Chloroethane

Genetic toxicity: in vitro

Administrative data

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

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference 1

Reference Type:

publication

Title:

Unnamed

Year:

1994

Reference 2

Reference Type:

secondary source

Title:

Chloroethane CAS: 75-00-3

Author:

OECD SIDS

Year:

2006

Bibliographic source:

SIDS Initial Assessment Report for SIAM 22

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes

Type of assay:

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

Test material

Test material information

Constituent 1

Reference substance name:

Chloroethane

EC Number:

200-830-5

EC Name:

Chloroethane

Cas Number:

75-00-3

Molecular formula:

C2H5Cl

IUPAC Name:

chloroethane

Details on test material:

- Name of test material (as cited in study report): chloroethane
- Physical state: gaseous
- Analytical purity: > 99% (Hüls AG, Marl, Germany)

Method

Target gene:

hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene

Species / strain

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from Aroclor-1254 induced livers derived from rats

Test concentrations with justification for top dose:

Trial I (-S9): 0.09, 0.38, 0.94, 1.36, 1.89 mg/mL (determined by GC-FID analyses)
Trial I (+S9): 0.10, 0.38, 0.96, 1.36, 2.34 mg/mL (determined by GC-FID analyses)
Trial II (-S9): 0.65, 0.91, 1.36, 1.72, 2.03 mg/mL (determined by GC-FID analyses)
Trial II (+S9): 1.02, 1.43, 1.80, 2.17, 2.48 mg/mL (determined by GC-FID analyses)

Controls

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, which was used as solvent of the positive control substances

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: methanesulfonate (without S9) and 3-methylcholanthrene (with S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium
CHO cells were seeded in gas-tight glass culture flasks equipped with a septum. After incubation at 37 °C for approx. 20 h, the medium was replaced by medium without FCS. The flasks were tightly closed and appropriate amounts of chloroethane were injected into the flasks through the septum.

DURATION
- Exposure duration: 4 h
- Expression time (cells in growth medium): 7 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 10 µg 6-thioguanine/mL

NUMBER OF REPLICATIONS: 2 independent experiments (each with duplicate exposure cultures per concentration)

DETERMINATION OF CYTOTOXICITY
- Method: After exposure period cells were harvested and seeded with 200 cells/dish in medium with FCS. After incubation for 7 days at 37 °C, colonies were fixed with methanol, stained with Giemsa and counted. Cell survival is expressed as the plating efficiency of treated cells relative to untreated cells.

CELL CULTURE MEDIUM
- Type and identity of media:
Ham's F12 medium supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 IU/mL penicillin and 50 µg/mL streptomycin
In the main study futher supplements were added to the media to reduce the number of cells harboring spontaneous mutations of the HPRT locus: 200 µM glycine, 5 µM thymidine, 10 µM hypoxanthine, 3.2 µM aminopterin)

Statistics:

The statistical significance of induced mutation frequencies was determined by means of t-test (two-sample analysis).

Results and discussion

Test results

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING:
The concentrations used in the main studies (trial I and II) based on the results of the cytotoxicity assays.

Any other information on results incl. tables

Target and measured chloroethane concentrations in the HPRT test with CHO cells and corresponding plating efficiencies

Trial	S9 mix	chloroethane target conc. (mg/vial)	chloroethane measured conc. (mg/vial)	chloroethane (mg/mL)	Absolute Plating efficiencies (%)
1	-	0	-	0	81
	-	2.5	2.9	0.09	85
	-	10.0	12.1	0.38	110
	-	25.0	29.7	0.94	87
	-	35.0	42.9	1.36	78
	-	50.0	59.3	1.89	59
1	+	0	-	0	86
	+	2.5	3.1	0.10	89
	+	10.0	12.0	0.38	78
	+	25.0	30.2	0.96	76
	+	35.0	42.6	1.36	78
	+	65.0	73.6	2.34	41
2	-	0	-	0	96
	-	25.0	20.3	0.65	85
	-	35.0	28.7	0.91	77
	-	45.0	42.9	1.36	70
	-	55.0	54.0	1.72	68
	-	65.0	63.7	2.03	39
2	+	0	-	0	78
	+	35.0	32.0	1.02	59
	+	45.0	45.0	1.43	62
	+	55.0	56.7	1.80	55
	+	65.0	68.2	2.17	49
	+	75.0	78.0	2.48	35

 

Induction of 6-thioguanine-resistent mutants in CHO cells after treatment with chloroethane for 4 h (without S9)

target dose (mg/vial) chloroethane^a	relative survival^b		mean number of colonies per plate^c		mutant colonies per 10^6clonable cells
	trial 1	trial 2	trial 1	trial 2	trial 1	trial 2
0	100	100	1 +/- 1^d	0 +/- 1^e	8 +/- 9	0 +/- 4
2.5	105	n.d.	0 +/- 1	n.d.	3+/- 4	n.d.
10	136	n.d.	0 +/- 0	n.d.	1+/- 3	n.d.
25	107	89	2 +/- 1	3 +/- 2	15 +/- 10	23 +/- 15**
35	96	80	2 +/- 1	2 +/- 1	13 +/- 9	15 +/- 8**
45	n.d.	73	n.d.	6 +/- 2	n.d.	48 +/- 16**
50	73	n.d.	6 +/- 2	n.d.	36 +/- 15**	n.d.
55	n.d.	71	n.d.	5 +/- 3	n.d.	46 +/- 28**
65	n.d.	51	n.d.	8 +/- 2	n.d.	74 +/- 19**
EMS	79	69	67 +/- 12	76 +/- 10	605 +/- 108**	585 +/- 77**

EMS: Ethylmethanesulfonate

n.d.: not determined

a: the actual chloroethane concentration were slighly different (see table above)

b: relative survival was calculated at the end of exposure as the plating efficiency of chloroethane-treated cells compared to negative control cells; 81% in Exp 1 (- S9) and 86% in Exp 2 (- S9)

c: in each trial, five flasks with 2E+5 cells/flask were plated with the exception of (d) [10 flasks] and (e) [15 flasks]

**: significantly higher than the negative control (p<0.01)

 

Induction of 6-thioguanine-resistent mutants in CHO cells after treatment with chloroethane for 4 h (with S9)

target dose (mg/vial) chloroethane^a	relative survival^b		mean number of colonies per plate^c		mutant colonies per 10^6clonable cells
	trial 1	trial 2	trial 1	trial 2	trial 1	trial 2
0	100	100	0 +/- 1	3 +/- 1^d	1 +/- 3	18 +/- 6
2.5	103	n.d.	2 +/- 2	n.d.	13 +/- 2	n.d.
10	91	n.d.	0 +/- 1	n.d.	1 +/- 3	n.d.
25	88	n.d.	1 +/- 1	n.d.	8 +/- 6	n.d.
35	91	76	2 +/- 3	3 +/- 1	12+/- 16	19 +/- 6
45	n.d.	80	n.d.	4 +/- 2	n.d.	27 +/- 14
55	n.d.	71	n.d.	4 +/- 2	n.d.	26 +/- 13
65	48	63	5 +/- 2	3 +/- 3	32 +/- 16**	24 +/- 24
75	n.d.	43	12 +/- 2	12 +/- 2	n.d.	74 +/- 12**
DMSO 1%	103	100	1 +/- 1	1 +/- 1	3+/- 3	7 +/- 7
MCA	99	97	59 +/- 10	47 +/- 11	348 +/- 55**	585 +/- 77**

MCA: 3-methylcholanthrene

n.d.: not determined

a: the actual chloroethane concentration were slighly different (see table above)

b: relative survival was calculated at the end of exposure as the plating efficiency of chloroethane-treated cells compared to negative control cells; 96% in Exp 1 (+ S9) and 78% in Exp 2 (+ S9)

c: in each trial, five flasks with 2E+5 cells/flask were plated with the exception of (d) [10 flasks]

**: significantly higher than the negative control (p<0.01)

 

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
The results of this test are ambiguous. On the one hand a significantly higher number of mutant colonies per 10exp6 clonable cells was observed with and without metabolic activation in boths trials, on the other hand no clear dose-response for this effect could be seen in any of the experiments.
Since according to the OECD testguideline 476 both, a statistically significant increase compared with the concurrent negative control and a concentration-dependency of this increase are necessary, the results of this test are considered as ambigous.