Tri-C18-22 (even numbered)-alkyl 2-hydroxypropane-1,2,3-tricarboxylate

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

from 1998-08-10 to 1998-08-28

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

Tri (hexyl, octyl, decyl) citrate

IUPAC Name:

Tri (hexyl, octyl, decyl) citrate

Constituent 2

Reference substance name:

2-Hydroxypropane-1,2,3-tricarboxylic acid, tri (hexyl, octyl, decyl) ester

IUPAC Name:

2-Hydroxypropane-1,2,3-tricarboxylic acid, tri (hexyl, octyl, decyl) ester

Constituent 3

Reference substance name:

-

EC Number:

430-290-8

EC Name:

-

IUPAC Name:

430-290-8

Details on test material:

- Name of test material (as cited in study report): Tri (hexyl, octyl, decyl) citrate
- Substance type: pure active substance
- Physical state: liquid
- Storage condition of test material: room temperature in a closed container

Method

Species / strain

Species / strain 1

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain 2

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Phenobarbital (80 mg/kg bw) and ß-Naphtoflavone (100 mg/kg bw) induced S9 liver microsomal fraction of male Wistar rats

Test concentrations with justification for top dose:

33.3 / 100.0 / 333.3 / 1000.0 / 2500.0 / 5000.0 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: The solvent was compatible with the survival of the bacteria and the S9 activity.

Controls

Controls 1

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: sodium azide, 10 µg/plate for S. typhimurium strains TA 1535, TA 100; 4-nitro-o-phenylene-diamine, 10 µg/plate for S. typhimurium strain TA 98 and 40 µg/plate for S. typhimurium strain TA 1537; methyl methane sulfonate, 1 µL/plate for E. coli strain WP2 u

Remarks:

without metabolic activation

Controls 2

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene, 2.5 µg/plate for all strains

Remarks:

with metabolic activation

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation) and preincubation in two independent experiments


DURATION
- Preincubation period: 60 minutes
- Exposure duration: 48 hours at least


NUMBER OF REPLICATIONS: For each strain and dose level, including the controls, three plates were used.


DETERMINATION OF CYTOTOXICITY
- Method: detected by a reduction in the number of revertants, a clearing of diminution of tht background lawn or by the degree of survival of treated cultures


OTHER EXAMINATIONS:
- Data recording: The colonies were counted using an Artek-counter Modell 880 ( Artek Systems Corpoporation, BIOSYS GmbH, Karben, Germany) at maximum sensitivity. If precipitation of test item precluded automatic counting the revertant colonies were counted by hand.


OTHER: The properties of the S. typhimurium and E. coli strains with regard to membrane permeability, ampicillin resistance and normal spontaneous mutation rates were checked regularly to Ames et al.

Evaluation criteria:

A test was considered acceptable if for each strain:
- the bacteria demonstrate their typical responses to crystal violet and ampicillin
- the control plates without S9 mix were within the range of spontaneous reversion frequencies
- corresponding background growth on both negative control and test plates was observed
- the positive controls showed a distinct enhancement over the control plates

A test item was considered as mutagenic if a dose-related increase in the number of revertants occured and/or a reproducible biologically relevant positive response for at least one the test points occured in at least one strain with or without metabolic activation.
A biologically relevant increase was described as:
- if in strain TA 100 the number of reversions was at least twice as high
- if in strains TA 1535, TA 1537, TA 98 and WP2 uvr A was at least three times higher
than the spontaneous reversion rate.
Also, a dose-dependent and reproducible increase in the number of revertants was regarded as an indication of possible existing mutagenic potential of the test item regardless of whether the highest dose induced the above described enhancement factors or not.

Statistics:

no statistical evaluation performed

Results and discussion

Test results

Test results 1

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Test results 2

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: not applicable
- Water solubility: no data
- Precipitation: nothing mentioned
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: Yes, performed using the same experimental conditions as described for the plate incorporation test to detemine the toxicity of the test item. 8 concentrations were tested for toxicity and induction of mutations with 3 plated each. No toxicity or genotoxicity was observed.


COMPARISON WITH HISTORICAL CONTROL DATA: nothing mentioned


ADDITIONAL INFORMATION TO CYTOTOXICITY: Normal background growth was observed up to the highest concentration of the test item with and without metabolic activation in all stains used.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Any other information on results incl. tables

Table #1:Number of revertants per plate (mean of 3 plates) of the plate incorporation test

	[Strain TA 1535]			[Strain TA 1537]			[Strain TA 98]
Conc. [µg/plate]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	12±2	9±2	-	11±1	7±3	-	40±7	59±21	-
33.3	10±2	8±3	no	8±3	9±2	no	59±3	64±8	no
100.0	11±1	10±2	no	10±4	6±2	no	57±12	65±10	no
333.3	10±3	9±3	no	15±8	9±2	no	47±2	67±6	no
1000.0	11±5	11±1	no	11±4	7±2	no	38±10	67±14	no
2500.0	11±6	8±4	no	8±2	4±1	no	62±7	67±4	no
5000.0	9±1	7±1	no	9±4	6±2	no	42±5	69±9	no
Positive control	1258±101	212±16	-	224±16	225±18	-	1090±34	247±151	-

*solvent control with DMSO

Table #2:Number of revertants per plate (mean of 3 plates) of the plate incorporation test

	[Strain TA 100]			[Strain WP2 uvrA]
Conc. [µg/plate]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	125±17	118±13	-	33±7	60±9	-
33.3	119±10	123±12	no	42±19	49±2	no
100.0	123±9	124±8	no	37±12	59±3	no
333.3	130±4	125±18	no	38±8	52±6	no
1000.0	135±9	138±12	no	42±7	58±11	no
2500.0	134±10	128±3	no	39±6	66±10	no
5000.0	146±12	122±10	no	36±3	54±7	no
Positive control	1321±8	2315±155	-	807±47	769±21	-

*solvent control with DMSO

Table #3:Number of revertants per plate (mean of 3 plates) of the preincubation test

	[Strain TA 1535]			[Strain TA 1537]			[Strain TA 98]
Conc. [µg/plate]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	16±4	13±2	-	11±2	10±2	-	37±3	50±5	-
33.3	15±5	11±4	no	13±2	11±2	no	51±7	51±2	no
100.0	11±4	12±3	no	14±5	10±1	no	49±2	64±6	no
333.3	17±1	14±2	no	13±3	12±4	no	50±4	60±13	no
1000.0	15±1	12±2	no	17±4	10±3	no	39±6	52±5	no
2500.0	17±9	11±4	no	8±3	8±1	no	33±6	50±12	no
5000.0	13±8	14±5	no	14±3	10±4	no	44±10	60±14	no
Positive control	1044±14	143±5	-	298±11	135±17	-	1163±58	1128±82	-

*solvent control with DMSO

Table #4:Number of revertants per plate (mean of 3 plates) of the preincubation test

	[Strain TA 100]			[Strain WP2uvrA]
Conc. [µg/plate]	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)	¿ MA	+ MA	Cytotoxic (yes/no)
0*	101±17	113±12	-	27±4	50±8	-
33.3	90±10	110±8	no	31±8	33±7	no
100.0	84±6	108±18	no	31±1	33±5	no
333.3	99±23	94±4	no	36±5	37±4	no
1000.0	99±7	104±10	no	27±5	36±2	no
2500.0	115±8	115±6	no	32±5	39±2	no
5000.0	144±30	98±13	no	30±9	41±15	no
Positive control	1335±103	1198±118	-	210±28	223±2	-

*solvent control with DMSO

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information):
negative

The reported data of this mutagenicity test showed that the test item did not induce gene mutations by base pair changes or frameshifts in the genome of the strains used. Therefore, Tri (hexyl, octyl, decyl) citrate was considered to be non-mutagenic in this bacterial reverse mutation assay.