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Administrative data

Description of key information

In a subchronic OECD 408 study, groups of male and female Sprague Dawley rats were dosed with 0, 60, 250 or 1000 mg/kg/day TM812 (1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters) in corn oil using a dose volume of 10mL/kg for a period of 90 days. Additional satellite group of recovery animals were included to understand the post-exposure recovery period in control and Group 4 (1000 mg/kg/day) animals.


Reduce body weight gain was observed in males and female at 1000 mg/kg/day and additionally in females at 250 mg/kg/day.  However, during the recovery period, the body weight gains of the animals in dose group 1000 mg/kg bw/day recovered to normal.  There was no effect on food consumption during the study.  Mortality was observed in 3 male animals in low, mid and high dose groups which two were attributed to gavage errors.  The cause of death for the other animal remained unknown.


There were no changes in ophthalmic examinations, no change to nerve function, no hematological and no urinalysis changes. Clinical chemistry changes were limited to an increase in liver enzymes ALP, ALT, AST and cholesterol with a decrease in total protein, albumin, globulin and T-Bilirubin in 1000 mg/kg bw males.  Potassium concentration was increased in males dosed with 1000 mg/kg and globulin values were decreased females in 1000 mg/kg dose group. The above changes were recovered to normal after stop of dosing withing the four weeks' recovery period.  The results of the coagulation parameter's analysis indicated that the test item at the dose of 1000 mg/kg had decreased fibrinogen in the males, but the animals recovered after stop of dosing within the four weeks's recovery period.


There were no test item-related toxicity in organ weights, gross and macro or microscopic changes


The NOEL was considered to be 250 mg/kg bw for the males and 60 mg/kg bw for the females. There was no significantly delayed occurrence of toxic effects during the four weeks recovery period. Under the conditions of the study, there were no toxicity pathology changes in Sprague Dawley rats with the test item.  The no-observed-adverse-effect-level (NOAEL) is therefore considered to be 1000 mg/kg bw.


The subchronic oral toxicity of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was investigated in a well-conducted study 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. 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. The liver was identified as the main target organ on the basis of the organ weight 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 a subacute 28-day oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d.


The adrenals were identified as the main target organs, based on the post-mortem examination.


The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL.


No effects were observed at the low dose level (NOEL = 100 mg/kg/d).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
supporting study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
read-across source
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
Limit test:
no
Specific details on test material used for the study:
N/A
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
N/A
Sex:
male/female
Details on test animals or test system 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)
Route of administration:
oral: gavage
Details on route of administration:
N/A
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
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
50 mg/kg/day (low dose)
Basis:
actual ingested
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
200 mg/kg/day (medium dose)
Basis:
actual ingested
Dose / conc.:
500 mg/kg bw/day (nominal)
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
Positive control:
N/A
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).
Optional endpoint(s):
N/A
Other examinations:
N/A
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.
Clinical signs:
no effects observed
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
no effects observed
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
no effects observed
Description (incidence and severity):
N/A
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
Description (incidence and severity):
N/A
Urinalysis findings:
no effects observed
Description (incidence and severity):
N/A
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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.
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
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
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.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2014
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
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
Limit test:
no
Specific details on test material used for the study:
N/A
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
N/A
Sex:
male/female
Details on test animals or test system 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)
Route of administration:
oral: gavage
Details on route of administration:
N/A
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
Dose / conc.:
50 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
50 mg/kg/day (low dose)
Basis:
actual ingested
Dose / conc.:
200 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
200 mg/kg/day (medium dose)
Basis:
actual ingested
Dose / conc.:
500 mg/kg bw/day (nominal)
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
Positive control:
N/A
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).
Optional endpoint(s):
N/A
Other examinations:
N/A
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.
Clinical signs:
no effects observed
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
N/A
Body weight and weight changes:
no effects observed
Description (incidence and severity):
N/A
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
N/A
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
no effects observed
Description (incidence and severity):
N/A
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
Description (incidence and severity):
N/A
Urinalysis findings:
no effects observed
Description (incidence and severity):
N/A
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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.
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
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.
Key result
Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
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
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.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study accordingt o OECD Guideline and GLP
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
adopted October 3, 2008
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
N/A
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
N/A
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Harlan Italy s.r.l.
- Age at study initiation: 27-29 days
- Weight at study initiation: 88-107 g
- Fasting period before study: no
- Housing: 5 of one sex in clear polycarbonate cages with a stainless steel mesh lid and floor
- Diet: rodent diet (4 RF 21, Mucedola S. r. l., Via G. Galilei, 4, 20019, 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): 15-25
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: June 4 2009 To: July 16, 2009
Route of administration:
oral: gavage
Details on route of administration:
N/A
Vehicle:
corn oil
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 20, 60, and 200 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:
Samples were diluted with 2-propanol and an internal standard was added. The dilutions were then analysed by GC/FID.

Analysis of samples of the formulations prepared on day 1 and week 4 to check the homogeneity and concentration
Duration of treatment / exposure:
28 days
Frequency of treatment:
once daily
Dose / conc.:
100 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
100 mg/kg bw
Basis:
actual ingested
Dose / conc.:
300 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
300 mg/kg bw
Basis:
actual ingested
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Doses / Concentrations:
1000 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5

additional 5 animals per sex in high dose and control group as satellite groups for recovery assessment
Control animals:
yes, concurrent vehicle
Details on study design:
- Post-exposure recovery period in satellite groups: 2 weeks
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily for mortality and once daily for clinical signs

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and once a week thereafter
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 AND COMPOUND INTAKE (if feeding study):
- Food consumption by each cage of rats was recorded at weekly intervals and mean daily diet consumption calculated as g food/animal/day.


OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters checked: 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, eosinophils, basophils, monocytes, large unstained cells), platelets, prothrombin time


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Animals fasted: Yes
- How many animals: all animals
- Parameters checked: alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, creatinine, glucose, triglycerides, bile acids, phosphorus, total bilirubin, total cholesterol, total protein, albumin, globulin, A/G ratio, sodium, potassium, calcium, chloride


URINALYSIS: Yes
- Time schedule for collection of urine: at the end of week 4 of treatment and at the end of week 2 of the recovery period
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters checked: appearance, volume, specific gravity, pH, protein, total reducing substances, glucose, ketones, bilirubin, urobilinogen, blood; epithelial cells, leucocytes, erythrocytes, chrystals, spermatozoa and precursors, other abnormal components


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once before commencement of treatment and once a week thereafter
- Dose groups that were examined: all

OTHER:
- motor activity assessment, sensory reactivity to stimuli of different modalities and assessment of grip strength (once during week 4 of treatment and once during week 2 of recovery)
- hormone determination (T3, T4 and THS)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, post mortem examination including examination of the external surface and orifices; determination of organ weights (see table 1)

HISTOPATHOLOGY: Yes (see table 2)

Histopathological evaluation was performed on all males and females of the control group and of the high dose group (receiving the test item at 1000 mg/kg/day), sacrificed at the end of treatment. All abnormalities from all main phase animals were also examined.
Particular attention was paid to the gonads (ovaries and testes), accessory sex organs (uterus including cervix, epididymides, seminal vesicles with coagulation glands, dorsolateral and ventral prostate), vagina, pituitary, thyroid and adrenal glands for the evaluation of possible endocrine-related effects. In addition, as indicated by the international test program, synchronization of the oestrus cycle was also evaluated to better identify eventual disturbances on female sex hormone homeostasis.
The histopathological evaluation was performed following the suggestions provided by the OECD Test Guideline 407 (which refers to "Endocrine sisruption: A guidance document for histologic evaluation of endocrin and reproductive tests", Draft 3, May 16 2008).
On the basis of the pathological changes noted in the adrenals of high dose males and in the stomach of a few animals in the same treated group, the examination was extended to include the stomach, from males and females receiving the test item at dosages of 100 and 300 mg/kg/day, and the adrenals from the males dosed at 100 and 300 mg/kg and in the males killed after the recovery period.
Optional endpoint(s):
N/A
Other examinations:
N/A
Statistics:
For continued variables the significance of the differences amongst groups was assessed by analysis of variance. Differences between each treatment group and the control group were assessed by Dunnet's test. The homogeneity of the data was verified by Bartlett's test before Dunnet's test.
Description (incidence and severity):
N/A
Mortality:
no mortality observed
Description (incidence):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Urinalysis findings:
no effects observed
Description (incidence and severity):
The differences of specific gravity between control and treated females were of low severity. In the recovery phase no changes were observed.
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortality occurred during the study. Clinical signs were limited to hair loss, observed in a total of 6/10 females dosed at 1000 mg/kg/day from Day 8 of treatment up to the end of the recovery period.

BODY WEIGHT AND WEIGHT GAIN (see table 3)
A trend towards a decrease in mean body weight, compared to controls, (from 5% to 9%) was recorded in the males receiving 1000 mg/kg/day from Day 8 throughout the administration period, up to the end of the recovery period. No changes of note were seen in the other male groups and in treated females. The body weight reductions noted in all animals on Day 29, when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

FOOD CONSUMPTION
Slight reductions in food intake were observed in the males dosed at 1000 mg/kg/day from Day 8 (-17%) to Day 29 (-7%). No changes were seen in the other male groups and in treated females. No differences between treated animals and controls were observed at the end of recovery.
The above changes were not considered to be toxicologically relevant as they were slight and comparable to the historical control data.
The food consumption reductions, noted in all animals on Day 29 when compared to Day 22, were due to the overnight fast preceding the bleeding procedures for clinical pathology analyses.

HAEMATOLOGY (see table 4a and 4b)
Leucocytosis was recorded in animals treated with 1000 mg/kg/day and in females dosed with 300 mg/kg/day. The increase in white blood cells mainly involved the lymphocytes and was 25% to 60% above controls.
The other statistically significant changes recorded, such as an increase in erythrocytes, haemoglobin and haematocrit, observed in males treated with 300 and/or 1000 mg/kg/day, a decrease in reticulocytes, seen in those receiving 1000 mg/kg/day, and of prothrombin time seen in males treated with 100 mg/kg/day and in females dosed with 1000 mg/kg/day, were of low magnitude and inconsistent between sexes, therefore they were considered to be of no toxicological significance. In the recovery group leucocytosis was still present in females treated with 1000 mg/kg/day (increased by 27% compared with controls.

CLINICAL CHEMISTRY (see table 5)
A moderate to severe increase in alkaline phosphatase (+67% and 6-fold higher for males of the mid- and high dose groups, +5-fold for high-dose females) and bile acids (+9 and 3-fold in high dose males and females, respectively) and a decrease in globulin were observed in males treated with 300 and 1000 mg/kg/day and in females dosed with 1000 mg/kg/day. In addition, increments of alanine aminotransferase (2-fold), gamma-glutamyl transferase (3-fold) and decrements of bilirubin (-27%), protein and sodium were observed in males dosed with 1000 mg/kg/day.
Females from the same groups showed additional increments of alanine aminotransferase (2.8-fold), aspartate aminotransferase (+36%), gamma-glutamyltransferase (+29%) and urea (+33%) and decrements of bilirubin (-40%), protein, sodium and calcium. Some of the above mentioned changes could be related to the modifications seen in the adrenals recorded at the histopathological examination and to the related increase in corticosteroids. The other statistically significant changes were considered to be incidental as they were not dose-related and/or inconsistent between sexes .

Changes recorded during the dosing phase showed a complete reversibility in the recovery groups. Even though phosphorus, in males dosed with 1000 mg/kg/day, and urea, in females from the same group, did not decrease, their mean values were comparable with controls.
The statistically significant changes detected (triglycerides, chloride and sodium in males, aspartate aminotransferase in females) were not seen during the dosing phase, therefore they were considered to be incidental.

URINALYSIS
No changes of toxicological significance were recorded. The differences of specific gravity between control and treated females were of low severity. In the recovery phase no changes were observed.

NEUROBEHAVIOUR
No treatment-related changes were found at the weekly neurotoxicity evaluation.

ORGAN WEIGHTS (see table 6)
Slight but statistically significant increases in absolute (+16% and 33%) and relative (+29% and 34%) liver weights were recorded at the end of the treatment period in both males and females of the highest dose group in comparison with the respective controls. In addition, the absolute and relative adrenal weights were increased in the males dosed at 1000 mg/kg/day (+8% and 20%, respectively). These changes were found to be completely recovered after 2 treatment-free weeks. The toxicological significance of the above changes is supported by the correlation with the clinical and microscopic pathology findings. No other changes of toxicological significance were reported.

GROSS PATHOLOGY
The only relevant change noted in treated animals, when compared with controls, during the post mortem examination, was a minimal (on one occasion moderate) hair loss on the skin of the head, detected in 4/5 females dosed at 1000 mg/kg/day. In the recovery group hair loss was again noted on the forelimbs and head region of females dosed at 1000 mg/kg/day and sacrificed after the recovery period. The remaining changes observed as enlarged ovaries, distended with clear fluid content in the uterus, swollen spleen or red colour of the thymus are considered to be incidental in origin and characteristically seen in untreated Sprague Dawley rats of the same age, under our experimental conditions.

HISTOPATHOLOGY:

Treatment-related changes were seen in the adrenals of the high dose males. This change consisted of cortical cell hypertrophy associated with vacuolation, as fatty change, primarily involving the zona fascicolata.
No similar changes were noted in the males receiving the test item at 100 and 300 mg/kg/day, sacrificed at the end of the treatment period or in those sacrificed at the end of the recovery period.
The evaluation of the reproductive system of male (testis, epididymis, prostate, coagulating gland and seminal vesicles) and female (ovary, uterus and vagina) animals did not show any pathological alteration in the spermatogenesis as well no irregularity in the oestrus cycle.

Squamous hyperplasia of the forestomach mucosa associated with gastric inflammation was only detected in one male and one female, dosed at 1000 mg/kg/day. Males and females dosed at 100 and 300 mg/kg/day did not show any alteration in the stomach.
This sporadic pathological change could be considered incidental and probably related to an individual stress-related change.
The remaining lesions reported were considered as an expression of spontaneous pathology or as incidental findings, commonly observed in this species at this age and under our experimental conditions.


OTHER FINDINGS
No significant differences between treated animals and controls were observed at the evaluation of sensory reaction performed at the end of treatment. Motor activity measurements performed at the end of the treatment period did not show changes which could be ascribed to treatment.

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified
Table 3: Body weights of males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
day 1 212.17 ± 8.62 207.60 ± 7.77 201.87 ± 6.58 206.30 ± 7.82
day 8 261.78 ± 12.72 254.79 ± 8.80 247.26 ± 9.25 247.53 ± 110.39*
day 15 309.27 ± 15.05 298.20 ± 11.60 288.67 ± 12.22* 284.52 ± 11.81**
day 22 339.44 ± 17.19 327.37 ± 9.40 320.53 ± 16.65 311.11 ± 15.12**
day 29 340.78 ± 19.25 329.45 ± 12.26 317.14 ± 19.98 309.66 ± 10.99**
Recovery phase
Day 1 365.39 ± 21.24 326.39 ± 17.49*
Day 8 382.19 ± 23.58 340.30 ± 19.49*
Day 15 377.09 ± 24.86 337.03 ± 22.06*
*p < 0.05; ** p < 0.01
Table 4a: Haematology findings in males (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d
Red blood cells (*106/µl) 7.316 ± 0.133 7.574 ± 0.248 7.635 ± 0.162 7.798 ± 0.258**
Haemaglobin (g/dl) 14.06 ± 0.29 14.44 ± 0.44 14.83 ± 0.43* 15.04 ± 0.29**
Haematocrit (%) 40.24 ± 0.62 41.30 ± 1.42 42.25 ± 1.08* 43.44 ± 1.06**
Reticulocytes 137.04 ± 26.46 123.04 ± 6.83 1325.25 ± 18.78 104.14 ± 12.13*
White cell blood count (*103/µl) 11.148 ± 2.220 9.3486 ± 0.805 10.458 ± 2.149 13.282 ± 1.959
Lymphocytes (*103/µl) 8.712 ± 2.307 7.332 ± 0.880 8.425 ± 1.941 10.866 ± 2.217
*p < 0.05; ** p < 0.01
Table 4b: Haematology findings in females (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
White cell blood count (*103/µl) 6.208 ± 1.028 5.684 ± 1.362 9.174 ± 1.010** 9.930 ± 1.261** 7.222 ± 0.704 9.198 ± 1.196
Lymphocytes (*103/µl) 5.196 ± 0.756 4.752 ± 1.196 7.838 ± 0.838** 8.410 ± 1.090** 6.016 ± 0.616 7.808 ± 1.190*
*p < 0.05; ** p < 0.01
Table 5: Clinical chemistry findings (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Alkaline phosphatase (U/l) males 357.96 ± 28.25 398.90 ± 97.75 597.06 ± 106.54** 2212.06 ± 429.06** 276.30 ± 29.77 301.44 ± 15.90
females 287.54 ± 26.85 336.94 ± 93.45 361.86 ± 61.66 1421.66 ± 631.98* 184.30 ± 20.87 189.12 ± 26.00
Alanine Amino-transferase (U/l) males  51.52 ± 17.46 35.70 ± 5.23 43.38 ± 5.97 103.24 ± 17.68** 40.56 ± 2.93 41.74 ± 5.05
females 30.68 ± 12.08 26.42 ± 3.94 25.32 ± 4.21 86.06 ± 26.53* 31.40 ± 1.57 30.66 ± 3.45
Aspartate Amino-transferase (U/l) females 57.70 ± 6.29 57.26 ± 4.39 60.26 ± 4.94 78.40 ± 4.99** 84.10 ± 5.43 73.94 ± 3.59**
¿-Glutamyl transferase (U/l) males 1.34 ± 1.02 0.88 ± 0.28 1.98 ± 0.56 4.12 ± 1.45* 2.08 ± 2.00 2.56 ± 1.03
females 0.70 ± 0.23 0.78 ± 0.13 0.60 ± 0.19 0.90 ± 0.16 2.50 ± 1.31 1.86 ± 1.02
Bile acids (µmol/l) males 10.08 ± 5.72 9.10 ± 5.25 16.12 ± 3.88 89.96 ± 29.39** 20.46 ± 11.16 8.54 ± 5.26
females 15.46 ± 8.78 16.14 ± 10.07 5.06 ± 2.75 43042 ± 32.32 9.44 ± 8.02 7.68 ± 3.80
Bilirubin (mg/dl) males 0.188 ± 0.018 0.192 ± 0.033 0.188 ± 0.026 0.138 ± 0.026* 0.160 ± 0.034 0.170± 0.035
females 0.226 ± 0.024 0.186 ± 0.026 0.232 ± 0.022 0.136 ± 0.027** 0.216 ± 0.038 0.230 ± 0.014
Protein (g/dl) males 5.96 ± 0.05 5.90 ± 0.14 5.88 ± 0.30 5.24 ± 0.18** 6.48 ± 0.26 6.60 ± 0.55
females 5.42 ± 0.11 5.42 ± 0.23 5.58 ± 0.27 4.78 ± 0.26** 6.34 ± 0.15 6.24 ± 0.11
Globulin males 2.30 ± 0.12 2.20 ± 0.07 2.08 ± 0.16* 1.64 ± 0.09** 2.38 ± 0.52 2.66 ± 0.53
females 1.94 ± 0.15 1.92 ± 0.16 1.96 ± 0.15 1.50 ± 0.14** 2.32 ± 0.08 2.38 ± 0.08
Albumin/Globulin ratio males 1.60 ± 0.13 1.68 ± 0.08 1.83 ± 0.14* 2.20 ± 0.12** 1.82 ± 0.57 1.53 ± 0.30
females 1.80 ± 0.18 1.83 ± 0.15 1.85 ± 0.13 2.20 ± 0.16** 1.74 ± 0.11 1.62 ± 0.09
Urea (mg/dl) females 37.54 ± 5.56 43.48 ± 3.56 39.34 ± 5.30 49.88 ± 3.81** 66.14 ± 16.19 66.00 ± 1.97
Sodium (mmol/l) males 149.76 ± 1.20 152.00 ± 1.12 149.50 ± 1.34 145.92 ± 1.76** 148.22 ± 1.53 151.22 ± 1.05**
females 150.28 ± 1.64 150.62 ± 1.68 148.92 ± 2.27 146.62 ± 0.52** 150.16 ± 1.25 148.18 ± 1.54
Calcium (mmol/l) females 2.322 ± 0.077 2.310 ± 0.022 2.342 ± 0.047 2.200 ± 0.063** 2.582 ± 0.058 2.578 ± 0.047
*p < 0.05; ** p < 0.01
Table 6: Organ weights (mean values and standard deviations)
Group Control 100 mg/kg/d 300 mg/kg/d 1000 mg/kg/d Recovery control group Recovery group 1000 mg/kg/d
Liver (absolute) males 10.279 ± 1.304 9.713 ± 0.500 9.874 ± 0.957 11.960 ± 1.125* 9.981 ± 0.826 9.013 ± 0.997
females 6.201 ± 0.371 6.646 ± 0.693 6.395 ± 0.413 8.229 ± 0.401** 5.907 ± 0.526 6.299 ± 0.430
Liver (relative) males 3.009 ± 0.217 2.954 ± 0.106 3.118 ± 0.165 3.868 ± 0.246** 2.671 ± 0.103 2.688 ± 0.135
females 2.765 ± 0.080 2.960 ± 0.201 2.934 ± 0.052 3.697 ± 0.194** 2.627 ± 0.200 2.727 ± 0.100
Adrenal (absolute) males 0.0536 ± 0.0080 0.0510 ± 0.0022 0.0480 ± 0.0031 0.0580 ± 0.0056 0.0552 ± 0.0053 0.0506 ± 0.0059
females 0.0674 ± 0.0084 0.0664 ± 0.0060 0.0656 ± 0.0051 0.0662 ± 0.0056 0.0584 ± 0.0047 0.0688 ± 0.0066*
Adrenal (relative) males 0.0157 ± 0.0022 0.0155 ± 0.0005 0.0152 ± 0.0016 0.0188 ± 0.0013* 0.0148 ± 0.0018 0.0152 ± 0.0019
females 0.0301 ± 0.0038 0.0297 ± 0.0027 0.0301 ± 0.0024 0.0297 ± 0.0018 0.0261 ± 0.0028 0.0298 ± 0.0019*
*p < 0.05; ** < 0.01
Conclusions:
On the basis of the results, some treatment-related effects were evident in males and, to a minor extent in females at the high dose level of 1000 mg/kg/d of the structural related substance 1,2,4-Benzenetricarboxylic acid, decyl octyl ester. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d. No effects were observed at the low dose level (100 mg/kg/d).
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2016-02-16 - 2016-10-25
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
published by the Ministry of Environmental Protection of People's Republic of China in the year of 2013
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
N/A
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Specific Pathoge Free (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Liao Ning Chang Sheng Biological Technology Co., LTD.
- Age at arrival: 42-49 days
- Weight at arrival: 144-176g (males), 120-160g (females)
- Fasting period before study: overnight, but water was available
- Housing: Animals were raised in suspended, stainless steel cages (32cmx28cmx20cm) on cage racks (167cmx70cmx171cm). There were 10 cages per layer, and 4 layers per rack. Animals were housed two per cage during the whole test.
- Diet: sterilized diet with complete nutrition supplied by Beijing keaoxieli Feed Co., Ltd., ad libitum
- Water: was purified by HT-R01000 purity system, ad libitum
- Acclimation period: 11 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.4-24.7 (target value 20-25)
- Humidity (%): 33 - 80 % (target value 40-70 %)
- Air changes (per hr): not mentioned
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2016-02-17 (allocation of animals) To: 2016-05-18 (males) and 2016-05-19 (females) (last day of necropsy)
Route of administration:
oral: gavage
Details on route of administration:
N/A
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.
According to the dose design, calculate the theoretical amounts of test item and weigh in a beaker. Put a small amount of vehicle into the beaker, and transfer the test item into the graduated cylinder. Wash the inner surfaces of the beaker for at least three times. Transfer the vehicle from the beaker to the graduated cylinder and subsequently dilute wtih vehicle from the beaker to the graduated cylinder and subsequently dilute with vehicle to the scale mark. Make the test item mixed and the prepared formulations were divided into the grinding jars.
Dosing formulations were shaked before dosing to make the test item mixed. The dosing volumes were adjusted according to the recent body weights. Formulations were gavaged to animals' stomach by using a standard gavage tube (1G-20G) attached to a disposal syringe.

VEHICLE
- Justification for use and choice of vehicle (if other than water): no justification given
- Concentration in vehicle: 6, 25 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg bw

VEHICLE
- Lot/batch no. (if required): 13173SCD03

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
All formulations were stable within eight days in room temperature (15-25 °C) after preparation.
The concentration of the formulations were determined by analysis the week before the first week, the 8th week, the 13th week of dosing. The formulations samples were collected for 100 µL for analysis, and at least three paralleled samples were collected each time. The vehicle were collected for 100 µL for analysis, and at least two paralleled samles were collected each time.
The actual results for the analysis of the dosing formulation were within +- 15 % of the nominal concentration.
Duration of treatment / exposure:
3 consecutive months followed by a recovery period of 4 weeks for 6 males and 6 females from the control and high dose group
Frequency of treatment:
once a day, 7 days a week, 3 months
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
16 animals per sex (control and high dose group)
10 animals per sex for the other groups
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
The study dose designation was based on the pre-study results. The results indicated that animals showed expected gains in body weights during the test and there were not statistical different compared with the control group at the dose of 1000 mg/kg mean bw. Animals showed no clinical symptoms and death after dosing for 18 days during the test.
- Rationale for selecting satellite groups: no rational given
- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
N/A
Observations and examinations performed and frequency:
Clinical observations: yes
At least once a day at about 1.5 h after dosing, in the recovery period, the time should be preferably at the same time each day. The healthy conditions and toxicity signs were recorded. All animals were inspected for signs of morbidity and mortality at the beginning and the end of work at least once daily.

Detailed clinical observations were made for all animals prior to the first exposure (at grouping) and once a week after doing during the treatment period and during the recovery period. General clinical observations will not be made on the day of detailled clinical observations. The animals were taken outside the home cage for observation, and all the findings were detailed recorded. Observation included, changes in skin, fur, eyes, mucous membranes, occurence of secretions and excretions and autonomic activity (e.g. lacrimation, pilo-erection, pupil size, unusual respiratory pattern), changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypes (e.g. excessive grooming, repetitive circling) or bizzarre behaviors (e.g. self-mutilation, walking backwards).

Body weights: yes
Animals were weighed the day before dosing (at grouping), once a week during the treatment period and recovery period and at moribund or death. Animals were fasted overnight (16-16 hours) prior to necropsy and empty stomach body weighs were collected before necropsy.

Food consumption: yes
The ration food were added weekly, added food weight were 500 ± 10g (including the food box weight). The food and food boxes were weighted again one day (20h ± 1.5h) later as surplus food weight. Mean food consumption for one animal per day is calculated based on the above data. Calculation formulation: Mean daily food consumption (g) = (added food weight (g, including weight of food box) - surplus food weigth (g, including weight of food box))/2.

Ophthalmic Examinations:
Ophthalmic examinations were conducted on the high-dose and control group animals prior to the dosing and at the end of treatment.

Nerve Function Observation:
in the eleventh week of the exposure period, nerve function observation for recovery animals in control and high dose group were made to assess general behavior, sensory reactivity by different types of stimulation, grip strength and motor activity.

Clinical Examination
Blood collection: All survivng animals at terminal dosing and recovery period were anestheiszed by CO2 inhalation, and blood were collected via abdominal aorta for hematology, coagulation and serum biochemistry. All animals were fasted overnight before blood collection.

Hematology: Yes
1-2 mL blood were drawn from abdominal aorta, and collected into vacuum tubes containing EDTA-K2 as anticoagulant and stored in broken ice.
The following parameters were determined by sysmex XT-2000iv automated hematology analyzer:
Erythrocyte Cound (RBC), hemaglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), hemoglobin concentration (MCHC), Platelet count (PLT), Mean platelet volumen (MPV), Total Leukocyte (WBC), Lymphocytes, Monocytes, Neutrophils, Eosinophils, Basophils, Lymphocytes ratio etc.

Coagulation: Yes
About 2.71 mL blood were drawn from abdominal aorta, and collected into vacuum tubes containg 3.8 % trisodium citrate as anticoagulant and stored at room temperature. Samples were centrifuged in half hour after blood collected, and the plasmas were assayed for the following items with STA-Compact automatic coagulometer:
prothrombin time
activated partial thromboplastin
time
fibrinogen

Clinical Chemistry: Yes
2-3 mL blood is collected into vacuum tube with accelator/seperated gel. The samples were stored at room temperature for 5-10 min and then stored in broken ice. Samples were centrifuged in 1 hour after blood collected, and the serum were assayed for the following items with Hitachi 7180 automated chemistry analyzer.
Parameters: Aspartate, Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Cholesterol (CHO), Total Bilirubin (T-BIL), Total Protein (TP), Albumin (ALB), Globulin (GLB), Albumin/Globulin Ratio (A/G), Urea (UREA), Creatinine (CREA), Glucose (GLU), Tiglycerides (TG), Na+, K+

Urinalysis: Yes
Urine collections: Before necropsy of terminal dosing and recovery period urine samples were collected from animals that morbibund and all surviving animals by abdominal extrusion. Urinalysis is conducted to measure following parameters using Uritest 500B automaric urinalyzer and test paper. In addition, the appearance of urine were recorded but not statistic analyzed.
Parameters: Appearance, pH, Urine Specific Gravity (SG), Occult blood (BLD), Urine Ketones (KET), Urine Glucose (GLU), Urine total protein (PRO), Urine bilirubin (BIL), URO, Nitrites (NIT), Leukocytes (WBC), Vitamin (Vc)

Gross Necropsy: Yes
During the study, animals that died were subjected to a full gross necropsy and general observation, animals that surviving to the end of the study were euthanized by CO2 inhalation follwed by exsanguinations from abdominal aorta and subjected to a full necropsy and general observation. Any death during the study whichs is not carried on a gross necropsy timely were maintained at 2-8 °C refrigerated. The time interval between death and necropsy will not exceed 24 hour: eye examination, external body orifices, the abdominal, thoracic, and cranial cavities and their contents of all animals, and the location, size, hardness and the color of the abnormal findings were recorded.
The gross lesions found and brain (including cerebrum, cerebellum, brain-pons and medulla), spinal cord (including cervical, thoracic and lumbar), pituitary gland, submandibular gland, sublingual gland, tongue, esophagus, stomach, small and large intestines (includig duodenoum jejunum, ileum, cecum, colon, rectum, Peyer's patches), liver, kidney, adrenal glands, spleen, heart, thymus thyroid with parathyroid, pancreas (including pancreas islet), lung, trachea, aorta, ovary, uterus, vagina, testis, epididymis, prostate, urinary bladder, lymph node (mesentric, submandibular), skeletal musle (including sciatic nerve), sternum, thigh (including joint), skin (including female mammary gland).

Organ weights: Yes
At sacrifies, the following organs as brain, heart, liver, spleen, lung, kidneys, adrenal glands, thymus, testis, epididymis, ovaries, uterus were weighed. Organs were trimmed of any adherent tissue as appropriate, and their wet weight were taken as soon as possible after dissection to avoid drying. Organ-to-body weight ratios were calculated.


Sacrifice and pathology:
Preservation: The above tissues from each animal were preserved in 10 % neutral-buffered formalin (testis were preserved in Bouin's solution). Lungs were infused with fixative prior to immersion in 10 % neutral-buffered formalin. Stomach intestine and bladder were infused with formalin prior to immersion in 10 % neutral-buffered formalin. After dissection, residual carcases were packed in a plastic bag and maintained at refrigerator temporarily. Their corpse treatment were entrusted to specialized agencies.

Speciment fabrication processing: Tissues and organs collected from the control and top dose group of animals and gross lesions found in the other dose group animals were sectioned, fixed and dehydrated with alcohol, embedded in paraffin after fixed, sliced up and stained with hematoxylin and eosin, clarity with dimethylbenzene, sealed with rosin and examimed microscopically.

Histopathology: was examined on the preserved organs and tissues of all animals in the control and the high dose group, and animals that died during the study.

Optional endpoint(s):
N/A
Other examinations:
N/A
Statistics:
Parameters were analyzed by Bartlett test for variance homogeneity. In case of heterogeneity of variance (p>0.05), a Kruskal-Wallis Non-parametric analysis of variance pairwise comparison were used to evaluate the difference between each treated group and the control group. If the test were significant (p>=0.04), non-parametric Dunnett's test were then used for pairwise comparisons between each treated group and control group. Statistic analysis were finished when the test is non-significant (p > 0.05). The data of neurotoxicity were used SPSS 17.0.
In the case of homogeneity of variance (p >= 0.05), one-way analysis of variance (ANOVA) were used to analyze above parameters. If the test is significant (p <= 0.05) and the number of animals of each group is same, Dunnett's test were conducted pairwise comparisons between each treated group and the control group. If the test is significant (p<= 0.05) and the number of animals of each group is different, Duncan's test were conducted. Statistic analysis were finished when the test were non-significant (p>0.05). The data of nominal data of neurotoxicity used one-way analysis of variance.
The data of grade data of neurotoxicity and urinalysis and grade data of neurotoxicity were analyzed by Mann-Whitney U-test. The data of clinical sign were analyzed by X2, and the incidences of pathological findings (gross pathological findings, non-graded histopathological findings were analyzed by the Fisher's exact test (one-tailed prohability level).
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
All symptoms were considered unrelated to the treatment because they occured in the control and in the treatment group or occured only sporadically in low incidence throughout the study with no correlation to the treatment.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
During the whole test, one male animal died on week 8, 9, 13 at the low, mid and high dose respectively.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reversible effects:
Males: Animals at the dose of 1000 mg/kg bw/d showed a significant decrease in mean body weight on week 6th to the end of the treatment period compared with 0 mg/kg bw/d, at the end of the treatment period, the total body weight gains for 0 to 13 weeks were significantly decreased. During the recovery period, there were no statistical significances between the 1000 mg/kg bw/d and 0 mg/kg bw/d, and the body weight gains of the animals in the dose of 1000 mg/kg bw/d recovered to normal.
Females: Animals at the dose of 1000 mg/kg bw/d and 250 mg/kg bw/d showed a decrease in mean body weight from week 6th to the end of the treatment period compared with the 0 mg/kg bw/d, at the end of the treatment period, the total body weight gains for 0 to 13 weeks were significantly decreased. During the recovery period, there were no statistical significances between the 1000 mg/kg bw/d and 0 mg/kg bw/d, and the body weight gains of the animals in the dose of 1000 mg/kg bw/d recovered to normal.
The results of the body weight measurement indicated that the body weight gains for males were inhibited by the test item at the dose of 1000 mg/kg bw/d and the body weight gains for females were inhibited by the test item at the doses of 1000 and 250 mg/kg bw/d.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
During the whole test, animals in the treatment groups showed a significant increase and decrease in mean food consumption on individual weeks compared with the control group. There was no dose-response relation with no toxicological meaning. The results of the statistical analysis indicated that the amount of food consumed of all animals in treated groups were not influenced by the test item.
Food efficiency:
not examined
Description (incidence and severity):
N/A
Water consumption and compound intake (if drinking water study):
not examined
Description (incidence and severity):
N/A
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No treatment-related changes in the periocular areas of the eyes, conjunctiva, iris, cornea, opticdisk and blood vessel of the eye fundus.
Haematological findings:
no effects observed
Description (incidence and severity):
No abnormal findings were observed in all treated animals, also not at the end of the recovery group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Reversible:
The males at the dose of 1000 mg/kg bw/d showed a significant decrease in ALP, ALT, AST and CHO compared to the control. Among males TP; ALB, GLB and T-Bil values were statistically significantly decreased and the ratio of A/G was statistically increased at 1000 mg/kg bw/d. The concentration of K+ was statistically increased at 1000 mg/kg bw/d in the males. Among females, GLB values were significantly increased at 1000 mg/kg bw/d compared with the control.
At the end of the recovery period no abnormal effects were found, means all above changes were recovered to normal after stop of dosing within the four weeks recovery group.

Coagulation: The males of the 1000 mg/kg bw/d group showed a significant decrease in fibrinogen which was 15 % lower than the control. At the end of the recovery period no abnormal differences were observed.
Endocrine findings:
not examined
Description (incidence and severity):
N/A
Urinalysis findings:
no effects observed
Description (incidence and severity):
no differences between control and treatment groups occured during the whole test.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the nerve function observation during week 11th of the treatment period indicated no neurotoxicity was found related to the test item treatment.
Immunological findings:
not examined
Description (incidence and severity):
N/A
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
reversible:
low absolute values of thymus weights were observed in both males and females compared with the control at 1000 mg/kg bw/d. There was a statistically significant increase in liver, brain and kidney relative weights at 1000 mg/kg bw/d for males. There was no statistically significant increase in liver relative weights at 1000 mg/kg bw/d for females.
At the end of the recovery period no abnormal findings were observed in absolute and relative organ weights of the animals. All organ weight changes noted above were considered to be affected by the lower body weight and unrelated to treatment due to lack of dose-response and/or microscopic or histopathological findings/correlations.
Gross pathological findings:
no effects observed
Description (incidence and severity):
one case of lung nodule was observed at 250 mg/kg bw/d at the end of the treatment period. One case of lung adhesion was observed in the control group at the end of the recovery period.
dead animals: in 2 of 3 animals lung nodule was observed.
The results indicated that there were no signs of toxicity related to gavage of the test item in gross necropsy and macroscopic observations during the whole test.
Neuropathological findings:
not examined
Description (incidence and severity):
N/A
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
At the end of the treatment period: low incidance of urinary bladder mucuos plug, lung necrotic pneumonia, lung mineralization, lung inflammatory cells infiltration, liver vacuole degeneration, thyroid gland c cell proliferation, kidney tube dilatation, kidney mineralization, oesophagus inflammation, fore stomach erosion, fore stomach inflammatory cells infiltration, heat necrosis were observed in the control and the 1000 mg/kg bw/d dose group at the end of the treatment period and the recovery period. One case of lung necrotic pneumonia was found only in males at the dose of 250 mg/kg bw/d. At the end of the recovery group: low incidence of lung hemorrhage, lung necrotic pneumonia, liver vacuole degeneration, liver single cell necrosis, kidney inflammatory cells and lung inflammatory cells infiltration were observed in the control and the 1000 mg/kg bw/d dose group. Lung inflammatory cell infiltration was related to anesthesia and bloodeletting.
dead animals: no changes were found in one animal. One animal had lung necrotic pneumonia and heart necrosis, one animal had lung necrotic pneumonia and liver vacuole degeneration. There were no statistically significant differences and no dose-related response by comparison of the ratios of the above abnormal findings between the control and the 1000 mg/kg bw/d for both sexes. So no test-item related changes were observed in histopathology examinations.
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
N/A
Description (incidence and severity):
N/A
Details on results:
N/A
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
Remarks on result:
other: after the recovery period no effects were observed
Key result
Dose descriptor:
NOEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
body weight and weight gain
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
Key result
Critical effects observed:
no

Table 1: mean body weight variations (male)

 Week     Dose/mg/kg bw/d         
 0  60  250  1000
 0  254.4 +-7.7  253.4 +-11.7  254.7 +- 9.4  254.9 +- 8.9
 1  296.8 +- 16.7  295.8 +-14.8  298.0 +-14.3  296.4 +-10.7
 2  325.5 +-27.1  329.7 +-21.0  326.8 +- 18.7  317.1 +-13.4
 3  356.8 +-31.9  363.2 +-21.7  357.7 +-27.9  345.7 +-18.4
 4  389.7 +-34.0  387.9 +-26.9  387.2 +-32.4  372.1 +-17.3
 5  416.8 +-36.0  427.7 +-28.0  414.1 +-39.4  394.5 +-20.6
 6  442.6 +-38.6  444.3 +-31.0  436.7 +-46.5  414.1 +-20.8*
 7  462.2 +-38.6  467.3 +-33.8  449.2 +-65.4  431.0 +-24.4*
 8  479.4 +-41.4  488.3 +-39.2  484.8 +-45.0  442.3 +-25.8*
 9  489.9 +-41.9  504.2 +-41.1  504.9 +-45.2  460.4 +-26.1*
 10  517.0 +-41.5  524.7 +-46.1  519.2 +-49.8  474.9 +-28.6*
 11  529.7 +- 43.7 543.4 +-57.6   538.3 +-54.5  488.5 +-34.2*
 12  544.7 +-43.4 556.6 +-53.7   551.4 +-61.4**  493.5 +-26.1**
 13  557.1 +-44.7  567.3 +-56.5  556.3 +-52.5  507.5 +-33.9*
 0 ->13 (G)  302.6 +-45 .9  315.7 +-63.4  299.7 +-53.5  251.8 +-32.0
 14  533.5 +-40.4  -  -  500.9 +-44.5
 15  543.6 +-42.7  -  -  513.2 +-41.2
 16  544.7 +-40.8  -  -  522.5 +-44.0
 17  553.1 +-45.1  -  -  534.2 +-43.4
 0 ->17 (G)  291.8 +-47.5  -  -  280.5 +-43.1

Data: Mean+-SD (g), G (Gain weight (g); P*<=0.05, P**<=0.01, P***<=0.001

Table 2: mean body weight variations (female)

 Week     Dose/mg/kg bw/d         
 0  60  250  1000
 0  187.2 +-2.7  189.7 +-4.0  186.9 +- 5.4  188.9 +- 4.6
 1  205.8+- 6.1  202.5+-5.8  198.6 +-6.9**  202.3+-5.9
 2 219.3+-8.4  213.2+-12.5  210.5+- 4.4  214.6+-14.4
 3  233.4+-7.7  229.7+-12.6  220.9+-5.3**  224.9+-9.4*
 4  245.1+-12.9  240.3+-12.0  230.5+-8.7**  232.9+-12.0*
 5  256.2+-13.1  248.9+-12.2

241.6+-10.7**

 241.9+-12.9**

 6

 266.5+-15.8

 259.5+-11.8

 249.0+-10.0**

 249.3+-15.4**

 7

 275.6+-14.3

 264.8+-13.6

 255.1+-10.9**

 258.1+-15.3**

 8

 279.2+-16.5

 271.5+-14.3

 260.9+-11.0**

 260.0+-12.1**

 9

281.0+-15.6

 273.1+-17.5

 264.1+-13.4**

 263.4+-14.3**

 10

 290.0+-17.8

 278.4+-18.0

269.5+-12.4**

 269.6+-15.1**

 11

294.3+- 16.9

282.4+-17.5

 272.5+-12.5**

 275.3+-16.2**

 12

 296.5+-18.1

284.6+-18.5 

273.3+-13.4**

 277.0+-15.5**

 13

 299.7+-20.6

288.4+-19.1

278.9+-15.9**

281.0+-14.9*

 0 ->13 (G)

 112.5 +-20.4

 98.7+-18.9

92.0+-16.1**

92.0+-13.2

 14

301.4+-20.2

 -

 -

 284.1+-9.3

 15

 305.5+-14.6

 -

 -

287.9+-11.0

 16

308.2+-17.5

 -

 -

287.0+-11.0

 17

312.0+-21.2

 -

 -

294.3+-14.6

 0 ->17 (G)

 122.0+-21.0

 -

 -

 104.8+-12.1

Data: Mean+-SD (g), G (Gain weight (g); P*<=0.05, P**<=0.01, P***<=0.001

Table 3: Serum biochemical examination statistics results (at the end of the treatment period, males)

 Dose  Unit  0  60  250  1000
 Animal quantity      9  9  9
 ALP  U/L  167 ± 46  167±27 244± 112 422± 110**
 ALT   U/L  40±4 39±5 35±4  67 ±12*
 AST   U/L  76±11 73±9 72± 6 89± 12*
 Urea  mmol/L  3.47±0.39  3.83±0.57 3.58±0.71  3.78±1.05
 Crea  µmol/L  32±3 33± 4 33± 4 32 ±5
 T-Bil   µmol/L  0.3±0.3 0.1± 0.4  0.4±0.3 -0.2± 0.5*
 Glu  nmol/L  10.47±1.47 11.06± 1.87 9.83± 1.73  9.38±2.15
 CHO  nmol/L  1.57±0.22 1.62± 0.18 1.35±0.25 1.83± 0.32*
 TP  g/L  63.5±2.6 64.8± 2.4  62.5±2.8  57.3±2.3**
 ALB  g/L  39.9±1.4 40.9± 1.5  40.0±1.7  37.9±0.7**
 TG  mmol/L  0.70±0.17 0.92± 0.25  0.63±0.19  0.82±0.25
 K  mmol/L  5.89±0.44 5.93±0.52   6.49±0.61* 6.98 ±0.63**
 NA  mmol/L  145±2 145±1   145±1  146±3
 GLB  g/L  23.6±1.5  23.9±1.4  22.4±1.7 19.4 ±1.8**
 A/G    1.8±0.2 1.8± 0.1  1.8±0.2  2.0±1.2**

P*<=0.05, P**<=0.01, P***<=0.001

Table 4: Serum biochemical examination statistics results (at the end of the treatment period, females)

 Dose  Unit  0  60  250  1000
 Animal quantity      10  10 10
 ALP  U/L  63 ± 14  82  ± 48 79± 18 74±20
 ALT   U/L 31±9 29±6 31±8  29 ±6
 AST   U/L 59±12

61±12

59±11

60±9

 Urea

 mmol/L

 5.99±1.11

6.55±1.58

5.88±1.48

 5.68±1.16

 Crea

 µmol/L

 37±5

45±94

42± 10

38 ±6

 T-Bil

  µmol/L

 0.3±0.4

0.5± 0.4

 0.5±0.2

0.6± 0.4

 Glu

 nmol/L

 8.65±1.32

9.38± 0.93

9.44± 1.04

 9.33 ± 0.61

 CHO

 nmol/L

 1.86±0.31

1.85± 0.32

1.84±0.25

1.96± 0.24

 TP

 g/L

 68.6±3.9

68.9± 2.9

 69.4±2.8

 66.8±3.2

 ALB

 g/L

 43.7±2.6

43.8± 2.2

 43.9±2.4

 43.8±1.5

 TG

 mmol/L

 0.37±0.09

0.40± 0.11

 0.38±0.08

 0.43±0.13

 K

 mmol/L

 5.64±0.45

5.30±0.46 

5.47±0.34

5.81 ±0.50

 NA

 mmol/L

 140±2

141±1 

 141±1

 142±1

 GLB

 g/L

 24.9±1.5

 25.1±1.5

25.5±1.1

23.0 ±1.9*

 A/G

 

 1.8±0.1

1.8± 0.1

 1.7±0.3

 1.9±0.1

P*<=0.05, P**<=0.01, P***<=0.001

Table 5:

Serum biochemical examination statistic results (at the end of the recovery group, males)

 Dose  Unit  0  1000
 Animal quantity     6
 ALP  U/L  94 ±22 110±22
 ALT   U/L 44±8 43 ±6
 AST   U/L 78±24

82±10

 Urea

 mmol/L

 5.18±0.83

 5.51±0.48

 Crea

 µmol/L

 34±5

33 ±3

 T-Bil

  µmol/L

 0.±0.3

0.6± 0.2

 Glu

 nmol/L

10.46±0.91

 9.85 ± 1.10

 CHO

 nmol/L

 1.85±0.47

1.77± 0.35

 TP

 g/L

 65.6±1.9

 65.4±1.3

 ALB

 g/L

 40.2±0.9

 40.3±0.3

 TG

 mmol/L

 0.47±0.17

 0.58±0.12

 K

 mmol/L

 6.20±0.49

6.03±0.27

 NA

 mmol/L

 143±2

 145±1

 GLB

 g/L

 25.5±1.7

25.1 ±1.4

 A/G

 

 1.50±0.3

1.7±0.2

P*<=0.05, P**<=0.01, P***<=0.001

Table 6:

Serum biochemical examination statistic results (at the end of the recovery group, females)

 Dose  Unit  0  1000
 Animal quantity    6 6
 ALP  U/L  67±14 50±12
 ALT   U/L

45±13

34±10

 AST

  U/L

88±28

71±17

 Urea

 mmol/L

 6.60±1.51

 7.20±1.76

 Crea

 µmol/L

40±6

36 ±3

 T-Bil

  µmol/L

 0.7±0.1

0.8± 0.3

 Glu

 nmol/L

9.93±1.96

 9.48 ± 1.41

 CHO

 nmol/L

 2.47±0.33

2.59± 0.36

 TP

 g/L

71.8±2.2

 75.2±2.7

 ALB

 g/L

 44.7±1.0

 46.6±1.6

 TG

 mmol/L

 0.47±0.04

 0.54±0.14

 K

 mmol/L

 6.11±0.50

5.83±0.28

 NA

 mmol/L

 141±2

 

141±2

 

 GLB

 g/L

 27.1±1.4

28.5±1.5

 A/G

 

 1.70±0.2

1.6±0.1

P*<=0.05, P**<=0.01, P***<=0.001

Conclusions:
According to the results the no-observed-effect-level (NOEL) for the test item 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters in repeated dose 90 -day oral toxicity study in SD rats under the condition of the study was considered to be 250 mg/kg bw for the males and 60 mg/kg bw for the females. There was no significantly delayed occurence of toxic effects during the four weeks recovery period. Under the conditions of the study, there were no toxicity pathology changes in SD rats with the test item. The no-observed-adverse-effect-level (NOAEL) is therefore considered to be 1000 mg/kg bw.
Executive summary:

The study was performed to assess the toxicity of 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters in Sprague Dawley rats according to OECD 408.

There were four groups at different dose levels, including the control group (0 mg/kg bw/d) and the test item groups (60; 250 and 1000 mg/kg bw/d). Animals in the three treatment groups were administered by the concentrations of 6 mg/ml, 25 mg/mL and 100 mg/ml of test item solution by the concentration of 6 mg/mL, 25 mg/mL and 100 mg/mL of test item solution by gavage once daily for three months. Animals in the control group were administered by the vehicle once daily for three months. Dosing volume was 10 ml/kg bw. The concentration of the formulations were determined by analysis the week before the first week, the 8th week, the 13th week of the dosing. Clinical symptom observation, body weight measurement, the food consumption measurement, opththalmic examinations, nerve function observation and the clinial pathology including urinalysis, hematological, coagulation, blood biochemistry, necropsy and anatomic pathology and histopathology were carried out after the end of the dosing period and recovery period.

The concentration of the formulations were determined by analysis the week before the first week, the 8th week, the 13th week of the dosing, and the actual results for the analysis of the dosing formulation were within +- 15 % of the nominal concentration. During the whole test, food consumption was not influenced by the gavage of the test item. The results of the body weight measurement indicated that the body weight gains for males were inhibited by the test item at the dose of 1000 mg/kg bw/d and the body weight gains for females were inhibited by the test item at the dose of 1000 mg/kg bw/d and 250 mg/kg bw/d. During the recovery period, there were no statistical significances between the 1000 mg/kg bw/d and 0 mg/kg bw/d, and the body weight gains of the animals in the dose of 1000 mg/kg bw/d recovered to normal. The ophthalmic examinations results indicated that there were no treatment-related changes in the periocular area of eyes, conjunctiva, iris, cornea, opticdisk and blood vessel of the eye fundus. The results of the nerve function observation during the eleventh week of the exposure indicated that no neurotoxicity was found related to the test item. The results of the hematological parameter's statistical analysis indicated that there were no treatment-related findings during the study. The serum biochemical statistical analysis indicated that ALP, ALT, AST and CHO values were increased and TP, ALB, GLB and T-Bil values were decreased in 1000 mg/kg bw for males. The concentration of K+ was increased by the test item at 1000 mg/kg dose in the males. GLB values were decreased in 1000 mg/kg dose group in the females. The above changes were recovered to normal after stop of dosing withing the four weeks' recovery period. The results of the coagulation paramether's analysis indicated that the test item at the dose of 1000 mg/kg had decreased FIB in the males, but the animals recovered after stop of dosing within the four weeks's recovery period. No differences between control and treatment groups of toxicological meaning were found during the whole test in hematological parameter and urinalysis. No test item-relaxed toxicity changes were found in organ weight, gross autopsy and macroscopic observation. Under the conditions of this study, there were no toxicity pathology changes in SD rats with the test item. During the whole test, one male animal was dead on week 8th, 9th, 13th at the low, mid and high dose respectively. To sum up the clinical observations, macroscopic observation and the histopathology results, two of the animals were dead by the reason of gavage trauma which casued the lesions in the lung. The cause of death for the other animal remained unknown. Futhermore there were not dose correlations and considered to be unrelated to the test item.

According to the above results the no-observed-effect-level (NOEL) for the test item 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters in repeated dose 90 -day oral toxicity study in SD rats under the condition of the study was considered to be 250 mg/kg bw for the males and 60 mg/kg bw for the females. There was no significantly delayed occurence of toxic effects during the four weeks recovery period. Under the conditions of the study, there were no toxicity pathology changes in SD rats with the test item. The no-observed-adverse-effect-level (NOAEL) is therefore considered to be 1000 mg/kg bw.

Under the conditions of the study, there were no toxicity pathology changes in SD rats with the test item.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

In a subchronic OECD 408 study, groups of male and female Sprague Dawley rats were dosed with 0, 60, 250 or 1000 mg/kg/day TM812 (1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters) in corn oil using a dose volume of 10mL/kg for a period of 90 days. Additional satellite group of recovery animals were included to understand the post-exposure recovery period in control and Group 4 (1000 mg/kg/day) animals.


Reduce body weight gain was observed in males and female at 1000 mg/kg/day and additionally in females at 250 mg/kg/day.  However, during the recovery period, the body weight gains of the animals in dose group 1000 mg/kg bw/day recovered to normal.  There was no effect on food consumption during the study.  Mortality was observed in 3 male animals in low, mid and high dose groups which two were attributed to gavage errors.  The cause of death for the other animal remained unknown.


There were no changes in ophthalmic examinations, no change to nerve function, no hematological and no urinalysis changes. Clinical chemistry changes were limited to an increase in liver enzymes ALP, ALT, AST and cholesterol with a decrease in total protein, albumin, globulin and T-Bilirubin in 1000 mg/kg bw males.  Potassium concentration was increased in males dosed with 1000 mg/kg and globulin values were decreased females in 1000 mg/kg dose group. The above changes were recovered to normal after stop of dosing withing the four weeks' recovery period.  The results of the coagulation parameter's analysis indicated that the test item at the dose of 1000 mg/kg had decreased fibrinogen in the males, but the animals recovered after stop of dosing within the four weeks's recovery period.


There were no test item-related toxicity in organ weights, gross and macro or microscopic changes


The NOEL was considered to be 250 mg/kg bw for the males and 60 mg/kg bw for the females. There was no significantly delayed occurrence of toxic effects during the four weeks recovery period. Under the conditions of the study, there were no toxicity pathology changes in Sprague Dawley rats with the test item.  The no-observed-adverse-effect-level (NOAEL) is therefore considered to be 1000 mg/kg bw.


The subchronic oral toxicity of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was investigated in a well-conducted study 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. 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. The liver was identified as the main target organ on the basis of the organ weight 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, those investigated were spermatogenic staging, oestrus cycle, weights of ovaries and testes, including microscopic and macroscopic observations.In a subacute oral toxicity study in rats some treatment-related effects were evident in males and to a minor extent in females at the high dose level of 1000 mg/kg/d. The adrenals were identified as the main target organs, based on the post-mortem examination. The observed effects were completely reversible over a 2-week recovery period in the high dose animals. Only mild effects were observed in the animals (essentially males) dosed at 300 mg/kg/d, therefore this dose is expected to be NOAEL. No effects were observed at the low dose level (NOEL = 100 mg/kg/d). In the subacute 28 -day study, a dose of 500 mg/kg bw/d was not used but adverse effects were seen at 1000 mg/kg bw/d (LOAEL). Therefore for repeated dose toxicity, it can be concluded in summary that the NOAEL is 500 mg/kg bw/d.

Justification for classification or non-classification

The results of the repeated dose oral toxicity study on 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters does not warrant classification according to EU regulations (Directive 67/548/EEC and Regulation (EC) No 1272/2008.