Sodium ethylenesulphonate

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: not mutagenic

Chromosome aberration test performed with human lymphocytes cells: not clastogenic

HPRT test performed with CHO cells: not mutagenic

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-03-15 to 1988-03-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

used dose level too low according to current requirements

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Species / strain / cell type:

E. coli WP2 uvr A

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
:
microsomal enzyme system (S-9 mix) from Aroclor 1254 induced Sprague-Dawley rat liver
- method of preparation of S9 mix:
Male Sprague Dawley rats (200 - 300 g) received a single intraperitoneal injection of Aroclor 1254 (500 mg/kg bodyweight) 5 days before sacrifice. Preparation was performed at 0 to 4 °C using cold sterile solution and glassware. The livers from at least 5 - 6 animals are removed and pooled, washed in 150 mM KCL (approximately 1 mL/g wet livers). The washed livers are cut into small pieces and homogenized in three volumes of KCL. The homogenate is centrifuged at 9000 g for 10 minutes. The supernatant is the S-9 fraction. It is divided into small portions, rapidly frozen and storage at -80 °C for not longer than three months.
- concentration or volume of S9 mix and S9 in the final culture medium: 10% S9-Mix

Test concentrations with justification for top dose:

Toxicity test (first and second experiment) and mutagenicity test (first experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30% sodium ethylene sulphonate solution.
Mutagenicity test (second experiment): 0, 4, 20, 100, 500, 2500, 10000 µg/plate of the 30% sodium ethylene sulphonate solution.

Vehicle / solvent:

aqua bidest

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 Mix (TA 100, TA 1535)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 Mix (TA 1537)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 Mix (TA 98, TA 1538)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

no

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 Mix (TA 98, TA 100, TA 1535, TA 1537, TA 1538, WPuvrA)

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: N-Methyl-N-nitro-N-nitrosoguanidine

Remarks:

without S9 Mix (WP2uvraA)

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments : two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium; in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 to 72 hours

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: thinning of the bacterial lawn

Evaluation criteria:

no data

Statistics:

no data

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

First experiment

Table 1: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	169	180	178	150
4	-	178	184	188	163
20	-	175	176	172	178
100	-	190	186	204	180
500	-	202	209	202	194
2500	-	185	199	172	185
10000	-	182	185	180	180
0	+	180	185	172	182
4	+	175	185	171	171
20	+	175	166	175	183
100	+	196	202	176	210
500	+	191	200	183	189
2500	+	177	179	182	169
10000	+	179	183	188	167

 

 

Table 2: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	10	12	14
4	-	13	15	10	15
20	-	13	14	15	11
100	-	13	11	12	17
500	-	12	15	12	10
2500	-	14	11	15	16
10000	-	11	11	11	10
0	+	13	15	11	13
4	+	13	14	13	12
20	+	12	13	12	11
100	+	14	17	13	11
500	+	15	11	16	17
2500	+	14	13	14	15
10000	+	13	11	15	12

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 3: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	10	11	8	11
4	-	10	11	9	11
20	-	8	9	7	8
100	-	8	7	9	7
500	-	9	8	9	9
2500	-	8	7	10	7
10000	-	8	9	7	8
0	+	10	10	9	11
4	+	10	10	10	11
20	+	11	11	9	12
100	+	11	10	12	12
500	+	12	11	13	11
2500	+	10	9	13	9
10000	+	10	10	10	11

 

 

Table 4: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	13	11		13
4	-	13	14	14		12
20	-	13	11	16		12
100	-	14	12	14		15
500	-	15	15	19		11
2500	-	13	15	14		9
10000	-	12	15	11		10
0	+	18	18		16	19
4	+	18	23		16	15
20	+	15	16		14	14
100	+	17	18		16	18
500	+	19	17		20	19
2500	+	18	14		16	21
10000	+	17	19		14	18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 5: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	25	23	26	25
4	-	26	21	28	28
20	-	25	22	24	28
100	-	23	25	25	20
500	-	23	23	27	20
2500	-	23	20	22	27
10000	-	25	25	28	23
0	+	30	29	32	29
4	+	34	34	35	33
20	+	31	33	32	29
100	+	33	30	38	32
500	+	35	34	32	38
2500	+	35	32	35	37
10000	+	32	31	34	30

 

 

Table 6: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	40	45	39		35
4	-	38	39	40		35
20	-	40	40	40		39
100	-	37	37	36		37
500	-	42	42	43		40
2500	-	41	43	37		42
10000	-	40	42	40		37
0	+	44	48		36	48
4	+	42	42		45	39
20	+	45	42		51	42
100	+	45	41		48	45
500	+	39	41		39	38
2500	+	42	43		43	39
10000	+	43	36		46	46

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Second experiment

Table 7: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 100

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 100

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate			Surviving fraction
0	-	143	152	143	133	1.0
4	-	147	150	136	154	1.0
20	-	142	143	119	164	1.0
100	-	156	161	150	157	1.0
500	-	148	158	119	168	1.0
2500	-	141	129	127	167	0.9
5000	-	142	140	126	160	1.0
0	+	183	183	195	172	1.0
4	+	165	155	158	182	1.0
20	+	174	181	158	182	1.0
100	+	178	197	163	173	0.9
500	+	155	187	155	154	1.0
2500	+	168	191	159	153	0.9
5000	+	172	200	158	159	0.9

 

 

Table 8: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1535

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1535

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	13	13	12		14
4	-	13	12	14		14
20	-	12	12	10		14
100	-	12	14	10		11
500	-	14	13	11		17
2500	-	11	10	12		10
5000	-	11	9	14		11
0	+	12	12		12	13
4	+	12	10		13	13
20	+	13	11		11	16
100	+	13	11		15	14
500	+	12	10		13	12
2500	+	12	13		15	9
5000	+	11	12		11	10

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 9: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1537

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1537

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	7	9	7	6
4	-	8	7	8	8
20	-	7	5	9	6
100	-	7	6	8	8
500	-	7	5	7	8
2500	-	7	6	9	6
5000	-	7	9	6	7
0	+	9	10	8	8
4	+	9	9	10	7
20	+	10	10	10	11
100	+	10	8	12	11
500	+	8	7	9	9
2500	+	9	9	9	8
5000	+	10	10	10	9

 

 

Table 10: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 1538

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 1538

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	12	12	11		13
4	-	12	13	12		10
20	-	14	15	15		13
100	-	12	13	13		12
500	-	11	9	11		13
2500	-	14	14	14		15
5000	-	12	10	11		14
0	+	19	16		21	20
4	+	17	15		17	19
20	+	19	17		20	20
100	+	17	18		19	15
500	+	17	17		18	15
2500	+	19	17		22	17
5000	+	17	15		18	18

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

Table 11: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

TA 98

Number of revertant colonies per plate and mean values using Salmonella typhimurium strain TA 98

Dose µg/plate	Metabolic activation	Mean value22	Colonies per plate
0	-	22	23	22	22
4	-	22	20	21	25
20	-	27	26	27	27
100	-	23	24	24	20
500	-	21	24	19	19
2500	-	24	30	20	21
5000	-	22	21	22	24
0	+	25	23	26	27
4	+	29	28	28	32
20	+	28	30	27	27
100	+	26	30	20	27
500	+	28	31	25	29
2500	+	27	23	31	27
5000	+	25	23	28	25

 

 

Table 12: Mutagenicity experiment with Vinylsulfonsaures Natrium 30%ig with and without metabolic activation

 

WP2uvrA

Number of revertant colonies per plate and mean values using Escherichia coli strain WP2uvrA

Dose µg/plate	Metabolic activation	Mean value	Colonies per plate
0	-	51	52	56		45
4	-	50	43	46		60
20	-	54	54	56		53
100	-	51	52	49		51
500	-	52	51	55		50
2500	-	53	51	57		50
5000	-	57	54	61		55
0	+	56	54		56	59
4	+	58	52		61	62
20	+	54	53		54	54
100	+	56	54		53	60
500	+	59	59		60	58
2500	+	57	56		60	56
5000	+	53	52		49	58

Compound dissolved in 100 microliter Aqua bidest.

- '       : absence

+        : presence

 

First experiment

Table 13: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	Sodium-azide	1	-	526	456	549	574
TA1535	Sodium-azide	1	-	409	410	398	419
TA1537	9-Aminoacridine	50	-	95	81	102	103
TA1538	2-Nitrofluorene	2.5	-	462	432	487	467
TA98	2-Nitrofluorene	2.5	-	327	294	355	333
WP2uvrA	MNNG	1.5	-	261	285	256	243
-	Vinylsulfon- saures Natrium 30%ig	10000	-	0	0	0	0

- : absence

 

 

Table 13a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose ug/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	2-Aminoanthracen	0.5	+	818	874	806	775
TA1535	2-Aminoanthracen	1	+	140	151	126	142
TA1537	2-Aminoanthracen	1	+	126	122	130	125
TA1538	2-Aminoanthracen	0.5	+	458	438	463	473
TA98	2-Aminoanthracen	0.5	+	426	407	444	426
WP2uvrA	2-Aminoanthracen	10	+	440	428	442	451
TA100	Benzo[a]pyrene	10	+	1311	1352	1298	1283
TA1535	Benzo[a]pyrene	10	+	18	15	18	21
TA1537	Benzo[a]pyrene	10	+	96	85	100	102
TA1538	Benzo[a]pyrene	10	+	176	206	173	149
TA98	Benzo[a]pyrene	10	+	550	535	585	531
WP2uvrA	Benzo[a]pyrene	10	+	87	97	75	88
-	S-9 mix	500 µl	+	0	0	0	0
-	Vinylsulfon- saures Natrium 30%ig	1000 µg	+	0	0	0	0

+ :presence

 

 

Second experiment

Table 14: mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	Sodium-azide	1	-	293	29+2	306	280
TA1535	Sodium-azide	1	-	272	277	287	252
TA1537	9-Aminoacridine	50	-	157	160	163	178
TA1538	2-Nitrofluorene	2.5	-	459	469	430	478
TA98	2-Nitrofluorene	2.5	-	340	314	379	328
WP2uvrA	MNNG	2.5	-	223	206	223	241
-	Vinylsulfon- saures Natrium 30%ig	5000	-	0	0	0	0

- : absence

 

 

Table 14a : mutability (positive controls) and sterility test of the experiment with Vinylsulfonsaures Natrium 30%ig

 

Number of revertant colonies per plate and mean values using Salmonella typhimurium strains and Escherichia coli

Strain	Compound	Dose µg/plate	Metab. Activ.	Mean Value	Colonies per plate
TA100	2-Aminoanthracen	0.5	+	465	399	497	502
TA1535	2-Aminoanthracen	1	+	88	77	84	103
TA1537	2-Aminoanthracen	1	+	90	79	108	84
TA1538	2-Aminoanthracen	0.5	+	494	474	549	458
TA98	2-Aminoanthracen	0.5	+	577	589	563	579
WP2uvrA	2-Aminoanthracen	10	+	481	463	496	483
TA100	Benzo[a]pyrene	10	+	685	746	670	639
TA1535	Benzo[a]pyrene	10	+	18	16	15	24
TA1537	Benzo[a]pyrene	10	+	86	81	96	80
TA1538	Benzo[a]pyrene	10	+	183	176	184	188
TA98	Benzo[a]pyrene	10	+	551	552	547	583
WP2uvrA	Benzo[a]pyrene	10	+	74	66	79	76
-	S-9 mix	500 ul	+	0	0	0	0
-	Vinylsulfon- saures Natrium 30%ig	5000 ug	+	0	0	0	0

+ :presence

 

Conclusions:

The test solution, containing 30% sodium ethylene sulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of sodium ethylene sulphonate was 3000 µg/plate.

Executive summary:

Sodium ethylene sulphonate was tested as a 30% aqueous solution in the Salmonella typhimurium reverse mutation assay (Hoechst AG (b), 1988) with the strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) similar to OECD guideline 471 and GLP. Two independent mutagenicity studies were conducted (plate incorporation method), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. For both studies each bacterial strain was exposed to 6 dose levels. In the first experiment concentrations ranged from 4 to 10000 µg/plate and in the second experiment from 4 to 5000 µg/plate.Control plates without mutagen showed that the number of spontaneous revertant colonies was similiar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test solution proved to be not toxic to the bacterial strains. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also in the presence of a metabolic activation system, treatment of the cells with the 30% sodium ethylene sulphonate solution did not result in relevant increases in the number of revertant colonies.In conclusion the test solution, containing 30% sodium ethylene sulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of sodium ethylene sulphonate was 3000 µg/plate.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-03-11 to 1997-04-25

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)

Version / remarks:

Adopted 4 April, 1984

Deviations:

yes

Remarks:

only 3 instead of 4 analysable concentrations were tested.

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5300 (Detection of Gene Mutations in Somatic Cells in Culture)

Version / remarks:

1983

Deviations:

yes

Remarks:

only 3 instead of 4 analysable concentrations were tested.

Qualifier:

according to guideline

Guideline:

other: EEC Directive 87/302/EEC

Version / remarks:

1988

GLP compliance:

yes

Type of assay:

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

Target gene:

hemizygous hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

CELLS USED
- Type and source of cells:
CHO-Kl-BH4 cells were obtained from the British Industrial Biological Research Association.
- Suitability of cells:
These cells are functionally hemizygous at the HPRT locus.

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature:
These cells are functionally hemizygous at the HPRT locus. Ham's F12 medium was supplemented with 2 mM L-glutamine and 50 µg/mL gentamicin.

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9
Aroclor stimulated S9 liver mix from male Sprague-Dawley rats
- method of preparation of S9 mix
S-9 fraction was prepared from a group of c. 10 animals. Mixed function oxidase systems in the rat liver were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg body weight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers aseptically removed. The following steps were carried out at 0 - 4 °C under aseptic conditions. The livers were placed in 250 mM sucrose solution (3 mL of sucrose solution : 1 g of liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation the homogenate was centrifuged at 9000 g for 10 minutes. The supernatant fraction (S-9 fraction) was dispensed into aliquots and stored at -80 °C until required.
- concentration or volume of S9 mix and S9 in the final culture medium
S-9 mix contains: S-9 fraction (25% v/v), isocitric acid (43.5 mM), NADP (8.0 mM) in ice cold medium. The co-factors were prepared on the morning of the test, neutralised with 1N NaOH and filter sterilised before adding to S-9 fraction.
- quality controls of S9: All batches of S-9 fraction were tested using 20-methylcholanthrene before use.

Test concentrations with justification for top dose:

Mutation tests (with and without S9 mix; two independent experiments)
1250, 2500, 5000 µg/mL of a 24.9% sodium ethylene sulphonate solution

Vehicle / solvent:

Positive controls: DMSO
Test material: cell medium

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 20-methylcholanthrene

Remarks:

with S-9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9 Mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: triplicate
- Number of independent experiments: two

METHOD OF TREATMENT/ EXPOSURE:
- Cell density at seeding: 7.5 x 10E5 cells/mL
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
Test substance, solvent or positive control were added to the flasks and incubated for 4 hours.
- Harvest time after the end of treatment:
At the end of the treatment period the cells were harvested, washed and seeded with 200 cells in medium. These plates were incubated at 37 °C for 7 days after which time the colonies growing in the plates were fixed, stained and counted.

FOR GENE MUTATION:
- Expression time (cells in growth medium between treatment and selection):
The cultures were sub-cultured once during the expression period on Day 4 or 5 and, after a total of 7 days, were harvested by trypsinisation. For each treatment group, three 60 mm culture plates were each seeded with 200 cells in H5 and five 100 mm plates with 2 x 10E5 cells in selective medium.
- Selection time:
Further cells from each culture were seeded in flasks containing medium and incubated for 7 days to allow for expression of the mutant phenotype. After 7 days the cells were harvested and 200 cells per culture plate were seeded in medium or selection medium for 7 days. At the end of this incubation period colonies growing in the plates were fixed, strained and counted.
- Selective agent: The selective medium, in which only HPRT deficient cells grow, consisted of heat-inactivated fetal calf serum supplemented with 6-TG (Sigma) at a final concentration of 10 µg/mL.

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: plating efficiency

Evaluation criteria:

The criteria for a positive response were:
- The demonstration of a statistically significant increase in mutant frequency following treatment with the test substance.
- Evidence of a dose relationship, over at least two dose levels, in any increase in mutant frequency.
- Demonstration of reproducibility in any increase in mutant frequency.
- In addition the mean mutant frequency must lie outside the upper limit of the historical control

Statistics:

The statistical significance of the data was analysed by weighted analysis of variance following the methods described by Arlett et al (1989), assuming the data are acceptable according to their criteria.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Some increases of pH were observed.
- Effects of osmolality: dose related increases in osmolarity were observed in all tests, both in the presence and the absence of S-9 mix.

TABLE 1

Test 1. Cytotoxicity in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)	Number of colonies on viability plates				Cell survival (% control)	Mean % survival
	1	2	3	Total
0	112	79	96	287	100	100
	87	98	85	270
(SDW control)	68	107	72	247
	47	56	57	160
1250	129	114	104	347	144	122
	81	77	84	242	100
2500	65	89	75	229	95	98
	66	97	79	242	100
5000	48	67	79	194	80	105
	108	94	111	313	130
Ems (250 µg/mL)	138	122	128	388	161	180
	180	154	145	479	199

 

TABLE 2

Test 1. Mutant frequency in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)	No. of colonies on non-selective plates				Plating efficiency (%)	No. of colonies on selective plates						Mutant frequency per 10^6 survivors	Mean mutant frequency per 10^6survivors
	1	2	3	Total		1	2	3	4	5	Total
0 (SDW control)	130	114	134	378	63	0	0	0	0	0	0	0
	113	120	105	338	56	0	1	0	1	0	2	4
	150	125	130	405	68	4	1	0	0	0	5	7	4
	128	131	114	373	62	1	1	1	0	0	3	5
1250	96	104	91	291	49	0	0	0	0	0	0	0	2
	113	109	107	329	55	0	0	1	1	0	2	4
2500	118	136	116	370	62	0	0	1	1	0	2	3	8
	117	114	103	334	56	1	0	0	4	2	7	13
5000	117	140	114	371	62	0	0	0	0	1	1	2	2
	112	114	104	330	55	0	0	1	0	0	1	2
Ems (250 µg/mL)	137	125	125	387	64	37	35	36	40	39	187	290	298
	115	134	111	360	60	39	34	34	37	39	183	305	***

*** - Significantly different from control P<0.001

 

 

TABLE 3

Test 2. Cytotoxicity in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)	Number of colonies on viability plates				Cell survival (% control)	Mean % survival
	1	2	3	Total
0	81	77	70	228	100	100
	73	81	80	234
(SDW control)	80	80	89	239
	80	75	105	260
1250	81	79	C	(240)	100	99
	78	C	C	(234)	98
2500	81	82	80	243	101	95
	71	83	56	210	88
5000	108	91	94	293	122	124
	87	107	105	299	125
Ems (250 µg/mL)	91	74	C	(248)	103	92
	73	52	68	193	80

C Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations

 

TABLE 4

Test 2. Mutant frequency in the absence of S-9 mix

Concentration of test item 24.9% (µg/mL)	No. of colonies on non-selective plates				Plating efficiency (%)	No. of colonies on selective plates						Mutant frequency per 10^6 survivors	Mean mutant frequency per 10^6survivors
	1	2	3	Total		1	2	3	4	5	Total
0 (SDW control)	117	164	123	404	67	1	0	1	0	0	2	3
	143	129	134	406	68	0	0	0	0	0	0	0
	120	110	127	357	60	0	0	0	0	2	2	3	3
	152	124	138	414	69	0	1	1	0	2	4	6
1250	116	143	102	361	60	0	3	1	2	0	6	10	9
	128	143	127	398	66	1	1	2	1	0	5	8
2500	132	118	124	374	62	0	0	0	0	1	1	2	2
	125	139	147	411	68	1	0	0	0	0	1	1
5000	101	122	115	338	56	0	0	0	0	0	0	0	1
	128	115	110	353	59	0	0	0	0	1	1	2
Ems (250 µg/mL)	106	118	101	325	54	23	23	19	21	20	106	196	228
	113	123	131	367	61	33	31	30	32	33	159	260	***

*** - Significantly different from control P<0.001

 

 

TABLE 5

Test 1. Cytotoxicity in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)	Number of colonies on viability plates				Cell survival (% control)	Mean % survival
	1	2	3	Total
0	109	91	70	270	100	100
	96	87	107	290
(SDW control)	117	117	93	327
	107	87	102	296
1250	245	224	248	717	242	168
	101	82	91	274	93
2500	80	91	92	263	89	94
	94	105	94	293	99
5000	85	90	99	274	93	92
	88	89	88	265	90
MC (5 µg/mL)	81	81	105	267	90	82
	80	50	89	219	74

 

 

TABLE 6

Test 1. Mutant frequency in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)	No. of colonies on non-selective plates				Plating efficiency (%)	No. of colonies on selective plates						Mutant frequency per 10^6 survivors	Mean mutant frequency per 10^6survivors
	1	2	3	Total		1	2	3	4	5	Total
0 (SDW control)	115	145	161	421	70	1	0	2	0	1	4	6
	139	142	122	403	67	3	0	0	0	1	4	6
	118	121	159	398	66	1	1	0	1	1	4	6	7
	159	137	124	420	70	1	3	1	1	0	6	9
1250	136	128	122	386	64	0	1	0	1	1	3	5	7
	125	121	134	380	63	3	0	0	1	1	5	8
2500	133	127	127	387	64	0	0	1	0	0	1	2	3
	122	131	121	374	62	0	0	0	0	2	2	3
5000	138	130	119	387	64	0	0	0	1	0	1	2	1
	127	134	107	368	61	0	0	0	0	0	0	0
MC (5 µg/mL)	147	133	140	420	70	40	38	39	38	41	196	280	299
	138	137	96	371	62	40	41	37	38	40	196	317	***

*** - Significantly different from control P<0.001

 

 

TABLE 7

Test 2. Cytotoxicity in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)	Number of colonies on viability plates				Cell survival (% control)	Mean % survival
	1	2	3	Total
0	90	102	92	284	100	100
	89	107	99	295
(SDW control)	97	83	90	270
	101	98	110	309
1250	128	106	116	350	121	117
	113	104	112	329	113
2500	108	113	113	334	115	119
	116	124	118	358	123
5000	135	135	120	390	134	125
	116	100	117	333	115
MC (5 µg/mL)	129	143	125	397	137	137
	131	136	129	396	137

 

 

TABLE 8

Test 2. Mutant frequency in the presence of S-9 mix

Concentration of test item 24.9% (µg/mL)	No. of colonies on non-selective plates				Plating efficiency (%)	No. of colonies on selective plates						Mutant frequency per 10^6 survivors	Mean mutant frequency per 10^6survivors
	1	2	3	Total		1	2	3	4	5	Total
0 (SDW control)	138	103	113	354	59	2	2	0	1	1	6	10
	124	184	111	419	70	1	0	1	1	2	5	7
	106	143	160	409	68	2	3	0	2	0	7	10	8
	160	145	C	(458)	76	1	1	0	0	2	4	5
1250	114	148	117	379	63	0	1	0	0	0	1	2	5
	113	121	117	351	59	0	0	1	0	3	4	7
2500	127	123	133	383	64	1	0	2	2	1	6	9	8
	135	115	135	385	64	0	0	0	3	1	4	6
5000	96	84	107	287	48	0	0	0	0	3	3	6	10
	121	120	117	358	60	2	0	3	1	2	8	13
MC (5 µg/mL)	102	96	112	310	52	56	65	58	46	54	279	540	521
	93	109	102	304	51	45	47	59	57	49	257	507	***

C - Contaminated. Figure in brackets is adjustment made for contaminated plates and is used in all calculations

*** - Significantly different from control P<0.001

 

 

TABLE 9

pH of the culture medium treated with test item - 0 hours

Concentration of test item 24.9% (µg/mL)	In the absence of S-8 mix		In the presence of S-9 mix
	Test 1	Test 2	Test 1	Test 2
0 (SDW control)	8.2	7.5	7.2	7.5
1250	8.4	7.9	7.2	7.6
2500	8.3	8.1	7.3	7.6
5000	8.6	8.4	7.3	7.6

 

Conclusions:

The test solution, containing 24.9% sodium ethylene sulphonate, did not demonstrate a mutagenic potential in mammalian cells. Under the conditions of the present mammalian cell gene mutation assay the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL. This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Executive summary:

A test solution, containing 24.9% sodium ethylene sulphonate, was tested in a mammalian gene mutation test in CHO cells (HPRT assay) according to OECD guideline 476 and GLP (Huntigdon Life Science Ltd. (b), 1997). In two independent experiments concentrations of 1250, 2500 and 5000 µg/mL of the 25% sodium ethylene sulphonate solution were tested with and without metabolic activation (S9 -mix). Some increases of pH values and dose related increases in osmolarity were observed in all tests. No significant increases in mutant frequency were observed after treatment with 24.9% sodium ethylene sulphonate in either test in the absence and the presence of S-9 mix. Ethyl methanesulphonate and 20-Methylcholanthrene, the positive controls, induced highly significant increases in mutant frequency in both tests. It is concluded that the test solution, containing 24.9% sodium ethylene sulphonate, did not demonstrate mutagenic potential in this in vitro gene mutation assay. Under the conditions of the present mammalian gene mutation test the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL (5000 µg/mL of a 25% aqueous sodium ethylene sulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1997-02-26 to 1997-04-29

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)

Version / remarks:

Adopted: 26 May 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

29.12.1992

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: UK Department of Health Repor on Health and Social Subjects No. 35. Guidelines for the testing of chemicals for mutagenicity (1989).

Version / remarks:

1989

Deviations:

no

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human lymphocytes

Details on mammalian cell type (if applicable):

CELLS USED
- Sex, age and number of blood donors:
male donors
- Whether whole blood or separated lymphocytes were used:
whole blood
- Whether blood from different donors were pooled or not:
pooled
- Mitogen used for lymphocytes:
stimulated to divide by addition of phytohaemagglutinin

MEDIA USED
- Type and composition of media, CO2 concentration, humidity level, temperature
37 °C, 5% CO2, RPMI 1640 tissue culture medium (Imperial) containing 10% foetal calf serum (PAA)

Metabolic activation:

with and without

Metabolic activation system:

Type and composition of metabolic activation system:
- source of S9: S9-mix (by Aroclor 1254 stimulated rat liver)

- method of preparation of S9 mix
Mixed function oxidase systems in the liver of a group of rats were stimulated by Aroclor 1254, administered as a single intraperitoneal injection in Arachis oil at a dosage of 500 mg/kg body weight. On the fifth day after injection, following an overnight starvation, the rats were killed and their livers asepticaly removed. The following steps were carried out at 0 - 4 °C under aseptic conditions. The livers were placed in 0.15 M KCl (3 ml KCl : 1 g liver) before being transferred to an Ultra-Turrax homogeniser. Following preparation, the homogenates were centrifuged at 9000 g for 10 minutes. The supernatant fraction (S9 fraction) was dispensed into aliquots and stored at -80 °C until required.

- concentration or volume of S9 mix and S9 in the final culture medium
S9 mix contained: S9 fraction (10% v/v), MgCI2 (8 mM), KCl (33 mM), sodium orthophosphate buffer pH 7.4 (100 mM), glucose-6-phosphate (5 mM), NADP (4 mM). All the cofactors were filter-sterilised before use.

- quality controls of S9: All batches of S9 fraction were tested for sterility and efficiency.

Test concentrations with justification for top dose:

1250, 2500 and 5000 µg/mL of the 24.9% sodium ethylene sulphonate solution.

Positive control:
+ S9 mix: 20, 25 and 30 µg/mL (Cyclophoshamide)
- S9 mix: 0.2, 0.4 and 0.8 µg/mL (Mitomycin C)

Vehicle / solvent:

- Justification for choice of solvent/vehicle:
Sodium ethylene sulphonate 25% was supplied as a 25% aqueous solution and on dosing at 1% v/v into aqueous tissue culture medium (RPMI 1640), giving a final concentration of 2500 µg/mL, no precipitate was observed. A concentration of 5000 µg/mL was achieved by the addition of undiluted Sodium etyhlenesulphonate (25%) at 2% v/v and again no precipitate was observed.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: duplicate
- Number of independent experiments
: two

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in medium

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment:
First test:
with and without S9 Mix: 21 hours (with S9 Mix: 3 hours exposure and 18 hours post-exposure)

Second test:
with and without S9 Mix: 21 hours (with S9 Mix: 3 hours exposure and 18 hours post-exposure)
with and without S9 Mix: 45 hours (with S9 Mix: 3 hours exposure and 42 hours post-exposure)

FOR CHROMOSOME ABERRATION:
- Spindle inhibitor: Colcemid at a final concentration of 0.1 µg/mL after 2 hours incubation

- Methods of slide preparation and staining technique used including the stain used:
Each cell suspension was transferred to a conical centrifuge tube and centrifuged for 5 minutes at 500 g. The cell pellets were treated with a hypotonic solution (0.075M KCl pre-warmed at 37 °C). After a 10 minute period of hypotonic incubation at 37 °C, the suspensions were centrifuged at 500 g for 5 minutes and the cell pellets fixed by addition of freshly prepared fixative (3 parts methanol : 1 part glacial acetic acid). The fixative was replaced several times. The pellets were re-suspended, then centrifuged at 200 g for 10 minutes and finally re-suspended in a small volume of fresh fixative. Two or three drops of the cell suspensions were dropped onto pre-cleaned microscope slides which were then allowed to air-dry. The slides were then stained in 10% Giemsa, prepared in buffered water (pH 6.8). After rinsing in buffered water the slides were left to air-dry and then mounted in DPX.

- Number of cells spread and analysed per concentration (number of replicate cultures and total number of cells scored):
The slides were then coded. Metaphase figures were identified, using a magnification of xl60 and examined at a magnification of x1000 using an oil immersion objective. Approximately 100 metaphase figures were examined, where possible, from each culture. Only cells with 44 - 46 chromosomes were analysed. The Vernier readings of all aberrant metaphase figures were recorded.
- Determination of polyploidy:
no data
- Determination of endoreplication:
no data

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: mitotic index (MI)

Statistics:

The number of aberrant metaphase figures in each treatment group was compared with the solvent control value using Fisher's test.

Key result

Species / strain:

lymphocytes: human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

absence of S9 mix at 5000 µg/mL of the 24.9% sodium etyhlene sulphonate solution, corresponding to 1250 µg/mL sodium ethylene sulphonate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

True negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH:
No substantial increases in pH values were observed.
- Data on osmolality:
Dose related increases in osmolality were observed both in the absence and presence of S9 mix.
- Precipitation and time of the determination:
no data

STUDY RESULTS
- see attached results

- Results from cytotoxicity measurements:
FIRST TEST:
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 55% of the solvent control value at 5000 µg/mL, corresponding to 1250 µg/mL sodium ethylene sulphonate. All three concentrations were therefore acceptable for metaphase analysis.
In the presence of S9 mix, the test item caused no reduction in the mitotic index compared to the solvent control. All three dose levels were again subject to metaphase analysis.
SECOND TEST (21 hour harvest):
In the absence of S9 mix, the test item caused a reduction in the mitotic index to 66% of the solvent control level at 5000 µg/mL. All three concentrations were acceptable for metaphase analysis.
In the presence of S9 mix, the test item caused no reduction in mitotic index compared to the untreated cultures. All three dose levels were again subject to metaphase analysis.
SECOND TEST (45 hour harvest):
In both the absence and presence of S9 mix, the test item caused no reduction in the mitotic index compared to the solvent control value. 5000 µg/mL, was chosen for metaphase analysis as this was the highest concentration analysed at the 21 hour harvest time.
- Genotoxicity results:
The test item caused no statistically significant increase in the proportion of metaphase figures with chromosome aberrations, at any concentrations analysed, in either the absence or presence of S9 mix.
Both positive control compounds, mitomycin C and cyclophosphamide, caused large, statistically significant increases (P<0.001) in the proportion of aberrant cells. This demonstrated the efficacy of the S9 mix and the sensitivity of the test system.

HISTORICAL CONTROL DATA
- see attached results

TABLE 1

Mitotic index data - first test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	80/1000	6.9	100
(De-ionised water)	57/1000
1250	51/1000	5.5	80
	58/1000
2500	41/1000	4.3	62
	45/1000
5000	40/1000	3.8	55
	35/1000

 

Mitotic index data - first test, 21 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	51/1000	5.5	100
(De-ionised water)	58/1000
1250	46/1000	3.8	69
	30/1000
2500	40/1000	3.9	71
	37/1000
5000	43/1000	5.9	107
	75/1000

 

TABLE 2

Metaphase analysis data - first test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100	1		1			1		2	2.0	3	2.5	100
(De-ionised water)	100	2							2		2
1250	100	1					1		1	0.5	2	1.0	80
	100								0		0
2500	100	1					2		1	1.0	1	2.0	62
	100	1							1		3
5000	100	4							3	3.5	3	3.5	55
	100	3		1					4		4
0.8	50	5	3	2			1		10	22.0	10	23.0	-
Mitomycin C	50	15	2	1					12	***	12	***

 

Metaphase analysis data - first test, 21 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL))	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100								0	1.0	0	1.0	100
(De-ionised water)	100	2							2		2
1250	100	1							1	1.0	1	1.0	69
	100	1							1		1
2500	100								0	2.0	0	2.5	71
	100	3		1			1		4		5
5000	100								0	0.5	0	0.5	107
	100	1							1		1
25	50	16	1	3					13	23.0	13	23.0	-
Cyclophosphamide	50	15		1					10	***	10	***

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01

TABLE 3

Mitotic index data - second test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	78/1000	7.0	100
(De-ionised water)	61/1000
1250	60/1000	6.1	87
	62/1000
2500	66/1000	6.1	87
	56/1000
5000	44/1000	4.6	66
	48/1000

 

 

Mitotic index data – second test, 21 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	78/1000	7.8	100
(De-ionised water)	78/1000
1250	82/1000	7.6	97
	70/1000
2500	90/1000	8.8	113
	85/1000
5000	76/1000	7.8	100
	80/1000

 

TABLE 4

Metaphase analysis data - second test, 21 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100								0	0.5	0	0.5	100
(De-ionised water)	100		1						1		1
1250	100	1							1	0.5	1	0.5	87
	100								0		0
2500	100	1							1	0.5	1	1.0	87
	100						1		0		1
5000	100								0	0.0	0	0.0	66
	100								0		0
0.8	100	9	1	10			1		17	21.5	17	22.5	-
Mitomycin C	50	6		16			2		13	***	14	***

 

Metaphase analysis data - second test, 21 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL)	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100	2							2	1.0	2	1.0	100
(De-ionised water)	100								0		0
1250	100								0	0.0	0	0.0	97
	100								0		0
2500	100								0	0.0	0	0.0	113
	100								0		0
5000	100								0	0.5	0	0.5	100
	100	1							1		1
25	100	13		1					11	10.0	11	10.0	-
Cyclophosphamide	100	10	1						9	***	9	***

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01

TABLE 5

Mitotic index data - second test, 45 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	66/1000	6.3	100
(De-ionised water)	60/1000
1250	73/1000	7.8	124
	82/1000
2500	79/1000	7.4	117
	68/1000
5000	64/1000	5.9	94
	54/1000

 

 

Mitotic index data – second test, 45 hour harvest -continued

With S9 mix

Concentration of test item 24.9% (µg/mL)	Mitotic index		Relative mitotic index (%)
	Incidence	% Mean
0	99/1000	9.3	100
(De-ionised water)	86/1000
1250	89/1000	8.6	92
	83/1000
2500	93/1000	8.7	94
	81/1000
5000	102/1000	9.7	104
	91/1000

 

TABLE 6

Metaphase analysis data - second test, 45 hour harvest

Without S9 mix

Concentration of test item 24.9% (µg/mL)	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100	1							1	0.5	1	0.5	100
(De-ionised water)	100								0		0
5000	100								0	1.0	0	1.0	94
	100	1		1					2		2

 

Metaphase analysis data - second test, 45 hour harvest - continued

With S9 mix

Concentration of test item 24.9% (µg/mL)	No. cells examined	Aberrations							No of aberrant cells				Relative MI (%)
		Chromatid type		Chromosome type		others	Gaps		Exc. gaps	Mean %	Inc. gap	Mean %
		ctb	cte	csb	cse		ctg	csg
0	100								0	0.5	0	0.5	100
(De-ionised water)	100		2						1		1		104
5000	100								0	0.0	0	0.0
	100								0		0

ctb     - Chromatid break                              cte - Chromatid exchange

csb    - Chromosome break                          cse - Chromosome exchange

ctg     - Chromatid gap                                  csg - Chromosome gap
others  - Cells with greater than 10 aberrations, pulverised cells and pulverised chromosomes

 

***      - P<0.001

Otherwise - P>0.01

 

Historical control data for human lymphocytes in whole blood culture,

treated with solvent controls from February 1995 until January 1997.

 

Aberrant cells

 

	Total no. of cells	Without gaps
		No. of aberrant cells	% mean	Range of means (%)		Upper 95% limit (%)
				min	max
-S-9	55980	317	0.57	0	3.68	1.5
+S-9	57600	304	0.53	0	3.0	1.5

 

 

	Total no. of cells	With gaps
		No. of aberrant cells	% mean	Range of means (%)		Upper 95% limit (%)
				min	max
-S-9	55980	403	0.72	0	4.47	2.0
+S-9	57600	391	0.68	0	3.0	2.0

 

 

Conclusions:

The test solution, containing 24.9% sodium ethylene sulphonate, did not induce chromosomal aberrations over a background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberration test the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL (5000 µg/mL of a 24.9% aqueous sodium ethylene sulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Executive summary:

A test solution, containing 24.9% sodium ethylene sulphonate, was assessed to its ability to induce chromosomal aberrations in human lymphocytes according to OECD guideline 473 and GLP (Huntingdon Life Science Ltd. (a), 1997). In two independent experiments at least 100 metaphase figures were analysed at concentrations of 1250, 2500 and 5000 µg/mL of the 24.9% sodium ethylene sulphonate solution. In the current version of the OECD TG 473 (2014) it is recommended to analyse at least 300 well-spread metaphases per concentration and control to conclude a test item as clearly negative. However, in the OECD TG 473 from 1983 it was sufficient to score 100 metaphases per concentration to come to a conclusion on clastogenicity. 

A mitotic index (MI) of 55 - 66% was observed after treatment with the highest concentration of test item in the absence of S-9 mix. No significant increases in mutant frequency were observed in cultures treated with the 24.9% sodium ethylene sulphonate solution in any of the tests either in the absence or the presence of S-9 mix. The positive controls induced highly significant increases in mutant frequency in all of the tests in both the absence and presence of S-9 mix thus demonstrating adequate sensitivity of the test system. In conclusion, the test solution, containing 24.9% sodium ethylene sulphonate, did not induce chromosomal aberrations over background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberration test the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL (5000 µg/mL of a 24.9% aqueous sodium ethylene sulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Ames test

Sodium ethylene sulphonate was tested as a 30% aqueous solution in the Salmonella typhimurium reverse mutation assay (Hoechst AG (b), 1988) with the strains of Salmonella typhimurium (TA1535, TA1537, TA1538, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA) similar to OECD guideline 471 and GLP. Two independent mutagenicity studies were conducted (plate incorporation method), each in the absence and in the presence of a metabolising system derived from a rat liver homogenate. For both studies each bacterial strain was exposed to 6 dose levels. In the first experiment concentrations ranged from 4 to 10000 µg/plate and in the second experiment from 4 to 5000 µg/plate. Control plates without mutagen showed that the number of spontaneous revertant colonies was similar to that described in the literature. All the positive control compounds gave the expected increase in the number of revertant colonies. The test solution proved to be not toxic to the bacterial strains. In the absence of the metabolic activation system the test compound did not show a dose dependent increase in the number of revertants in any of the bacterial strains. Also, in the presence of a metabolic activation system, treatment of the cells with the 30% sodium ethylene sulphonate solution did not result in relevant increases in the number of revertant colonies. In conclusion the test solution, containing 30% sodium ethylene sulphonate, did not demonstrate mutagenic potential. Under the conditions of the present bacterial reverse mutation assay the maximum test concentration of sodium ethylene sulphonate was 3000 µg/plate.

As this maximum concentration, in absence of precipitation, did not fulfill the maximum dose requirement for the active substance in an Ames test, the OECD 471 study was repeated, recently, using Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli (WP2uvrA pKM101) with concentrations up to 5000 µg/plate active substance (sodium ethylene sulphonate). The outcome of this study confirmed the findings of the study mentioned above and no significant increase of revertants was found in any of the strains tested, with or without metabolic activation.

In conclusion sodium ethylene sulphonate solution, when tested up to 5000 µg/plate, did not demonstrate mutagenic potential.

 

In vitro mammalian chromosome aberration test

A test solution, containing 24.9% sodium ethylene sulphonate, was assessed to its ability to induce chromosomal aberrations in human lymphocytes according to OECD guideline 473 and GLP (Huntingdon Life Science Ltd. (a), 1997). In two independent experiments at least 100 metaphase figures were analysed at concentrations of 1250, 2500 and 5000 µg/mL of the 24.9% sodium ethylene sulphonate solution. In the current version of the OECD TG 473 (2014) it is recommended to analyse at least 300 well-spread metaphases per concentration and control to conclude a test item as clearly negative. However, in the OECD TG 473 from 1983 it was sufficient to score 100 metaphases per concentration to come to a conclusion on clastogenicity.

A mitotic index (MI) of 55 - 66% was observed after treatment with the highest concentration of test item in the absence of S-9 mix. No significant increases in mutant frequency were observed in cultures treated with the 24.9% sodium ethylene sulphonate solution in any of the tests either in the absence or the presence of S-9 mix. The positive controls induced highly significant increases in mutant frequency in all of the tests in both the absence and presence of S-9 mix thus, demonstrating adequate sensitivity of the test system. In conclusion, the test solution, containing 24.9% sodium ethylene sulphonate, did not induce chromosomal aberrations over background when tested up to cytotoxic concentrations. Under the conditions of the present mammalian chromosome aberration test the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL (5000 µg/mL of a 24.9% aqueous sodium ethylene sulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

 

In vitro mammalian cell gene mutation test

A test solution, containing 24.9% sodium ethylene sulphonate, was tested in a mammalian gene mutation test in CHO cells (HPRT assay) according to OECD guideline 476 and GLP (Huntingdon Life Science Ltd. (b), 1997). In two independent experiments concentrations of 1250, 2500 and 5000 µg/mL of the 25% sodium ethylene sulphonate solution were tested with and without metabolic activation (S9-mix). Some increases of pH values and dose related increases in osmolarity were observed in all tests. No significant increases in mutant frequency were observed after treatment with 24.9% sodium ethylene sulphonate in either test in the absence and the presence of S-9 mix. Ethyl methanesulphonate and 20-Methylcholanthrene, the positive controls, induced highly significant increases in mutant frequency in both tests. It is concluded that the test solution, containing 24.9% sodium ethylene sulphonate, did not demonstrate mutagenic potential in this in vitro gene mutation assay. Under the conditions of the present mammalian gene mutation test the maximum test concentration of sodium ethylene sulphonate was 1250 µg/mL (5000 µg/mL of a 25% aqueous sodium ethylene sulphonate solution). This concentration is in the range of the maximum test concentration of 1300 µg/mL (0.01M) recommended according to the guideline.

 

In the present four in vitro genotoxicity studies (Ames test, HPRT assay and chromosome aberration test) no mutagenic potential was indicated. Under the conditions of the mammalian chromosome aberration and gene mutation test the maximum test concentration of sodium ethylene sulphonate was in the range of the maximum test concentration recommended according to the guideline. In the Ames test the maximum test concentration of sodium ethylene sulphonate was minimally lower (3000 µg/plate instead of 5000 µg/plate) than specified in the guideline. Hence, this study was repeated with a sodium ethylene sulphonate maximum concentration of 5000 µg/plate, confirming the results of absence of mutagenic potential in all strains tested (with and without metabolic activation). Hence, the results of the two Ames tests were in line with the mammalian chromosome aberration and gene mutation test and overall no signs of genotoxicity were indicated. Thus, it is concluded that sodium ethylene sulphonate will not demonstrate mutagenicity in vitro. Of note, in a QSAR analysis by means of the software toxtree v. 2.5.0 (Benigni/Bossa rulebase for mutagenicity and carcinogenicity) sodium ethylene sulphonate was no potential S.typhimurium TA 100 mutagen based on available structure attributes.

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on the available results sodium ethylene sulphonate was not classified and labelled for genotoxicity according to Regulation (EC) No 1272/2008 (CLP).