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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:
May 2013 to March 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): LINPLAST 810 TM
- Substance type: pure active substance
- Physical state: liquid
- Expiration date: April 2015
- Storage condition of test material: at ambient temperature (18 ± 4°C) in a dry place (humidity < 65%)
- Other: For historical reasons another CAS number / EINECS name is used for the same substance: 1,2,4-Benzenetricarboxylic acid, decyl octyl ester, EC number 268-007-3, CAS number 67989-23-5. Both EC numbers / CAS numbers refer to the identical substance.

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 41-43 days
- Weight at study initiation: 191.7 - 222.6g (males), 152.4 - 178.5g (females)
- Fasting period before study: no
- Housing: 5 of one sex in clear polysulphone solid bottomed cages measuring 59.5x38x20 cm (Code 1354 G, Techniplast Gazzada S.a.r.l., Varese, Italy)
- Diet: commercially available laboratory rodent diet (4 RF 21, Mucedoly S.r.l., Settimo Milanese (MI), Italy) ad libitum
- Water: drinking water ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 55 ± 15
- Air changes (per hr): 15 - 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2013-05-30 (allocation of animals) To: 2013-10-03 (last day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The formulations were prepared daily. Concentrations were calculated and expressed in terms of test item as supplied.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: 10, 40 and 100 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in RTC Study no. 77280 in the range from 10 to 200 mg/mL. Linearity, accuracy and precision were within the limits for suspensions (r > 0.98; accuracy 95-105 %; precision CV< 5 %).
Stability after 24 hours at room temperature was verified in the range from 10 to 200 mg/mL in RTC study no. 77280. Suspensions are considered to be stable if concentration and homogeneity, after the defined period of storage, are still acceptable (90 %-110% for concentration and CV<10% for homogeneity). Results obtained at the end of the stability tests were still within the acceptability limits.
The proposed formulation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) during the pre-treatment period to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (85-115 %) and homogeneity (CV<10 %).
Samples of the formulations prepared on Weeks 1 and 13 were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV<10% for homogeneity).
Duration of treatment / exposure:
13 consecutive weeks followed by a recovery period of 4 weeks for 5 males and 5 females from the control and high dose group
Frequency of treatment:
once a day, 7 days a week
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
50 mg/kg/day (low dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
200 mg/kg/day (medium dose)
Basis:
actual ingested
Remarks:
Doses / Concentrations:
500 mg/kg/day (high dose)
Basis:
actual ingested
No. of animals per sex per dose:
10, 5 additional animals per sex for the satellite groups (control and high dose group)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels were selected in consultation with the study sponsor based on information from preliminary studies
- Rationale for selecting satellite groups: no rational given
- Post-exposure recovery period in satellite groups: 4 weeks

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes (morality, clinical signs)
- Time schedule:
Mortality check: early each working day and again in the afternoon, at weekends and Public Holidays the second check were done at approx. mid-day
Clinical signs: daily, once before commencement of treatment and once during the study (performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions)

DETAILED CLINICAL OBSERVATIONS AND NEUROTOXICITY ASSESSMENT: Yes
- Time schedule: weekly, once before commencement of treatment and once a week thereafter
- Neurotoxicity assessment: animals were examined in an open arena for a minimum of 3 minutes, observed parameters: removal from cage, handling reactivity, lachrymation, palpebral closure, salivation, piloerection, rearing, spasms, myoclonia, mobility impairment, arousal (animal activity), vocalisation, stereotypies, unusual respiratory pattern, bizarre behaviour, urination, defecation, Tremors, gait (one of the following options: normal, ataxia, hunched, pronation forlimbs drag, hindlimbs drag)

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION:
The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was calculated.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes, by means of an ophthalmoscope, and by a slitlamp microscope after the instillation of 0.5% Tropicamide
- Time schedule for examinations: prior to commencement of treatment, re-examination during week 13 of treatment
- Dose groups that were examined: prior treatment: all groups, re-examination: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during week 13 of treatment and during week 4 of the recovery period
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group)
- Parameters examined: haematocrit, haemoglobin, red blood cell count, reticulocyte count, mean red blood cell volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, white blood cell count, differential leucocyte count (neutrophils, lymphocytes, eocinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during week 13 of treatment and during week 4 of the recovery period
- Animals fasted: Yes
- How many animals: 10 each sex from all groups during week 13, from all surviving animals of the recovery groups (control, high dose group)
- Parameters examined: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, gamma-glutamyltransferase, urea, creatinine, glucose, trigycerides, phosphorous, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride

URINALYSIS: Yes
- Time schedule for collection of urine: overnight during week 13 of treatment and during week 4 of the recovery period
- Metabolism cages used for collection of urine: Yes / No / No data
- Animals fasted: Yes and water bottles were removed, each animal received approx. 10 mL/kg bw of drinking water by gavage
- Parameters examined: appearance, volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood, sediment (obtained from centrifugation at approx. 3000 rpm for 10 min, examined microscopically for: epithelial cells leucocytes, crystals, spermatozoa and precursors, other abnormal components)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during weeks 12/13 of treatment and once during week 4 of recovery
- Dose groups that were examined: all dose groups, all animals from recovery groups (control and high dose group)
- Battery of functions tested: sensory activity (auditory, visual and propriocetive stimuli), grip strength, motor activity (measured over a period of 5 min/animal)

OTHER: Oestrous cycle and spermatogenic cycle
From the first day of Week 12 and up to the end of the treatment period, vaginal smears were taken daily in the morning. The vaginal smear data were examined to determine potential anomalies of the oestrous cycle.
A detailed qualitative evaluation of testes was performed on all control and high dose males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. Identification of the stages of the spermatogenic cycle was carried out as described by Leblond and Clermont, 1952 and referred to the comprehensive reviews on the subject Russell, 1990; Creasy, 1997; Creasy, 2002. The PAS-H stained sections were used to identify the spermatogenic stages.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table below)
Samples of all tissues were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which were fixed in Modified Davidson's fluid and preserved in 70% ethanol)

HISTOPATHOLOGY: Yes (see table below)
After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.
In addition, the testes and epididymides of main group animals were cut at 2-3 micrometre thickness and stained with Periodic Acid Schiff (PAS). The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed on all animals in the control and high dose groups killed after 13 weeks of treatment.
The examination was performed as detailed below:
- Tissues specified in table from all animals in the control and high dose groups killed at the end of the 13 weeks of treatment.
- Tissues specified in table from all animals killed or dying during the treatment period.
- All abnormalities in all main phase groups.
The examination was then extended to include the liver from all animals of low and mid dose groups killed after 13 weeks of treatment and those of the recovery groups (control and high dose).
Statistics:
For continuous variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treated group and the control group were assessed by Dunnett’s test. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If data were found to be inhomogeneous a Modified t test (Cochran and Cox) was applied. The mean values, standard deviations and statistical analysis were calculated. Statistical analysis of histopathology findings was carried out by means of the non-parametric Kolmogorov-Smirnov test.

Results and discussion

Results of examinations

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
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
considered unrelated to treatment
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
considered of no toxicological importance
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Changes no longer observed at the end of the recovery period, therefore not considered to be adverse.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
mild treatment related findings in the liver of both sexes, and full reversible, therefore not considered to be adverse.
Details on results:
CLINICAL SIGNS AND MORTALITY
One female receiving 200 mg/kg/day was sacrificed for humane reasons (damaged hind limb) on Day 82 and one male of the same group was found dead on Day 40. The cause of death of this animal is not known.
Clinical signs in surviving animals were limited to rales and/or dyspnoea in a single female animal from Day 42 up to the end of the study.

BODY WEIGHT AND WEIGHT GAIN
No significant differences in body weights and body weight changes were noted between treated animals and controls during treatment and recovery periods.

FOOD CONSUMPTION
No treatment-related changes were observed in either sex during the experimental period.

OPHTHALMOSCOPIC EXAMINATION
No significant findings were detected at the ophthalmic examinations performed during the study.

HAEMATOLOGY
Dosing phase: Neutrophilia was recorded in some animals dosed with 500 mg/kg/day, males being more sensitive than females. Neutrophils mean group values were increased by 87% in males and 16% in females. Large unstained cells were also increased in males receiving 200 mg/kg/day and 500 mg/kg/day, with no dose-relation (32% and 23 %, respectively). The reason for this change is unclear. The statistically significant differences between control and treated animals recorded for mean corpuscular haemoglobin concentration in both sexes and eosinophils in females were of minimal severity and/or not dose- or sex-related, therefore considered unrelated to treatment.
Recovery phase: Neutrophilia was completely recovered in animals of both sexes after 4 weeks of recovery.

CLINICAL CHEMISTRY
Dosing phase: In general, changes of liver/metabolic parameters were recorded in animals dosed with 500 mg/kg/day and in males receiving 200 mg/kg/day. In particular, males dosed with 500 mg/kg/day showed increases of alkaline phosphatase, alanine aminotransferases, cholesterol, triglycerides and creatinine and decreases of bilirubin and glucose. Those receiving 200 mg/kg/day showed most of the above findings, often with less severity. Females from the same groups showed some of the above changes, such as increases of alkaline phosphatase, alanine aminotransferases, creatinine and decreases of glucose and bilirubin (see table #1 below). The other statistically significant changes recorded, such as: increase of protein, albumin and sodium in males, decrease of chloride and increase of sodium in females, were of minimal severity and/or not dose-related, therefore considered of no toxicological importance.
Recovery phase: The changes recorded during the dosing phase showed an almost complete reversibility. Total bilirubin and glucose were still slightly lower than control in some treated animals. However, due to the low severity and the absence of other related changes, the above findings were considered of no toxicological importance (see table #2 below).

URINALYSIS
No changes were recorded.

NEUROBEHAVIOUR
No differences between treated animals and controls, which could be considered of toxicological relevance, were observed at evaluations of sensory reaction performed at the end of treatment.
A slight reduction of grip strength was observed in the high dose animals of both sexes at the end of treatment and recovery periods. However, due the low magnitude, to the high individual variability of the measured data and to the absence of other correlated signs this variation was not considered of toxicological relevance.
Motor activity measurements performed at the end of the treatment or recovery periods did not show any toxicologically significant differences between treated animals and controls.

ORGAN WEIGHTS
A slight increase in the absolute weight of the liver was observed at the end of the treatment period in the males dosed at 200 and 500 mg/kg/day (+9% and +8 %, respectively) and in the females dosed at 500 mg/kg/day (+19 %, significant at statistical analysis). The relative weights of the liver were also significantly increased, at statistical analysis, in the mid- and high dose males (+6% and +9 %) and in the females from all treated groups (+9 %, +9% and +22 %). No other significant changes were observed at the end of treatment.

GROSS PATHOLOGY
No apparent tretament-related changes were noted. All observed changes were considered as incidental findings.

HISTOPATHOLOGY: NON-NEOPLASTIC
Final sacrifice: Treatment-related findings were found in the liver of both sexes treated with the high dose, and in the males treated with the mid-dose. The changes included: diffused centrilobular hepatocytic hypertrophy, which consisted of increased cytoplasmic eosinophilia and relative reduction in the cytoplasmic pallor (i.e., glycogen). The hypertrophy was of mild degree in males and of minimal degree in females, treated with the high dose, and of minimal degree in the males treated with the mid-dose.
All other observed changes had comparable incidence in the control and treated groups and/or are known to occur spontaneously in untreated Sprague Dawley rats of the same age, under our experimental conditions.
Recovery sacrifice: No treatment-related changes were noted

OTHER FINDINGS
Spermatogenic cycle: Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. No treatment-related changes were seen in the testes at spermatogenic staging.
Oestrus cycle: No significant differences in oestrous cycle were observed between treated and control animals.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs

Target system / organ toxicity

Key result
Critical effects observed:
not specified

Any other information on results incl. tables

Table #1: Clinical chemistry in dosing phase (control data expressed in absolute values and
data from treated groups as percentage differences with controls)
Treatment AP ALT BIL CHOL TRI GLU CREA
[mg/kg/day] [U/l] [U/I] [mg/dl] (mg/dl] [mg/dl] [mg/dl] [mg/dl]
0 (control) males 229.74 37.29 0.053 69.07 44.67 136.86 0.337
50 11 -5 -25 6 45 -10* 9**
200 65** 6 -25 17** 24 -13** 10**
500 241** 94** -51** 15** 76** -12** 17**
0 (control) females 175.99 33.39 0.072 96.48 35.19 112.37 0.403
50 9 9 -11 -4 -6 -4 7
200 38** 8 -6 -7 1 -8* 1
500 100** 46** -33 -1 6 -9* 8*
Table #2: Clinical chemistry in recovery phase (control data expressed in absolute values and
data from treated groups as percentage differences with controls)
Treatment AP ALT BIL CHOL TRI GLU CREA
[mg/kg/day] [U/l] [U/I] [mg/dl] [mg/dl] [mg/dl] [mg/dl] [mg/dl]
0 (control) males 172.02 46.80 0.08 69.30 61.82 158.12 0.34
500 -8 -13** -30 1 6 -7 -6
0 (control) females 120.10 44.20 0.090 87.36 73.80 120.74 0.404
500 -8 -10 4 -1 25 -8 3
* Indicates group mean is significantly different from control at level p < 0.05
** Indicates group mean is significantly different from control at level p < 0.01

Applicant's summary and conclusion

Conclusions:
Signs of treatment-related effects of the test item were observed in males and females dosed at 200 and 500 mg/kg/day. These effects included some changes in clinical pathology parameters and some post mortem findings. A main target organ, the liver, was identified on the basis of the organ weights data and histopathological findings. However, these changes were mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse.
No significant changes were observed in the animals dosed at 50 mg/kg/day. It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day. In the study there were not seen any indications for reproductive effects, investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.
Executive summary:

The toxicity of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was investigated in Sprague Dawley rats after daily oral administration at dose levels of 50, 200 and 500 mg/kg/day for 13 weeks and recovery from any treatment-related effects during a recovery period of 4 weeks.

One female receiving 200 mg/kg/day was sacrificed for humane reasons on Day 82 for a damaged hind limb and one male of the same group was found dead on Day 40. The cause of this last death cannot be clearly established.

No significant signs of toxic or neurotoxic effects were seen during the “in-life” phase of the study.

No lesions were recorded at ophthalmological examination.

No significant differences in oestrous cycle were observed between treated and control animals, no treatment-related changes were seen in the testes at spermatogenic staging.

Neutrophilia was recorded in both male and female rats dosed with 500 mg/kg/day.

Changes of liver/metabolic parameters (increases of alkaline phosphatase, alanine aminotransferase, cholesterol, triglycerides and creatinine and decreases of bilirubin and glucose) were recorded in animals dosed with 500mg/kg/day and in males receiving 200 mg/kg/day. The majority of the above changes were reversible at the end of the recovery period.

No effects in body weight or body weight changes were seen in any of the treated groups of rats.

The absolute and relative liver weights were slightly but significantly increased in the mid- and high dose males and in all treated females. This change was correlated with the hepatocytic hypertrophy seen at the end of treatment in males from these dose groups and in the high dose females. No changes were observed at the end of the 4 week recovery period.

Minimal to mild centrilobular hepatocytic hypertrophy was observed at microscopic examination in the males dosed at 200 and 500 mg/kg/day and in the females dosed at 500 mg/kg/day. These changes were no longer present at the end of recovery. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum (see Maronpot et al, 20106). No other changes were noted in the hepatocytes (i.e., degeneration and/or necrosis) and, therefore, the changes were considered as potentially adaptive.

No treatment-related changes were seen in the testes at spermatogenic staging.

On the basis of the above mentioned results, the liver was the main target organ. However, the changes observed at 200 and 500 mg/kg/day were minimal to mild and fully reversible. The morphological aspect of the hepatocytic hypertrophy was consistent with proliferation of endoplasmic reticulum. As such, the hepatic effects were not considered to be adverse. No significant changes were observed in the animals dosed at 50 mg/kg/day. In the study there were not seen any indications for reproductive effects, investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.

It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 500 mg/kg/day.