Dipropoxymethane

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental start date 28 July 2017 Experimental completion date 17 November 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

Diethoxymethane

EC Number:

207-330-6

EC Name:

Diethoxymethane

Cas Number:

462-95-3

Molecular formula:

C5H12O2

IUPAC Name:

Diethoxymethane

Test material form:

liquid

Details on test material:

Identification: Diethoxymethane
Synonym: Ethylal
CAS Number: 462-95-3
Physical State/Appearance: Clear colourless liquid
Storage Conditions: Approximately 4 °C, in the dark, under nitrogen
No correction for purity was required.

Specific details on test material used for the study:

Identification: Ethylal
Chemical name: Diethoxymethane
CAS Number: 462-95-3
Physical state/Appearance: Clear colorless liquid
Storage Conditions: Approximately 4 °C in the dark under nitrogen
Intended use/Application: Not supplied
No correction for purity was made.

Method

Target gene:

not applicable

Species / strain

Species / strain / cell type:

primary culture, other: whole blood

Details on mammalian cell type (if applicable):

Cells
For each experiment, sufficient whole blood was drawn from the peripheral circulation of a non-smoking volunteer (18-35) who had been previously screened for suitability. The volunteer had not knowingly been exposed to high levels of radiation or hazardous chemicals and had not knowingly recently suffered from a viral infection. Based on over 20 years in-house data for cell cycle times for lymphocytes using BrdU (bromodeoxyuridine) incorporation to assess the number of first, second and third division metaphase cells to calculate the average generation time (AGT) for human lymphocytes it is considered to be approximately 16 hours. Therefore using this average the in-house exposure time for the experiments for 1.5 x AGT is 24 hours.

The details of the donors used are:
Preliminary Toxicity Test: male, aged 29 years
Main Experiment: female, aged 25 years

Cell Culture
Cells (whole blood cultures) were grown in Eagle's minimal essential medium with HEPES buffer (MEM), supplemented “in-house” with L-glutamine, penicillin/streptomycin, amphotericin B and 10% fetal bovine serum (FBS), at approximately 37 ºC with 5% CO2 in humidified air. The lymphocytes of fresh heparinized whole blood were stimulated to divide by the addition of phytohaemagglutinin (PHA).

Cytokinesis block (if used):

Cytochalasin B

Metabolic activation:

with and without

Metabolic activation system:

standard metabolizing system (S9)

Test concentrations with justification for top dose:

Preliminary Toxicity Test
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.

Main Experiment
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Vehicle / solvent:

The test item was miscible in aqueous media (MEM) at 10.42 mg/mL by mixing on a vortex for approximately 10 seconds in a solubility check performed in-house. The highest concentration of the test item tested in this study was 10.42 mg/mL.

Controls

Controls 1

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.2 μg/mL for 4-hour exposure

Positive control substance:

mitomycin C

Remarks:

Absence of S9-mix

Controls 2

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

0.075 μg/mL for 24-hour continuous exposure

Positive control substance:

other: Demecolcine

Remarks:

Absence of S9-mix

Controls 3

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

Minimal Essential Medium

True negative controls:

no

Positive controls:

yes

Remarks:

5 μg/mL for 4-hour exposure

Positive control substance:

cyclophosphamide

Remarks:

Presence of S9-mix

Details on test system and experimental conditions:

Culture conditions
Duplicate lymphocyte cultures (A and B) were established for each dose level by mixing the following components, giving, when dispensed into sterile plastic flasks for each culture:
8.05-9.05 mL MEM, 10% (FBS)
0.1 mL Li-heparin
0.1 mL phytohaemagglutinin
0.75 mL heparinized whole blood

4-Hour Exposure With Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC, 5% CO2 in humidified air, the cultures were transferred to tubes and centrifuged. Approximately 9 mL of the culture medium was removed, reserved, and replaced with the required volume of MEM (including serum) and 1.0 mL of the appropriate solution of vehicle control or test item was added to each culture. For the positive control, 0.1 mL of the appropriate solution was added to the cultures. 1.0 mL of 20% S9-mix (i.e. 2% final concentration of S9 in standard co-factors) was added to the cultures of the Preliminary Toxicity Test and the Main Experiment. All cultures were then returned to the incubator. The nominal total volume of each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

4-Hour Exposure Without Metabolic Activation (S9)
After approximately 48 hours incubation at approximately 37 ºC with 5% CO2 in humidified air, the cultures were decanted into tubes and centrifuged. Approximately 9 mL of the culture medium was removed and reserved. The cells were then resuspended in the required volume of fresh MEM (including serum) and dosed with 1.0 mL of the appropriate vehicle control, test item solution or 0.1 mL of positive control solution. The nominal total volume for each culture was 10 mL.
After 4 hours at approximately 37 ºC, the cultures were centrifuged, the treatment medium was removed by suction and replaced with an 8 mL wash of MEM culture medium. After a further centrifugation the wash medium was removed by suction and replaced with the reserved original culture medium, supplemented with Cytochalasin B, at a final concentration of 4.5 μg/mL, and then incubated for a further 24 hours.

24-Hour Exposure Without Metabolic Activation (S9)
The exposure was continuous for 24 hours in the absence of metabolic activation. Therefore, when the cultures were established the culture volume was a nominal 9 mL. After approximately 48 hours incubation the cultures were removed from the incubator and dosed with 1.0 mL of vehicle control, test item dose solution or 0.1 mL of positive control solution. The nominal total volume of each culture was 10 mL. The cultures were then incubated for 24 hours, the tubes and the cells washed in MEM before resuspension in fresh MEM with serum. At this point Cytochalasin B was added at a final concentration of 4.5 μg/mL, and then the cells were incubated for a further 24 hours.
The extended exposure detailed above does not follow the suggested cell treatment schedule in the Guideline. This is because it avoids any potential interaction between Cytochalasin B and the test item during exposure to the cells and any effect this may have on the activity or response. Additionally, as the stability or reactivity of the test item is unknown prior to the start of the study this modification of the schedule is considered more effective and reproducible due to the in-house observations on human lymphocytes and their particular growth characteristics in this study type and also the significant laboratory historical control data using the above format.
The preliminary toxicity test was performed using the exposure conditions as described for the Main Experiment but using single cultures only, whereas the Main Experiment used replicate cultures.

Preliminary Toxicity Test
Three exposure groups were used:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used was 0, 4.07, 8.14, 16.28, 32.56, 65.13, 130.25, 260.5, 521 and 1042 μg/mL.
Parallel flasks, containing culture medium without whole blood, were established for the three exposure conditions so that test item precipitate observations could be made. Precipitate observations were recorded at the beginning and end of the exposure periods.
Using a qualitative microscopic evaluation of the microscope slide preparations from each treatment culture, appropriate dose levels were selected for the evaluation of the frequency of binucleate cells and to calculate the cytokinesis block proliferation index (CBPI). Coded slides were evaluated for the CBPI. The CBPI data were used to estimate test item toxicity and for selection of the dose levels for the experiments of the main test.

Main Experiment
Three exposure groups were used for Main Experiment:
i) 4-hour exposure to the test item without S9-mix, followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
ii) 4-hour exposure to the test item with S9-mix (2%), followed by a 24 hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
iii) 24-hour continuous exposure to the test item without S9-mix, followed by a 24-hour incubation period in treatment-free media, in the presence of Cytochalasin B, prior to cell harvest.
The dose range of test item used for all three exposures was 0, 65.13, 130.25, 260.5, 521, 781.5, 1042 μg/mL.

Cell Harvest
At the end of the Cytochalasin B treatment period the cells were centrifuged, the culture medium was drawn off and discarded, and the cells resuspended in MEM. The cells were then treated with a mild hypotonic solution (0.0375M KCl) before being fixed with fresh methanol/glacial acetic acid (19:1 v/v). The fixative was changed at least three times and the cells stored at approximately 4 ºC prior to slide making.

Preparation of Microscope Slides
The lymphocytes were re-suspended in several mL of fresh fixative before centrifugation and re-suspension in a small amount of fixative. Several drops of this suspension were dropped onto clean, wet microscope slides and left to air dry. Each slide was permanently labelled with the appropriate identification data.

Staining
When the slides were dry they were stained in 5% Giemsa for 5 minutes, rinsed, dried and a cover slip applied using mounting medium.

Evaluation criteria:

Providing that all of the acceptability criteria are fulfilled, a test item is considered to be clearly negative if, in most/all of the experimental conditions examined:
1. None of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is no dose-related increase.
3. The results in all evaluated dose groups should be within the range of the laboratory historical control data.

Providing that all of the acceptability criteria are fulfilled, a test item may be considered to be clearly positive, if in any of the experimental conditions examined, there is one or more of the following applicable:
1. At least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control.
2. There is an increase which can be considered to be dose-related.
3. The results are substantially outside the range of the laboratory historical negative control data.

When all the criteria are met, the test item is considered able to induce chromosome breaks and/or gain or loss in this test system.
There is no requirement for verification of a clear positive or negative response.
In case the response is neither clearly negative nor clearly positive as described above or in order to assist in establishing the biological relevance of a result, the data should be evaluated by expert judgement and/or further investigations.
Test items that induce micronuclei in the MNvit test may do so because they induce chromosome breakage, chromosome loss, or a combination of the two. Further analysis using anti-kinetechore antibodies, centromere specific in situ probes, or other methods can be used to determine whether the mechanism of micronucleus induction is due to clastogenic and/or aneugenic activity.

Statistics:

The frequency of binucleate cells with micronuclei was compared, where necessary, with the concurrent vehicle control value using the Chi-squared Test on observed numbers of cells with micronuclei. Other statistical analyses may be used if appropriate (Hoffman et al., 2003). A toxicologically significant response was recorded when the p value calculated from the statistical analysis of the frequency of binucleate cells with micronuclei was less than 0.05 and there was a dose-related increase in the frequency of binucleate cells with micronuclei which was reproducible.

Results and discussion

Test results

Species / strain:

primary culture, other: whole blood

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

Preliminary Toxicity Test (Table 1)
The dose range for the Preliminary Toxicity Test was 4.07 to 1042 μg/mL. The molecular weight of the test item was given as 104.15, therefore, the maximum dose level was 1042 μg/mL, which was calculated to be equivalent to the 10mM concentration (the maximum recommended dose level).
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
Microscopic assessment of the slides prepared from the exposed cultures showed that binucleate cells were present at up to the maximum concentration in all three exposure groups. The test item induced no evidence of toxicity in any of the exposure groups.
The selection of the maximum dose level for the Main Experiment was, therefore, based on the maximum recommended dose level (1042 μg/mL) for all three exposure group.

Micronucleus Test – Main Experiment
As during the Preliminary Toxicity Test, there were binucleate cells suitable for scoring at the maximum dose level of test item, 1042 μg/mL, for all three exposure groups.
No precipitate of the test item was observed in the parallel blood-free cultures at the end of the exposure at any dose level tested in any of the exposure groups.
The CBPI data confirm the qualitative observations in that no dose-related inhibition of CBPI was observed Tables 2 to 6).
The maximum dose level selected for analysis of binucleate cells was the maximum recommended dose level (1042 μg/mL) for all three exposure groups.
The vehicle control cultures had frequencies of cells with micronuclei within the expected range. The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.
The test item did not induce any statistically significant increases in the frequency of binucleate cells with micronuclei, either in the absence or presence of metabolic activation.

Any other information on results incl. tables

 

The dose levels of the controls and the test item are given in the table below:

 

Group	Final concentration of test item Ethylal (µg/mL)
4-hour without S9	0*,65.13, 130.25, 260.5, 521*, 781.5*, 1042*,MMC0.2*
4-hour with S9 (2%)	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,CP5*
24-hour without S9	0*, 65.13, 130.25, 260.5, 521*, 781.5*, 1042*,DC0.075*

 

*   = Dose levels selected for analysis of micronucleus frequency in binucleate cells

MMC  = MitomycinC

CP = Cyclophosphamide

DC            =Demecolcine

 

 

Table 1: CBPI - Preliminary Toxicity test

4-hour exposure without S9						4-hour exposure with S9						24-hour exposure without S9
Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis	Dose Level μg/mL	Nucleate Cells/500 Cells			CBPI	% Cytostasis
	Mono	Bi	Multi				Mono	Bi	Multi				Mono	Bi	Multi
0	248	228	24	1.55	0	0	229	244	27	1.60	0	0	83	313	104	2.04	0
4.07	-	-	-	-	-	4.07	-	-	-	-	-	4.07	-	-	-	-	-
8.14	-	-	-	-	-	8.14	-	-	-	-	-	8.14	-	-	-	-	-
16.28	-	-	-	-	-	16.28	-	-	-	-	-	16.28	-	-	-	-	-
32.56	-	-	-	-	-	32.56	-	-	-	-	-	32.56	-	-	-	-	-
65.13	-	-	-	-	-	65.13	-	-	-	-	-	65.13	-	-	-	-	-
130.25	-	-	-	-	-	130.25	-	-	-	-	-	130.25	-	-	-	-	-
260.5	244	229	27	1.57	0‡	260.5	270	210	20	1.50	17	260.5	101	306	93	1.98	6
521	296	193	11	1.43	22	521	201	282	17	1.63	0‡	521	54	354	92	2.08	0‡
1042	228	255	17	1.58	0‡	1042	259	224	17	1.52	13	1042	66	345	89	2.05	0‡

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

Table 2: CBPI Data - Main Experiment - 4HOUR Exposure With and Without Metabolic Activation (S9)

4-Hour exposure without S9								4-hour exposure with S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis	Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		M ono	Bi	Multi						M ono	Bi	Multi
0	A	125	316	59	1.87	1.89	0	0	A	227	241	32	1.61	1.65	0
	B	109	333	58	1.90				B	203	251	46	1.69
65.13	A	-	-	-	-	-	-	65.13	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
130.25	A	-	-	-	-	-	-	130.25	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
260.5	A	-	-	-	-	-	-	260.5	A	-	-	-	-	-	-
	B	-	-	-	-				B	-	-	-	-
521	A	150	306	44	1.79	1.80	10	521	A	231	244	25	1.59	1.59	10
	B	150	302	48	1.80				B	233	244	23	1.58
781.5	A	125	331	44	1.84	1.84	5	781.5	A	217	253	30	1.63	1.59	9
	B	113	353	34	1.84				B	258	209	33	1.55
1042	A	144	325	31	1.77	1.76	15	1042	A	265	209	26	1.52	1.58	12
	B	172	286	42	1.74				B	218	247	35	1.63
MMC 0.2	A	268	227	5	1.47	1.48	46	CP 5	A	356	142	2	1.29	1.31	53
	B	258	238	4	1.49				B	346	146	8	1.32

MMC    = Mitomycin C

CP      = Cyclophosphamide

-        = No selected for scoring

 

 

Table 3: CBPI Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

24-Hour exposure without S9
Dose level (µg/mL)	Replicate	Nucleate cells /500 cells			CBPI	Mean CBPI	% Cytostasis
		Mono	Bi	Multi
0	A	82	383	65	1.97	1.96	0
	B	96	340	64	1.94
65.13	A	-	-	-	-	-	-
	B	-	-	-	-
130.25	A	-	-	-	-	-	-
	B	-	-	-	-
260.5	A	-	-	-	-	-	-
	B	-	-	-	-
521	A	91	346	63	1.94	1.96	0‡
	B	89	336	75	1.97
781.5	A	75	366	59	1.97	1.99	0‡
	B	74	345	81	2.01
1042	A	69	366	65	1.99	1.97	0‡
	B	88	346	57	1.94
DC 0.075	A	196	259	45	1.70	1.66	31
	B	237	218	45	1.62

DC  = Demecolcine

-     = Not selected for scoring

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

 

 

Table 4: CBPI and Micronucleus Data - Main Experiment - 4-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr-S9	0	A	125	316	59	1.87	0	5	0	0	0.50	0.65
		B	109	333	58	1.90		7	1	0	0.80
	521	A	150	306	44	1.79	10	3	0	0	0.30	0.40
		B	150	302	48	1.80		5	0	0	0.50
	781.5	A	125	331	44	1.84	5	6	0	0	0.60	0.50
		B	113	353	34	1.84		4	0	0	0.40
	1042	A	144	325	31	1.77	15	4	1	0	0.50	0.45
		B	172	286	42	1.74		3	1	0	0.40
	MMC 0.2	A	268	227	5	1.47	46	62	5	0	6.70	9.7***
		B	258	238	4	1.49		116	8	3	12.70

MMC   = Mitomycin C

***    = P<0.001

 

Table 5: CBPI and Micronucleus Data - Experiment - 4-Hour Exposure With Metabolic Activation (S9)

Exposure Time +/-S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
4Hr+S9	0	A	227	241	32	1.61	0	5	0	0	0.50	0.45
		B	203	251	46	1.69		4	0	0	0.40
	521	A	231	244	25	1.59	10	1	1	0	0.20	0.25
		B	233	244	23	1.58		3	0	0	0.30
	781.5	A	217	253	30	1.63	9	5	1	1	0.70	0.55
		B	258	209	33	1.55		4	0	0	0.40
	1042	A	265	209	26	1.52	12	4	0	0	0.40	0.50
		B	218	247	35	1.63		6	0	0	0.60
	CP 5	A	356	142	2	1.29	53	35	2	0	3.70	4.15***
		B	346	146	8	1.32		44	2	0	4.60

CP   = Cyclophosphamide

*** = P<0.001

 

Table 6: CBPI and Micronucleus Data - Main Experiment - 24-Hour Exposure Without Metabolic Activation (S9)

Exposure Time +/- S9	Dose Level (μg/mL)	Replicate	Nucleate cells /500 cells			CBPI	% Cytostasis	Micronuclei (MN) Per 1000 Binucleate cells			% Binucleate Cells with MN	Mean % Binucleate Cells with MN
			Mono	Bi	Multi			1 MN	2 MN	>2 MN
24Hr-S9	0	A	82	383	65	1.97	0	6	0	1	0.70	0.55
		B	96	340	64	1.94		3	0	1	0.40
	521	A	91	346	63	1.94	0‡	5	0	0	0.50	0.65
		B	89	336	75	1.97		6	0	2	0.80
	781.5	A	75	366	59	1.97	0‡	7	0	0	0.70	0.90
		B	74	345	81	2.01		11	0	0	1.10
	1042	A	69	366	65	1.99	0‡	0	0	0	0.00	0.40
		B	88	346	57	1.94		2	0	6	0.80
	DC 0.075	A	196	259	45	1.70	31	40	9	5	5.40	5.75***
		B	237	218	45	1.62		36	19	6	6.10

DC   = Demecolcine

***  = P<0.001

‡    = Cytosis reported as 0 as the CBPI value is equal to or higher than the solvent control

Applicant's summary and conclusion

Conclusions:

Under the conditions of this test, the test item, Ethylal, did not induce a statistically significant increase in the frequency of binucleate cells with micronuclei in either the absence or presence of a metabolizing system. The test item was therefore considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.

Executive summary:

 Introduction

 

This report describes the results of an in vitro study for the detection of the clastogenic and aneugenic potential of the test item on the nuclei of normal human lymphocytes.

 

 Methods

 

Duplicate cultures of human lymphocytes, treated with the test item, were evaluated for micronuclei in binucleate cells at three dose levels, together with vehicle and positive controls. Three exposure conditions in a single experiment were used for the study using a 4-hour exposure in the presence and absence of a standard metabolizing system (S9) at a 2%

final concentration and a 24-hour exposure in the absence of metabolic activation. At the end of the exposure period, the cell cultures were washed and then incubated for a further 24 hours in the presence of Cytochalasin B.

 

The dose levels used in the Main Experiment were selected using data from the preliminary toxicity test where the results indicated that the maximum concentration should be the maximum recommended limit (1042 µg/mL). The dose levels selected for the Main Test were as follows:

 

Group	Final concentration of test item Ethylal (µg/mL)
4-hour without S9	0, 65.13, 130.25, 260.5, 521, 781.5, 1042
4-hour with S9 (2%)
24-hour without S9

 

Results

 

All vehicle (Eagle's minimal essential medium with HEPES buffer (MEM)) controls had frequencies of cells with micronuclei within the range expected for normal human lymphocytes.

 

The positive control items induced statistically significant increases in the frequency of cells with micronuclei. Thus, the sensitivity of the assay and the efficacy of the S9-mix were validated.

 

The test item was non-toxic to human lymphocytes and did not induce any statistically significant increases in the frequency of cells with micronuclei, using a dose range that included a dose level that was the maximum recommended dose level.

 

Conclusion

 

Under the conditions of this study, the test item, Ethylal, was considered to be non-clastogenic and non-aneugenic to human lymphocytes in vitro.