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

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

30th of July 2015 - 7th of September 2015

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 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

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:

mouse

Strain:

ICR

Details on species / strain selection:

N/A

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory animal technology Co., Ltd.
- Initial age at start of acclimatisation: 49-62 days
- Age at start of treatment: 55-68 days
- Weight at study initiation: 29.95-34.61 g (males)
- Assigned to test groups randomly: yes
- Fasting period before study: not mentioned
- Housing: 1 animal per cage (plastic cages (L29.0×W18.0×H16.0cm) lied on shelf (L167.0×W70.0×H171.0cm) in the SPF grade barrier system (room D124). There were 7 cages per layer, and 5 layers per rack)
- Bedding: Corn cob bedding was supplied by Beijing Keao Xieli Feed Co., Ltd. (Batch No.: 15049811)
- Diet (e.g. ad libitum): ad libitum (SPF rodent growth and breeding feed bought from Beijing Keao Xieli Feed Co., Ltd (Product License No: SCXK (jing) 2012-0001, Batch No. was 15033113)
- Water (e.g. ad libitum): ad libitum (drinking water, purified by HT-R01000 purity system)
- Acclimation period: adequate duration, not specified

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.8-24.4
- Humidity (%): 54 - 70
- Air changes (per hr): 17
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Justification for choice of solvent/vehicle: relatively non-toxic to the animals, result of solubility test
- Amount of vehicle: 0.2 mL/10 g bw
- Lot/batch no. (if required): MKBF8603V

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
4.000g/4.000g (the first administration/ the second administration) of the test item were weighed using the electronic balance and mixed with vehicle to the certain volume (40mL) as the concentration of 100mg/ml, then the suspension were continuously mixed round with homogenate machine for one minute at least. The test suspension of other concentrations were prepared by dilution by factor 2.
All test suspensions were prepared just before on the day of each administration. When prepared, the test item was weighed accurately and mixed with the vehicle thoroughly, in addition, the test item was applied to the test system within two hours after being formulated. It is assumed that the formulation was prepared exactly, homogeneously and was stable during the test. So no analysis was carried out to determine the homogeneity, concentration and stability of the test item formulation.

The order of administration was 0, 500mg/kg, 1000mg/kg, 2000mg/kg and CP (cyclophosphamide monohydrate = positive control) group. The accuracy of injector used was 0.02mL and the max volume was 1mL. Dosing formulations and vehicle were stirred on the magnetic stirrer at least 5 minutes prior to dosing and continuously stirred during dosing. At the first administration, academic dosing volume was calculated based on the body weight as grouping, and the corresponding volume based on the accuracy of injector was dosed to the animal. At the last administration, animals were weighed firstly, then academic volume was calculated and corresponding volume based on the accuracy of injector was dosed to the animal. All data during the treatment were recorded accurately.

Duration of treatment / exposure:

N/A

Frequency of treatment:

twice with the test item with an interval approximately 24 hours

Post exposure period:

none

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 (6 (3 males and 3 females) in pre-experiment)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide monohydrate (CP)
- Justification for choice of positive control(s): not mentioned
- Supplier: Sigma-Aldrich
- Batch no.: SLBG4216V
- Doses / concentrations: 50 mg/kg bw, single dose (2.5 mg/mL)

In this test, cyclophosphamide monohydrate (CP) was used as positive control item at the dose level of 50mg/kg (the concentration was 2.5mg/mL). 0.025g of CP was weighed and prepared in 10mL water as the positive control solution. The CP solution was prepared on the day of each treatment.

Examinations

Tissues and cell types examined:

N/A

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
Prior to the start of this study, a preliminary test had been performed to determine the maximum tolerated dose (MTD) in this lab. The test item came from the same supplier (Batch No.: 04698/MA; Content:＞98%). Based on the result of the preliminary test, if the test item did not produce toxicity at 2000 mg/kg/body weight/day, the highest dose for an administration period should be 2000 mg/kg/body weight/day in this micronucleus test. However, if the test item does cause toxicity, the maximum tolerated dose (MTD) should be the highest dose administered in this micronucleus test.
In the preliminary test, the same species animals with the micronucleus test were used. Before administration, based on the result of solubility of the test item and designed dose, the test item was prepared to suspend in corn oil. Because corn oil was used as vehicle, the route of administration by gavage was used. In the preliminary test, two dose levels were set, include: 2000 and 1000 mg/kg/body weight/day. There were three males and three females in each group. The animals were dosed twice with an interval of 24 hours. The dose volume of 0.2ml/10g bw by gavage. After the second administration, all the animals continued to be observed one day. The results indicated that all animals had no obvious toxic symptoms or were death during the administration of the test item.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Sampling of peripheral blood was carried out on all animals (7 per dose group)

DETAILS OF SLIDE PREPARATION:
All animals were sacrificed by cervical dislocation nearly 20 hours after the second administration. Bone marrow from sternum was harvested and mixed with a drop of fetal calf serum on a side of a slide. Then the slides were smeared. After air-dried, the slides were fixed in methanol for nearly 10 minutes, and stained with Giemsa’s solution for nearly 30 minutes, flushed and air dried.

METHOD OF ANALYSIS:
The slides were analysed by the manual method using microscopy. All slides were coded randomly so the manual scorer was unaware of the treatment condition. 2000 polychromatic erythrocytes (PCE) were scored per animal for the number of micronucleated polychromatic erythrocytes (MNPCE) and the incidence of micronuclei was shown in permillage. At the same time, the PCE in 200 red blood cells (RBC) were scored and the ratio between PCE and RBC was calculated for each animal.
Observed under immersion objective, both PCE and NCE (normochromatic erythrocytes) are anucleate cells. After Giemsa staining, PCE shows bice and NCE shows light jacinth.
Identification of micronuclei:
Circle is the main form, and oval, ring, hemicycle is found occasionally. Outline of micronuclei is clear and flat. The staining of Micronuclei should be same as the nucleus of an adjacent nucleated cell.
Usually, the upper limit is half of a diameter of an erythrocyte. PCE with two or more micronuclei is counted as one MNPCE.

Evaluation criteria:

If the incidences of MNPCE in the treated groups show statistically significant increase (P<0.01) compared to the values of the NG and the increase is dose-related or very clear in one dose level, the results are deemed as positive.
The result is evaluated as negative if none of the above criteria is met.
Where small significant increase in the incidences of MNPCE in the treated groups occurs, additional cells may be scored and the biological relevance of the result will be considered. Results of this type will be reported as equivocal if the results maintain unclear.

Statistics:

Before each administration and sampling, the mean and standard deviation of the body weight in each group was calculated, and plotted graphically respectively; the ratio between PCE and RBC of each animal was calculated, and the mean and standard deviation of the ratios in each group were calculated at the same time (the ratio in the treated groups should not be less than 20% of NG (control). The incidence of MNPCE of each animal was calculated, and the mean and standard deviation in each group were calculated at the same time, then the data were evaluated with two tailed t-test method in Excel to observe if there were statistical significant differences in the treated groups and CP group (positive control) as compared to the value of NG.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 mg/kg bw and 1000 mg/kg bw
- Clinical signs of toxicity in test animals: none observed
- Evidence of cytotoxicity in tissue analyzed: no tissue analyzed
- Rationale for exposure: 2000 mg/kg was chosen as MTD


RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay): No biologically relevant increase of micronuclei was found, nor were any significant differences between the negative control groups, dose groups and the historical control data found.
- Ratio of PCE/NCE (for Micronucleus assay): No significant difference between the negative control groups, dose groups and the historical control data found.
- Appropriateness of dose levels and route: Both, MTD and oral administration, were appropriate to investigate the genotoxic potential of the test item. The positive control groups showed a significantly increased micronucleus frequency.
- Statistical evaluation: No statistically significant differences between the negative control groups and the dose group were found.

Any other information on results incl. tables

Table 1: Statistics results of microscopic analysis

Group (mg/kg)	Animal number	Number of PCE	MNPCE/PCE (‰) Mean±SD	P value	PCE/RBC Mean±SD
0	7	14000	2.3±0.6	-	0.55±0.03
500	7	14000	2.2±0.6	> 0.05	0.59#±0.07
1000	7	14000	2.3±0.7	> 0.05	0.58#±0.04
2000	7	14000	1.8±0.9	> 0.05	0.54#±0.08
CP	7	14000	25.9±3.8	<0.01*	0.47#±0.04

Note: * statistically significant difference compared to negative control (P<0.01);

# the ratio was more than 20 percent of the ratio in the negative control group.

During the test, the change of animal body weights was determined, there was no obvious decrease as compared with the concurrent NG during the administration.

The clinical observation results showed that all animals had no toxic symptom and death.

Applicant's summary and conclusion

Conclusions:

Under the conditions of this study, the results were negative, so the study suggested that 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.

Executive summary:

This study was conducted to detect the possibility that 1,2,4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters could induce the increase of the incidence of micronucleated polychromatic erythrocytes (PCE) in mice, in order to provide genotoxicity related data for the test item according to OECD 474. This study was conducted in ICR mice. All animals were SPF grade. According to the related information about the test item and the results of the preliminary test, in the micronucleus test, the animals were treated at 2000, 1000 and 500mg/kg. Negative control group (NG, corn oil) and positive control (Cyclophosphamide, CP, 50mg/kg) group were performed at the same time with the same method. All animals were randomly grouped based on body weight with 7 male animals in each group. Mice were administered the test item orally by gavage twice, with a 24 hours interval between doses. All animals were sacrificed by cervical dislocation nearly 20 hours after the last administration. Bone marrow from sternum was harvested. Bone marrow smear was prepared and analysed with microscope. The data were evaluated with t-test (two-tail) in Excel.

During the administration, no obvious decrease in the body weights of the mice was found in all designed dose groups as compared with the NG. No animal exhibited obvious toxic symptoms and death during the administration.

The frequencies of micronucleated PCE were 2.3±0.6‰ in the negative control group, 2.2±0.6‰ in the 500mg/kg group, 2.3±0.7‰ in the1000 mg/kg group, 1.8±0.9‰ in the 2000mg/kg group and25.9±3.8‰ in the CP group. The results of t-test indicated that statistically no significant difference (P>0.05) in the incidence of micronucleated PCE was found in the treated groups as compared with NG. At the same time, a statistically significant difference (P<0.01) in the incidence of micronucleated PCE was found in the CP group as compared with NG. Furthermore, the PCE/RBC ratios in all treated groups and positive control group were more than twenty percent of the ratio in NG.

Under the conditions of this study, the results were negative, so the study suggested that 1, 2, 4-Benzenetricarboxylic acid, mixed dodecyl and octyl triesters did not induce an increase of the incidence of micronucleated PCE in mice.