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

Developmental toxicity / teratogenicity

Administrative data

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2016

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 414 (Prenatal Developmental Toxicity Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

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 2

Reference substance name:

1,2,4-Benzenetricarboylic acid, mixed dodecyl and octyl triesters

IUPAC Name:

1,2,4-Benzenetricarboylic acid, mixed dodecyl and octyl triesters

Constituent 3

Reference substance name:

Linplast 812 TM

IUPAC Name:

Linplast 812 TM

Details on test material:

Smiles notation (if other than submission substance): O=C(c1ccc(cc1C(=O)OCCCCCCCC)C(=O)OCCCCCCCC)OCCCCCCCCCCCC

Specific details on test material used for the study:

N/A

Test animals

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Beijing Vital River Laboratory, China
- Age at study initiation: female approx. 84-97 days
- Weight at study initiation: 199-265 g female, males 359-445 g
- Fasting period before study: no
- Housing: the test was conducted in the barrier system. Male rats were housed in the suspensory stainless cages (32cmx28cmx20cm) fixed on the shelf (167cmx70cmx171cm). There are 4 layers on the frame and 10 cages per layer. Female rats were housed in plastic cages (46cmx 31.5cmx20 cm). There were 2 rats at most in per cage. Bedding: The corn cob bedding from Beijing Keao Xieli Co, Ltd was used for the animals.
Diet: sterilized diet with complete nutrition supplied by Beijing keao Xieli Feed Co., Ltd., ad libitum
Water: was made in that center and was checked daily, ad libitum

- Acclimation period: 12 days before the start of the treatment

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

IN-LIFE DATES: From 2015-09-10 To: 2015-10-30

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Remarks on MMAD:

N/A

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The required amount of 1,2,4-Benzenetricarboxylic acid, decyl octyl ester was dissolved/suspended in the vehicle corn oil. 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: 25, 75, 250 mg/ml
- Amount of vehicle (if gavage): 4 ml/kg bw

Analytical verification of doses or concentrations:

yes

Remarks:

study nr. G1563B0010

Details on analytical verification of doses or concentrations:

Concentration of the prepared test item was conducted at the first and the last preparation. The prepared test items were considered as solution according to their properties, so the analytical check for homogeneity was not performed in this test. As being checked the prepared test items of different concentrations and the vehicle control were sampled. After the treatment, each concentration was analyzed for three times.

In the study, the dose formulations were sampled accurately and diluted with linear range (4.785-76.56 mg/L) and determined with GC/FID. The results showed that the analytical concentrations of at least two-thirds samples of each concentration were within the range of 85-115% of the theoretical concentrations, so it was considered the concentrations of the prepared test item had met the requirements of the study.

The results of the determined concentrations were in accordance with the study plan.

Details on mating procedure:

During the mating period, two females and one male rat were put together after 16:00 each day and separated in the next morning. Then the female rats were examined for the presence of sperm or vaginal plug using vaginal smear method. The day of finding the sperm or vaginal plug was defined as Day 0 of gestation (GD0). The female rats that failed to mate were mated again until the number of pregnant rats met the number of required for the test.

Duration of treatment / exposure:

days 5 to 19 post coitum

Frequency of treatment:

once daily

Duration of test:

up to day 19 of gestation

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

300 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

vehicle control

No. of animals per sex per dose:

24

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels were in agreement with the Sponsor based on a previous study in rats
The results in the preliminary test indicate that, no significant toxic symptoms or death were observed in the dose group of 1000 mg/kg bw/d, and the body weight change of the animals has no obvious decrease compared with the animals in the control group. No dead foetuses or absorptive foetuses were found in all pregnant rats, at the same time, all viable foetuses in the dose group of 1000 mg/kg bw/d had no external abnormality and slow growth compared with the foetuses in the control group.

Examinations

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before the beginning of the administration, a general clinical observation was made each day. During the administration, a cage-side observation was made at least once a day; an observation with holding was made when the animal was weight. All animals were observed for morbidity and mortality twice daily (once daily on non-working day).
Appearance, fur, activity, reaction, breathing, posture, excrement and urine etc.
Observation with holding: hold out the animal from the cage and put on a table, observe any abnormalilty of neck, head (including eyes, ears, mouth and nose), back, abdomen, skin color around perineum, muscle tension, any trauma and tumour.

BODY WEIGHT: Yes
- Time schedule for examinations: all pregnant animals were weighed on Days 0, every 3 days during the dosing period (GD 5-19), and on the day of the scheduled kill.

FOOD CONSUMPTION: Yes
During the administration, all pregnant rats were provided with known quantity feed on the day before body weight determination, and the remaining feed was weight on the next day. The average food consumption of each animal was calculated.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20 (euthanized with CO2)
- Organs examined: uterus was removed immediately. The pregnancy status of the animals was ascertained firstly. For the pregnant animals, the gravid uteri and total placentas were weighed. The numbers of corpora lutea, absorptive fetuses (early and terminal seperately), dead fetuses (early and terminal seperately) and viable fetuses were determined.

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Blood sampling:

N/A

Fetal examinations:

- External examinations: Yes: all live foetuses (sex and body weight of each foetus was determined, additionally head, face trunk, limbs and external genitalia were investigated)
- Soft tissue examinations: Yes: approx. half per litter (after being fixed at Bouin's solution in at least two weeks) Four specimens from the skull were cut to examine the structural alterations of the head. The Thorax and abdomen of each foetus was opened to examine the size, shape and position of organs. The uteruses or testicles were examined to determine the sex of each foetus.
- Skeletal examinations: Yes: approx. half per litter: The foetuses were removed from the skin and soft tissue after the external examination, dipped in absolute alcohol, and stained using alizarin red staining method. The prepared foetal samples were examined for effects on the skeleton, including the skull, mandible, vertebra, sternum, ribs, scapula, limb, bones and pelvis.

Statistics:

All data were evaluated by the single factor analysis of variance, and if there was significant difference, multi group comparison was done. The data about abnormal clinical sign, ratio of absorptive fetus and viable fetus, and the incidence of individual anomalies etc. were evaluated by Kruskal-Wallis test. All data analysis was done with SPSS software.

Indices:

N/A

Historical control data:

no historical data were used for comparison with test data

Results and discussion

Results: maternal animals

General toxicity (maternal animals)

Clinical signs:

no effects observed

Description (incidence and severity):

N/A

Dermal irritation (if dermal study):

not examined

Description (incidence and severity):

N/A

Mortality:

no mortality observed

Description (incidence):

N/A

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The body weights (GD11 and GD 14) and body weight changes (corrected for gravid uterine weight) of the pregnant rats were significantly decreased.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Food consumption was decreased from GD 16 to 17 only in all dose groups, however, this decrease reached statistical significance in the mid-and high dose group only (P < 0.05). Food consumption was not significantly decreased at any other time throughout the gestation period.

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:

not examined

Description (incidence and severity):

N/A

Haematological findings:

not examined

Description (incidence and severity):

N/A

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

N/A

Endocrine findings:

not examined

Description (incidence and severity):

N/A

Urinalysis findings:

not examined

Description (incidence and severity):

N/A

Behaviour (functional findings):

not examined

Description (incidence and severity):

N/A

Immunological findings:

not examined

Description (incidence and severity):

N/A

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

N/A

Gross pathological findings:

not examined

Description (incidence and severity):

N/A

Neuropathological findings:

not examined

Description (incidence and severity):

N/A

Histopathological findings: non-neoplastic:

not examined

Description (incidence and severity):

N/A

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

N/A

Description (incidence and severity):

N/A

Details on results:

N/A

Maternal developmental toxicity

Number of abortions:

no effects observed

Description (incidence and severity):

N/A

Pre- and post-implantation loss:

no effects observed

Description (incidence and severity):

N/A

Total litter losses by resorption:

no effects observed

Description (incidence and severity):

N/A

Early or late resorptions:

no effects observed

Description (incidence and severity):

N/A

Dead fetuses:

no effects observed

Description (incidence and severity):

N/A

Changes in pregnancy duration:

no effects observed

Description (incidence and severity):

N/A

Changes in number of pregnant:

no effects observed

Description (incidence and severity):

N/A

Description (incidence and severity):

N/A

Details on maternal toxic effects:

Maternal toxic effects: yes

Details on maternal toxic effects:
No death were observed during the course of this study. In the 1000 mg/kg bw/d group, both the body weights (GD11 and GD 14) and body weight changes (corrected for gravid uterine weight) of the pregnant rats were significantly decreased compared with the solvent group (P < 0.05). However, no statistically significant differences in the body weights or body weight changes were observed in the mid- and the low-dose groups (P > 0.05) at any observation period. Food consumption was decreased from GD 16 to 17 only in all dose groups, however, this decrease reached statistical significance in the mid-and high dose group only (P < 0.05). Food consumption was not significantly decreased at any other time throughout the gestation period.

Effect levels (maternal animals)

1

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Basis for effect level:

body weight and weight gain

2

Key result

Dose descriptor:

LOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

body weight and weight gain

Maternal abnormalities

Key result

Abnormalities:

no effects observed

Results (fetuses)

Fetal body weight changes:

no effects observed

Description (incidence and severity):

N/A

Reduction in number of live offspring:

no effects observed

Description (incidence and severity):

N/A

Changes in sex ratio:

no effects observed

Description (incidence and severity):

N/A

Changes in litter size and weights:

no effects observed

Description (incidence and severity):

N/A

Description (incidence and severity):

N/A

Changes in postnatal survival:

no effects observed

Description (incidence and severity):

N/A

External malformations:

no effects observed

Description (incidence and severity):

N/A

Skeletal malformations:

no effects observed

Description (incidence and severity):

N/A

Visceral malformations:

no effects observed

Description (incidence and severity):

N/A

Description (incidence and severity):

N/A

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects: no effects

No statistically significant difference in the number of copora lutea and implentations, gravid uterine weight, percent of live and dead fetuses and resorptions was observed in all dose groups compared with the solvent control groups.
No statistically significant difference in the foetal body weight and sex ratio (the ratio of female fetuses in the total fetuses) was observed in all dose groups compared with the solvent control group.
No statistically significant difference in placenta weight was observed in all dose groups compared with the solvent control group.
In all dose groups, including the solvent control, no foetuses with external and soft tissue malformations or any other alterations was observed. No statistically significant differences in the percent of foetuses with skleletal alterations were observed in all dose groups including the solvent control group.

Effect levels (fetuses)

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Developmental Toxicity

Fetal abnormalities

Key result

Abnormalities:

no effects observed

Overall developmental toxicity

Key result

Developmental effects observed:

no

Any other information on results incl. tables

Table 1: Body weight and gravid uteri weight of pregnant rats: Gestation day (GD)

Gestation day (GD)
Dose mg/kg/d/ control	Number of pregnant rats	GD0	GD5	GD8	GD11	GD14	GD17	GD20	Gravid uteri weight (g)
Solvent control	22	266.9  ± 18.3	292.5± 19.3	303.3±19.6	317.2±20.9	333.1±22.2	355.3±22.2	398.6±45.8	64.0±   35.4
100	24	266.3±17.4	294.5± 22.0	303.5± 18.8	313.0±27.9	335.9± 18.8	360.8± 32.3	408.7±35.9	68.2± 24.1
300	21	267.6±20.1	297.6±25.1	303.3± 26.5	316.8±25.3	330.2±25.7	356.3±32.2	386.4±46.4	51.6± 39.8
1000	20	267.9± 23.4	283.9± 30.4	293.9±29.1	301.7±30.3	313.3±34.8	336.2±38.6	374.0±55.5	39.4±29.7*

Note: compare to solvent control, *P<0.05; data expressed as mean  ± SD

Table 2: Food consumption of gestation period rats (mean ± SD)

Food consumption (g)
Dose/mg/kg/d/control	Number of female rats	GD8	GD11	GD14	GD17
Solvent control	22	20±5.5	20±7.8	21±3.4	26±7.2
100	24	20±8.1	18±5.2	20±5.3	23±5.1
300	21	18±6.0	20±6.7	21±5.7	21±5.1*
1000	20	19±3.9	19±6.3	21±4.3	22±6.1*

Note: compare to solvent control, *P<0.05; data expressed as mean  ± SD

Table 3 The pregnant upshot of pregnant rats and the growth results of fetuses

Parameter	Solvent control	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
Mated female (n)	24	24	24	24
Pregnant female (n)	22	24	21	20
total number of corpora lutea	388	325	276	276
total number of implementation	322	313	265	272
Average number of implementation	14.6 ±2.2	13.0±4.0	12.6± 4.4	13.6±3.7
Gravid uterine weight	82.9±26.0	79.0±30.6	73.4±36.4	84.3±29.5
Live fetuses
Litter	21	24	19	19
Total numer of live fetuses	288	280	232	257
Average number of live fetuses (%)	13.1± 4.8	11.7 ± 4.9	11.0± 5.9	12.9± 4.8
Ratio of live fetuses (%)	89.4 (288/322)	89.5 (280/313)	87.5 (232/265)	94.5 (257/272)
Absorptive fetuses
Litter	4	6	7	2
Number of absorptive fetuses	33	23	27	9
Ratio of absorptive fetuses (%)	10.2 (33/322)	7.3 (23/313)	10.2 (27/265)	3.3 (9/272)
Dead fetuses
Litter	1	5	1	3
Number of dead fetuses	1	10	6	6
Ratio of dead fetuses (%)	0.3 (1/322)	3.2 (10/313)	2.3 (6/265)	2.2 (6/272)
Sex
Number of male fetuses	145	129	127	125
Number of female fetuses	143	151	105	132
Ratio of sex (female/totality)	0.48 ±0.15	0.53  ±0.21	0.50±0.18	0.52±0.13
Fetuses
Body weight of fetuses (g)	3.90  ±1.08	4.22±0.85	4.06±0.85	4.21±0.67
Placenta weight	11.2±2.8	9.4±3.6	9.9±4.1	10.4±3.3

Table 4 Examination of fetuses

Parameter	Solvent control	100 mg/kg bw/d	300 mg/kg bw/d	1000 mg/kg bw/d
External
Number of fetuses/nest	288/21	280/24	232/19	257/19
Number of external normal fetuses	288	280	232	257
Number of external malformations (%)	0	0	0	0
Soft tissue
Number of fetuses/nest	146/21	148/24	120/19	133/19
Number of normal soft tissue fetuses	146	148	120	133
Ratio of soft tissues malformations (%)	0	0	0	0
Skeleton
Number of fetuses/nest	144/21	132/22	112/18	124/19
Ratio of sternum dystosis in the first sternebra (%)	0.7 (1/142)	0 (0/132)	0 (0/112)	0 (0/124)
Ratio of sternum defect in the second sternebra (%)	2.1 (3/142)	0 (0/132)	0 (0/112)	0 (0/124)
Ratio of sternum dystosis in the second sternebra (%)	0 (0/142)	0 (0/132)	0.9 (1/112)	0.6 (1/124)
Ratio of sternum dystosis in the third sternebra (%)	0.7 (1/142)	0 (0/132)	0.9 (1/112)	0 (0/124)
Ratio of sternum dystosis in the fourth sternebra (%)	0.7 (1/142)	0 (0/132)	0.9 (1/112)	0 (0/124)
Ratio of sternum defect in the fifth sternebra (%)	12.8 (19/142)	6.1 (8/132)	14.3 (16/112)	6.5 (8/124)
Ratio of sternum dystosis in the fifth sternebra (%)	1.4 (2/142)	0 (0/132)	1.8 (2/112)	0 (0/124)
Ratio of sternum defect in the sixth sternebra (%)	6.3 (9/142)	1.5 (2/132)	0.9 (1/112)	0.6 (1/124)
Number of skeletal alteration fetuses/nest	21/5	8/3	18/7	8/3
skeletal alteration fetuses/nest (%)	23.8 (5/21)	13.6 (3/22)	38.9 (7/18)	15.8 (3/19)
Ratio of skeleteral alteration (%)	14.8 (21/142)	6.1 (8/132)	16.1 (18/112)	6.5 (8/124)

Applicant's summary and conclusion

Conclusions:

1,2,4 -Benzenetricarboxylic acid, dodecyl and octyl triesters was tested in rats according to OECD 414.

Exposure of pregnant rats to 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters by oral gavage on GD5 -19 resulted in a statistically significant decrease in body weight change corrected for gravid uterine weight, and the food consumption had obvious decrease during the late gestation compared with the solvent control group at 1000 mg/kg bw/d level. These results showed some maternal toxicity; in all groups treated with the test item, no statistically significant difference on the number and the indexes about the growth and the development of the foetus compared with the solvent control group, at the same time, no external, soft tissue and skeleton alteration was observed in all foetuses.

Based on the results above, it is concluded that the LOAEL for the maternal effects is 1000 mg/kg bw/d and the NOAEL is 300 mg/kg bw/d. The NOAEL for the developmental effects is 1000 mg/kg bw/d. The test item under the conditions of the study did not induce any malformation in rats at 1000 mg/kg bw/d and the lower dose levels.

Executive summary:

1,2,4 -Benzenetricarboxylic acid, dodecyl and octyl triesters was tested in rats to detect the effects on pregnant animals, when the material was administered during the period of organogenesis. The study design was based on OECD Guideline of Testing of Chemicals No. 414.

Based on the results of a preliminary range finding test, the animals were treated in the main study with test substances at 100, 300 and 1000 mg/kg bw/d. A concurrent solvent control group was also included in the study. There were 24 mated female rats in each group which resulted in 22, 24, 21, 20 pregnant rats being included in the 1000, 300, 100 mg/kg bw/d and solvent control group. All animals were administered the test item by gavage daily during the 5 -19 of pregnancy (GD5 -19) and were euthanized with C02 on GD 20. After that, the uterus of each animals was removed immediately and weighed. The number of corpora lutea, absorptive fetuses, dead fetuses and viable fetuses were conducted for each animal. The sex and body weight of each fetus was determined. Each fetus was examined for external alterations, and soft tissue alteration or skeleton alteration after the treatment. A statistic analysis was conducted for all data.

No death were observed during the course of this study. In the 1000 mg/kg bw/d group, both the body weights (GD11 and GD 14) and body weight changes (corrected for gravid uterine weight) of the pregnant rats were significantly decreased compared with the solvent group (P < 0.05). However, no statistically significant differences in the body weights or body weight changes were observed in the mid- and the low-dose groups (P > 0.05) at any observation period. Food consumption was decreased from GD 16 to 17 only in all dose groups, however, this decrease reached statistical significance in the mid-and high dose group only (P < 0.05). Food consumption was not significantly decreased at any other time throughout the gestation period.

No statistically significant difference in the number of copora lutea and implentations, gravid uterine weight, percent of live and dead fetuses and resorptions was observed in all dose groups compared with the solvent control groups.

No statistically significant difference in the foetal body weight and sex ratio (the ratio of female fetuses in the total fetuses) was observed in all dose groups compared with the solvent control group.

No statistically significant difference in placenta weight was observed in all dose groups compared with the solvent control group.

In all dose groups, including the solvent control, no foetuses with external and soft tissue malformations or any other alterations was observed. No statistically significant differences in the percent of foetuses with skleletal alterations were observed in all dose groups including the solvent control group.

Exposure of pregnant rats to 1,2,4 -Benzenetricarboxylic acid, mixed dodecyl and octyl triesters by oral gavage on GD5 -19 resulted in a statistically significant decrease in body weight change corrected for gravid uterine weight, and the food consumption had obvious decrease during the late gestation compared with the solvent control group at 1000 mg/kg bw/d level, these results showed some maternal toxicity, in all groups treated with the test item, no statistically significant difference on the number and the indexes about the growth and the development of the foetus compared with the solvent control group, at the same time, no external, soft tissue and skeleton alteration was observed in all foetuses.

Based on the results above, it is concluded that the LOAEL for the maternal effects is 1000 mg/kg bw/d and the NOAEL is 300 mg/kg bw/d. The NOAEL for the developmental effects is 1000 mg/kg bw/d. The test item under the conditions of the study did not induce any malformation in rats at 1000 mg/kg bw/d and the lower dose levels.