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Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Available data support the fact that BCEE induce a weak mutagenic effect in bacteria and in mammalian cells in the presence of metabolic activation. Very limited data support a mutagenic effect of the substance in the absence of metabolic activation, which suggest that mutagenicity might be induced by a metabolite of BCEE. BCEE did not induce chromosome damage or damage to the cell division apparatus of mammalian cells in an OECD 490 test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
Very similar to OECD Guideline 471. However, no E. coli WP2 uvrA strain was included. The study on Bis(2-chloroethyl) ether has been conducted by 2 different laboratories and some inconsistencies between the results have been observed. The laboratoires were Case Western Reserve University (CWR) and SRI International (SRI).
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Source: Aldrich
- Name ( as cited) : Bis(2-chloroethyl) ether
- Purity: 98.8%
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Dr. Bruce Ames (University of California, Berkeley)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction obtained from the liver of male Sprague-Dawley rats (RLI) and male Syrian hamsters (HLI) treated with Aroclor 1254 (200 mg/ml in corn oil) by ip at 500 mg/kg 5 days prior to decapitation. S9 mix contanied 10% S9 fraction.
Test concentrations with justification for top dose:
Flexibility was allowed in the protocol of the repeat test to adjust doses to better define or clarify a mutagenic response and/or toxicity.
Vehicle / solvent:
- Ssolvent used: DMSO
- Justification for choice of solvent/vehicle: solubility reasons
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
9-aminoacridine
sodium azide
other: 4-nitro-o-phenylenediamine; 2-aminoanthracene
Remarks:
sodium azide (TA1535 and TA 100); 4-nitro-o-phenylenediamine (TA98), 9-aminoacridine (TA97 and TA1537); 2-aminoanthracene (all strains)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 48 hr

SELECTION AGENT: histidine

DETERMINATION OF CYTOTOXICITY
- Identified based on one of the following criteria: appearance of his- pinpoint colonies, reduced numbers of revertant colonies per plate, or thinning or absence of the bacterial lawn in the preliminary dose setting assay.

NUMBER OF REPLICATIONS: at least 3 plates per dose levels.
Evaluation criteria:
The criteria used for data evaluation were as follows:
1) mutagenic response: a dose-related, reproducible increase in the number of revertants over background, even if the increase was less than twofold;
2) nomutagenic response: when no increase in the number of revertants was elicited by the chemical;
3) questionable response: when there was an absence of a clear-cut dose-related increase in revertants; when the dose-related increases in the number of revertants were not reproducible; or when the response was of insufficient magnitude to support a determination of mutagenicity
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid

- Results from CWR laboratory:

  TA100 TA1535 TA1537 TA98
Dose No act. 10% HLI 10% RLI No act. 10% HLI 10% RLI  No act. 10% HLI 10% RLI   No act. 10% HLI 10% RLI 
µg/plate

Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

 Mean±SEM

0 122 +/-8.3 176 +/-9.0 157 +/-13.8 15 +/-1.2 8 +/-1.7 15 +/-4.0 11 +/-1.0 22 +/-2.6 18 +/-2.2 23 +/-2.8 26 +/-1.7 15 +/-1.2
1 139 +/-1.5 159 +/-9.0 130 +/-3.2 15 +/-1.2 12 +/-1.3 12 +/-1.7 12 +/-1.2 19 +/-1.8 17 +/-0.6 29 +/-3.2 35 +/-3.2 12 +/-1.7
3.3 142 +/-5.7 145 +/-4.7 140 +/-5.4 14 +/-2.1 17 +/-2.6 12 +/-2.2 15 +/-1.9 15 +/-1.5 17 +/-0.0 41 +/-2.6 39 +/-4.1 11 +/-2.3
10 145 +/-3.7 146 +/-3.4  129 +/-6.3 13 +/-0.3 18 +/-1.9 12 +/-1.5 8 +/-0.9 22 +/-4.2 16 +/-0.6 31 +/-1.5 37 +/-7.0 14 +/-0.6
33 141 +/-7.5 162 +/-2.9 142 +/-6.4 8 +/-0.7 17 +/-2.7 13 +/-3.5 8 +/-1.2 12 +/-0.9 7 +/-0.9 36 +/-6.1 26 +/-2.5 14 +/-0.9
100 145 +/-7.9 167 +/-15.9 154 +/-5.2 10 +/-1.5 11 +/-2.4 15 +/-2.2 11 +/-2.0 13 +/-1.5 16 +/-2.8 26 +/-1.5 32 +/-7.1 10 +/-3.5
Pos 1518 +/-31 2615 +/-100 2333 +/-153 900 +/-48  296 +/-16 249 +/-18 297 +/-53 171 +/-20 268 +/-23 679 +/-35 1411 +/-48 1036 +/- 63

No act.: without metabolic activation; Pos: positive control ; NA: data not available ; HLI: Hamster liver S9 (Aroclor 1254 -induced) ; RLI: Rat liver S9 (Aroclor 1254 -induced)

- Results from SRI laboratory:

TA100 TA1535
Dose No act. No act. 10% HLI 10% HLI 10% RLI 10% RLI No act. No act. 10% HLI 10% HLI 10% RLI 10% RLI
µg/plate

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

0 96 +/-2.8 103 +/-5.3 120 +/-7.0 110 +/-7.6 110 +/-7.3 87 +/-5.5 23 +/-4.0 30 +/-1.5 7 +/-0.3   15 +/-1.7 9 +/-1.5  9 +/-1.5 
33 NA 85 +/-3.1 NA NA NA NA NA  19 +/-0.6  NA  NA  NA  NA 
100 98 +/-6.9 84 +/-3.0 137 +/-1.8 NA 110 +/-3.8 NA 22 +/-2.8 16 +/-2.3  7 +/-0.6  NA  6 +/-1.2  NA 
333 114 +/-7.3 87 +/-2.3 129 +/-7.1 98 +/-6.4 115 +/-4.7 116 +/-7.3  18 +/-5.9 16 +/-1.7  15 +/-1.8  14 +/-2.3  8 +/-2.7  15 +/-1.5 
1000 132 +/-12.2 106 +/-4.5 135 +/-4.7 93 +/-7.9 125 +/-5.5  138 +/-7.2 27 +/-3.1 16 +/-1.5 16 +/-2.0   17 +/-1.2  10 +/-3.6  11 +/-1.8
3333  134 +/-10.2 98 +/-4.7 149 +/-10.8 123 +/-7.4 138 +/-13.4 147 +/-8.1 17 +/-6.1 22 +/-3.4 25 +/-2.6 22 +/-1.0 16 +/-1.5 17 +/-1.3
6666 86 +/-8.1 NA NA 150 +/-5.8 NA 141 +/-14.5 NA NA NA 22 +/-1.0 NA 16 +/-2.0
10000  NA NA 160 +/-8.8

146 +/-0.6

149 +/-9.4 152 +/-3.1 NA NA 18 +/-9.1 20 +/-2.0 21 +/-2.1 12 +/-2.0
Pos  348 +/-10.4 346 +/-2.0 1067 +/-33 1123 +/-40 464 +/-5.2 385 +/-12.3 366 +/-31.1 344 +/-11.1  452 +/-6.0 358 +/-9.8 257 +/-29.8 163 +/-6.2

TA1537

TA98

Dose

No act.

10% HLI

10% HRI

No act.

10% HLI

10% HRI

µg/plate

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

Mean±SEM

0

3 +/-0.7

9 +/-1.3

4 +/-0.6 

17 +/-1.2 

25 +/-3.3 

30 +/-6.3 

33

5 +/-1.7

NA

NA 

17 +/-1.5

NA

NA 

100

3 +/-0.3

NA

NA

14 +/-0.6

NA 

NA 

333

3 +/-1.5

8 +/-0.6

5 +/-2.1

14 +/-1.2

18 +/-5.2

30 +/-5.2

1000

5 +4 -1.7

6 +/-1.5

7 +/-0.6

14 +/-1.5

20 +/-3.5

26 +/-0.6

3333

3 +/-0.7

7 +/-2.4

6 +/-1.2 

9 +/-2.0

24 +/-3.8

21 +/-3.2

6666

NA

7 +/-2.7

4 +/-1.5

NA

19 +/-3.5

15 +/-1.5

10000

NA

3 +/-1.2

3 +/-0.7

NA

20 +/-2.6

18 +/-3.2

Pos

156 +/-22.4

320 +/-6.1

169 +/-2.6

315 +/-7.8

973 +/-13.7

230 +/-8.8

No act.: without metabolic activation ; Pos: positive control ; NA: data not available ; HLI: Hamster liver S9 (Aroclor 1254 -induced) ; RLI: Rat liver S9 (Aroclor 1254 -induced)

Conclusions:
In the conditions of this test, Bis(2-chloroethyl) ether is considered to be mutagenic activity based on the result of one laboratory (SRI), while the mutagenic activity of the substance is questionable based on the results of the other laboratory (CWR).
Executive summary:

This study presents the results of a Salmonella preincubation assay, which is a modification of the standard plate incorporation assay, is used by the National Toxicology Program (NTP) to screen chemicals for mutagenicity. The tests were conducted by three independent laboratories on 270 chemicals, including Bis(2-chloroethyl) ether.

An appropriate methodology was used and the raw data on the test substance are reported. It is judged acceptable for assessment. Bis(2-chloroethyl) ether was independently studied by Case Western Reserve University (CWR) and SRI International (SRI). Data from CWR showed questionable results suggesting that the substance is mutagenic in TA100 without metabolic activation (slight dose-dependent increase in the number of revertants over background). A slight increase over background was also observed in TA1535 and TA98 (with metabolic activation). Data from SRI showed a dose-dependent increase in the number of revertants with metabolic activation (either with 10% RLI or 10% HLI) for TA100. A slight increase with metabolic activation was also observed for TA1535. The results were negative (with or without metabolic activation ) for TA 1537 or TA98.

Overall, in the conditions of this test, Bis(2-chloroethyl) ether is considered to have mutagenioc activity from the result of one laboratory (SRI) and the results are questionable based on the results of the other laboratory (CWR).

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
significant methodological deficiencies
Remarks:
Single S. typhimurium strain tested (TA100)
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
only one strain tested (TA100); methanol was used as solvent; no negaive control; no positive control
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and of test material: Merck Co. (D-6100 Darmstadt, FRG)
- Purity: 99%
Species / strain / cell type:
S. typhimurium TA 100
Additional strain / cell type characteristics:
other: provided by Prof. B. N. Ames of the University of California at Berkeley
Metabolic activation:
with
Metabolic activation system:
Liver homogenates were prepared according to the Ames method (1975) after induction of liver enzymes by a single i. p. injection of Aroclor 1254 (in Mazola oil) into each 150-200 g male Sprague-Dawley rat.
Test concentrations with justification for top dose:
0; 5; 10; 20; 40 µg/plate (no justification of the top dose)
Vehicle / solvent:
- Solvent used: metanol
- Justification for choice of solvent: none provided
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Positive controls:
no
Details on test system and experimental conditions:
Substance tested for mutagenicity in the presence and absence of an NADPH generating system, before and after exposure to daylight.
Evaluation criteria:
Statistically significant increase in the number of revertants.
Statistics:
"Commonly" used statistical approaches to analysis of test data and Jonckheere's distribution-free rank variation test.
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
other: weak positive
Cytotoxicity / choice of top concentrations:
cytotoxicity

Effect of daylight exposure on mutagenicity and cytotoxicity of 2,2'-DDE in the Salmonella/oxygenase test (Ames test) employing S. typhimurin TAI00 in the presence and absence of an NADPH generating system, in triplicate experiments. Cytotoxicity assays were performed using a histidine-rich medium (complete agar) where the number of initial bacterial cells used was about 1E+06 times lower than that employed in the mutagenicity tests.

Daylight        
Dose With NADP    Without NADP   
(µg/plate) Minimal agar Complete agar Minimal agar Complete agar
0 125.0 +/-2.8 22.4 +/-2.4 105.8 +/-1.9 20.2 +/-3.5 
5 141.7 +/-5.7 11.7 +/-1.6  124.6 +/-4.4 10.3 +/-1.2 
10 147.3 +/-2.3 10.3 +/-1.7 115.7 +/-7.3 7.0 +/-2.9 
20 155.0 +/-2.4 2.6 +/-1.2 124.3 +/-6.9 4.6 +/-1.7 
Darkness
Dose With NADP Without NADP 
(µg/plate) Minimal agar Complete agar  Minimal agar Complete agar
0 125.0 +/-2.8 22.4 +/-2.8  105.8 +/-1.9  20.2 +/-3.5 
5 133.0 +/-4.0 7.5 +/-0.5  126.7 +/-8.4  6.0 +/-1.6 
10 143.5 +/-1.5 3.0 +/-1.4  124.0 +/-8.6  5.3 +/-0.9 
20 150.3 +/-13.1 2.0 +/-0.8  127.7 +/-4.1  3.0 +/-1.6

Effect of UV irradiation on mutagenicity of 2,2'-DDE tested with the Salmonella/oxygenase test (Ames test) using S. typhimurium TA100 in the presence and absence of an NADPH generating system, in triplicate experiments. Cytotoxicity assays were performed using a histidine-rich medium (complete agar) where the number of initial bacterial cells used was about 1E+06 times lower than that employed in the mutagenicity tests.

Before UV radiation
Dose With NADP    Without NADP   
(µg/plate) Minimal agar Complete agar Minimal agar Complete agar
0 97.6 +/-6.8 168.4 +/-6.8 80.6 +/-3.7 128.4 +/-5.1
5 107.0 +/-2.9 63.7 +/-1.8 83.0 +/-2.8 103.0 +/-3.2 
10 122.7 +/-3.4 47.6 +/-1.8 86.3 +/-5.4 94.6 +/-1.7 
20 140.4 +/-3.7 44.0 +/-2.1 88.0 +/-6.1 94.6 +/-1.7
40 171.3 +/-2.8 14.0 +/-2.4  128.4 +/-2.8  12.3 +/-2.8
Dose After UV radiation
(µg/plate) With NADP  Without NADP  
(µg/plate)  Minimal agar Complete agar  Minimal agar Complete agar
0 97.6 +/-6.8 168.4 +/-6.8 80.6 +/-3.7 128.4 +/-5.1
5 122.0 +/-2.1 52.7 +/-1.2 95.8 +/-7.6 70.6 +/-0.5
10 131.7 +/-3.1 52.0 +/-1.4 101 +/-2.4 67.0 +/-4.9
20 153.1 +/-4.1 17.6 +/-2.8  127.7 +/-2.0  62.3 +/-1.7 
40  225.0 +/-2.7 16.5 +/-1.5  118.6 +/-0.4  60.0 +/-2.4 
Conclusions:
Under the conditions of this test, Bis(2-chloroethyl) ether was found to be weakly mutagenic with metabolic activation (rat liver S9).
Executive summary:

Based on the Ames test conducted using a single S. typhimurium strain (TA 100) with metabolic activation, Bis(2-chloroethyl) ether was found to be weakly mutagenic. The influence of light and/or UV irradiation were also evaluated as they create conditions under which ether peroxide may be produced from Bis(2-chloroethyl) ether. The presence or absence of light and/or UV irradiation did not seem to affect the weak mutagenicity of Bis(2-chloroethyl). The substance was found to be cytotoxic even at very low dose.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23-sept-2015 to 18-apr-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
GLP compliance:
yes (incl. QA statement)
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No.: 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), protected from light
Target gene:
hisD3052; hisG46; hisC3076; trpE
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. The S9 mix comprised 10% S9 fraction.
Test concentrations with justification for top dose:
78.1; 156.2; 312.5; 625; 1250; 2500; 5000 µg/plate. No inhibitory, cytotoxic effect of the test item was observed in the preliminary experiment.
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble at 100 mg/mL concentration in Distilled water (stock solution); but the formulation at the same concentration using DMSO as vehicle (solvent) was a clear solution.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Remarks:
Without activation: 4-nitro-1,2-phenylene-diamine (TA98 only) / Na-azide (TA100, TA 1535) / 9-aminoacridine (TA1537) / Methyl-methanesulfonate (WP2 uvrA) With activation: 2-aminoanthracene (all Solmonella strains and E. coli WP2 uvrA)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); preincubation
- Cell density at seeding: 1.89-4.63E+09 CFU/mL

DURATION
- Preincubation period: 20 min
- Exposure duration: 48h
Evaluation criteria:
A reproducible and dose-related increase in mutant counts of at least one strain is considered to be a positive result. For TA 1535, TA 100 and TA 98 this increase should be about twice that of negative controls, whereas for TA 1537, at least a threefold increase should be reached. For TA 102 an increase of about 100 mutants should be reached. Otherwise, the result is evaluated as negative. However, these guidelines may be overruled by good scientific judgment.
In case of questionable results, investigations should continue, possibly with modifications, until a final evaluation is possible.
Statistics:
No statistics perfomed; evaluation based on criteria mentioned above
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Remarks:
but relatively low number of revertants
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: Dose-dependent increase in the observed mutation factor values at 5000 and 2500 μg/plate (mutation factor values of 2.78 and 2.12, respectively; threshold value: 2)

In the Initial Mutation Test (using the plate incorporation method), slight positive effect was observed in E. coli WP2 uvrA strain with metabolic activation. The observed mutation factor value (MF=2.12) at the highest examined concentration of 5000 μg/plate was above the threshold limit of 2 of this strain, and dose dependence was also observed. In the Confirmatory Mutation Test, a clear positive effect was seen: the observed mutation factor values at the two highest examined concentrations of 5000 and 2500 μg/plate were above the threshold limit of 2 of this strain (mutation factor values of 2.78 and 2.12, respectively) and dose dependence was also observed. Additionally, dose-dependent increase in the number of revertant colonies was also observed in this strain with metabolic activation using the pre-incubation method, although the mutation factor values remained slightly below the relevant threshold limit (MF=2) in that case (highest mutation factor value was 1.83).

In the Confirmatory Mutation Test using the pre-incubation method, biologically relevant increases were also observed in S. typhimurium TA1535 strain with metabolic activation. The observed mutation factor values were 4.91 and 3.18 at 2500 and 1250 μg/plate concentration, respectively (i.e. above the threshold limit of 3 of this strain). The observed values in the examined concentration range were compatible with a dose response curve (slightly lower value was detected at 5000 μg/plate due to the observed cytotoxicity on the plates). These values also indicate a mutagenic effect, although the numbers of revertant colonies seen in the DMSO control plates of this strain were relatively low compared to the untreated control (as indicated by the MF: 1.50 value of the untreated control). However, even compared with the untreated control, the mutation factor at 2500 μg/plate had a positive result with a mutation factor of 3.27.

Higher numbers of revertant colonies compared to the vehicle (solvent) control were detected in the main test in some other sporadic cases. However, no dose-dependence was observed in those cases and they were below the biologically relevant threshold value. The numbers of revertant colonies were within the historical control range in each case, so they were considered as reflecting the biological variability of the test. Sporadically, lower revertant counts compared to the vehicle (solvent) control were observed in the main test at some non-cytotoxic concentrations. However, no background inhibition was recorded and the mean numbers of revertant colonies were in the historical control range in all cases, thus they were considered as biological variability of the test system.

No inhibitory, cytotoxic effect of the test item was observed in the Initial Mutation Test using the plate incorporation method in any of the examined strains. However, inhibitory, cytotoxic effect of the test item (reduced / slightly reduced background lawn development, pinpoint colonies were also detected in some cases) was observed in the Confirmatory Mutation Test using the pre-incubation method in all examined strains at 5000 μg/plate concentration with and without metabolic activation and in Salmonella typhimurium TA1537 strain at 2500 μg/plate without metabolic activation.

Conclusions:
Under the conditions of this test, the test item 2,2’-Dichlorodiethyl ether exhibit a weak mutagenic activity in S. typhimurium TA 1535 and E. coli WP2 uvrA with metabolic activation.
Executive summary:

The test substance was evaluated in an Ames Test on Salmonella typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and Escherischia coli WP2 uvrA, performed according to OECD TG 471. In the Initial Mutation Test a weak positive effect of the test item was obtained in Escherichia coli WP2 uvrA strain with metabolic activation using the plate incorporation method (the observed revertant colony numbers were above the respective biological threshold value, slight dose response was also indicated). Although the effect was weak, it was reproducible in the Confirmatory Mutation Test using the same test conditions. Furthermore, a weak positive effect of the test item was also observed in the Confirmatory Mutation Test in Salmonella typhimurium TA1535 strain with metabolic activation using the pre-incubation method.

The reported data of this mutagenicity assay show that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains Escherichia coli WP2 uvrA and Salmonella typhimurium TA1535 strains in the presence of metabolic activation. In conclusion, the test item 2,2’-Dichlorodiethyl ether exhibit a weak mutagenic activity under the test conditions of this study.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 23-Oct-2015 to 11-Jul-2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
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
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No.: 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), protected from light
Target gene:
Not applicable (not a gene mutation assay).
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: ATCC

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 mediumcontaining 10% inactivated horse serum, L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL); 5% CO2/95% air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction was purchased from Moltox (Molecular Toxicology, INC, Boone, NC 28607, USA) and obtained from the liver of rats treated with Aroclor 1254 (500 mg/kg) by intraperitoneal route.
Test concentrations with justification for top dose:
With a treatment volume of 0.5% (v/v) in culture medium, the test concentrations were as follows: 4, 40, 200, 400, 1000 and 2000 μg/mL.
At the highest dose-level , the pH of the culture medium was approximately 7.4 (as for the vehicle control) and the osmolality equal to 364 mOsm/kg H2O (349 mOsm/kg for the vehicle control). Therefore, this dose-level was not considered to produce extreme culture conditions.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO; batch K46301950506
- Justification for choice: Based on available solubility data, the test item was dissolved in DMSO at 400 mg/mL. Therefore, using this stock solution at 400 mg/mL and a treatment volume of 0.5% (v/v) in culture medium, the highest recommended dose-level of 10 mM (corresponding to 1430 µg/mL) was achievable.
Positive controls:
yes
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: Colchicine
Remarks:
See section "Any other information on materials and methods incl. tables"
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 3E+05 cells/mL

DURATION:
- Preliminary and main experiment: 3h treatment + 24h recovery (with and wthout S9 mix); 24h treatment + 0h recovery (without S9 mix)

DETERMINATION OF CYTOTOXICITY
- Method: % decrease in Population Doubling (PD)
Evaluation criteria:
A test item is considered to have clastogenic and/or aneugenic potential, if all the following criteria were met: - a dose-related increase in the frequency of micronucleated cells was observed,- for at least one dose-level, the frequency of micronucleated cells of each replicate culture was above the corresponding vehicle historical range,- a statistically significant difference in comparison to the corresponding vehicle control was obtained at one or more dose-levels.The biological relevance of the results was considered first. Evaluation of a negative response: a test item is considered negative if none of the criteria for a positive response were met.
Statistics:
For each condition of the cytogenetic experiment, the frequency of micronucleated cells in treated cultures was compared to that of the vehicle control cultures. This comparison was performed using the Chi2 test unless treated culture data are lower than or equal to the vehicle control data. P = 0.05 is used as the lowest level of significance. To assess the dose-response trend, a linear regression was performed between the frequencies of micronucleated cells and the dose-levels.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

VALIDITY CRITERIA:

The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

TREATMENT WITHOUT S9 mix:

Cytotoxicity:

- 3 h treatment: slight to moderate cytotoxicity was induced at dose-levels ≥ 500 μg/mL, as shown by a 25 to 47% decrease in the PD.

- 24 h treatment: moderate cytotoxicity was induced at dose-levels ≥ 500 μg/mL, as shown by a 42 to 49% decrease in the PD.

Micronucleus analysis:

The dose-levels selected for micronucleus analysis were 250, 1000 and 2000 μg/mL, the latter corresponding to the highest recommended dose-level. The dose-levels of 250 and 1000 μg/mL were selected for reading in order to analyse dose-levels which showed different levels of cytotoxicity (i.e. from moderate to slight or no cytotoxicity). Increases in the frequency of micronucleated cells were observed at all the analyzed dose-levels after the 3-hour treatment period and at 1000 μg/mL after the 24-hour treatment period. However, these increases were neither statistically significant when comparing to the respective vehicle control, nor dose-related. Furthermore, the values obtained for each replicate culture were included in the historical negative data range. These results met the criteria of a negative response.

TREATMENT WITH S9 mix:

Cytotoxicity: Moderate to severe cytotoxicity was induced at dose-levels ≥ 10 μg/mL, as shown by a 53 to 100% decrease in the PD

Micronucleus analysis:

The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 μg/mL. The dose-level of 10 μg/mL was selected as high dose for micronucleus analysis because it induced the recommended level of cytotoxicity (i.e. 53% decrease in the PD). The two immediately tested dose-levels of 2.5 and 5 μg/mL were also selected for analysis, even if they did not induced any cytotoxicity. Increases in the frequency of micronucleated cells were observed at all the analyzed dose-levels. However, these increases were neither statistically significant when comparing to the vehicle control, nor dose-related. Furthermore, the values obtained for each replicate culture were included in the historical negative data range. These results met the criteria of a negative response.

Conclusions:
Under the experimental conditions of the study, the test item 2,2’-Dichlorodiethyl ether did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or presence of a rat liver metabolizing system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce an increase in the frequency of micronucleated cells in the mouse cell line L5178 TK+/-. This study was conducted in compliance with the OECD Guideline No. 487 under GLP. The mean population doubling and the mean frequencies of micronucleated cells for the vehicle controls were as specified in the acceptance criteria. Also, positive control cultures showed clear statistically significant increases in the frequency of micronucleated cells. The study was therefore considered to be valid.

Experiments without S9 mix :

The dose-levels selected for micronucleus analysis were 250, 1000 and 2000 μg/mL, the latter corresponding to the highest recommended dose-level. The dose-levels of 250 and 1000 μg/mL were selected for reading in order to analyse dose-levels which showed different levels of cytotoxicity (i.e. from moderate to slight or no cytotoxicity). Increases in the frequency of micronucleated cells were observed at all the analyzed dose-levels after the 3-hour treatment period and at 1000 μg/mL after the 24-hour treatment period. However, these increases were neither statistically significant when comparing to the respective vehicle control, nor dose-related. Furthermore, the values obtained for each replicate culture were included in the historical negative data range. These results met the criteria of a negative response.

Experiments with S9 mix :

The dose-levels selected for micronucleus analysis were 2.5, 5 and 10 μg/mL. The dose-level of 10 μg/mL was selected as high dose for micronucleus analysis because it induced the recommended level of cytotoxicity (i.e. 53% decrease in the PD). The two immediately tested dose-levels of 2.5 and 5 μg/mL were also selected for analysis, even if they did not induced any cytotoxicity. Increases in the frequency of micronucleated cells were observed at all the analyzed dose-levels. However, these increases were neither statistically significant when comparing to the vehicle control, nor dose-related. Furthermore, the values obtained for each replicate culture were included in the historical negative data range. These results met the criteria of a negative response.

Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells, using L5178Y TK+/- mouse lymphoma cells, either in the absence or in the presence of a rat liver metabolising system.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 08-feb-2016 to 02-Feb-2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. QA statement)
Type of assay:
other: mammalian cell gene mutation test
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): 2,2`- Dichlorodiethyl ether
- Purity: 99.76%

SOURCE OF TEST MATERIAL
- Batch No.: 20150706
- Expiration date of the lot/batch: 01 June 2016
- Purity test date: 28 August 2015

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Controlled Room Temperature (15-25°C, below 70 RH%), protected from light
Target gene:
tk+/- (thymidine kinase) locus in L5178Y cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
CELLS USED
- Cell line: L5178Y TK+/- 3.7.2 C mouse lymphoma
- Source of cells: ATCC

MEDIA USED
- Type and identity of media: RPMI-10 medium; 5% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix was made from the livers of male Sprague Dawley rats, which were treated with phenobarbital (PB) and β-naphthoflavone (BNF) at 80 mg/kg/day by oral gavage for three consecutive days. The S9 mix comprised 10% S9 fraction.
Test concentrations with justification for top dose:
MAIN TEST:
- Assay 1: 3h treatment period (with S9 mix): 1.25; 2.5; 5; 10; 20; 40; 60; 80; 100; 120 µg/L ; 3h treatment period (without S9 mix): 46.88; 93.75; 187.5; 375; 750; 1500 µg/L
- Assay 2: 3h treatment period (with S9 mix): 1.25; 2.5; 5; 10; 20; 40; 60; 80; 100; 120 µg/L ; 24h treatment period (without S9 mix): 46.88; 93.75; 187.5; 375; 750; 1500 µg/L
Vehicle / solvent:
- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: The test item was insoluble at 100 mg/mL concentration in Distilled water (stock solution); but the formulation at 150 mg/mL using DMSO as vehicle (solvent) was a clear solution.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
cyclophosphamide
Remarks:
4-Nitroquinoline-N-oxide (without metabolic activation): 0.15 μg/mL in DMSO for 3-hour treatment and 0.1 μg/mL in DMSO for 24-hour treatment. Cyclophosphamide (with metabolic activation): 4 μg/mL in DMSO.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding: 1E+07 cells/mL in the 3-h treatments; 6E+06 cells/mL in the 24-h treatments; 2E+05 cells/mL in the expression period

DURATION:
- Preliminary experiment: 3h treatment (with and without S9 mix); 24h (without S9 mix)
- Main experiment: 3h treatment (with and without S9 mix) in Assay 1; 3h treatment (with S9 mix) and 24h (without S9 mix) in Assay 2.
- Expression period: 48h

SELECTION AGENT: trifluorothymidine (TFT)

DETERMINATION OF CYTOTOXICITY
- Method: Relative Total Growth (RTG)
Evaluation criteria:
A test item is considered to have mutagenic potential, if all the following criteria were met:
1. At least one concentration exhibited a statistically significant increase (p<0.05) compared with the concurrent negative control and the increase was biologically relevant (i.e. the mutation frequency at the test concentration showing the largest increase was at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (vehicle/solvent) control value).
2. The increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions).
3. The increase is concentration-related (p < 0.05) as indicated by the linear trend analysis.
Statistics:
Not specified
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid

PRELIMINARY EXPERIMENT:

Treatment concentrations for the mutation assay were selected based on the results of a short Preliminary Toxicity Test. 3-hour treatment in the presence and absence of metabolic activation system (S9-mix) and 24-hour treatment in the absence of metabolic activation system was performed with a range of test item concentrations to determine toxicity immediately after the treatments. The highest concentration tested in the preliminary experiment was 1500 μg/mL (approx. 10 mM concentration). No insolubility was observed in the preliminary experiment, but cytotoxicity was recorded in the absence of metabolic activation. Therefore, concentrations up to the cytotoxicity limit or the highest recommended concentration were selected for the main experiments according to the instructions of the relevant OECD guideline.

MUTATION ASSAYS:

- ASSAY 1:

In the presence of S9-mix (3-hour treatment), excessive cytotoxicity of the test item was observed: cells of the 120 and 100 μg/mL concentrations died during the treatment or in the expression period. Extremely low number of cells survived the expression period at 80 and 60 μg/mL concentrations; no plating could be performed in those cases. The relative total growth at 40 μg/mL was still less than 1%; therefore this concentration was excluded from the evaluation. An evaluation was made using data of 20 μg/mL (relative total growth: 16%) and four lower concentrations (a total of five concentrations). Biologically relevant and statistically significant increase was seen at 20 μg/mL concentration, although due to high degree of cytotoxicity this result should be handled with care. Statistically significant increase was also detected at 10 μg/mL concentration, however in this case the effect was biologically non-relevant (the difference compared to the negative (vehicle) control mutation values was smaller than the Global Evaluation Factor). Significant dose-response to the treatment was indicated by the linear trend analysis.

In the absence of S9-mix (3-hour treatment), no marked cytotoxicity of the test item was observed. An evaluation

was made using all the six examined concentrations (including the maximum concentration of 1500 μg/mL). No biologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significant dose-response to the treatment was indicated by the linear trend analysis.

- ASSAY 2:

In the presence of S9-mix (3-hour treatment), similarly to the first test, excessive cytotoxicity of the test item was observed: cells of the 120, 100 and 80 μg/mL concentrations died during the treatment or in the expression period . Extremely low number of cells survived the expression period at 60 μg/mL concentration; no plating could be performed in that case. The relative total growth at 40 μg/mL was still less than 1%; therefore this concentration was excluded from the evaluation. An evaluation was made using data of 20 μg/mL (relative total growth: 19%) and four lower concentrations (a total of five concentrations). Biologically relevant and statistically significant increase was seen at 20 μg/mL concentration, although due to high degree of cytotoxicity this result should be again handled with care. Significant dose-response to the treatment was indicated by the linear trend analysis. This result was considered as confirming the positive effect observed in the first main test.

In the absence of S9-mix (24-hour treatment), no marked cytotoxicity was observed. An evaluation was made using all the six tested concentrations. No biologically relevant and statistically significant increase in the mutation frequency was observed at the evaluated concentrations (including the maximum concentration of 1500 μg/mL, where slight cytotoxicity was observed as indicated by relative total growth value of 37%). Although statistically significant increase was seen at 187.5 μg/mL concentration; the difference compared to the negative control mutation factor value was less than the global evaluation factor, so it was not considered as biologically relevant, furthermore none of the three higher concentrations showed similar statistically significant increase. No significant dose-response to the treatment was indicated by the linear trend analysis.

Conclusions:
Under the conditions of this Mouse Lymphoma Assay, a reproducible mutagenic effect of 2,2’-Dichlorodiethyl ether was observed in the presence of metabolic activation system, although the effect was seen only at a concentration with marked cytotoxicity. No mutagenic activity of the test item was detected in the absence of metabolic activation system.
Executive summary:

The objective of this study was to evaluate the potential of the test item to induce genetic mutations in cells in the mouse cell line L5178 TK+/-. This study was conducted in compliance with the OECD Guideline No. 490 under GLP. The experiments were performed using appropriate untreated, negative (vehicle) and positive control samples in all cases. The spontaneous mutation frequency of the negative (vehicle) controls was in the appropriate range. The positive controls gave the anticipated increases in mutation frequency over the controls. The plating efficiencies for the negative (vehicle) controls at the end of the expression period were within the acceptable range in all assays. The evaluated concentration ranges were considered to be adequate. The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

In the mutation assays, cells were exposed to the test item for 3 hours with or without metabolic activation (±S9-mix) and for 24 hours without metabolic activation (-S9-mix). The cells were plated for determination of survival data and in parallel subcultured

without test item for approximately 2 days to allow expression of the genetic changes. At the end of the expression period, cells were allowed to grow and form colonies for approximately 2 weeks in culturing plates with and without selective agent (TFT) for determination of mutations and viability.

Overall, statistically significant and biologically relevant increases were seen in the experiments with metabolic activation in Assay 1 and Assay 2. However, the dose response relationships for both cytotoxicity and mutagenicity were quite steep in these assays and the positive effect was only observed at a concentration with high degree of cytotoxicity. No similar effects were seen in the experiments without metabolic activation; those experiments were clearly negative (some minor sporadic increases in the mutation frequency values in those cases were considered not being test item related effects, but showing the biological variability of the test system).

In conclusion, under the conditions of this Mouse Lymphoma Assay, a reproducible mutagenic effect of 2,2’-Dichlorodiethyl ether was observed in the presence of metabolic activation system, although the effect was seen only at a concentration with marked cytotoxicity. No mutagenic activity of the test item was detected in the absence of metabolic activation system.

Endpoint:
in vitro DNA damage and/or repair study
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
- Principle of test: E. coli multitest developped by Toman et al. (1987).
- Short description of test conditions: The method allows the measurement of all major known effects of DNA damage: mutation, recombination and SOS induction.
GLP compliance:
no
Type of assay:
other: E. coli multitest
Specific details on test material used for the study:
TEST MATERIAL:
- Name (as cited): bis(2-chloroethyl)ether (BCEE)
- Source: Aldrich-Chemie.
- Purity: not specified
Species / strain / cell type:
E. coli, other: MT 103
Remarks:
see section "Any other informations on materials and methods incl. tables"
Species / strain / cell type:
E. coli, other: MT 119
Remarks:
see section "Any other informations on materials and methods incl. tables"
Species / strain / cell type:
E. coli, other: MT 126
Remarks:
see section "Any other informations on materials and methods incl. tables"
Test concentrations with justification for top dose:
One-ml aliquots were incubated with the test chemical at different concentrations ranging from non-toxic to highly toxic doses leaving < 0.01 surviving cells.
Vehicle / solvent:
No vehicle
Untreated negative controls:
yes
Details on test system and experimental conditions:
METHOD OF APPLICATION:
One-ml aliquots were incubated with the test chemical at different concentrations Incubation was performed at 37 °C for 10 min; bacteria were washed twice, resuspended in 0.01 M MgSO4 and plated at appropriate dilutions on MacConkey medium containing galactose or lactose. After 19 h incubation at 37°C the plates were scored for survival and Lac+ and Gal+ colonies. Spontaneous events occurred at a frequency of 1E-05 for mutation, and 1E-04 for recombination and SOS induction.

DURATION
- Preincubation period: 10min
- Selection time: 19h

SELECTION AGENT: galactose or lactose
Evaluation criteria:
Following the treatment with a chemical, dose-effect curves were obtained for each type of induced event (Mutagenicity, Recombinagenicity and SOS inducing potency). The dose was expressed in terms of lethal hits (z) calculated as -Ln survival, and the effect was expressed in terms of yield, i.e. the fraction of treated cells giving rise to altered colonies. The yield of mutants, recombinants and SOS inductants was calculated as (nx-n0(Sx))/N0, where nx is the number of altered colonies (per ml of cell culture) at dose x (the induced events), n0 is the number of altered colonies (per ml of cell culture) at dose 0 (the spontaneous events), Sx is the survival fraction at dose x and N0 is the number of colonies (per ml of cell culture) at dose 0.

The parameters were used to evaluate the results:
- The potency (P) calculated as the integral under the dose-effect curve
- Ymax calculated as the yield of altered colonies at the maximal effective treatment
- Zmax calculated as the number of lethal hits giving the maximum yield (Ymax).
Species / strain:
E. coli, other: MT 103, MT126 and MT 119
Metabolic activation:
without
Genotoxicity:
negative

- Genotoxicity spectrum of Bis(2-chloroethyl) ether in E. coli:

   P  Ymax  Zmax
 Mutation  nd  nd nd 
 Recombination  nd  nd nd 
 SOS induction  nd  nd nd 

nd: not detected

Conclusions:
In the conditions of this test, Bis(2-chloroethyl) ether did not exhibit any detectable mutagenic, recombinagenic or SOS inducing potency.
Executive summary:

In this study, and E. coli multitest was used to evaluate the genotoxic potential of bifunctional alkylating agents. The test allows measuring the mutagenic, recombinagenic and SOS inducing potencies of the tested chemicals. This potency corresponds to the cumulative yield of the affected cell population over the entire effective dose range of the chemical treatment. No metabolic activation was used.

Bis(2-chloroethyl) ether did not induce mutations in the selected E. coli strains. This forward mutation test is considered by the authors to have low sensitivity towards weak mutagens (low rate of spontaneous mutations in the selected strains). In addition, the lack of recombigenic and SOS inducing potential may be due to low sensitivity of E. coli to Bis(2-chloroethyl) ether or the need of metabolic activation for this compound.

In the conditions of this test, Bis(2-chloroethyl) ether did not exhibit any detectable mutagenic, recombinagenic or SOS inducing potency.

Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
4 (not assignable)
Rationale for reliability incl. deficiencies:
abstract
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
Reverse mutation systems: (a) E. coli WP2 B/r hcr + and WP2 hcr-, (b) Ames's Salmnonella 4 TA strains, and (c) B. subtilis GSY1035
GLP compliance:
no
Remarks:
pre-GLP
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): bis(2-chloroethyl) ether
- Purity: not specified
Species / strain / cell type:
S. typhimurium, other: not specified
Species / strain / cell type:
E. coli WP2
Additional strain / cell type characteristics:
other: WP2 B/r hcr + and WP2 hcr-
Species / strain / cell type:
bacteria, other: B. subtilis GSY1035
Metabolic activation:
not specified
Test concentrations with justification for top dose:
Not specified
Vehicle / solvent:
Not specified
Untreated negative controls:
not specified
Negative solvent / vehicle controls:
not specified
Positive controls:
not specified
Details on test system and experimental conditions:
Not specified
Rationale for test conditions:
Not specified
Evaluation criteria:
Not specified
Statistics:
Not specified
Genotoxicity:
positive
Conclusions:
Bis(2-chloroethyl) ether was mutagenic in various strains of Escherichia coli, Bacillus subtilis and S. typhimurium, although experimental details were not provided in the published account of this study.
Executive summary:

Bis(2-chloroethyl) ether was mutagenic in various strains of Escherichia coli, Bacillus subtilis and S. typhimurium, although experimental details were not provided in the published account of this study. In this test system, Bis(2-chloroethyl) ether was found to be a direct acting, base-change mutagen.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Study performed according to standardised protocol, but test conditions and results were not sufficiently detailed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Principles of method if other than guideline:
As described in Ames et al. 1975
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
TEST MATERIAL
- Name (as cited): Bis(2-chloroethyl)ether
- Highest available purity
Target gene:
Histidine-revertant
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
provided by Dr. B. N. Ames, University of California at Berkeley
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
provided by Dr. B. N. Ames, University of California at Berkeley
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
provided by Dr. D. K. McCalla, McMaster University, Hamilton
Species / strain / cell type:
Saccharomyces cerevisiae
Details on mammalian cell type (if applicable):
provided by Dr. M. S. Legator, University of
Texas at Galveston
Additional strain / cell type characteristics:
other: D3
Metabolic activation:
with and without
Metabolic activation system:
S9 mix as described in Ames et al. 1975
Test concentrations with justification for top dose:
Not specified
Vehicle / solvent:
Not specified
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
not specified
Details on test system and experimental conditions:
METHOD OF APPLICATION:
***1/ Standard Ames test: Assays with E. coli WP2 were performed according to the standard test except 0.125% oxoid broth was incorporated in the bottom agar
***2/ Assays in suspension: Incubation mixture contianing 0.2 ml of bacterial suspension, 0.2 ml of the chemical solution, 0.5 ml of S9 mix, in 0.067 M sodium phosphate buffer (pH 7.2, final volume: 2.0 mL). Incubated at 37° C on a roller drum for 2 hours. Aliquots (0. 5 ml) were a) added to 2 ml top agar containing histidine and biotin which was overlayed in 30 Vogel-Bonner (V-B) salts containing 2% agar to determine the number of revertants per plate or b) added to 2 ml top agar containing biotin (but no histidine) which was overlayed on V-B salts containing 2% agar. In the latter procedure, an aliquot was diluted (1E-05) and 0.5 ml was added to 2 ml top agar which was overlayed on nutrient broth agar to determine survival of the treated cells.
***3/ Assays in dessicator: Agar overlay plates containing bacteria (and optionally S9 mix) were placed uncovered in a 9-liter desiccator. The desiccators were placed on magnetic stirrers in a 37°C room for 7 to 10 hours. The plates were removed, their lids were replaced, and they were incubated approximately 40 hours at 37°C before being counted.
***4/ Assays with S. cerevisiae D3: These assays were conducted in suspension at 30°C for 4 hours on a roller drum. The suspension contained c.a. 1E+08 cells, 0.2 ml of a solution of the test chemical in dimethylsulfoxide, 0.5 ml of S9 mix (optional) in a final volume of 2 ml of 0.067 M phosphate buffer (pH 7.4). Five 0.2 ml aliquots of a 1E-03 dilution and three aliquots of 1E-05 dilution were spread on plates to determine the number of mitotic recombinants,and survivors, respectively. The plates were incubated at 30° C for two days, refrigerated for two days at 4 ° C to enhance the development of the red pigment indicative of mitotic recombination (ade- homozygosity), and then counted.
Rationale for test conditions:
Not specified
Evaluation criteria:
Not specified
Statistics:
Not specified
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
Dessicator experiment
Cytotoxicity / choice of top concentrations:
not specified
Vehicle controls validity:
not specified
Untreated negative controls validity:
not specified
Positive controls validity:
not specified
Conclusions:
Bis(2-chloroethyl)ether was found to be weakly mutagenic when incorporated into agar and very mutagenic when assayed in desiccators or in suspension. The substance did not require S9 mix for mutagenic activity.
Executive summary:

Bis(2-chloroethyl)ether was found to be weakly mutagenic when incorporated into agar and very mutagenic when assayed in desiccators or in suspension. The substance did not require S9 mix for mutagenic activity. Very limited results are reported in the original publication.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Based on the evaluation of available evidences, no data support the fact that Bis(2-chloroethyl) ether induce in vivo genetic toxicity in mammals. The substance was negative in a Heritable Tanslocation Test in mice (Jorgensen et al. 1977) and was no found to form DNA adducts or to induce hepatocellular ATPase-deficient foci in rats exposed to BCEE by inhalation for 18h (Gwinner et al. 1983). BCEE was found to induce recessive lethals in a sex‐linked recessive lethal (SLRL) assay following injection (irrelevant exposure route) but no effect was found in a feeding study. Based on current knowledge, no data support the fact that BCEE induce genetic toxicity in vivo.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Additional information

GENOTOXICITY IN VITRO:

 

8 studies have been performed to assess the potential of Bis(2-chloroethyl) ether (BCEE) to induce genetic toxicity in vitro. Published accounts regarding studies dealing with the evaluation of the in vitro genetic toxicity of BCEE have yielded somewhat equivocal results and guideline studies were recently conducted to clarify the degree to which BCEE induces genetic toxicity. While detailed descriptions of the laboratory conditions were not provided in some older studies (leading to a reliability score of 3 or 4), they were included in this assessment in order to provide a comprehensive view of available data.

 

Most available studies have been conducted to evaluate the mutagenic activity of BCEE in bacteria. Shirasu et al. (1975) reported that BCEE was mutagenic in various strains of Escherichia coli, Bacillus subtilis and Salmonella typhimurium. In this study, BCEE was found to be a direct acting, base-change mutagen. However, the information originates from a conference abstract (reliability 4) and a comprehensive description of this study has not been published. Simmon et al. (1977) reported that BCEE, when tested in a desiccator containing the vapor, was strongly mutagenic to S. typhimurium strain TA 100, with the number of revertants increasing with the duration of exposure. BCEE was also found to be weakly mutagenic when incorporated into agar (bacterial strains not specified). BCEE did not require S9 mix for mutagenic activity. The study of Simmon et al. (1977) includes a comparative assessment of forty-five chemicals identified in drinking water. It provides only limited information on the results of the Ames assay conducted with BCEE (reliability 3). While it appears that BCEE was mutagenic in S. typhimurium strain TA 100 without metabolic activation, no information is provided on the mutagenic activity of BCEE on the other tested bacterial strains (S. typhimurium strain TA 98, TA 1535, TA 1537, TA 1538; E. coli WP2; S. cerevisiae D3).Norpoth et al. (1986) reported "weak" mutagenic activity inS. typhimurium TA 100 (in the presence of a metabolic activating system) when BCEE (up to 40 μg/dish) was added directly to culture plates.The influence of light and/or UV irradiation was also evaluated as it may lead to conditions under which ether peroxide is produced from BCEE. The presence or absence of light and/or UV irradiation did not seem to affect the weak mutagenicity of BCEE. This study only reports limited details on the methodology and results and has been assigned a reliability score of 3.Mortelmans et al. (1986) reported the results of a Salmonella pre-incubation assay, which is a modification of the standard plate incorporation assay used by the National Toxicology Program (NTP). The tests on BCEE were conducted by two independent laboratories. Data from one laboratory (CWI) showed questionable results suggesting that the substance is mutagenic in TA100 without metabolic activation (slight dose-dependent increase in the number of revertants over background). A slight increase over background was also observed in TA 1535 and TA 98 (with metabolic activation). Data from the other laboratory (SRI) showed a dose-dependent increase in the number of revertants with metabolic activation (either with 10% Rat Liver S9 or 10% Hamster Liver S9) for TA 100. A slight increase with metabolic activation was also observed for TA 1535. The results were negative (with or without metabolic activation) for TA 1537 or TA98. Quinto & Radman (1987) reported the results of an E. coli multitest to evaluate the genotoxic potential of bifunctional alkylating agents. The test allows measuring the mutagenic, recombinagenic and SOS inducing potencies of the tested chemicals. No metabolic activation was used. BCEE did not induce mutations in the MT 103, MT 119 and MT126 E. coli tester strains. This forward mutation test is considered by the authors to have low sensitivity towards weak mutagens (low rate of spontaneous mutations in the selected strains). In addition, the lack of recombigenic and SOS inducing potential may be due to low sensitivity of E. coli to BCEE or the need of metabolic activation for this compound. BCEE was recently evaluated in an Ames Test on S. typhimurium strains TA 1535, TA 100, TA, 1537, TA 98, and E. coli WP2 uvrA, performed according to OECD Guideline No. 471 under GLP. In the Initial Mutation Test a weak positive effect of the test item was obtained in E. coli WP2 uvrA strain with metabolic activation using the plate incorporation method (the observed revertant colony numbers were above the respective biological threshold value, slight dose response was also indicated). Although the effect was weak, it was reproducible in the Confirmatory Mutation Test using the same test conditions. Furthermore, a weak positive effect of the test item was also observed in the Confirmatory Mutation Test in S. typhimurium TA 1535 strain with metabolic activation using the pre-incubation method. No mutagenicity was observed with or without metabolic activation in S. typhimurium TA 98, TA 100 or TA 1537.

 

Publication

Bacterial test system

Metabolic activation

Result

Shirasu et al (1977)

Various strains of E. coli, B. subtilis and S. typhimurium

Not specified

Positive

Simmon et al. (1977)

S. typhimurium TA 100; Effect on other strains not detailed

Without activation

Strong in desiccator; Weak in suspension

Norpoth et al. (1986)

S. typhimurium TA 100

With activation

Weak

Mortelmans et al. (1986)

S. typhimurium TA 100

With activation

Positive

 

S. typhimurium TA 1535

With activation

Weak

 

S. typhimurium TA 98, TA 1537

With or without

Negative

Quinto & Radman (1987)

E. coli MT 103, MT 119, MT126

Without activation

Negative

OECD 471 test (2016)

S. typhimurium TA 1535

With activation

Weak

 

E. coli WP2 uvrA

With activation

Weak

 

S. typhimurium TA 98, TA 100, TA 1537

With or without

Negative

 

Data on the in vitro genetic toxicity to mammalian cells are limited to two recent guideline studies. In a study conducted in compliance with the OECD Guideline No. 487 under GLP, the potential of BCEE induce an increase in the frequency of micronucleated cells in the mouse cell line L5178 TK+/- was evaluated. Under the experimental conditions of the study, the test item did not induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells either in the absence or in the presence of a rat liver metabolizing system. In a study conducted in compliance with the OECD Guideline No. 490 under GLP, the induction of genetic mutations in cells of the mouse cell line L5178 TK+/- was tested. Statistically significant relevant increases were seen in the experiments with metabolic activation. However, the positive effect was only observed at a concentration with high degree of cytotoxicity. No similar effects were seen in the experiments without metabolic activation.

Conclusion:

Available data support the fact that BCEE induce a weak mutagenic effect in bacteria and in mammalian cells in the presence of metabolic activation. Very limited data support a mutagenic effect of the substance in the absence of metabolic activation, which suggest that mutagenicity is induced by a metabolite of BCEE. BCEE did not induce chromosome damage or damage to the cell division apparatus of mammalian cells in an OECD 490 test.

GENOTOXICITY IN VIVO:

 

3 studies have been performed to assess the potential of Bis(2-chloroethyl) ether (BCEE) to induce genetic toxicity in vivo.

 

In a Heritable Tanslocation Test (Jorgensen et al. 1977), male adult mice were exposed daily to BCEE at three dose levels for 8 weeks. Following exposure, the test animals were mated to two virgin females to produce an F1 generation. One hundred F1 males per treatment groups were bred to three virgin females each. The pregnant females were evaluated against predetermined selection criteria to identify suspect F1 males. These suspect F1 males were then rebred to three additional virgin females each. Presumptive sterile F1 males after two breedings were examined cytogenetically. All breeding data were evaluated and correlated with cytogenetic examinations. No heritable translocations occurred in the BCEE treated groups. The results of this study were published in a conference abstract. Accordingly, it was assigned a reliability score of 4.

 

In Gwinner et al. (1983), analysis of hydrolysates of liver DNA isolated after exposure (by inhalation) of male Sprague-Dawley rats to [14C]BCEE (amount not clearly specified) for 18h gave no indication for the formation of either 7-N-(2-oxoethyl)guanine, 1,N6-ethenodeoxyadenosine or 3,N4-ethenodeoxycytidine DNA adducts. In addition, under the conditions of this test, the substance did not induce the formation of hepatocellular ATPase-deficient foci.

 

In Foureman et al. (1994), a sex‐linked recessive lethal assay on male Drosophila melanogaster was conducted to assess the genotoxicity of BCEE. Adult Canton-S males were subjected to a 3 day feeding exposure. They were the mated to Bnsc females using a 2 to 3 day brooding pattern for a total of three broods spanning 7 days. An injection exposure was also performed. As in the feeding exposure, a 2 to 3 day brooding pattern for three broods was used. An RT test was performed on the most sensitive brood. BCEE did not induce recessive lethals in the feeding exposures. However, it was detected as mutagenic following injection exposures, as shown by a significant increase in recessive lethal mutations in comparison to control. However, the substance did not induce a significant number of reciprocal translocations. Based on this result, the substance is positive in Drosophila SLRL translocation assay.

Conclusion:

Based on the evaluation of available evidences, no data support the fact that Bis(2-chloroethyl) ether induce in vivo genetic toxicity in mammals. The substance was negative in a Heritable Tanslocation Test in mice (Jorgensen et al. 1977) and was not found to form DNA adducts or to induce hepatocellular ATPase-deficient foci in rats exposed to BCEE by inhalation for 18h (Gwinner et al. 1983). BCEE was found to induce recessive lethals in a sex‐linked recessive lethal (SLRL) assay following injection (irrelevant exposure route) but no effect was found in a feeding study. Based on current knowledge, no data support the fact tha BCEE induce genetic toxicity in vivo.

Justification for classification or non-classification

BCEE was not found to induce any chromosome damage, or damage to the cell division apparatus, in cultured mammalian somatic cells either in the absence or in the presence of a rat liver metabolizing system. BCEE induced a significant increase in genetic mutations in cells of the mouse cell line L5178 TK+/- but only at a concentration leading to a high degree of cytotoxicity. No experimental data support the fact that BCEE induce somatic cell mutagenicity in vivo in mammals.

According to the classification criteria of EC regulation 1272/2008, Bis(2-chloroethyl) ether should not be classified for germ cells mutagenicity.