Tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate

Endpoint summary

Description of key information

Based on all pieces of weight of evidence it is clear that tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate is of very low  toxicity after repeated administrations.
1) A GLP test equivalent to OECD 408 resulted in a NOAEL (rat) of 1000 mg/kg bw . [Read-across data from tributyl-O-acetylcitrate (CAS 77-90-7)]
2) Finkelstein, 1959: Tributyl citrate did not induce deleterious effects at a concentration of 5%, but the 10%-diet tended to depress the growth, an effect which may be due to frequent diarrhoea. Sections taken from the treated animals were indistinguishable from the controls. [Read-across data from tributyl citrate (CAS 77-94-1)]
3) Finkelstein, 1959: There were no symptoms of toxicity and the tests showed no abnormalities.  [Read-across data from tributyl citrate (CAS 77-94-1)]

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

migrated information: read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to Guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Principles of method if other than guideline:

Range-finding study: Only limited histopathology performed and no special neurotoxicity examination included

GLP compliance:

no

Remarks:

no GLP required (range-finding study for long-term toxicity)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Analytical verification of doses or concentrations:

yes

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

Daily via the diet

Remarks:

Doses / Concentrations:
0, 96.02, 287.50 and 961.16 mg/kg
Basis:
actual ingested

No. of animals per sex per dose:

10 m / 10 f

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
Observation for mortality/viability were recorded twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
Time schedule: At least once daily. Palpation for tissue masses were performed at least once weekly

BODY WEIGHT: Yes
Time schedule for examinations: Weekly during pre-test, treatment and before necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day

FOOD EFFICIENCY: no

WATER CONSUMPTION AND COMPOUND INTAKE: no

OPHTHALMOSCOPIC EXAMINATION: Yes
Time schedule for examinations: During acclimatization and during week 13
Dose groups that were examined: All groups during acclimatization and all animals of the control and high dose group during week 13

HAEMATOLOGY: Yes
Time schedule for collection of blood: week 13 weeks
Anaesthetic used for blood collection: yes (light isoflurane anaesthesia)
Animals fasted: Yes
How many animals: all (10 m / 10 f per group)
Parameters examined: Erythrocyte count, haemoglobin, haematocrit, MCV, RDW, MCH, MCHC, HDW, reticulocyte, reticulocyte maturity index, leukocyte count, differential leukocyte count, prothrombin time, partial thromboplastin time

CLINICAL CHEMISTRY: Yes
Time schedule for collection of blood: week 13
Animals fasted: Yes
How many animals: all (10 m / 10 f per group)
Parameters examined: Glucose, urea, creatinine, bilirubin (total), cholesterol, tryglicerides, phospholipides, ASAT, ALAT, LDH, CK, ALP, GGT, Sodium, potassium, chloride, calcium, inorganic phosphorus, protein (total), albumin, globulin, albumin/globulin ration

URINALYSIS: no

NEUROBEHAVIOURAL EXAMINATION: no

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all organs, all groups
HISTOPATHOLOGY: Yes.

Statistics:

Dunnett-test (many to one t-test) based on a pooled variance estimate if variables can be assumed to follow a normal distribution
Steel-test (many-one rank test) was applied instead of the Dunnett-test when data were not assumed to follow a normal distribution
Fisher's exact test was applied for ophthalmoscopic data and macroscopic findings
Armitage/Cochran Trend Test was used for non-neoplastic lesions, if appropriate

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

CLINICAL CHEMISTRY
In m and f at 1000 mg/kg bw/day, decreased bilirubin levels, decreased aspartate aminotransferase and lactate dehydrogenase activity, increased sodium level, decreased chloride and calcium levels, decreased globulin levels (which resulted in increased albumin/globulin ratio) were evident in m at 300 and 1000 mg/kg bw/d

ORGAN WEIGHTS
1000 mg/kg: increased liver weights in m and f

GROSS PATHOLOGY
1000 mg/kg: enlarged livers in 2 f

HISTOPATHOLOGY: NON-NEOPLASTIC
1000 mg/kg: minimal hepatocellular hypertrophy (non-adverse) in several animals

Dose descriptor:

NOAEL

Remarks:

1000 mg/kg bw/day

Effect level:

>= 1 000

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Critical effects observed:

not specified

Table: Summary of test item-related findings for ATBC

 

Intended test item intake (mg/kg bw/day)	0	100	300	1000
Clinical chemistry
Males (means)
Glucose (mmol/L)	4.886	6.124**	5.895**	5.784*
Bilirubin (µmol/L)	2.055	1.955	1.767	1.305**
ASAT (U/L)	82.37	76.37	71.90**	66.08**
LDH (U/L)	136.50	120.88	92.29**	91.27**
Sodium (mmol/L)	144.58	145.00	154.52**	153.72**
Chloride (mmol/L)	100.94	101.33	89.18**	89.25**
Calcium (mmol/L)	2.834	2.811	2.666**	2.694**
Globulin (g/L)	26.356	26.474	24.431**	24.123**
Alb/Glob ratio (rel., %)	1.590	1.640	1.727*	1.755**
Females (means)
Bilirubin (µmol/L)	2.795	2.346	2.057	1.642**
Organ weights
Liver (means)
Males (abs., gram)	8.83	9.30	9.58	10.86**
Males (rel., %)	2.34	2.38	2.51	2.86**
Females (abs., gram	5.88	6.12	5.80	7.10**
Females (rel., %)	2.65	2.71	2.76	3.03*
Macroscopic findings
Liver (no. affected/10)	0/10	0/10	0/10	2/10
Females: Enlarged
Histopathology
Liver (no. affected/10)
Males: Hepatocellular hypertrophy	1/10	0/10	6/10	5/10
Females: Hepatocellular hypertrophy	0/10	0/10	1/10	2/10

 

• * = 5% level (Anova and/or Dunnett-Test)
• ** = 1% level (Anova and/or Dunnett-Test)

 

Conclusions:

Based on the results of this study, dose levels of 100, 300 and 1000 mg/kg bw/d were proposed for the subsequent 2-year combined chronic/carcinogenicity study. Slight effects seen at 1000 mg/kg bw/d were considered due to hepatic metabolism adaption. Thus the NOAEL for m and f in this study was given with 1000 mg/kg bw/d.

Executive summary:

This 13-weeks dietary toxicity study with acetyl tributyl citrate (ATBC) in Wistar rats was designed as dose range finding study for a subsequent combined chronic/carcinogenicity study. In principle, the study was performed according to OECD Guideline 408, but with restricted histopathological organ examinations. The administration at target doses of 100, 300 and 1000 mg/kg bw/d resulted in no unscheduled deaths. No test item-related clinical signs were observed. Treatment-related findings were restricted to slight changes in clinical biochemistry (males were more affected than females) and slightly increased liver weights accompanied by minimal hepatocellular hypertrophy at 1000 mg/kg bw/d. The findings were considered to be due to hepatic metabolic adaption rather than as sign of toxicity. The highest dose of 1000 mg/kg bw can be regarded as NOAEL. The proposed dose levels for a subsequent combined chronic/carcinogenicity study were 100, 300 and 1000 mg/kg bw/d. It can be assumed that the same applies to tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate (CAS 144 -15 -0) as it is a near analogue to the test substance acetyl tributyl citrate.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

chronic

Species:

rat

Quality of whole database:

good quality

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: inhalation

Data waiving:

exposure considerations

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

repeated dose toxicity: dermal

Data waiving:

other justification

Justification for data waiving:

other:

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

There is no study available for this endpoint for the target substance tris(2-ethylhexyl) 2-(acetyloxy)propane-1,2,3-tricarboxylate, therefore data on its structural analogues is taken into account (please refer to the separate read-across statement for further explanation of this procedure):

A 13-weeks dietary toxicity study with acetyl tributyl citrate (ATBC) in Wistar rats was designed as dose range finding study for a subsequent combined chronic/carcinogenicity study. In principle, the study was performed according to OECD Guideline 408, but with restricted histopathological organ examinations. The administration at target doses of 100, 300 and 1000 mg/kg bw/d resulted in no unscheduled deaths. No test item-related clinical signs were observed. Treatment-related findings were restricted to slight changes in clinical biochemistry (males were more affected than females) and slightly increased liver weights accompanied by minimal hepatocellular hypertrophy at 1000 mg/kg bw/d. The findings were considered to be due to hepatic metabolic adaption rather than as sign of toxicity. The highest dose of 1000 mg/kg bw can be regarded as NOAEL. The proposed dose levels for a subsequent combined chronic/carcinogenicity study were 100, 300 and 1000 mg/kg bw/d.

The repeated dose toxicity via the oral route of tributyl citrate was studied in 22 rats (Finkelstein & Gold, 1959). A diet representing a 5 % concentration of tributyl citrate had no deleterious effect on growth. The concentration of 10 % seemed to depress the growth curve. In view of the fact that the higher concentration tended to produce diarrhoea, it may well be that this factor accounts for the impairment in growth. A blood count was done before treatment, after feeding for 1 month, and again after feeding for 2 months. There are very wide fluctuations in the red cell count, white cell count, and differential count in both the control and treated animals. There is no conspicuous difference between the values in the controls and the treated animals. Eight rats were sacrificed after this period of feeding the diets containing 5 and 10 % tributyl citrate, and 2 control rats. The gross examination of the thoracic and abdominal viscera showed no abnormalities. Forty histological sections were made representing the following organs: heart, lungs, gastrointestinal tract, liver, pancreas, spleen, kidneys. The sections taken from the treated animals were indistinguishable from the controls. Further, in another test two cats were fed 5 mL/kg bw/day by stomach tube for 2 months. In addition to observations on the general behaviour and appearance of the animals, the following were done at approximately weekly intervals: weight, urine, blood NPW; blood creatinine; blood sugar and blood counts. Similar observations were made on the 2 untreated cats which were maintained under similar conditions during the same period. Each of the treated animals received approximately 50 doses of the compound.

There were no symptoms of toxicity and the tests showed no abnormalities. The treated animals developed loose bowel movements, and it is likely that the factor is responsible for the loss of approximately 30 % in body weight.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable subchronic study conducted with a structural analogue ATBC.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Inhalation is not a relevant route of exposure. An oral study is available, the oral NOAEL can be extrapolated to an inhalation NOAEC.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Inhalation is not a relevant route of exposure. An oral study is available, the oral NOAEL can be extrapolated to an inhalation NOAEC.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Oral study is available. The oral NOAEL can be extrapolated to a dermal NOAEL.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Oral study is available. The oral NOAEL can be extrapolated to a dermal NOAEL.

Justification for classification or non-classification

Repeated dose toxicity: oral The test material does not meet the criteria for classification and will not require labelling in accordance with European Regulation (EC) No. 1272/2008.