2-Butenedioic acid (Z)-, ester with 1,2-propanediol, compd. with 2-(dibutylamino)ethanol

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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: Well documented and reported study fully adequate for assessment. The study was conducted according to internationally accepted technical guidelines and in compliance with GLP in a recognized contract research organization.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Test guideline 3

Qualifier:

no guideline available

Principles of method if other than guideline:

For the Comet assay no internationally accepted guideline is available. This study was conducted according to the procedures indicated by the following recommendations: Tice, R.R. et al. (2000): Single Cell Gel/Comet Assay: Guidelines for ln Vitro and ln Vivo Genetic Toxicology Testing. Environmentaland Molecular Mutagenesis 35: 206-221 and Hartmann, A. et al. (2003): Recommendations for conducting the in vivo alkaline Comet assay. Mutagenesis 18 (1): 45-51

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Micronucleus Assay and Comet Assay

Test material

Test material information

Constituent 1

Test animals

Species:

rat

Strain:

Wistar

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS:
- Strain: rat (Wistar HsdCpb: WU)
- Source: Charles River Laboratories, Research Models and Services Germany GmbH,Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Age at study initiation: 8-11 weeks
- Weight at study initiation: 271.8 g (SD +/- 6.7 g)
- Housing: in groups in cage type Makrolon Type IV, with wire mesh top
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/-2
- Humidity (%): 45 - 65
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: sterile water
- Justification for choice of solvent/vehicle: It shows relative non-toxicity for the animals.
- Amount of vehicle (if gavage or dermal): 10 mL/kg b.w.

Details on exposure:

oral administration

Duration of treatment / exposure:

48h

Frequency of treatment:

All animals received three times (48h, 24h, 4h prior preparation) orally a single standard volume.
The animals of the positive control groups received the positive control substances once orally.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

200 mg/kg bw/day (actual dose received)

Doses / concentrations 2

Dose / conc.:

400 mg/kg bw/day (actual dose received)

Doses / concentrations 3

Dose / conc.:

800 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

7 male rats/dose

Control animals:

yes, concurrent vehicle

Positive control(s):

For the Micronucleus Assay: Cyclophosphamide (CPA)
- Administration: orally, once, 24h prior to preparation
- Doses / concentrations: CPA dissolved in sterile wate; 20 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.

For the Comet Assay: Methylmethanesulphonate (MMS)
- Administration: orally, once, 4h prior to preparation
- Doses / concentrations: MMS dissolved in 0,9% NaCl solution; 25 mg/kg b.w.
- Volume administered: 10 mL/kg b.w.

Examinations

Tissues and cell types examined:

Micronucleus Assay: bone marrow cells of tibias/femur
Comet Assay: hepatocytes and cells from the small intestine

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: A preliminary study on acute toxicity of WS400402 was performed with at least two animals per sex and test group under identical conditions as in the mutagenicity study concerning the following parameters: animal strain; vehicle; route, frequency, and volume of administration. Since no gender-specific differences in toxicity were observed, the main experiment was performed using male animals only treated with doses of 200, 400, and 800 mg/kg b.w. WS400402, respectively.

TREATMENT AND SAMPLING TIMES: Samples were taken at 24h and 48h - covering the interval in which maximum frequencies of micronuclei occur. This could be too late for the assessment of the DNA strand breaks, as these might have undergone repair. For this purpose a shorter additional treatment interval has to be considered in order detect damaged DNA in the correct time frame. For those reasons the animals were treated three times with three doses of the test item at intervals of 48 h, 24 h and 4 h prior to preparation.

DETAILS OF SLIDE PREPARATION: Three slides per organ of the animals were prepared with 10 % cell suspension and 90 % 0.7 % (w/v) agarose (low melting point agarose). 100 µL was applied per slide. The slides were cooled before being submerged in lysis buffer.

METHOD OF ANALYSIS:
Micronucleus Assay: At least 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei by manual inspection. The micronucleated PCE´s per 2000 PCE´s and the ratio of polychromatic erythrocytes to the total number of erythrocytes (NCE + PCE) are presented for each animal.

Comet Assay: DNA strand breaks were analysed using the alkaline single cell gel electrophoresis. One hundred cells per animal were evaluated on coded slides with a fluorescence microscope and the damage of each nucleus was measured and recorded by an image analysis programme (Comet Assay IV, Perceptive Instruments).

Evaluation criteria:

Micronucleus Assay: A test item was classified as mutagenic if it induced either a dose-related increase in the number of micronucleated polychromatic erythrocytes or a statistically significant positive response for at least one of the tested dose levels.
Comet Assay: A test item was classified as mutagenic if it induced either a dose-related increase or a biologically relevant increase in the tail % intensity in a single dose group as compared to the historical control range.

Statistics:

Micronucleus Assay: This was confirmed by means of the nonparametric Mann-Whitney test.
Comet Assay: Normally distributed data was analysed using a one-tailed student´s t-test. Not normal distributed data were analysed using a Mann-Whitney rank sum test. However, the primary point of consideration was the biological relevance of the results.

Results and discussion

Test results

Test results 1

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Micronucleus assay in bone marrow cells

Toxicity:

no effects

Remarks:

no cytotoxicity observed in bone marrow cells

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 2

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Comet assay in liver cells

Toxicity:

no effects

Remarks:

no cytotoxicity observed in liver cells

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Test results 3

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

the result of the Comet Assay in cells of the small intestine at the highest concentration (800 mg/kg b.w.) is disregarded (further discussion see in "applicant's conclusion")

Toxicity:

yes

Remarks:

cytotoxicity observed in cells of small intestine at each dose level without showing a dose-response effect

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 800 and 1250 mg/kg b.w.
- Clinical signs of toxicity in test animals: Severe toxic symptoms after the second treatment at the high concentration; the moribund animals were sacrificed. Animals treated three times with 800 mg/kg b.w. showed clear signs of toxicity, e.g. hunchback, ruffled fur. This concentration was taken as the highest one in the definitve study.

RESULTS OF DEFINITIVE STUDY
- Micronucleus assay: There was no biologically relevant or statistically significant enhancement in the frequency of the detected micronuclei after administration of the test material and with any dose level used. The observed values in all test material treated groups are below the concurrent solvent controls; no micronuclei induced at none of the tested concentrations.
- Comet assay:
The comet assay on cells of the liver did not reveal a biologically relevant or statistically significant increase in DNA damage at any of the tested dose levels compared to the corresponding vehicle controls on the evaluated parameter (% Tail Intensity). All obtained test material group values were below or near to the values of the vehicle control group and additionally within the historical vehicle control data range.

The comet assay on cells of the small intestine, i.e. the first site of contact of cells with the substance, did not reveal an increase in DNA damage at 200 and 400 mg/kg b.w. of the tested item compared to the corresponding vehicle control on the evaluated parameter (% Tail Intensity). However, with the highest dose applied (800 mg/kg b.w.) an increased value of % Tail Intensity was observed as compared to the solvent control. This result is considered as artefact as discussed in "applicant's conclusion". The number of dead cells was twice as high as the vehicle control value in cells of the small intestine treated with test material - without showing a dose response effect.
Results for the comet assay in cells of the small intestine see Table 1 ("Any other information on results incl. tables").

Any other information on results incl. tables

Table 1a: Analysis of Dead Cell Index and % Tail Intensity in Cells of the Small Intestine

Test Group	Dose (mg/kg b.w.)	Dead Cell Index		Comet Assyay Data
		apototic/dead cells per 500 cells	Standard Deviation	Mean % Tail Intensity	Standard Deviation	Number of evaluable animals
Negative Control	0	15.3	11.1	4.55	1.7	7
WS400402	3x200	30.3	9.1	2.85	0.8	7
	3x400	31.3	11.1	2.18	0.4	6*
	3x800	30.3	10.3	10.35	4.9	7
Methyl methane sulphonate	1x25	32.0	12.5	21.98	6.2	7

* one animal scarified due to an application error

Table 1b: Biometry (Cells of the Small Intestine)

Negative controll versus test group	Mean % Tail Intensity
Dose of WS400402	Significance	p
3 x 200 mg/kg b.w.	n.t.	-
3 x 400 mg/kg b.w.	n.t.	-
3 x 800 mg/kg b.w.	+	<0.011
1 x 25 mg/kg b.w. Methylmethanesulphonate	+	<0.001

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
This in vivo genotoxicity test was performed because in two in vitro tests positive results were obtained for clastogenicity and for gene mutation.
In the micronucleus test-part of this study induction of micronuclei in bone marrow cells was not detected up to the highest dose. For evaluation of an in vivo potential for gene mutation in liver cells the Comet assay was performed instead of a test for unscheduled DNA synthesis (UDS). The positive control substance induced genotoxicity in liver cells whereas no effects were observed up to the highest dose of WS400402. From these results it can be stated that under the experimental conditions reported, WS400402, up to the maximum dose level tested, did neither induce micronuclei as determined by the micronucleus test with bone marrow cells nor primary DNA damage in the liver as determined by the Comet assay.
Next to these two endpoints in bone marrow cells and liver cells also cells of the small intestine, i.e. the first site of contact with the test substance, were investigated for DNA damage by the Comet assay. In these cells cytotoxicity was observed at each dose level whereas in bone marrow and liver cells no cytotoxicity was observed. The severity of cytotoxicity seen as apoptotic and dead cells was independent of the applied dose. At the highest dose, i.e. 800 mg/kg body weight applied by gavage, an increase of DNA damage was observed. At the two lower doses no effects were seen. However, this result of DNA damage is regarded as artefact for the following reason. The test substance WS400402 was applied by oral gavage at a concentration of 80 mg/ml with a dosing volume of 10 ml/kg body weight. This concentration is 40 times higher than the maximum recommended concentration of test substances in in vitro genotoxicity tests. A significant dilution of the test substance concentration in the stomach and small intestine does not occur. Thus, the cells were exposed to a very high concentration of WS400402 which already at a dose of 200 mg/kg b.w. (corresponding to 20 mg/ml) produced significant cytotoxicity to cells of the small intestine. Therefore, the observed increase of DNA damage at the highest dose of 800 mg/kg is considered an artefact due to too high test substance concentration and not as result of true genotoxic/mutagenic activity of WS400402. No effects in the cells of the small intestine were observed at the two lower dose concentrations. It is concluded that WS400402 does not have genotoxic/mutagenic activity.