1,2,4-Benzenetricarboxylic acid, mixed lauryl and octyl triesters

Repeated dose toxicity: oral

Administrative data

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

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2016

Report date:

2016

Materials and methods

Test guideline

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

Test material

Test material information

Constituent 1

Reference substance name:

1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octy triesters

IUPAC Name:

1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octy triesters

Constituent 2

Reference substance name:

tris(mixed dodecyl and octyl)benzene-1,2,4-tricarboxylate

EC Number:

700-342-7

Cas Number:

1163775-81-2

Molecular formula:

C14H47O6 to C18H55O6

IUPAC Name:

tris(mixed dodecyl and octyl)benzene-1,2,4-tricarboxylate

Constituent 3

Reference substance name:

Linplast 812 TM

IUPAC Name:

Linplast 812 TM

Specific details on test material used for the study:

N/A

Test animals

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)

Administration / exposure

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

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

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

Examinations

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).

Results and discussion

Results of examinations

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

Effect levels

1

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

2

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

3

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

Target system / organ toxicity

Key result

Critical effects observed:

no

Any other information on results incl. tables

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

Applicant's summary and conclusion

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.