body weights, food consumption, ophthalmic examinations, and functional observational battery and oestrus cycle (females only) assessments. At the end of the main study phase, blood samples were collected for haematology, coagulation, clinical chemistry, and thyroid hormone assessments. Necropsies were performed on all animals following 90 days of dosing, and any macroscopic abnormalities were recorded. Selected organs were also weighed. Microscopic examinations were performed on selected tissues from control and high dose animals. A test article-related effect was noted in the liver and thyroid glands; therefore, these tissues were also examined from low and intermediate dose animals to establish a no observed adverse effect level (NOAEL).

No test article-related deaths occurred. Salivation was evident for all test article-treated groups, and on occasion, for control females. Paddling behaviour was observed postdose for up to five animals administered 300 or 1000 mg/kg/day, and a dose response was observed. In the absence of any microscopic findings that suggested irritancy, these findings were considered attributable to an unpalatable or unpleasant tasting test article formulation and considered not to represent systemic toxicity of the test article. No adverse effect on body weight change or food consumption was noted for test article-treated animals, compared with controls. Weekly open-field arena observations did not reveal any toxicologically significant effects of the test article. Grip strength, foot splay, and locomotor activity were unaffected by the test article at all dose levels. No difference in the stage of oestrus was noted for test article-treated females, compared with controls and no ocular changes were noted at any dose level at the end of the dosing phase. No toxicologically important haematology changes were noted for test article-treated animals, compared with controls.

Creatinine levels for males administered 1000 mg/kg/day were significantly lower than controls. Blood urea nitrogen and urea levels were also significantly lower for males administered 1000 mg/kg/day, compared with controls. No such effects were noted for females administered 1000 mg/kg/day. Elevated cholesterol levels were evident for males administered 300 or 1000 mg/kg/day and for females administered 1000 mg/kg/day, compared with controls.These increases may be associated with the liver and thyroid gland changes noted.

Total thyroxine was reduced and thyroid stimulating hormone was increased for both sexes administered 1000 mg/kg/day, compared with controls; the effect was more pronounced in males. Thyroid/parathyroid weights were higher for males administered 300 or 1000 mg/kg/day and females administered 100 or 1000 mg/kg/day, compared with controls. Microscopic examination revealed follicular cell hypertrophy for all males and most females administered 1000 mg/kg/day, with the effect also evident following 300 mg/kg/day administration.

Enlarged livers and corresponding elevated liver weights were noted for males administered 1000 mg/kg/day. Liver weights were also higher for females administered 1000 mg/kg/day, males and females administered 300 mg/kg/day, and males administered 100 mg/kg/day, compared with controls. Microscopic examination revealed centrilobular hepatocyte hypertrophy and all animals administered 1000 mg/kg/day, all males and some females administered 300 mg/kg/day, and most males and some females administered 100 mg/kg/day.

In conclusion, once daily oral gavage administration of Benzyl 3-isobutyryloxy-1-isopropyl-2,2-dimethylpropyl phthalate a Reaction Mass of Benzyl (1R,1S) 2,2,4-trimethyl-1-[(2-methylpropanoyl)oxy]pentan 3-yl benzene-1,2-dicarboxylate and Benzyl (3R,3S) 2,2,4-trimethyl-3-[(2-methylpropanoyl)oxy]pentyl benzene-1,2-dicarboxylate at 100, 300, or 1000 mg/kg/day, for up to 90 consecutive days resulted in test article‑related changes at all dose levels, which generally consisted of changes in the liver and thyroid gland, however, these were considered to be adaptive changes and not adverse. Therefore, the no observed adverse effect level (NOAEL) was considered to be 1000 mg/kg/day.