Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vivo

Description of key information
Based on all pieces of weight of evidence it is clear that triethyl citrate is not mutagenic. Genetic toxicity in vitro: 1) ATBC was negative without metabolic activation in a bacterial reverse mutation assay (Ames test) according to OECD 471 (GLP). [Read-across data from tributyl-O-acetylcitrate (CAS 77-90-7)] 2) ATCB was negative with and without metabolic activation in a mammalian cell gene mutation assay according to OECD 476 (GLP). [Read-across data from tributyl-O-acetylcitrate (CAS 77-90-7)] 3) TNO BIBRA, 1998: Triethyl citrate was negative with and without metabolic activation in a bacterial reverse mutation assay (Ames test). Genetic toxicity in vivo: 1) ATBC was tested in a chromosomal aberration study according to OECD 475 and found to be negative [Read across data from tributyl-O-acetylcitrate (CAS 77-90-7)]
Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration
Remarks:
Type of genotoxicity: chromosome aberration
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:
guideline study with acceptable restrictions
Qualifier:
according to
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
chromosome aberration assay
Species:
rat
Strain:
Wistar
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
Vehicle(s)/solvent(s) used: polyethylene glycol
Amount of vehicle (if gavage or dermal): 10 mL/kg bw
Purity: PEG 400
Duration of treatment / exposure:
24 and 48 hours after treatment, the bone marrow cells were collected for chromosome aberration analysis
Frequency of treatment:
One single oral treatment
Post exposure period:
24 and 48 hours
Remarks:
Doses / Concentrations:
2000 mg/kg bw
Basis:
nominal conc.
No. of animals per sex per dose:
10 animals (6 male and 6 female rats, 5 of each sex were evaluated)
Control animals:
yes, concurrent vehicle
Positive control(s):
Cyclophosphamide (CPA)
- Justification for choice of positive control(s): CPA is known to induce a distinct increase in induction of aberration frequency
- Route of administration: Once oral by gavage
- Dose: 15 mg/kg bw
Tissues and cell types examined:
Bone marrow: at least 100 well spread metaphases per animals were scored for cytogenetic damage
Evaluation criteria:
The test item is classified as mutagenic if it induces either a dose-related increase in the number of structural chromosomal aberrations and a reproducible statistically significant positive response for at least one of the test points.
Statistics:
non-parametric Mann-Whitney test
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
The mitotic index was slightly reduced after 24 h
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RESULTS OF RANGE-FINDING STUDY
A single dose was administered at 2000 mg/kg bw to 4 animals (2 males/2 females)
Solubility: Formulated in PEG 400
Clinical signs of toxicity in test animals: Signs recorded 1 - 48 h post-treatment. All animals showed reduction of spontaneous activity at all time points.
Evidence of cytotoxicity in tissue analyzed: The mitotic indices were slightly reduced at 2000 mg/kg bw in the main study indicating that the test item had a slight cytotoxic effect to the bone marrow
Rationale for exposure: 2000 mg/kg bw was the highest dose as required by regulatory Guidelines and expresses the MTD

RESULTS OF DEFINITIVE STUDY
Types of structural aberrations for significant dose levels: No enhancement of the aberrations frequencies
Appropriateness of dose levels and route: The single dose selected of 2000 mg/kg bw was appropriate, as it reflected the MTD and had a slight cytotoxic effect to the bone marrow after 24 h
Statistical evaluation: No significant differences for the test item as compared to the control group

Table: Experimental results

Experimental group

Dose
mg/kg bw

Preparation hours post administra-tion

Number of cells scored

% aberrant cells

Incl. Gaps Excl. gaps

Mean mitotic index (%)

PEG 400

0

24

1000

0.3

0.3

4.87

o-Acetytributyl citrate

2000

24

1000

0.6

0.6

3.52

Cyclophosphamide

15

24

1000

12.1***

11.8***

3.09

o-Acetytributyl citrate

2000

48

1000

0.3

0.3

5.30

 

*** = p<0.0001

 

Conclusions:
Interpretation of results (migrated information): negative
O-Acetyltributyl citrate did not induce chromosomal aberration in the rat in vivo
Executive summary:

A chromosomal aberration study according to OECD 475 was performed in rats in vivo after a single dose of 2000 mg/kg bw O-Acetyltributyl citrate. Five animals per sex and group, i.e. one vehicle control group, two test item groups and one positive control group were employed for the study. The test item was formulated in PEG 400. 24 and 48 h post-treatment, bone marrow cells were collected and 100 well-spread metaphases/animal were scored for chromosomal aberrations. Test item-treated animals showed reduction of spontaneous activity.

The mitotic index was slightly reduced after 24 h, but the test substance did not induce chromosomal aberrations at any time point investigated.

In contrast, the positive control substance induced a highly significant effect documenting the sensitivity of the test system. It can be assumed that the same applies to triethyl citrate (CAS 77-93-0) as it is a near analogue to the test substance acetyl tributyl citrate.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Genetic toxicity in vitro

The following data is available on its structural analogue acetyl tributyl citrate (ATBC; please refer to the separate read-across statement for further explanation of this procedure): ATBC was tested in an Ames test similar to OECD Guideline 471 with limited information on test conditions (Heath & Reilly, 1982). The test substance gave no indication for mutagenic activity in S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100 when tested without S9 mix. The Evaluation of ATBC in a Mouse Lymphoma Assay (L5178Y TK +/-) (Bigger and Harbell, 1991; cited in US EPA (2003) HPV Chemicals Challenge Program) has been carried out according to OECD Guideline 476. The test substance gave no indication for mutagenic activity (with and without metabolic activation).

It can be assumed that the same applies to triethyl citrate (CAS 77 -93 -0) as it is a near analogue to the test substance acetyl tributyl citrate.

Further, in the "toxicity profile of triethyl citrate" (TNO BIBRA, 1998) the genetic toxicity in a bacterial reverse mutation assay is summarized as follows:

Triethyl citrate was not mutagenic in Salmonella typhimurium and in the yeast Saccharomyces cerevisiae, either with or without a mammalian liver metabolic activation system (Litton Bionetics Inc., 1976).

Genetic toxicity in vivo

A chromosomal aberration study according to OECD 475 was performed in rats in vivo after a single dose of 2000 mg/kg bw O-Acetyltributyl citrate (Bigger and Harbell, 1991). Five animals per sex and group, i.e. one vehicle control group, two test item groups and one positive control group were employed for the study. The test item was formulated in PEG 400. 24 and 48 h post-treatment, bone marrow cells were collected and 100 well-spread metaphases/animal were scored for chromosomal aberrations. Test item-treated animals showed reduction of spontaneous activity.

The mitotic index was slightly reduced after 24 h, but the test substance did not induce chromosomal aberrations at any time point investigated.

In contrast, the positive control substance induced a highly significant effect documenting the sensitivity of the test system.

Taking all these result into account, it can be concluded that triethyl citrate is not mutagenic.


Justification for selection of genetic toxicity endpoint
GLP and guideline study conducted on mammalian cells with the structural analogue ATBC.

Justification for classification or non-classification

ATBC was tested in a variety of valid in vitro and in vivo test systems and in all of these tests negative results were obtained. Therefore, there is no need for classification. Triethyl citrate (CAS 77-93-0) was also negative in an Ames test and as the substance is a near analogue to ATBC it can be considered to be also not classified.