Butanedioic acid, 2,3-dihydroxy- [R-(R*,R*)]-,C12-13-branched alkyl esters

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test material Butanedioic acid, 2,3 -dihydroxy-[R-(R*,R*)]-, C12 -13 -branched alkyl esters and the source substance were in all Ames tests negative, means did not induce an increase in the number of revertant colonies compared to the negative controls and was considered as non-mutagenic in this test-system.

It can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Butanedioic acid, 2,3-dihydroxy-[R-(R*,R*)]-, C12-13-branched alkyl esters did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line. Therefore, the test item Butanedioic acid, 2,3-dihydroxy-[R-(R*,R*)]-, C12-13-branched alkyl esters is considered to be non-clastogenic in this chromosome aberration test.

The similar substance Diethyl hexyl adipate was not genotoxic in the Mouse lymphoma test. It is likely that the same is valid for the test substance.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1992-06-22-1992-06-26

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

No testing of phenotypic characteristics mentioned (no ampicillin resistance, sensitivity to crystal violet and ultraviolet light), no justification for missing follow-up experiment given.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

TA 1535 rfa His- uvrB
TA 1537 rfa His- uvrB
TA 1538 rfa His- uvrB
TA 98 rfa His- uvrB R. Amp
TA 100 rfa His- uvrB R.Amp

Species / strain / cell type:

S. typhimurium, other: Strains TA 1535, TA 1537, TA 1538, TA 100 and TA 98

Metabolic activation:

with and without

Metabolic activation system:

S9 mix, derived from the livers of male adult rats induced with Aroclor 1254. AROCLOS was used as a dilution in soya bean oil containing 200 mg Aroclor/ml. The S9 mix was checked for sterility.

Test concentrations with justification for top dose:

Concentration range in the main test (with metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate
Concentration range in the main test (without metabolic activation): 1, 10, 100, 1000 and 10000 µg/plate

Vehicle / solvent:

- Solvent: DMSO
- Justification for choice of solvent/vehicle: no data

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene 1 µg/plate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

Test solution: The test material was dissolved in DMSO at a concentration of 100 mg/ml.

Plate test without metabolic activation:
0.1 ml of the test solution and 0.1 ml of a bacterial suspension containing approximately 10exp8 - 10exp9 cfu/ml were added in this sequence to 2 ml of molten top agar. The ingredients were thouroughly mixed and immediately poured into plates containing minimal medium. At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.

Plate test with metabolic activation
0.1 ml of the test solution, 0.1 ml of bacterial suspensions and 0.5 ml of the metabolic activation system were added in this sequence to 2 ml of molten top agar. The ingredients were thoroughly mixed and immediately poured into plates containing minimal medium. At the same time, negative control and positive controls using the chosen concentration of the reference mutagens were performed.

DURATION
- Preincubation period: not applicable
- Exposure duration: 48 - 72 h
After incubation the revertant colonies obtained in the plates were counted.

NUMBER OF REPLICATIONS: triplicates plates were used for the test material, two for positive controls




DETERMINATION OF CYTOTOXICITY
- Method: determination of toxicity by semiquantitative evaluation of the background lawn and the number of spontaneous revertant colonies

OTHER EXAMINATIONS:
Sterility test

Evaluation criteria:

- at the end of the assay, a sterility check on S9 mix must show less than two viable colonies per 0.5 mL
- at the end of the test, a sterility check of the test material nust show less than two viable colonies per plate
- the positive conrols must produce at least a threefold increase in the number of revertant colonies with regard to the mean value for the respective negative control
- the mean number of spontaneous revertant colonies in the negative controls must correspond to the following values:
strain TA 1535: 20 ± 15; strain TA 1537: 20 ± 15; strain TA 1538: 15 ± 10; strain TA 98: 40 ± 25; strain TA 100: 150 ± 90

Statistics:

no statistics performed

Key result

Species / strain:

other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

other: Salmonella typhimurium: strains TA 1535, TA 1537, TA 1538, TA 98 and TA 100

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

> 10000 µg/plate

Vehicle controls validity:

other: vehicle controls valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

- Preliminary test: The genetic characteristics of the bacterial strains were found to be unaltered.
- Test material toxicity: The test material did not induce toxic effects at a concentration of 10 mg/plate both in the absence and presence of the metabolic activation system.
- Negative control: The number of revertant colonies on the negative control plates fell within the normal range.
- Positive control: The positive controls induced an increase in the number of colonies.
- Sterility test: The sterility test performed on the test material and on S9 mix did not show any bacterial contamination.

Table #1: Salmonella typhimurium reverse mutation assay without metabolic activation
Concentration [µg/plate]	Revertant colonies / plate mean ± SD
	Strain TA 1535	Strain TA 1537	Strain TA 1538	Strain TA 98	Strain TA 100
0	12 ± 3	17 ± 2	13 ± 3	9 ± 4	105 ± 19
1	14 ± 3	16 ± 5	13 ± 2	12 ± 2	166 ± 23
10	16 ± 7	17 ± 2	12 ± 2	10 ± 2	101 ± 21
100	18 ± 6	16 ± 2	9 ± 1	16 ± 5	114 ± 17
1000	22 ± 8	17 ± 1	13 ± 4	15 ± 2	112 ± 16
10000	15 ± 3	16	10 ± 4	13 ± 2	114 ± 14
Positive control	433 ± 47	96 ± 283	264 ± 31	385 ± 18	152 ± 37
Table #2: Salmonella typhimurium reverse mutation assay with metabolic activation
Concentration [µg/plate]	Revertant colonies / plate mean ± SD
	Strain TA 1535	Strain TA 1537	Strain TA 1538	Strain TA 98	Strain TA 100
0	25 ± 3	24 ± 2	19 ± 2	16 ± 3	113 ± 12
1	22 ± 3	21 ± 2	19 ± 2	19 ± 6	121 ± 13
10	25 ± 4	19 ± 3	15 ± 2	19 ± 4	121 ± 19
100	19 ± 2	24 ± 5	18 ± 1	18 ± 4	117 ± 17
1000	21 ± 1	21 ± 4	15 ± 5	17 ± 2	121 ± 6
10000	26 ± 3	22 ± 3	16 ± 4	17 ± 4	133 ± 17
Positive control	66 ± 3	480 ± 23	658 ± 14	216 ± 5	335 ± 21

SD = Standard Deviation

Conclusions:

Interpretation of results:
negative with metabolic activation
negative without metabolic activation

The test material induced the development of as many revertant colonies as those induced by the negative control, both in the absence and in presence of the rat liver metabolizing system, at the maximum dose tested (10 mg/plate).

The test material did not induce an increase in the number of revertant colonies compared to the negative controls and was considered as non-mutagenic in this test-system.

Executive summary:

A test was performed to verify the mutagenic potential of the test material Butanedioic acid, 2,3-dihydroxy-[R-(R*, R*)], C12-13-branched alkyl esters on Salmonella typhimurium.

The test was carried out on 5 strains of Salmonella typhimurium (TA 1535, TA 1537, TA1538, TA 98, TA 100). The mutagenic activity of the material was assessed by comparing the number of revertant colonies induced with the positive control. This activity was tested in the presence and absence of a metabolizing system and done directly in a petri dish. The test material was tested at concentrations of 100000, 10000, 1000, 100 and 10 µg/mL corresponding to doses of 10000, 1000, 100, 10 and 1 µg/petri dish. The results of the study can be summatized as follows: The test material at maximum concentration 10 mg/petri dish produced an increase in number of revertant colonies similar to the negative control so the test material Butanedioic acid, 2,3-dihydroxy-[R-(R*, R*)], C12-13-branched alkyl esters is unable to induce mutation under these conditions.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

October 2016 - March 2017

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)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Bayerisches Landesamt für Gesundheit und Lebensmittelrecht, Date of Inspection 2015-03-18/19

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

The S9 liver microsomal fraction was prepared at Eurofins Munich. Male Wistar rats were induced with phenobarbital (80 mg/kg bw) and ß-naphthoflavone (100 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

Pre-experiment for toxicity:
5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 µg/mL with and without S9 mix

Experiment I:
without S9 mix: 10 /20 / 50P µg/mL (4 h treatment, 21 h preparation intervall)
with S9 mix: 10 /20 / 50P µg/mL (4 h treatment, 21 h preparation intervall)

Experiment II:
without S9 mix: 10 / 30 / 75P µg/mL (21 h treatment, 21 h preparation intervall)

P Precipitation was observed at the end of treatment

It could not be used a higher dose because of precipitation.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: MEM cell culture medium (MEM + 0% FBS). After a treatment by ultrasound for 30 min at 37°C a homogenous emulsion was obtained. The solvent was compatible with the survival of the cells and the S9 activity. The osmolality and pH of the stock solution were within the physiological range.
- Justification for choice of solvent/vehicle: A solubility test was performed with different solvents and vehicles up to the maximum recommended concentration of 5 mg/mL. The solvent was compatible with survival of the cells and S9 actvity.

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 mix

Untreated negative controls:

yes

Remarks:

culture medium

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Pre-experiment and Experiment I: 4 h with and without S9 mix
Experiment I with and without S9 mix
- recovery time: 17 h
Experiment I with and without S 9 mix and Experiment II without S9 mix:
- Preparation interval: 21 h
Experiment II: 21 h
- Fixation time (start of exposure up to fixation or harvest of cells): 21 h

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, 0.2 µg/mL culture medium, was added to the cultures around 17.5 h after the start of the treatment
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 300 well spread metaphases(containing 22 +/- 1 centromeres) per concentration and validity controls

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (% cells in mitosis; determined by counting the number of cells in mitosis in 1000 cells) and the cell count (RICC) were determined
The RICC was calculated as follows: RICC (%)= N-N0(treated)/N-N0 (untreated) x 100
N0: initial cell number; N: cell number at end of treatment

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
Additionally the number of polyploid cells is scored. Polyploid means a near tetraploid karyotype in the case of this aneuploid cell line.

- Determination of endoreplication: no

Evaluation criteria:

Criteria for determining a positive result:
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- the increase is dose-related when evaluated with an appropriate trend test,
- any of the results are outside the distribution of the historical negative control data
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend test,
- all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured mammalian cells in this test system.

Statistics:

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the concentrations evaluated.
The Chi² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in all experimental conditions.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

see details below

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: not determined
- Effects of osmolality: not determined
- Evaporation from medium: not applicable
- Water solubility: low water solubility, no further details mentioned
- Precipitation: yes
- Other confounding effects: none


RANGE-FINDING/SCREENING STUDIES: A pre-experiment was conducted under identical conditions as described for the main experiment. The following concentrations were tested without and with S9 mix:
5, 10, 20, 50, 100, 200, 500, 1000, 2000 and 5000 µg/mL
Cytotoxicity was characterised by the relative increase in cell count (RICC) in comparison with the controls. In general the culturing and experimental conditions are the same as described for the main experiment. 
On the basis of the data and the observations from the pre experiment and taking into account the recommendations of the guidelines, the concentrations for the main experiments I and II were selected.
The dose group selection for microscopic analyses of chromosomal aberrations was based on recommendations of the guidelines.

COMPARISON WITH HISTORICAL CONTROL DATA: All values for the microscopically evaluated concentrations of the test item and the controls were in the range of the historical control data of the testing laboratory.

Table 1: Pre-experiment for toxicity
Dose group	concentration [mM]	Cell count		Precipitate
		Culture	RICC [%]	(+/-)
without metabolic activation
control	0	110.32	100	-
1	5	123.65	115	-
2	10	130.21	122	-
3	20	103.77	93	-
4	50	104.89	94	-
5	100	74.08	60	+
6	200	76.30	62	+
7	500	50.71	34	+
8	1000	34.80	16	+
9	2000	11.98	0	+
10	5000	11.21	0	+
with metabolic activation
control	0	110.60	100	-
1	5	126.75	118	-
2	10	129.95	121	-
3	20	122.93	114	-
4	50	111.32	101	-
5	100	110.38	100	+
6	200	94.22	82	+
7	500	87.26	74	+
8	1000	58.62	43	+
9	2000	61.09	45	+
10	5000	51.48	35	+

Table 2: Experiment I- Summary of Cytotoxicity Data

without metabolic activation

Dose group	Concentration [mg/mL]	Cell Count			RICC [%]			Precipitate
		1  /2 /  Mean
control	0	90.62/86.20/88.41			100			-
1	5	81.31/77.45/79.38			88			-
2	10	79.73/84.59/82.16			92			-
3	20	90.72/89.42/90.07			102			-
4	50	80.46/77.30/78.88			88			+
5	100	68.42/56.67/62.55			67			+
6	200	57.50/51.20/54.35			57			+
7	500	44.10/46.80/45.45			45			++
EMS	900	73.27/68.79/71.03			78			-

Table 3: Experiment I- Summary of Cytotoxicity Data

with metabolic activation

Dose group	Concentration [mg/mL]	Cell Count			RICC [%]			Precipitate
		1  /2 /  Mean
control	0	101.34/91.13/96.24			100			-
1	5	76.28/84.33/80.31			82			-
2	10	86.55/84.20/85.38			87			-
3	20	96.84/91.08/93.96			97			-
4	50	82.29/85.95/84.12			86			+
5	100	72.85/74.71/73.78			74			+
6	200	64.10/66.86/65.48			64			+
7	500	55.70/51.72/53.71			51			++
CPA	0.83	79.00/81.32/80.16			81			-

Table 4: Experiment II- Summary of Cytotoxicity Data

Dose group	Concentration [mg/mL]	Cell Count			RICC [%]			Precipitate
		1  /2 /  Mean
control	0	208/286/247			100			-
1	5	215/254/235			95			-
2	10	330/222/276			112			-
3	20	206/184/195			78			-
4	30	221/193/207			83			-
5	50	215/194/205			82			-
6	75	199/206/203			81			+
7	100	191/170/181			72			+
8	200	178/201/190			76			+
9	300	192/182/187			75			+
10	400	70/148/109			42			+
11	500	133/139/136			53			+
EMS	400	185/216/201			80			-

Table 5: Experiment I - Summary of Aberration Rates

Dose group	Concentration [µg/mL]	Treatment Time	Fixation Interval	mean aberration cells			Precipitation
				incl. Gaps	excl. Gaps
without metabolic activation
Control	0	4	21	2.3	1.3		-
2	10	4	21	0.7	0.7		-
3	20	4	21	4.0	3.3		-
4	50	4	21	2.7	1.0		+
EMS	900	4	21	9.2	8.4		-
with metabolic activation
Control	0	4	21	3.3	2.3		-
2	10	4	21	3.7	1.7
3	20	4	21	2.3	1.7
4	50	4	21	3.7	2.0		+
CPA	0.83	4	21	9.7	7.3		-

Table 6: Experiment II: Summary of Aberration Rates

Dose Group	Concentration [µg/mL]	Treatment Time	Fixation Interval	mean aberrant cells		Precipitation
				incl. Gaps	excl. Gaps
without metabolic activation
Control	0	21	21	1.7	0.3	-
2	10	21	21	1.0	0.7	-
4	30	21	21	3.0	1.3	-
6	75	21	21	1.3	1.0	+
EMS	400	21	21	9.7	7.0	-

300 cells evaluated for each concentration

EMS: Positive Control (without metabolic activation: Ethylmethanesulfonate)

Conclusions:

Interpretation of results:
negative

In conclusion, it can be stated that during the described in vitro chromosome aberration test and under the experimental conditions reported, the test item Butanedioic acid, 2,3-dihydroxy-[R-(R*,R*)]-, C12-13-branched alkyl esters did not induce structural chromosomal aberrations in the V79 Chinese hamster cell line.
Therefore, the test item Butanedioic acid, 2,3-dihydroxy-[R-(R*,R*)]-, C12-13-branched alkyl esters is considered to be non-clastogenic in this chromosome aberration test.

Executive summary:

To investigate the potential of Butanedioic acid, 2,3-dihydroxy-[R-(R*,R*)]-, C12-13-branched alkyl esters to induce structural chromosome aberrations in Chinese hamster V79 cells, an in vitro chromosome aberration assay was carried out.

The metaphases were prepared 21 h after start of treatment with the test item. The treatment interval was 4 h without and with metabolic activation in experiment I. In experiment II, the treatment interval was 21 h without metabolic activation. Duplicate cultures were treated at each concentration. 150 metaphases per culture were scored for structural chromosomal aberrations, except for the positive control EMS (250 cells) in experiment I without metabolic activation due to a clearly positive result.

The following concentrations were evaluated for the microscopic analysis of chromosomal aberrations:

Experiment I:

without and with metabolic activation: 10, 20 and 50 µg/mL

Experiment II:

without metabolic activation: 10, 30 and 75 µg/mL

In experiment I, precipitation of the test item was noted without and with metabolic activation at concentrations of 50 µg/mL and higher with the unaided eye. In experiment II, precipitation of the test item was seen without metabolic activation at concentrations of 75 µg/mL and higher.

In experiment I without metabolic activation, cytotoxic effects of the test item were noted at concentrations of 100 µg/mL and higher judged by the RICC. With metabolic activation cytotoxic effects of the test item were noted at concentrations of 200 µg/mL and higher.

In experiment II without metabolic activation, cytotoxic effects of the test item were observed at concentrations of 400 µg/mL and higher.

In both experiments, no biologically relevant increase of the aberration rates was noted after treatment with the test item without and with metabolic activation. The aberration rates of all dose groups treated with the test item were within the historical control data of the negative control.

As neither in experiment I nor in experiment II an increase in chromosomal aberrations was observed, an additional sampling time after 28 h was not regarded as necessary. Furthermore, the highest concentration evaluated was selected by precipitation, which was already noticed at a concentration of 50 µg/mL respectively 75 µg/mL. At these concentrations, cytotoxic effects were not present compared to the negative control. Therefore, extensive mitotic delay was not induced by the test item.

The Fisher´s exact test was performed to verify the results in the experiment. No statistically significant increase (p < 0.05) of cells with chromosomal aberrations was noted in the concentrations evaluated.

The CHI² Test for trend was performed to test whether there is a concentration-related increase in chromosomal aberrations. No statistically significant increase was observed in all experimental conditions.

EMS (400 and 900 µg/mL) and CPA (0.83 µg/mL) were used as positive controls and induced distinct and biologically relevant increases of chromosomal aberrations, thus proving the efficiency of the test system to indicate potential clastogenic effects.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable publication that meets basic scientific principles.

Justification for type of information:

Please see Analogue Approach

Reason / purpose for cross-reference:

read-across source

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

no

Type of assay:

mammalian cell gene mutation assay

Target gene:

Thymidine kinase locus.

Key result

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:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of di(2-ethylhexyl)adipate was observed at 1000 µg/ml, but testing was continued up 5000 µg/ml. In the absence of S9 mix there was no indication of a mutagenic response in two experiments. In the presence of S9 mix, one experiment similarly failed to show any mutagenic effect, while significant increases in mutant fraction occured in a second experiment. The LOED in this experiment was 2,000 µg/ml, higher than the precipitation concentration. SIgnificant toxicity was observed in all experiments. It was concluded that di(2-ethylhexyl) adipate was not mutagenic in the system.

Conclusions:

The substance DEHA was in the Mouse lymphoma test not genotoxic.

Executive summary:

The substance DEHA was in the Mouse lymphoma test not genotoxic.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In in vivo Micronucleus tests the similar substances Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters and Bis(C12-C13)alkyl-2-hydroxybutandioate did not cause damage to this mitotic system under the conditions of the studies. It is likely that the same is valid for the test substance.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

July 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Please see Analogue Approach

Reason / purpose for cross-reference:

read-across source

Route of administration:

intraperitoneal

No. of animals per sex per dose:

5

Tissues and cell types examined:

P

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

The difference between the number of micronucleated polychromatic erythrocytes in the treatment group and negative control is not significant.
The difference between the number of micronucleated polychromatic erythrocytes in the treatment group and positive control is significant.
The difference between the number of micronucleated polychromatic erythrocytes in the negative control and positive control is significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and negative control is not significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and positive control is significant.
The difference between the proportion of polychromatic/normochromatic erythrocytes in the negative control and positive control is significant.

Table #1: Mean of micronucleated polychromatic erythrocytes

Concentration [mg/kg bw]

Sampling time

24 h

48 h

72 h

male

female

male

female

male

female

12500

1.40 ± 1.14

1.40 ± 0.89

1.20 ± 0.83

1.20 ± 0.44

1.00 ± 0.71

0.80 ± 0.45

Positive control

61.2 ± 6.06

59.4 ± 2.61

62.2 ± 6.22

61.8 ± 3.35

64.4 ± 5.86

63.4 ± 2.61

Negative control

1.60 ± 1.51

1.80 ± 0.83

1.80 ± 0.83

1.20 ± 1.09

2.00 ± 1.41

0.40 ± 0.89

Table #2: Ratio polychromatic/normochromatic erythrocytes

Concentration [mg/kg bw]

Sampling time

24 h

48 h

72 h

male

female

male

female

male

female

12500

2.30 ± 0.28

2.47 ± 0.30

2.16 ± 0.41

2.41 ± 0.39

2.20 ± 0.31

2.35 ± 0.41

Positive control

0.99 ± 0.10

0.96 ± 0.08

0.98 ± 0.18

0.94 ± 0.12

0.85 ± 0.11

0.81 ± 0.07

Negative control

2.20 ± 0.40

2.30 ± 0.34

2.23 ± 0.23

2.38 ± 0.32

2.23 ± 0.33

2.26 ± 0.43

* = only platelets and blasts were observed

Conclusions:

Interpretation of results: negative
The test material Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters did not cause damage to this mitotic system under the conditions of the study.

Executive summary:

In order to verify its possible mutagenic effect, the test material Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters was administered to 90 mice C57 (45 males and 45 females) according to EEC Directive 92/69. 30 were treated with the test material (12500 mg/kg), 30 treated with Cyclophosphamide (75 mg/kg bw) as positive control and 30 treated with sesame seed oil (50 mL/kg bw) as negative control. The 90 mice were sacrificed at the following times:

- 30 (10 from each group) 24 hours after the beginning of treatment

- 30 (10 from each group) 48 hours after the beginning of treatment

- 30 (10 from each group) 72 hours after the beginning of treatment.

After the sacrifice, the bone marrow of each animal was extracted, smeared on a slide, stained and observed with a microscope. For each animal 1000 polychromatic erythrocytes were counted and the frequence of normochromatic and micronucleated polychromatic erythrocytes were recorded. The data obtained from the proportional relationship between the polychromatic erythrocytes and the normochromatic erythrocytes was statistically evaluated by means of Student's t-test. The following results 24 and 48 hours after the beginning of the test were obtained:

- the difference between the number of micronucleated polychromatic erythrocytes in the treatment group and negative control is not significant.

- the difference between the number of micronucleated polychromatic erythrocytes in the treatment group and positive control is significant

- the difference between the number of micronucleated polychromatic erythrocytes in the negative control and positive control is significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and negative control is not significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the treatment group and positive control is significant

- the difference between the proportion of polychromatic/normochromatic erythrocytes in the negative control and positive control is significant.

It was concluded that the test material Butanedioic acid, 2,3-dihydroxy-di-C14-C15 alkyl esters did not have a mutagenic effect on this mitotic system under the conditions of the study.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

In all available tests the test substance and the similar substances were not mutagenic. No classification for genetic toxicity is indicated according to the classification, labeling and packaging (CLP) regulation (EC 1272/2008).