Diethyl malonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Two studies were performed on diethylmalonat: in vitro gene mutation study in bacteria according to EU B. 13/14 and in vitro gene mutation study in mammalian cells according to OECD 476.

Additionally one study is provided on dimethylmalonate: in vitro cytogenicity / chromosome aberration study in mammalian cells according to OECD 473.

In non of 3 studies the substance was found to have a mutagenic potential.

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

other: TA98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Test concentrations with justification for top dose:

Concentrations tested: 8, 40, 200, 1000, 5000 μg/plate
5000 μg/plate (maximal concentration)

Vehicle / solvent:

DMSO

Details on test system and experimental conditions:

- Species/cell type: Salmonella typhimurium TA 1535, TA 1537, TA 1538, TA 98, TA 100
- Metabolic activation system: phenobarbiturate induced rat liver S9 fraction
- Solvent: DMSO
- Number of replicates: 2 main studies, one plate incorporation, one pre-incubation test. Three replicates per experiment.
- Concentrations tested: 8, 40, 200, 1000, 5000 μg/plate
- Positive and negative controls:
- Negative control: solvent DMSO
- Positive control:
without S9 mix:
- TA 98, 1538: Nitrofluorene (111-304 μg/plate)
- TA 100, 1535 Sodium azide (295-372 μg/plate)
- TA 1537: Aminocridine (78-194 μg/plate)
With S9 mix:
Cyclophosphamide (93-114 μg/plate)
- Pre-incubation time: 30 min at 37 °C
STATISTICAL METHODS: Mean values and standard deviations by Biosys software.

Key result

Species / strain:

other: Salmonella typhimurium TA98, TA 100, TA 1535, TA 1537, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Remarks on result:

other: all strains/cell types tested

Conclusions:

No cytotoxicity was observed up to the maximum test. Thus the test substance does not fulfil the criteria for a classification as genetic toxic according to the study.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

24/03/2003-02/12/2003

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 (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Postmitochondrial (S9) fraction of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500, 5000 µg/mL medium

In the preliminary experiment, complete cytotoxicity was noted at the top concentration of 5000 μg Dimethyl malonate/ml in both experiments without and with metabolic activation. No signs of cytotoxicty were noted at concentrations up to 2500 μg Dimethyl malonate/ml in both experiments without and with metabolic activation.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

POSITIVE CONTROLS:
Without S9: Mitomycin C, 0.1 and 0.2 µg/mL medium
With S9: Cyclophosphamide, 10 to 20 µg/mL medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 20 h
- Selection time: Addition of colcemid (2h)
- Fixation time (start of exposure up to fixation or harvest of cells): 26 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN AND FIXATION: Giemsa; The cells were harvested, fixed in freshly prepared methanol: glacial acetic acid (4: 1) and slides prepared.

NUMBER OF REPLICATIONS: Duplicate (Cells from different donors)

NUMBER OF CELLS EVALUATED: For each treatment and culture 100 metaphases per plate were examined. For the determination of cytotoxicity 1000 cells were scored and the mitotic index determined as percentage of cells in metaphase.

DETERMINATION OF CYTOTOXICITY
-Preliminary cytotoxicity test:
With concentrations from 10 to 5000 µg/mL medium with and without metabolic acitvation, 48 h after culture establishment, 24 h incubation. Examination of 1 slide per culture, 1000 lymphocytes per culture.
- Method: mitotic index

Statistics:

Fisher Exact Test

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a dose of 5000 µg/mL with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS:
- With metabolic activation:
The mean incidence of chromosomal aberrations excluding gaps at concentrations from 625 to 5000 µg/mL ranged from 1.5% to 3.5% and was comparable to control rates and within the historical control range of 0 to 5%. There was no dose related increase in chromosomal aberrations. No polyploidy was noted.
- Without metabolic activation: The mean incidence of chromosomal aberrations excluding gaps at concentrations from 625 to 5000 µg/mL ranged from 1.0% to 3% and was comparable to control rates and within the historical control range of 0 to 5%. There was no dose related increase in chromosomal aberrations. No polyploidy was noted.
All positive and negative controls gave the expected results that were within the ranges of the historical controls of the laboratory and consistent with those reported in the literature.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
MITOTIC INDEX:
Pretest.
Without S9 mix, 24 h exposure:
At concentrations up to 250 µg/mL >= 1
1000 µg/mL: 0.44
2500 µg/mL: 0.72
5000 µg/mL: 0
With S9, 4 h exposure:
At concentrations up to 1000 µg/mL: 0.72 to 1.0 (not concentration dependent)
2500 µg/mL: 0.44
5000 µg/mL: 0

Main experiment:
Without S9, 4 h exposure: not significantly reduced (0.88 to 1.0)
Without S9, 24 h exposure: >=1 up to 625 µg/mL
1250 µg/mL: 0.79
2500 µg/mL: 0.54
With S9:
Not significantiy reduced up to 1250 µg/mL: 0.92 to 1.22
2500 µg/mL 0.72 and 0.87
5000 µg/mL: 0 in both experiments

CYTOTOXIC CONCENTRATION:
- With metabolic activation: Pretest and main test: 5000 µg/mL
- Without metabolic activation: Pretest and main test: 5000 µg/mL after 24 h.

The incidence of chromosomal aberrations (excluding gaps) was not significantly different from the controls with and without metabolic activation at all tested dose levels. The positive controls induced a statistically significant increase in chromosomal aberrations excluding gaps.

Conclusions:

Under the present test conditions, Dimethyl malonate tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without 59) and one exposure time (with 59) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

Executive summary:

Treatment with concentrations of 2500 or 5000 μg Dimethyl malonate/ml medium in the experiment without metabolic activation (24-h exposure) resulted in pronounced to complete cytotoxicity. In the experiments with metabolic activation complete cytotoxicity was observed at the top concentration of 5000 μg Dimethyl malonate/ml.

Tests without metabolic activation (4- and 24-hour exposure):

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dimethyl malonate at concentrations from 625 to 5000 μg/m L or 31 2 .5 to 1250 μg/ml medium (4-h and 24-h exposure, respectively) in the absence of metabolic activation ranged from 1.0% to 3.0%. No polyploidy was noted. The results obtained are considered to be within the normal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 0.0% or 2 .5 % was observed after a 4-hour and 24-hour exposure, respectively.

Test with metabolic activation (4-hour exposure):

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dimethyl malonate at concentrations from 625 to 2500 μg/mL medium in the presence of metabolic activation ranged from 1.5% to 3.5%. No polyploidy was noted. The results obtained are considered to be within the normal range of the negative controls where a mean incidence of chromosomal aberrations (excluding gaps) of 0.5% or 1 .0% were observed. The incidence of chromosomal aberrations (excluding gaps) of the controls at Dimethyl malonate concentrations from 625 to 2500 μg/ml medium were statistically not significant at (p s 0.05), using the exact test of R.A. FISHER as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society). The incidence of chromosomal aberrations (excluding gaps) of the controls without and

with metabolic activation ranged from 0.0% to 5 .0% for the last 30 experiments.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Study period:

24/03/2003-02/12/2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

A study on the structural similar dimethyl malonate is available.
Please see the section 13.2 of the IUCLID file (OECD SIAM 20 Assessment report) for the read-across justification.

This document stated (page 5):
"Because it is
impossible to reach blood levels of ethanol which are associated with reproductive/developmental toxicity as a
consequence of the manufacture and normal use of DEM, it can be expected that malonic acid will be the
metabolite that determines the toxicity of DEM. Taking also into account that DEM is not likely to be more toxic
than DMM, which has shown no potential for reproductive and developmental toxicity, it is overall concluded
that there is no indication for a relevant reproductive and/or developmental toxicity of DMM and DEM."

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

Postmitochondrial (S9) fraction of rats treated with Arochlor 1254.

Test concentrations with justification for top dose:

312.5, 625, 1250, 2500, 5000 µg/mL medium

In the preliminary experiment, complete cytotoxicity was noted at the top concentration of 5000 μg Dimethyl malonate/ml in both experiments without and with metabolic activation. No signs of cytotoxicty were noted at concentrations up to 2500 μg Dimethyl malonate/ml in both experiments without and with metabolic activation.

Vehicle / solvent:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive control substance:

cyclophosphamide

mitomycin C

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

POSITIVE CONTROLS:
Without S9: Mitomycin C, 0.1 and 0.2 µg/mL medium
With S9: Cyclophosphamide, 10 to 20 µg/mL medium

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 20 h
- Selection time: Addition of colcemid (2h)
- Fixation time (start of exposure up to fixation or harvest of cells): 26 h


SPINDLE INHIBITOR (cytogenetic assays): colcemid

STAIN AND FIXATION: Giemsa; The cells were harvested, fixed in freshly prepared methanol: glacial acetic acid (4: 1) and slides prepared.

NUMBER OF REPLICATIONS: Duplicate (Cells from different donors)

NUMBER OF CELLS EVALUATED: For each treatment and culture 100 metaphases per plate were examined. For the determination of cytotoxicity 1000 cells were scored and the mitotic index determined as percentage of cells in metaphase.

DETERMINATION OF CYTOTOXICITY
-Preliminary cytotoxicity test:
With concentrations from 10 to 5000 µg/mL medium with and without metabolic acitvation, 48 h after culture establishment, 24 h incubation. Examination of 1 slide per culture, 1000 lymphocytes per culture.
- Method: mitotic index

Statistics:

Fisher Exact Test

Key result

Species / strain:

lymphocytes: human peripheral blood lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at a dose of 5000 µg/mL with and without metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

GENOTOXIC EFFECTS:
- With metabolic activation:
The mean incidence of chromosomal aberrations excluding gaps at concentrations from 625 to 5000 µg/mL ranged from 1.5% to 3.5% and was comparable to control rates and within the historical control range of 0 to 5%. There was no dose related increase in chromosomal aberrations. No polyploidy was noted.
- Without metabolic activation: The mean incidence of chromosomal aberrations excluding gaps at concentrations from 625 to 5000 µg/mL ranged from 1.0% to 3% and was comparable to control rates and within the historical control range of 0 to 5%. There was no dose related increase in chromosomal aberrations. No polyploidy was noted.
All positive and negative controls gave the expected results that were within the ranges of the historical controls of the laboratory and consistent with those reported in the literature.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
MITOTIC INDEX:
Pretest.
Without S9 mix, 24 h exposure:
At concentrations up to 250 µg/mL >= 1
1000 µg/mL: 0.44
2500 µg/mL: 0.72
5000 µg/mL: 0
With S9, 4 h exposure:
At concentrations up to 1000 µg/mL: 0.72 to 1.0 (not concentration dependent)
2500 µg/mL: 0.44
5000 µg/mL: 0

Main experiment:
Without S9, 4 h exposure: not significantly reduced (0.88 to 1.0)
Without S9, 24 h exposure: >=1 up to 625 µg/mL
1250 µg/mL: 0.79
2500 µg/mL: 0.54
With S9:
Not significantiy reduced up to 1250 µg/mL: 0.92 to 1.22
2500 µg/mL 0.72 and 0.87
5000 µg/mL: 0 in both experiments

CYTOTOXIC CONCENTRATION:
- With metabolic activation: Pretest and main test: 5000 µg/mL
- Without metabolic activation: Pretest and main test: 5000 µg/mL after 24 h.

The incidence of chromosomal aberrations (excluding gaps) was not significantly different from the controls with and without metabolic activation at all tested dose levels. The positive controls induced a statistically significant increase in chromosomal aberrations excluding gaps.

Conclusions:

Under the present test conditions, Dimethyl malonate tested up to cytotoxic concentrations in the absence and in the presence of metabolic activation employing two exposure times (without 59) and one exposure time (with 59) revealed no indications of mutagenic properties with respect to chromosomal or chromatid damage.

Executive summary:

Treatment with concentrations of 2500 or 5000 μg Dimethyl malonate/ml medium in the experiment without metabolic activation (24-h exposure) resulted in pronounced to complete cytotoxicity. In the experiments with metabolic activation complete cytotoxicity was observed at the top concentration of 5000 μg Dimethyl malonate/ml.

Tests without metabolic activation (4- and 24-hour exposure):

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dimethyl malonate at concentrations from 625 to 5000 μg/m L or 31 2 .5 to 1250 μg/ml medium (4-h and 24-h exposure, respectively) in the absence of metabolic activation ranged from 1.0% to 3.0%. No polyploidy was noted. The results obtained are considered to be within the normal range of the negative control where a mean incidence of chromosomal aberrations (excluding gaps) of 0.0% or 2 .5 % was observed after a 4-hour and 24-hour exposure, respectively.

Test with metabolic activation (4-hour exposure):

The mean incidence of chromosomal aberrations (excluding gaps) of the cells treated with Dimethyl malonate at concentrations from 625 to 2500 μg/mL medium in the presence of metabolic activation ranged from 1.5% to 3.5%. No polyploidy was noted. The results obtained are considered to be within the normal range of the negative controls where a mean incidence of chromosomal aberrations (excluding gaps) of 0.5% or 1 .0% were observed. The incidence of chromosomal aberrations (excluding gaps) of the controls at Dimethyl malonate concentrations from 625 to 2500 μg/ml medium were statistically not significant at (p s 0.05), using the exact test of R.A. FISHER as recommended by the UKEMS guidelines (The United Kingdom Branch of the European Environmental Mutagen Society). The incidence of chromosomal aberrations (excluding gaps) of the controls without and

with metabolic activation ranged from 0.0% to 5 .0% for the last 30 experiments.

Reference 4

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

The V79 cell line has been used successfully in in vitro experiments for many years be-cause of its sensitivity to chemical mutagens. Especially the high proliferation rate (dou-bling time 12 – 16 h in stock cultures) and a high cloning efficiency of untreated cells both necessary for the appropriate performance of the study, recommend the use of this cell line. The cells have a stable karyotype with a modal chromosome number of 22 (Bradley et al., 1981). The cells were purchased by CLS (Eppelheim, Germany) and were sold un-der the name V79-4. The modal chromosome number was analysed and confirmed by the supplier of the cells.

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

liver S9 mix from male rats, treated with Aroclor 1254

Test concentrations with justification for top dose:

Test Item Concentrations in the Pre-Test [mM]: 10, 5, 2.5, 1.25, 0.63, 0.31, 0.16
The highest nominal concentration (experiment I +S9: 10 mM; -S9: 10 mM; experiment II -S9: 10 mM) applied was chosen with regard to the solubility of the test item in organic solvents and aqueous media as well as the results of the pre-test.

Vehicle / solvent:

The test item was completely insoluble in medium but completely soluble in acetone, etha-nol and DMSO. Therefore, DMSO will be used as solvent and a stock solution containing 2000 mM will be prepared for the pre-test.

Untreated negative controls:

yes

Remarks:

DMEM without supplements was used as solvent control for the positive control Ethylme-thane sulfonate (EMS).

Negative solvent / vehicle controls:

yes

Remarks:

DMSO was used as solvent control for the test item and the positive control 7,12-dimethylbenz(a)anthracene (DMBA).

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

First 1 * 106 cells per 10 cm culture dish and 500 cells (for the determination of the cyto-toxicity) per 6 cm culture dish were seeded per tested concentration as well as for the sol-vent and positive controls and incubated for 24 h. The incubation conditions during the whole assay were 37.0° 1.5 °C in 5.0 ± 0.5 % CO2.
The cells were treated with the test item after incubation. Each concentration was pre-pared in duplicate. Directly after the treatment period the cells were washed with PBS Dul-becco (2.5 % HS) twice (not in experiment II –S9). Fresh complete culture medium (5 % HS) was added to the cells before the following incubation. The further implementation of the experiment was divided into the determination of the survival as well as the viability and the mutagenicity.
For the determination of a cytotoxic effect of the test item, the survival of the cells was measured. For this purpose, the cells in the 6 cm dishes were stained with 0.1 % Löffler’s methylene blue solution in 0.01 % KOH solution after a 7 day-incubation time. The colo-nies were counted and the cloning efficiency (absolute and relative) was calculated.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

not specified

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 1        Summary of Results of Experiment I

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[mM]		[h]		[%]		Mean
Solvent Control Test Item (DMSO)	-	+	4	A	-	12	13
				B	-	14
Solvent Control DMBA (DMSO)	-	+	4	A	-	6	9
				B	-	12
Positive Control (DMBA)	1.5µg/mL	+	4	A	55.0	371**	358**
				B	90.5	345**
Test item	10	+	4	A	83.6	16	16
				B	93.3	17
Test item	5	+	4	A	64.2	21	22
				B	77.3	22
Test item	2.50	+	4	A	89.9	22	20
				B	88.1	18
Test item	1.25	+	4	A	61.6	21	22
				B	76.6	23
Test item	0.63	+	4	A	73.0	n/e	n/e
				B	87.2	n/e
Test item	0.31	+	4	A	62.0	n/e	n/e
				B	71.5	n/e
Solvent Control Test Item (DMSO)	-	-	4	A	-	35+	29
				B	-	23
Solvent Control EMS (DMEM)	-	-	4	A	-	37	36
				B	-	34
Positive Control (EMS)	300 µg/mL	-	4	A	74.2	271**+	233**
				B	80.2	194**
Test item	10	-	4	A	132.5	16	19
				B	103.0	22
Test item	5	-	4	A	103.1	19	29
				B	88.1	38
Test item	2.50	-	4	A	75.0	31	28
				B	97.9	25
Test item	1.25	-	4	A	106.5	9	12
				B	99.7	16
Test item	0.63	-	4	A	97.1	n/e	n/e
				B	106.0	n/e
Test item	0.31	-	4	A	46.5	n/e	n/e
				B	120.0	n/e

n/e = not evaluated because the OECD 476 guideline requires only 4 concentrations

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

+ value is marginal out of the historical data (95.5 % control limit)

Table 2        Summary of Results of Experiment II

	Concentration	S9 mix	Treatment time	Culture	Relative Survival	mutant frequency per 10^6cells	mutant frequency per 10^6cells
	[mM]		[h]		[%]		Mean
Solvent Control Test Item (DMSO)	-	-	24	A	-	14	14
				B	-	14
Solvent Control EMS (DMEM)	-	-	24	A	-	11	10
				B	-	8
Positive Control (EMS)	150 µg/mL	-	24	A	98.5	239**	246**
				B	105.9	253**
Test item	10	-	24	A	97.9	11	11
				B	82.4	11
Test item	5	-	24	A	94.1	11	9
				B	88.6	8
Test item	2.50	-	24	A	100.2	9	11
				B	90.2	12
Test item	1.25	-	24	A	95.1	11	10
				B	91.3	10
Test item	0.63	-	24	A	98.9	n/e	n/e
				B	87.5	n/e
Test item	0.31	-	24	A	96.8	n/e	n/e
				B	95.0	n/e

 

n/e = not evaluated because the OECD 476 guideline requires only 4 concentrations

Asterisks indicate statistically significant differences to solvent control, with * p < 0.05, ** p < 0.01

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)