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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

ATBC was tested in valid in vitro test systems and in all of these tests negative results were obtained.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
21.01.2001-26.02.2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
his-operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
-S9: 0, 156.3, 312.5, 625, 1250, 2500, 5000 µg/plate (five strains)
+S9: 0, 156.3, 312.5, 625, 1250, 2500, 5000 µ/g/plate (five strains)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Choice based on information from sponsor
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: -S9 mix: 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (TAlOO, TA98, WP2uvrA), sodium azide (TA1535) and 9-aminoacridine(TA1537) +S9 mix: 2-arninoanthracene (five strains).
Details on test system and experimental conditions:
Test Design:
Procedure
(1) Plate incorporation test with and without S9 using five strains
(2) Pre-incubation test with and without S9 using five strains
Number of replicates: 2
Plates/dose: 3
Rationale for test conditions:
A confirmation test was carried out in pre-incubation method as negative mutagenic effect (-) was noted in plate incorporation test.
Evaluation criteria:
The test substance was judged positive ( +) when the number of revertant colonies in the test substance treated plates increased dose dependently and became two-fold or more compared to that of the negative control and this effect was reasonably reproducible or significant reproducible increase was noted at one or more concentrations at least in one strain with or without metabolic activation system and negative (-) when any of the above criteria were not fulfilled.
Statistics:
No statistical method was followed.
Key result
Species / strain:
bacteria, other: Salmonella typhimurium TAlOO, TA1535, TA98, TA1537 Escherichia coli Wp2uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Conclusions:
ATBC has been tested according OECD TG 471 and 472 in compliance with GLP. Under conditions of the test, no mutagenic effect was observed for ATBC tested up to cytotoxic concentration in any of the test strains (S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2) without and with metabolic activation. ATBC is non-mutagenic in test strains used.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Principles of method if other than guideline:
Mammalian cell forward mutation assay
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Target gene:
Forward mutation assay TK+/TK-
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
other: TK +/-
Metabolic activation:
with and without
Metabolic activation system:
S9 mix: S-9 mix obtained from the liver of Aroclor-induced rats
Test concentrations with justification for top dose:
1st initial toxicity test:
0.1 to 5140 μg/mL (+/- S-9 activation)

Main test:
-S9-activation: 10, 70, 150, 230, or 310 µg/mL
+S9-activation: 200, 270, 340, 410, 480, or 550 µg/mL

Based on the results of the initial toxicity test, cultures were exposed to the test article over a range of concentrations from 10 to 550 μg/ml for the nonactivated cultures and from 54 to 550 μg/mL for the S-9 activated cultures.
After a two-day expression period, two cultures per concentration were selected for cloning based upon their degree of toxicity.
Vehicle / solvent:
DMSO
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
not specified
Positive controls:
yes
Positive control substance:
other: 3-methylcholanthrene (+S9) and ethyl methanesulfonate (-S9)
Evaluation criteria:
A 2-fold increase in the mutant frequency over the concurrent solvent-treated control value was interpreted as an indication of a positive effect, together with evidence of a dose-related increase.
Only doses yielding total growth values >= 10% were used in the analysis of induced mutant frequency. Doses yielding <= 10% total growth were used in determining dose response.
Statistics:
Details on statistical methods are not reported. In the version of OECD 476 (1984) no specific statistical methods is listed and both statistical effect as well as biological relevance should be considered for evaluation.
The criteria of a 2-fold increase in mutant frequency would be significant at the <0.02 level, under the appropriate statistical assumption that the counts are a Poisson mean (see Clive et al. 1979 Mut Res 59, 61-108).
In the study without S9-mix, only two concentrations had an RTG >=10%. In the first experiment an increase in mutation frequence is observed while in the second experiment it is the opposite. All value are below the frequency of the solvent control.
In the study with S9-mix, four concentrations had an RTG >=10%. In the both experiment no increase in mutation frequence is observed, rather a decrease (linear regression model with negative coefficient). All value are below the frequency of the solvent control.
Therefore, the statistical evaluation would not lead a different interpretation of the study. In this case, the reported evaluation criteria (see above) are regarded sufficient to identify biological relevant increase. The drawn conclusion can be regarded as valid.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Complete toxicity was observed during the initial toxicity test at concentrations of 514 μg/ml and above for cultures without S9 mix and from 1028 μg/ml and above for cultures with S9 mix.
For cultures without S9 mix a dose-dependent increase in toxicity was observed in the mutagenicity assay, with an average Relative Total Growth of 16%, 6% and 3% at concentrations of 70, 150, and 230 μg/mL, respectively (complete toxicity at 310 μg/mL and above).
For cultures with S-9 mix, the average Relative Total Growth was 16% and 8% at 410 and 480 μg/mL, respectively (complete toxicity at 550 μg/mL).

The mutant frequencies in the ATBC treated cultures without metabolic activation ranged from 2.5 to 1.0 times the mean mutant frequency of the solvent controls.
Total Growth of these cultures ranged from 3% to 89%. No cultures with ≥10% Total Growth exhibited a two-fold increase in mutant frequency over the average solvent control mutant frequency required for a positive result. There was a dose-dependent increase in cytotoxicity, but there was not a dose-related increase in mutant frequency.
The frequency of small colonies in these treated cultures was comparable to the solvent controls. The colony size distribution for ethylmethanesulfonate treated cultures showed the characteristic increase in larger colonies expected with this compound. The absolute size of the colonies was shifted to a more medium size due to metabolic inhibition resulting from the high number of colonies per plate.

The mutant frequencies in the ATBC treated cultures with metabolic activation ranged from 1.2 to 0.6 times the mean mutant frequency of the solvent controls.
Total Growths of these cultures ranged from 7% to 50%.
There was a dose-dependent increase in cytotoxicity, but there was not a dose-related increase in mutant frequency.
There was not an increase in the frequency of small colonies when the treated cultures were compared to the solvent control cultures.
3-Methylcholanthrene treated cultures showed both an increase in small colonies and a prominent increase in large colonies.

VC = Viable counts (cloning efficiency)

TFT = Counts after TFT-selection

RTG = Relative total growth

Non-Activated Cultures S9-Activated Cultures
Dose (ug/mL) Average TFT Average VC Mut Freq RTG Dose (ug/mL) Average TFT Average VC  Mut Freq RTG
or ul/mL or ul/mL
10 35 158 0,44 86 200 55 165 0,67 49
44 165 0,53 89 61 160 0,76 50
70 42 167 0,5 16 270 39 151 0,52 34
38 157 0,48 16 52 182 0,57 21
150 45 106 0,85 5 340 63 173 0,73 26
39 128 0,61 6 56 161 0,7 27
230 42 79 1,06 3 410 68 170 0,8 14
37 78 0,95 3 47 149 0,63 18
Solvent 44 161 0,55 480 59 157 0,75 9
Positive 446 108 8,26 38 66 138 0,96 7
Solvent 69 153 0,91
Positive 292 103 5,67 34
Conclusions:
ATBC gave no indication for mutagenic activity in the mouse lymphoma L5178Y assay. The test was negative with and without metabolic activation.
Executive summary:

For the endpoint in vitro mammalian gene mutation, a Mouse Lymphoma Assay has been carried out according to OECD Guideline 476. In this assay. L5178Y TK +/- cells were exposed to ATBC at concentrations up to 310 µg/ml -S9 mix and up to 550 µg/ml +S9 mix. No significant increase in the mutant frequencies were observed with or without metabolic activation. ATBC is non-mutagenic in mammalian cells in vitro, under the conditions of this assay.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11.12.2000-23.02.2001
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
Chinese hamster lung (CHL/IU)
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital and 5,6-benzoflavone induced rat liver S9
Test concentrations with justification for top dose:
-S9: 0, 0.125, 0.250, 0.500, 1 µLg/mL (short term treatment)
+S9: 0, 0.125, 0.250, 0.500, 1 µg/mL (short term treatment)
24 hr: 0, 0.019, 0.038, 075, 0.15 μg/mL (continuous treatment)
48 hr: 0, 0.02, 0.03, 0.04, 0.05 μg/mL ( continuous treatment)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Choice based on information from sponsor
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
mitomycin C
Details on test system and experimental conditions:
Test Design:
Procedure
In the case of short term treatment without (-S9) and with ( + S9), the cells were treated for 6 hrs without and with S9 and cultivated with fresh medium for 18 hrs. In the case of continuous treatment, the cells
were treated for 24 and 48 hrs without S9.
In the case of confirmation test, the cells were treated for 6 hrs with S9 and cultivated with fresh medium for 24 hrs
Frequency of dosing: One time
Plates/dose: 2

Description of confirmation study:
A confirmation test was carried out in short-term treatment with S9 with the recovery period of 24 hr instead of 18 hr to assess whether the test substance in the presence of S9 increased the chromosomally
aberrant cells by changing the cell proliferation time.

Number of metaphases analyzed: 100 metaphases/plate or specimen
Evaluation criteria:
The test substance was judged positive (+) when the incidence of cells with chromosomally aberration increased dose-dependently as compared with those of concurrent negative controls or a reproducible increase in the incidence of cells with chromosomally aberration at one or more concentrations and the others were judged negative (-).
Statistics:
No statistical method was followed
Species / strain:
other: CHL/IU
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
ATBC was tested in a valid and reliable test performed according to OECD TG 473 and in compliance with GLP. ATBC did not induce structural chromosomal aberrations in the CHL/IU cells in short term treatment without metabolic activation (-S9) and with metabolic activation (+S9) or in continuous treatment for 24 hrs and 48 hrs. Appropriate solvent and positive controls were included and gave expected results. It is concluded that ATBC is negative for the induction of chromosome aberrations under the conditions of the test.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

ATBC was tested in valid in vivo test systems and negative results were obtained.

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:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 475 (Mammalian Bone Marrow Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
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
Vehicle controls validity:
valid
Negative controls validity:
not applicable
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. 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.

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

Additional information

Additional information from genetic toxicity in vitro and vivo:

In vitro studies:

Ames test similar to OECD Guideline 471 (S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100): negative (San & Wagner, 1991)

Ames test similar to OECD Guideline 471 (S. typhimurium TA1535, TA1537, TA1538, TA98 and TA100): negative (Heath & Reilly, 1982)

Mammalian cell forward mutation assay with L5178Y TK+/- mouse lymphoma cells comparable to OECD Guideline 476: negative (Bigger & Harbell, 1991)

Ames test according to OECD Guideline 471 and 472 (S. typhimurium TA1535, TA1537, TA98 and TA100 and E.coli Wp2uvrA): negative (Nite 2001)

Chromosome aberration test according to OECD Guideline 473 (CHL/IU cells): negative (Nite 2001)

In vivo studies:

Chromosomal aberration study according to OECD Guideline 475 in rats: negative (Honarvar, 2002)


Justification for selection of genetic toxicity endpoint
GLP and guideline study conducted with mammalian cells.

Justification for classification or non-classification

ATBC was tested in 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.