Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In-vitro Ames test

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats

The test item had slight mutagenic activity in Salmonella typhimurium TA1535 strain under the test conditions of this study. No mutagenic activity was observed in the other four examined bacterium strains under the test conditions of the study.

In-vitro Mammalian Cell Chromosome Aberration V79 Chinese Hamster Lung fibroblasts

Assay 1 and 2, both with and without metabolic activation, showed a significant increase in aberration levels in at least one concentration when compared with the appropriate negative (solvent) control values. The effects were considered to be repeatable and treatment related.

In conclusion, the test substance (PREPOLYMER D) induced a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, the test substance (PREPOLYMER D) is considered clastogenic in this test system.

In-vitro Mouse Lymphoma L5178 TK +/- 3.7.2 C (mammalian cell gene mutation test)

In conclusion, mutagenic effect of the test substance (PREPOLYMER D) was observed in the presence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. No mutagenic effect of the test substance (PREPOLYMER D) was observed in the absence of metabolic activation system under the conditions of this study.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
08 August 2013 - 06 September 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine/tryptophan operon
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
other: see table 1
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
other: see table 1
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
Based on the solubility test, 100 mg/mL stock formulation was prepared in the vehicle, which was diluted in 6 steps by factors of 2, 2.5 and approximately √10. The revertant colony numbers and the inhibition of the background lawn of auxotrophic cells of two of the tester strains (Salmonella typhimurium TA98, TA100) were determined at the concentrations of 5000, 2500, 1000, 316, 100, 31.6 and 10 μg/plate of the test item. In the Preliminary Concentration Range Finding Test the plate incorporation method (5.6.4) was used.

Based on the results of the preliminary tests, 100 mg/mL stock solution was prepared from the test item with DMSO, which was diluted by serial dilutions in six steps to obtain seven dosing solutions for lower doses. The maximum test concentration was 5000 μg test item/plate.
Examined concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000, 1581, 500, 158.1, 50, 15.81 and 5 μg/plate.

Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: See table 2
Details on test system and experimental conditions:
METHOD OF APPLICATION: In the Range Finding Test and in the Initial Mutation Test, the plate incorporation method was used. In the Confirmatory Mutation Test, the pre-incubation method was used.

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): histidine/tryptophan

NUMBER OF REPLICATIONS:

NUMBER OF CELLS EVALUATED:

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Other:

OTHER:
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Species / strain:
other: S. typhimurium TA 98, TA 100, TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Conclusions:
Interpretation of results (migrated information):
other: Positive with and without metabolic activation in TA1535

The test item had slight mutagenic activity in Salmonella typhimurium TA1535 strain under the test conditions of this study. No mutagenic activity was observed in the other four examined bacterium strains under the test conditions of the study.
Executive summary:

The test item was tested for potential mutagenic activity using the Bacterial Reverse Mutation Assay.

The experiments were carried out using histidine-requiring auxotroph strains of Salmonella typhimurium (Salmonella typhimurium TA98, TA100, TA1535 and TA1537) and the tryptophan-requiring auxotroph strain of Escherichia coli (Escherichia coli WP2 uvrA) in the presence and absence of a post mitochondrial supernatant (S9 fraction) prepared from the livers of phenobarbital/β-naphthoflavone-induced rats.

The study included a Preliminary Compatibility Test, a Preliminary Range Finding Test (Informatory Toxicity Test), an Initial Mutation Test (Plate Incorporation Method) and a Confirmatory Mutation Test (Pre-Incubation Method).

Based on the available information, the test item was formulated in DMSO. Concentrations of 5000; 2500; 1000; 316; 100; 31.6 and 10 μg/plate were examined in the Range Finding Test. Based on the results of the Range Finding Test, the test item concentrations in the Initial Mutation Test and Confirmatory Mutation Test were 5000; 1581; 500; 158.1; 50; 15.81 and 5 μg/plate.

In the Initial Mutation Test (using the plate incorporation method) and Confirmatory Mutation Tests (using the pre-incubation method), a clear repeatable positive effect of the test item was obtained in Salmonella typhimurium TA1535 strain as a dose-related increase in the number of revertants occurred and the observed revertant colony numbers were above or near the respective biological threshold value.

No insolubility was detected in the main tests. Inhibitory, cytotoxicity of the test item was observed in the Confirmatory Mutation Test in all strains at 5000 μg/plate concentration without metabolic activation; and in Salmonella typhimurium TA98, TA100 and TA1537 strains at 5000 μg/plate concentration with metabolic activation.

The mean values of revertant colonies of the solvent control plates were within the historical control range, the reference mutagens showed the expected increase in the number of revertant colonies, the viability of the bacterial cells was checked by a plating experiment in each test. At least five analyzable concentrations were presented in all strains of the main tests. The tests were considered to be valid.

The reported data of this mutagenicity assay show (see Appendix 2 to 5) that under the experimental conditions applied the test item induced gene mutations by base pair changes or frameshifts in the genome of the strains used.

In conclusion, the test item had slight mutagenic activity in Salmonella typhimurium TA1535 strain under the test conditions of this study. No mutagenic activity was observed in the other four examined bacterium strains under the test conditions of the study.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 August 2013 - 22 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
Chromosomes
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
V79: Chinese hamster lung, male
ECACC: 86041102
Lot. Number: 10H016*
Supplier: ECACC (European Collection of Cells Cultures)
Morphology: Fibroblast
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
see tables below
Vehicle / solvent:
see tables below
Remarks:
see tables below
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium on plate

DURATION
- Exposure duration: 3 hours or 20 hours
- Expression time (cells in growth medium): Harvesting was performed after 20 hours (approximately 1.5 normal cell cycles) or 28 hours (approximately 2 normal cell cycles) from the beginning of treatment.
- Fixation time (start of exposure up to fixation or harvest of cells): 23/48 hours

SPINDLE INHIBITOR (cytogenetic assays): Colchicine
STAIN (for cytogenetic assays): 5 % Giemsa solution

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: At least one hundred metaphases* with 22±2 chromosomes (dicentric chromosomes were counted as two chromosomes) from each culture were examined for the presence or absence of chromosomal aberrations (approximately 1000x magnification), where possible. Where there were insufficient metaphases in one replicate, the total was made up to 200 cells examined per concentration using the other replicate. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately.
*Note: The examination of slides from a culture was halted when 15 or more metaphases with aberrations (excluding gaps) have been recorded for that culture.

DETERMINATION OF CYTOTOXICITY
- Method: For concurrent measurement of cytotoxicity an extra dish was plated for each sample and treated in the same manner. At the scheduled harvesting time, the number of surviving cells was determined using a haemocytometer. Results are expressed compared to the negative (solvent) control as % relative survival.
Evaluation criteria:
The assay will be considered valid, if the following criteria are met:
- The negative (solvent) control data are within the laboratory’s normal range for the spontaneous aberration frequency.
- The positive controls induce increases in the aberration frequency, which are significant.
The test item will be considered to have shown clastogenic activity in this study if all of the following criteria are met:
- Increases in the frequency of metaphases with aberrant chromosomes are observed at one or more test concentrations (only data without gaps will be considered).
- The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
- The increases are statistically significant.
- The increases are not associated with large changes in pH or osmolarity of the treated cultures.

The historical control data for this laboratory will also be considered in the evaluation. Evidence of a dose-response relationship will be considered to support the conclusion.
The test item will be concluded to have given a negative response if no reproducible, statistically significant increases in the aberration frequency are observed.
Statistics:
For statistical analysis, Fisher’s exact test will be used. The parameter evaluated for statistical analysis will be the number of cells with one or more chromosomal aberrations (excluding gaps).
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
see tables below
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Summary table of Chromosome Aberration Assay 1 without metabolic activation

Concentration
(
μg/mL)

Time of Treatment / Sampling

Relative Survival#
(%)

Insolubility##

Mean % aberrant cells###

PREPOLYMER D without metabolic activation (-S9)

Negative (solvent) control

3h / 20h

100

+a

5.5

800 µg/mL

3h / 20h

33

NE

600 µg/mL

3h / 20h

39

13.7**

400 µg/mL

3h / 20h

58

6.5

200 µg/mL

3h / 20h

87

1.0

100 µg/mL

3h / 20h

92

NE

50 µg/mL

3h / 20h

95

NE

Positive control

3h / 20h

72

13.3**

Negative (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 1 µL/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

a: minimal amount of insolubility

 

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Summary table of Chromosome Aberration Assay 1 with metabolic activation

Concentration
(
μg/mL)

Time of Treatment / Sampling

Relative Survival#
(%)

Insolubility##

Mean % aberrant cells###

PREPOLYMER D with metabolic activation (+S9)

Negative (solvent) control

3h / 20h

100

3.5

80 µg/mL

3h / 20h

26

NE

60 µg/mL

3h / 20h

38

NE

40 µg/mL

3h / 20h

37

NE

30 µg/mL

3h / 20h

49

43.5***

20 µg/mL

3h / 20h

76

43.5***

10 µg/mL

3h / 20h

95

6.0

5 µg/mL

3h / 20h

97

NE

Positive control

3h / 20h

68

96.8***

Negative (solvent) control: 1% (v/v) DMSO

Positive control (+S9): Cyclophosphamide, 6 µg/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

 

Summary table ofChromosome Aberration Assay 2 without metabolic activation

Concentration
(
μg/mL)

Time of Treatment / Sampling

Relative Survival#
(%)

Insolubility##

Mean % aberrant cells###

PREPOLYMER D without metabolic activation (-S9)

Negative (solvent) control

20h / 28h

100

1.0

600 µg/mL

20h / 28h

28

NE

500 µg/mL

20h / 28h

37

NE

400 µg/mL

20h / 28h

45

6.5**

300 µg/mL

20h / 28h

54

NE

200 µg/mL

20h / 28h

74

3.0

100 µg/mL

20h / 28h

93

1.0

50 µg/mL

20h / 28h

88

NE

Positive control

20h / 28h

51

29.4***

Negative (solvent) control: 1% (v/v) DMSO

Positive control (-S9): Ethyl methanesulfonate, 0.4 µL/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

 

 

**: p<0.01 comparing numbers of aberrant cells excluding gaps with corresponding negative control

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Summary table ofChromosome Aberration Assay 2 with metabolic activation

Concentration
(
μg/mL)

Time of Treatment / Sampling

Relative Survival#
(%)

Insolubility##

Mean % aberrant cells###

PREPOLYMER D with metabolic activation (+S9)

Negative (solvent) control

3h / 28h

100

3.5

60 µg/mL

3h / 28h

18

NE

40 µg/mL

3h / 28h

24

NE

30 µg/mL

3h / 28h

48

41.7***

20 µg/mL

3h / 28h

63

6.5

10 µg/mL

3h / 28h

77

6.0

5 µg/mL

3h / 28h

87

NE

2.5 µg/mL

3h / 28h

94

NE

Positive control

3h / 28h

73

34.1***

Negative (solvent) control: 1% (v/v) DMSO

Positive control (+S9): Cyclophosphamide, 6 µg/mL

NE: not evaluated

#: compared to the negative (solvent) control

##: in the final treatment medium at the end of the treatment

###: excluding gaps

 

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

 

 

 

***: p<0.001 comparing numbers of aberrant cells excluding gaps with corresponding negative control

Conclusions:
Interpretation of results (migrated information):
positive

The test item PREPOLYMER D was tested for potential clastogenic activity using the Chromosome Aberration Assay. The study included two Concentration Selection Cytotoxicity Assays and two Chromosome Aberration Assays.

The performed experiments were considered to be valid and to reflect the real potential of the test item to cause structural chromosomal aberrations in the cultured V79 Chinese hamster cells used in this study.

Treatment with the test item resulted in a statistically and biologically significant, repeatable, dose-dependent increase in the frequency of the cells with structural chromosome aberrations without gaps either in the presence or absence of a metabolic activation system which was a cofactor-supplemented post-mitochondrial S9 fraction prepared from the livers of phenobarbital/alpha-naphthoflavone induced rats.

In conclusion, PREPOLYMER D induced a significant level of chromosome aberrations in the performed experiments with or without metabolic activation. Therefore, PREPOLYMER D is considered clastogenic in this test system.

Executive summary:

PREPOLYMER D was testedin vitroin a Chromosome Aberration Assay using Chinese hamster V79 lung cells. The test item was formulated in DMSO and it was examined up to cytotoxic concentrations according to the OECD guideline recommendations. In the performed independent Chromosome Aberration Assays using duplicate cultures at least 200 well-spread metaphase cells (or until a clear positive response was detected) were analysed for each test item treated, negative (solvent) and positive control sample.

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 3-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 20 hours after the beginning of the treatment in both cases. The examined concentrations of the test itemwere 800, 600, 400, 200, 100 and 50 µg/mL without metabolic activation; and80, 60, 40, 30, 20, 10 and 5 µg/mL with metabolic activation.

In Assay 1, insolubility was detected at the end of the treatment period in the final treatment medium at the highest concentration (800 µg/mL) without metabolic activation. There were no large changes in the pH and osmolality. Marked cytotoxicity was observed at800 and 600 µg/mL concentrations without metabolic activation (relative survival values were 33 and 39%, respectively); and at80, 60, 40 and 30 µg/mL concentrations with metabolic activation (relative survival values were 26, 38, 37 and 49%, respectively). Therefore, concentrations of 600, 400 and 200 µg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation; and concentrations of30, 20 and 10 µg/mL (a total of three) were chosen for evaluation with metabolic activation.

In Chromosome Aberration Assay3-hour treatment with metabolic activation (in the presence of S9-mix) and a 20-hour treatment without metabolic activation (in the absence of S9-mix) were performed. Sampling was performed 28 hours after the beginning of the treatment in both cases. The examined concentrations of the test itemwere 600, 500, 400, 300, 200, 100 and 50 µg/mLwithout metabolic activation; and 60, 40, 30, 20, 10, 5 and 2.5 µg/mL with metabolic activation.

In Assay 2, no insolubility was detected at the end of the treatment period in the final treatment medium. No large changes in the pH and osmolality were observed. Marked cytotoxicity was observed in this assay at 600, 500 and 400 µg/mL concentrations without metabolic activation (relative survival values of 28, 37 and 45%, respectively); and at60, 40 and 30 µg/mL concentrations with metabolic activation (relative survival values were 18, 24 and 48%, respectively). Therefore, concentrations of 400, 200 and 100 µg/mL (a total of three) were chosen for evaluation in the experiment without metabolic activation; and concentrations of 30, 20 and 10 µg/mL (a total of three) were chosen for evaluation in the experiment with metabolic activation.

In Assay 1 and 2 without metabolic activation, a significant increase in aberrations was observed at the highest evaluated concentration (600 and 400 µg/mL, respectively)and a dose response was observed across the 3 concentrations analysed. In Assay 1 and 2 with metabolic activation, a highly significant increase in aberrations was observed at the highest evaluated concentration (30 µg/mL), and additionally in Assay 1, the next highest concentration analysed (20 µg/mL)also showed a highly significant increase. Thus Assay 1 and 2, both with and without metabolic activation, showed a significant increase in aberration levels in at least one concentration when compared with the appropriate negative (solvent) control values. The effects were considered to be repeatable and treatment related.

 

The occurrence of polyploid and endoreduplicated metaphases was recorded in the main tests. Polyploid metaphases (1-5) were found in some cases in the negative (solvent) control or test item treated samples in Assay 1. No endoreduplicated metaphases were found in any of the samples except of the 400 µg/mLsample in Assay 2 without metabolic activation and the 20 µg/mLsample in Assay 2 with metabolic activation (one and three endoreduplicated metaphases were detected in these samples, respectively).

 

The negative (solvent) control data were within the acceptable range for the spontaneous aberration frequency, the positive control substances caused a statistically significant increase in the number of structural aberrations excluding gaps in the experiments with or without metabolic activation demonstrating the sensitivity of the test system. The evaluated concentration range was considered to be adequate; three test item treated concentrations were evaluated in each assay. The tests were considered to be valid.

 

In conclusion, PREPOLYMER D induced a significant level of chromosome aberrations in the performed experiments with or without metabolic activation.Therefore,PREPOLYMER D is considered clastogenic in this test system.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
key study
Study period:
05 August 2013 - 21 October 2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study was performed according to OECD guidelines and GLP.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
tk locus
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
Name of Cell Line: L5178Y TK+/- 3.7.2 C mouse lymphoma
Product No.: CRL-9518
Lot. No.: 1661603
Supplier: American Type Culture Collection (Manassas,Virginia, USA)
Date of arrival: 22 January 2004
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
Assay 1, 3-hour treatment with metabolic activation: 20; 18; 17; 16; 15; 14; 13; 12; 11; 10; 5; 2.5 and 1.25 µg/mL
Assay 1, 3-hour treatment without metabolic activation: 700; 600; 500; 400; 300; 200; 100; 50; 25; 12.5 and 6.25 µg/mL
Assay 2, 3-hour treatment with metabolic activation: 20; 18; 17; 16; 15; 14; 13; 12; 11; 10; 5; 2.5 and 1.25 µg/mL
Assay 2, 24-hour treatment without metabolic activation: 300; 250; 225; 200; 175; 150; 125; 100; 50; 25; 12.5 and 6.25 µg/mL µg/mL
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium on a plate

DURATION
- Exposure duration: 3 or 24 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 14 days
- Fixation time (start of exposure up to fixation or harvest of cells): 18 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: duplicate

NUMBER OF CELLS EVALUATED: small and large colonies were counted

DETERMINATION OF CYTOTOXICITY
- Method: relative survival; colony survival of the control is compared with the survival of the treated cells.

Evaluation criteria:
Valid if:
1. The mutant frequency in the negative (solvent) control cultures fall within the normal range (50-170 mutants per 106 viable cells).
2. The positive control chemicals induce a statistically significant increase in the mutant frequency.
3. The plating efficiency (PEviability) of the negative (solvent) controls is within the range of 65% to 120% at the end of the expression period.
4. At least four test concentrations are present, where the highest concentration produces approximately 80-90% toxicity (measured by %RS or RTG), results in precipitation, or it is 5 mg/mL, 5 μL/mL or 0.01 M (whichever is the lowest), or it is the highest practical concentration.

Mutagenic if:
1. The assay is valid;
2. statistically significant (p < 0.05) and biologically relevant increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations;
3. the increases in mutation frequency are reproducible between replicate cultures and/or between tests (under the same treatment conditions);
4. there is a significant concentration-relationship as indicated by the linear trend analysis (p < 0.05);
5. the mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative (solvent) control value.
Results, which only partially satisfy the acceptance and evaluation criteria, will be evaluated on a case-by-case basis. Similarly, positive responses seen only at high levels of cytotoxicity will require careful interpretation when assessing their biological significance. Caution will be exercised with positive results obtained at levels of survival (or RTG) lower than 10%. Equivocal results need to be verified in a follow-up experiment. Modification of test conditions in this experiment will be considered.
Statistics:
Statistical significance of mutant frequencies (total wells with clones) will be carried out using Microsoft Excel 2000 software. The control log mutant frequency (LMF) will be compared to the LMF from each treatment dose, based on Dunnett's test for multiple comparisons, and secondly the data will be checked for a linear trend in mutant frequency with treatment dose using weighted regression. The test for linear trend is one-tailed, therefore negative trend will not be considered significant. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
with metabolic activation
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Cytotoxicity and insolubility were detected in the preliminary experiments
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
see tables below

Mutagenicity Results of Assay 1 (I.)

(3 hour treatment in the presence of S9-mix)

 

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)¨

Mutation frequency

+

3

20 µg/mL

ND

ND

ND

ND

ND

18 µg/mL

ND

ND

ND

ND

ND

17 µg/mL

ND

ND

ND

ND

ND

16 μg/mL

NE

NE

NE

NE

NE

15 µg/mL

NE

NE

NE

NE

NE

14 µg/mL

NE

NE

NE

NE

NE

13 µg/mL

371/768

163/768

234/768

56.18

545.1*

12 µg/mL

320/768

200/768

248/768

64.61

606.5*

11 µg/mL

456/768

122/768

190/768

36.87

404.6*

10 µg/mL

513/768

120/768

135/768

22.42

302.3*

5 µg/mL

661/768

57/768

50/768

5.96

183.8*

2.5 µg/mL

674/768

56/768

38/768

0.62

79.4

1.25 µg/mL

679/768

39/768

50/768

0.30

84.7

Negative control

662/768

70/768

36/768

--

98.5

Untreated control

672/768

53/768

43/768

--

89.2

Positive control
(CP: 4 μg/mL)

226/768

216/768

326/768

¨¨
2.19E-12

1411.8*

 

In linear trend analysisβ2/var (β) = 115.30,significant.

 

* = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05.

 

Dn= Difference of log mutant frequency of dose “n” and that of the negative (solvent) control

var(Dn) = variance of Dn                             β = slope of the curve                    var(β) = variance of the slope

 

+ = in the presence of S9-mix

Negative (solvent) control = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

ND = No data (No cells were plated for colony growing due to the excessive cytotoxicity observed in the expression period.)

NE = Not evaluated due to the marked cytotoxicity

 

Note: Mutation frequency refers to 106viable cells

Mutagenicity Results of Assay 1 (II.)

(3 hour treatment in the absence of S9-mix)

 

S9 mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)¨

Mutation frequency

-

3

700 µg/mL

NE

NE

NE

NE

NE

600 µg/mL

618/768

72/768

78/768

6.86

160.5*

500 µg/mL

610/768

93/768

65/768

6.46

156.2*

400 μg/mL

614/768

83/768

71/768

4.06

135.1

300 µg/mL

670/768

62/768

36/768

0.22

88.6

200 µg/mL

685/768

43/768

40/768

0.08

84.5

100 µg/mL

676/768

60/768

32/768

0.44

94.2

50 µg/mL

676/768

52/768

40/768

0.68

99.0

25 µg/mL

689/768

44/768

35/768

0.08

84.3

12.5 µg/mL

690/768

50/768

28/768

n.d.

80.2

6.25 µg/mL

699/768

39/768

30/768

n.d.

65.7

Negative control

686/768

54/768

28/768

--

77.1

Untreated control

679/768

57/768

32/768

--

89.7

Positive control
(NQO: 0.15 μg/mL)

402/768

235/768

131/768

¨¨
1.04E-10

673.6*

 

In linear trend analysisβ2/var (β) = 10.87,significant.

 

* = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.66(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05.

 

Dn= Difference of log mutant frequency of dose “n” and that of the negative (solvent) control

var(Dn) = variance of Dn      β = slope of the curve        var(β) = variance of the slope

 

- = in the absence of S9-mix

Negative (solvent) control = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

NE = Not evaluated due to the marked cytotoxicity

n.d. = no data (Concentration was not used for linear trend analysis)

 

Note: Mutation frequency refers to 106viable cells

Mutagenicity Results of Assay 2 (I.)

(3 hour treatment in the presence of S9-mix)

 

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)¨

Mutation frequency

+

3

20 µg/mL

ND

ND

ND

ND

ND

18 µg/mL

ND

ND

ND

ND

ND

17 µg/mL

ND

ND

ND

ND

ND

16 μg/mL

NE

NE

NE

NE

NE

15 µg/mL

NE

NE

NE

NE

NE

14 µg/mL

NE

NE

NE

NE

NE

13 µg/mL

282/768

190/768

296/768

69.76

694.0*

12 µg/mL

366/768

151/768

251/768

46.75

498.9*

11 µg/mL

441/768

129/768

198/768

36.35

424.5*

10 µg/mL

462/768

112/768

194/768

25.66

339.7*

5 µg/mL

625/768

58/768

85/768

7.48

205.6*

2.5 µg/mL

670/768

49/768

49/768

0.07

96.6

1.25 µg/mL

679/768

47/768

42/768

0.49

85.3

Negative control

670/768

46/768

52/768

--

103.7

Untreated control

674/768

47/768

47/768

--

87.9

Positive control
(CP: 4 μg/mL)

218/768

204/768

346/768

¨¨
8.40E-16

1379.5*

 

In linear trend analysisβ2/var (β) = 126.91,significant.

 

* = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.48(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

 

Dn= Difference of log mutant frequency of dose “n” and that of the negative (solvent) control

var(Dn) = variance of Dn      β = slope of the curve        var(β) = variance of the slope

 

+ = in the presence of S9-mix

Negative (solvent) control = DMSO

DMSO = Dimethyl sulfoxide

CP = Cyclophosphamide

ND = No data (No cells were plated for colony growing due to the excessive cytotoxicity observed in the expression period.)

NE = Not evaluated due to the marked cytotoxicity

 

Note: Mutation frequency refers to 106viable cells

Mutagenicity Results of Assay 2 (II.)

(24 hour treatment in the absence of S9-mix)

 

S9-mix

Treatment period (hours)

Test item or control concentration

Number of empty wells/total number of wells

Number of large colonies/total number of wells

Number of small colonies/ total number of wells

Dn2/var(Dn)¨

Mutation frequency

-

24

300 µg/mL

ND

ND

ND

ND

ND

250 µg/mL

ND

ND

ND

ND

ND

225 µg/mL

648/768

72/768

48/768

0.09

104.1

200 µg/mL

650/768

76/768

42/768

0.44

113.9

175 µg/mL

653/768

56/768

59/768

0.80

120.7

150 µg/mL

667/768

47/768

54/768

0.04

101.9

125µg/mL

667/768

60/768

41/768

0.02

100.5

100 μg/mL

654/768

55/768

59/768

0.11

105.1

50 µg/mL

666/768

55/768

47/768

<0.01

98.0

25 µg/mL

660/768

48/768

60/768

0.12

105.7

12.5 µg/mL

660/768

51/768

57/768

 

89.4

6.25 µg/mL

667/768

46/768

55/768

 

100.5

Negative control

655/768

63/768

50/768

--

96.8

Untreated control

662/768

47/768

59/768

--

97.8

Positive control
(NQO: 0.1 μg/mL)

293/768

291/768

184/768

¨¨
5.38E-13

808.6*

 

In linear trend analysisβ2/var (β) =0.41,notsignificant.

 

* = Statistically significant.

¨= Evaluated by Dunnett’s test for multiple comparisons. Significant if Dn2/var(Dn) >5.66(at p<0.05).

¨¨= Evaluated by T-test for independent samples. Significant at p<0.05 compared to the untreated control.

 

Dn= Difference of log mutant frequency of dose “n” and that of the negative (solvent) control

var(Dn) = variance of Dn      β = slope of the curve        var(β) = variance of the slope

 

- = in the absence of S9-mix

Negative (solvent) control = DMSO

DMSO = Dimethyl sulfoxide

NQO = 4-Nitroquinoline-N-oxide

ND = No data (No cells were plated for colony growing due to the excessive cytotoxicity observed in the expression period.)

n.d. = no data (Concentration was not used for linear trend analysis)

 

Note: Mutation frequency refers to 106viable cells

Conclusions:
Interpretation of results (migrated information):
positive

The Mouse Lymphoma Assay with PREPOLYMER D on L5178Y TK +/- 3.7.2 C cells was considered to be valid and to reflect the real potential of the test item to cause mutations in the cultured mouse cells used in this study.

Treatment with the test item did result in a biologically relevant and statistically significant increase in mutation frequency in the presence of a rat metabolic activation system (S9 fraction) in the Mouse Lymphoma Assay. Therefore, mutagenic activity of the test item could be observed in the performed experiments with metabolic activation. However, no similar effect was observed in the performed experiments without metabolic activation.

In conclusion, mutagenic effect of PREPOLYMER D was observed in the presence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. No mutagenic effect of PREPOLYMER D was observed in the absence metabolic activation system under the conditions of this study.

Executive summary:

Anin vitromammalian cell assay was performed in mouse lymphoma L5178Y TK+/-3.7.2 C cells at the tk locus to test the potential ofPREPOLYMER Dto cause gene mutation and/or chromosome damage. Treatment was performed for 3 hours with and without metabolic activation (±S9 mix) and for 24 hours without metabolic activation (-S9 mix).

 

Dimethyl sulfoxidewas used as solvent of the test item in this study. The test item was examined up to 5000 µg/mL (the recommended maximum concentration) in the Preliminary Toxicity Test. Based on the results of the preliminary experiment, the following test item concentrations were examined in the mutation assays:

Assay 1, 3-hour treatment with metabolic activation:20; 18; 17; 16; 15; 14; 13; 12; 11; 10; 5; 2.5 and 1.25 µg/mL

Assay 1, 3-hour treatment without metabolic activation:700; 600; 500; 400; 300; 200; 100; 50; 25; 12.5 and 6.25 µg/mL

Assay 2, 3-hour treatment with metabolic activation:20; 18; 17; 16; 15; 14; 13; 12; 11; 10; 5; 2.5 and 1.25 µg/mL

Assay 2, 24-hour treatment without metabolic activation:300; 250; 225; 200; 175; 150; 125; 100; 50; 25; 12.5 and 6.25 µg/mL µg/mL

 

In Assays 1 and 2, no insolubility was detected in the final treatment medium at the end of the treatment. There were no large changes in pH and osmolality after treatment in any cases.

In Assay 1,following a3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 20, 18 and 17 µg/mL concentrations, cells of these samples did not survive the expression period. Marked cytotoxicity was observed at 16, 15 and 14 µg/mL concentrations (relative total growth of 1, 3 and 4%, respectively); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of the following concentration of 13 µg/mL (relative total growth of 12%) and the next six concentrations (a total of seven concentrations). Biologically relevant and statistically significant increase in the mutation frequency was observed at 13, 12 11 and 10 µg/mLconcentrations. Statistically significant increase in the mutation frequency was also detected at 5 µg/mLconcentration, althoughthe observed mutation frequency value was below the limit of the biological relevance.Dose-response to the treatment was also indicated by the linear trend analysis.Overall this experiment was considered to show a clear genotoxic response.

In Assay 1, following a 3-hour treatment without metabolic activation, excessive cytotoxicity of the test item was observed in one replicate of the highest concentration (700 µg/mL), cells of this sample did not survive the expression period. An evaluation was made using data of following concentration of 600 µg/mL (relative total growth of 13%) and the next seven concentrations (a total of eight concentrations). Statisticallysignificant increases in the mutation frequency were observed at600 and 500 µg/mLconcentrations.

However, the difference between the mutation frequency of the test item treated sample and the corresponding vehicle control value did not exceed the global evaluation factor, thus they were considered as biologically non relevant increases. Slight doseresponse to the treatment was also indicated by the linear trend analysis but based on the individual mutation frequency values this fact had no relevance. This experiment was considered as being negative.

In Assay 2,following a3-hour treatment with metabolic activation, excessive cytotoxicity of the test item was observed at 20, 18 and 17 µg/mL concentrations, cells of these samples did not survive the expression period. Marked cytotoxicity was observed at 16, 15 and 14 µg/mL concentrations (relative total growth of 3, 4 and 6%, respectively); therefore, these concentrations were excluded from the evaluation. An evaluation was made using data of the following concentration of 13 µg/mL (relative total growth of 14%) and the next six concentrations (a total of seven concentrations). Biologically relevant and statistically significant increase in the mutation frequency was observed at 13, 12 11 and 10 µg/mLconcentrations. Statistically significant increase in the mutation frequency was also detected at 5 µg/mLconcentration, althoughthe observed mutation frequency value was below the limit of the biological relevance.Dose-response to the treatment was also indicated by the linear trend analysis. This experiment was considered to confirm the positive effect of the test item observed in the first assay under identical experimental conditions.

In Assay 2,following a 24-hour treatment without metabolic activation), excessive cytotoxicity of the test item was observed at the two highest examined concentrations (300 and 250 µg/mL concentrations), cells of these samples did not survive the expression period. An evaluation was made using data of following concentration of 225 µg/mL (relative total growth of 31%) and the next seven concentrations (a total of eight concentrations). Nobiologically relevant or statistically significant increase in the mutation frequency was observed at the evaluated concentrations. No significantdose-response to the treatment was indicated by the linear trend analysis. This experiment was considered as being negative (confirming the results of the experiment without metabolic activation in the first assay).

The experiments were performed using appropriate untreated, negative (solvent) and positive control samples in all cases. The spontaneous mutation frequency of the negative (solvent) controls was in the recommended range in each test. The positive controls gave the anticipated increases in mutation frequency over the negative controls. The plating efficiencies for the negative (solvent) 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 (concentrations were tested up to the cytotoxic range in each test). The number of test concentrations met the acceptance criteria. Therefore, the study was considered to be valid.

 

In conclusion, mutagenic effect of PREPOLYMER D was observed in the presence of metabolic activation system under the conditions of this Mouse Lymphoma Assay. No mutagenic effect of PREPOLYMER D was observed in the absence metabolic activation system under the conditions of this study.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

In-vivo Micronucleus test in rats

Dose-related, decreases in the PCE/NCE ratio were observed in the 200 mg/kg test item dose groups when compared to the vehicle control group. These decreases, together with the observation of clinical signs, which include two deaths at the 200mg/kg top dose, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group (cyclophosphamide) showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusion

In the Micronucleus Test the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non genotoxic under the conditions of the test.

 

In-vivo Comet test in rats

In the Comet Assay, the positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in all tissues analyzed indicating that the test method itself was operating as expected. The vehicle control groups all had percentage tail intensity and median percentage tail intensity values within the expected range.

There was no statistically significant marked increase in percentage tail intensity or median percentage tail intensity for any of the test item dose groups in the liver, duodenum or glandular stomach when compared to the vehicle control, confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.

Conclusion

In the Comet Test, the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 did not induce any increases in the percentage tail intensity values in the liver, duodenum or glandular stomach. The test item was considered to be non-genotoxic to the liver, duodenum or glandular stomachin vivounder the conditions of the test.

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 23 November 2016 Experimental completion date 02 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian germ cell cytogenetic assay
Specific details on test material used for the study:
Identification: Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4
Physical state/Appearance: Extremely pale yellow liquid
Batch: 1603101
Purity: 100%
Expiry Date: 19 June 2017
Storage Conditions: Room temperature in the dark
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 186.8 to 216.1 g, and were approximately 7 to 12 weeks old. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card.

The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.

The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
A range-finding test was performed to find suitable dose levels of the test item following a triple oral administration at approximately 0, 24 and 44 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. Based on toxicology information supplied by the Sponsor the initial dose level was limited to 200 mg/kg. Additionally bone marrow slides were prepared and scored for PCE/NCE ratio as an indicator of toxicity to bone marrow.
Duration of treatment / exposure:
44 hours
Frequency of treatment:
Daily
Post exposure period:
Animals were observed approximately 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 18-24
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 25-31
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Remarks:
Animal numbers 32-38
No. of animals per sex per dose:
7 males rats per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
Dose level Concentration Dose volume Kill time Animal numbers
(mg/kg) (mg/mL) (mL/kg) (hours after
initial dosing)
Positive control
(Cyclophosphamide) 25 2.5 10 24‡ 8 - 12

Positive Control
(N-Nitroso-N-methylurea) 25 2.5 10 24‡ 13 -17
Tissues and cell types examined:
Bone marrow
Details of tissue and slide preparation:
Micronucleus Slide Preparation
A 15 mL centrifuge tube was filled with approximately 2-3 mL of foetal bovine serum (FBS) and, using a suitable hypodermic needle mounted on a 2 mL syringe, a portion of the serum was withdrawn from the centrifuge tube into the syringe. The needle was pushed a few millimeters into the bone marrow canal and the marrow from 1 femur was flushed into the serum in the centrifuge tube.
Approximately 1 mL of the bone marrow suspension was transferred to a microfuge tube and centrifuged at approximately 200 g and the supernatant was removed using a Pasteur pipette leaving sufficient serum to just cover the top of the cell pellet. The cells were mixed to give a homogenous suspension. A small drop of each bone marrow suspension was transferred to the frosted end of four glass microscope slides and smears were made and air-dried. Two slides were stained for scoring of micronuclei and two reserved as spares.
The smears were fixed in absolute methanol for 20 minutes and stained with May Grünwald stain and then counterstained with Giemsa stain and subsequently rinsed with pH 6.8 buffer solution for 3 minutes and then rinsed in distilled water. After air drying the smears had a cover slip applied using a mounting medium.
Evaluation criteria:
Micronucleus Scoring
The stained slides were "blind" coded and examined using light microscopy at x 1000 magnification. 4000 polychromatic erythrocytes per animal were scored for the incidence of micronuclei.
In addition, the number of normochromatic erythrocytes within a total of at least 1000 erythrocytes were counted and these cells were also scored for the incidence of micronuclei. The ratio of polychromatic/normochromatic erythrocytes (PCE/NCE), group mean values and standard deviations were calculated.
Statistics:
For the Micronucleus Test statistical analysis of MN/4000 PCE and PCE/NCE ratio data was performed where necessary using a t test following a  (x+1) transformation of the data.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Please see "Any other information on results incl. tables" for a summary of the group mean data for the Micronucleus Test

                         Micronucleus Test – Summary of Group Mean Data

TREATMENT GROUP

NUMBER OF PCE WITH MICRONUCLEI PER 4000 PCE

PCE/NCE RATIO

GROUP MEAN

SD

GROUP MEAN

SD

1.  Vehicle Control

2.4

1.6

1.34

0.49

      48-hour sampling time

2.   Positive Control

34.0*

22.2

0.67**

0.10

      24-hour sampling time

3.   List No 940-783

3.6

2.1

0.98

0.54

      200 mg/kg

      48-hour sampling time

4.   List No 940-783

3.6

2.4

1.36

0.72

      100 mg/kg

      48-hour sampling time

5.   List No 940-783

2.7

1.3

1.29

0.74

      50 mg/kg

      48-hour sampling time

PCENCESD******



PCE      =Polychromatic erythrocytes

NCE     =Normochromatic erythrocytes

SD       =Standard deviation

*          = P < 0.05

**        =P <0.01

 

Range-Finding Toxicity Test

The mortality data are summarized as follows:

Dose Level
(mg/kg)

Sex

Number of Animals Treated

Route

Deaths on Day

Total Deaths

0

1

2

200

Male

2

oral

0

0

0

0/2

240

Male

2

oral

0

0

2

2/2

200

Male

2

oral

0

0

0

0/2

 

The following clinical signs were observed in the animals dosed with the test item at 200 mg/kg via the oral route: hunched posture, lethargy, ataxia, splayed gait and diuresis. The dose level was increased to 240 mg/kg and the following clinical signs were observed, hunched posture and lethargy. Both animals were found dead at the 48-hour observation time, indicating that lethal toxicity occurred after the third administration of test item at 240 mg/kg. A confirmatory range-finding test was performed on two animals at 200 mg/kg and the observations were considered to be relatively moderate (hunched posture and ataxia), confirming this was an acceptable maximum dose level to use in the main test.

Bone marrow slides were prepared for quantitative assessment from the three range-finding experiments. The slides were scored per 1000 cells and the data is presented below.  

Animal Code

Dose level (mg/kg)

Number of polychromatic erythrocytes

Number of Normochromatic erythrocytes

PCE/NCE ratio

1-0

200

460

540

0.85

1-1

351

649

0.54

2-0

240

290

710

0.41

2-1

254

746

0.34

3-0

200

417

583

0.72

3-1

348

652

0.53

 

The quantitative assessment revealed that bone marrow toxicity had occurred at both dose levels. The bone marrow toxicity that occurred at 200 mg/kg was considered to be moderate whilst at 240mg/kg was considered to be too marked.

Based on the above data the maximum tolerated dose (MTD) of the test item, 200 mg/kg, was selected for use in the main test, with 100 and 50 mg/kg as the lower dose levels. 

Micronucleus Main Test

Mortality Data and Clinical Observations

One premature death was observed in the 200 mg/kg (MTD) dose group. A second animal in the 200 mg/kg group died just prior to sampling and although it was processed for comet and micronucleus evaluation it was subsequently decided that any data would be compromised and it was therefore excluded from analysis. 

 

The following clinical signs were observed in animals dosed with test item at 200 mg/kg: hunched posture, ataxia, pilo erection, prostration, increased respiration, gasping respiration, labored respiration, ptosis and splayed gait. The more severe observations were most apparent at the 48 hour observation, approximately 4-hours after the last dose.

 Evaluation of Micronucleus Slides

Dose-related, decreases in the PCE/NCE ratio were observed in the 200 mg/kg test item dose groups when compared to the vehicle control group. These decreases, together with the observation of clinical signs, were taken to indicate that systemic absorption had occurred and exposure to the target tissue had been achieved.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.

Conclusions:
In the Micronucleus element of the test, the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non-genotoxic under the conditions of the test.
Executive summary:

Summary

Introduction

The Micronucleus Assay is a standard follow-upin vivo study. 

The micronucleus test is a mammalianin vivotest which detects chemical-induced damage to the chromosomes or the mitotic apparatus. Polychromatic erythrocytes in the bone marrow of rats are used in this study. When the erythroblast develops into an erythrocyte, the main nucleus is extruded and may leave micronuclei in the cytoplasm. Observation of micronuclei is facilitated in these cells because they lack a nucleus. As micronuclei occur under normal conditions, this study is based on detecting an increase in the frequency of micronucleated polychromatic erythrocytes in the bone marrow of treated animals. 

Bone Marrow was sampled for micronucleus slides.

Methods

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 200 mg/kg with 100 mg/kg and 50 mg/kg as the lower dose levels. Groups, each of seven rats, were dosed three times at 0, 24 and 44 hours with the test item. Animals were killed 4 hours after the third dose administration. 

In addition, three further groups of rats were included in the study; one group (seven rats) was dosed via the oral route with the vehicle alone (arachis oil) at 0, 24 and 44 hours, and a second group (five rats) was dosed orally with cyclophosphamide (CP), once only, at the 24-hour time-point, to act as a positive control for the micronucleus element of the study. The third group of rats was dosed with N-Nitroso-N-methylurea (NMU) at 24 and 44-hours to act as the positive control for the Comet element of the study. 

 

The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 48 hours after the start of the test. The glandular stomach, duodenum, liver, bone marrow and testes were processed for comet slides. Samples of liver, duodenum, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. Comet slides were not prepared for the positive control animals dosed with CP.

Bone marrow was then extracted from the second femur, and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Micronucleus slides were not prepared for the positive control animals dosed with MNU.

Results

One premature death was observed in the 200 mg/kg (MTD) dose group. A second animal in this 200 mg/kg group died just prior to sampling and although it was processed for comet and micronucleus evaluation it was subsequently decided that any data may be compromised and this animal was therefore excluded from the final analysis. The following clinical signs were observed in animals dosed with test item at 200 mg/kg: hunched posture, ataxia, pilo erection, prostration, increased respiration, gasping respiration, labored respiration, ptosis and splayed gait. The more severe observations were most apparent at the 48 hour observation, approximately 4-hours after the last dose.

 

In the micronucleus test, dose-related, decreases in the PCE/NCE ratio were observed in the 200 mg/kg test item dose group when compared to the vehicle control group. Although not statistically significant, these decreases, together with the observation of clinical signs, were taken to indicate that systemic absorption had occurred and exposure to the target tissues had been achieved.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

 

The positive control group (cyclophosphamide) showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.


 

Conclusion

In the Micronucleus Test the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non genotoxic under the conditions of the test.


 

Endpoint:
in vivo mammalian cell study: DNA damage and/or repair
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date 23 November 2016 Experimental completion date 02 March 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian comet assay
Specific details on test material used for the study:
Identification: Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4
Physical state/Appearance: Extremely pale yellow liquid
Batch: 1603101
Purity: 100%
Expiry Date: 19 June 2017
Storage Conditions: Room temperature in the dark
Species:
rat
Strain:
Wistar
Sex:
male
Details on test animals and environmental conditions:
5.1.1 Animals and Animal Husbandry
Sufficient male Wistar Han™ (HsdRCCHan™WIST) rats were supplied by Envigo (UK). At the start of the main test the males weighed 186.8 to 216.1 g, and were approximately 7 to 12 weeks old. After a minimum acclimatization period of five days the animals were selected at random and given a number unique within the study by tail marking and a number written on a color coded cage card. A table of the animal weights, with means and standard deviations is presented in Table 1.
The animals were housed in groups of up to five in solid-floor polypropylene cages with woodflake bedding. Free access to mains drinking water and food (Envigo Teklad 2014 Rodent Pelleted Diet) was allowed throughout the study.
The temperature and relative humidity were set to achieve limits of 19 to 25 ºC and 30 to 70% respectively. Any occasional deviations from these targets were considered not to have affected the purpose or integrity of the study. The rate of air exchange was approximately fifteen changes per hour and the lighting was controlled by a time switch to give twelve hours light and twelve hours darkness.
Route of administration:
oral: gavage
Vehicle:
Arachis oil
Details on exposure:
A range-finding test was performed to find suitable dose levels of the test item following a triple oral administration at approximately 0, 24 and 44 hours. The upper dose level selected should ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg. Based on toxicology information supplied by the Sponsor the initial dose level was limited to 200 mg/kg. Additionally bone marrow slides were prepared and scored for PCE/NCE ratio as an indicator of toxicity to bone marrow.

All animals were dosed three times (approximately) 0, 24 and 44 hours at the appropriate dose level by gavage using a metal cannula attached to a graduated syringe. The volume administered to each animal was calculated according to its bodyweight at the time of the initial dosing.
Animals were observed approximately 1 hour after each dosing and immediately prior to termination. Any deaths and evidence of overt toxicity were recorded at each observation. No necropsies were performed.
Duration of treatment / exposure:
44 hours
Frequency of treatment:
0, 24 and 44 hours
Post exposure period:
1 hour
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
7 males per dose
Control animals:
yes, concurrent vehicle
Positive control(s):
N-Nitroso-N-Methylurea (MNU)

(Cyclophosphamide (CP) for micronucleus element of study)
Tissues and cell types examined:
The primary target tissues of the comet phase assay are liver, glandular stomach, and duodenum. Comet slides were prepared from the testes and bone marrow but not scored.
Details of tissue and slide preparation:
. The glandular stomach, duodenum, liver, bone marrow and testes were processed for comet slides. Samples of liver, duodenum, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. Comet slides were not prepared for the positive control animals dosed with CP.

The remaining tissue samples were processed to provide single cell suspensions, providing sufficient cells for scoring, for the Comet Assay as follows:

Liver - A small piece of liver was excised (approximately 1 cm3) and washed in liver buffer, (Hanks balanced salt solution supplemented with EDTA), before being minced and filtered to provide a single cell suspension.

Glandular Stomach and Duodenum – The stomach was removed and cut longitudinally to allow the stomach contents to be removed. Half the stomach was removed for possible histopathology and the remaining stomach was immersed in stomach buffer (Hanks balanced salt solution supplemented with EDTA and EGTA) and incubated for approximately 15 minutes on ice. The mucosal layer of the stomach was removed by scraping and a single cell suspension was obtained by scraping the underlying glandular stomach tissue and suspending it in stomach buffer. The resulting cell suspension was filtered through gauze prior to use for the comet slides. The duodenum was removed and cleaned. A section was retained for possible histopathology and the remaining section was immersed in stomach buffer for approximately 15 minutes on ice. A cell suspension was obtained by scraping the tissue of the duodenum into stomach buffer and filtering it through gauze.

Bone Marrow –A femur was dissected from each animal and aspirated with phosphate buffered saline (PBS) to provide a single cell suspension. The cell suspension was filtered through gauze prior to use for the Comet slides. Bone marrow was not taken from the animals dosed with cyclophosphamide for Comet assay processing.

Testes – These were dissected from each animal. One was retained for possible histopathology. The second one was placed in liver buffer and chopped with scissors to provide a single cell suspension which was filtered through gauze before use for the comet slides.

The above procedures were performed under subdued lighting and the Comet Assay tissues/cells were processed at approximately 4 °C.

Comet Slide Preparation
Adequate numbers of slides were pre-coated with 0.5% normal melting point agarose and stored at room temperature prior to the start of the experiment. Prior to use in the study the slides were labelled for animal number, project number and tissue type.

Once the cell suspensions had been obtained, 30 µL was mixed with 270 µL of molten 0.5% low melting point (LMP) agarose, mixed thoroughly and 50 µL of this cell suspension/agarose mix placed onto a pre coated slide. Four slide gels per animal for each tissue, two gels per pre-prepared agarose slide were prepared. The volumes of cell suspension and low melting point agarose used may be adjusted depending on the tissue type and cell density. The cell suspension/agarose mix was immediately covered with a glass coverslip and kept at approximately 4 °C in the dark for approximately 20 minutes to allow the gel to solidify.

All the comet slides were processed through the subsequent procedure. Once the LMP agarose has set the coverslips were removed and the slides gently lowered into freshly prepared lysing solution (pH 10) and refrigerated in the dark overnight.

After the lysis phase had been completed the slides were removed from the lysing buffer, rinsed with neutralization buffer (0.4M Tris pH 7.5), to remove residual detergents and salts and then placed randomly into an electrophoresis unit. The electrophoresis unit was filled with chilled electrophoresis buffer (pH >13) until the slide surface was just covered. The slides were left for approximately 20 minutes to allow the DNA to unwind. When the DNA unwinding period has finished electrophoresis was performed at approximately 0.7 V/cm (calculated between the electrodes), for 20 minutes. The buffer in the bath was maintained at a low temperature (approximately 2-10 C) during the electrophoresis period. The temperature of the electrophoresis solution at the start of unwinding, the start of electrophoresis, and the end of electrophoresis was recorded. The time will depend on the tissue type and the above settings may also be adjusted. The voltage and current at the start and end of the electrophoresis period was recorded. The aim is to induce sufficient migration of the DNA so that minimal sized comets are produced in the nuclei of vehicle control cells.

At the end of the electrophoresis period the bath was switched off, the slides gently removed and rinsed three times with neutralization buffer for approximately 5 minutes each time. The slides were carefully drained and fixed in cold 100% methanol for 5 minutes and allowed to air dry. Once dry the slides are stored prior to staining and scoring. Two of the four processed slides were scored (A and B replicates) and the remaining two slides (C and D replicates) are stored as backup slides.

Evaluation criteria:
6.6 Comet Scoring
Comet slides from the liver, glandular stomach and duodenum were scored. Comet slides from the testes and bone marrow were prepared but not scored.
The slides were coded prior to scoring to allow “blind” scoring. The slides are stained just prior to analysis for comets. To each dry slide, 75 L of propidium iodide (20 g/mL) was placed on top of the slide and then overlaid with a clean cover slip. After a short period to allow hydration and staining of the DNA the slide was placed onto the stage of a fluorescence microscope and scored for comets using a CCD camera attached to a PC-based image analysis program, i.e. Comet IV version 4.3.1.
Two slide gels for each tissue per animal were scored with a maximum of 100 cells per slide gel giving an accumulative total of 200 cells per tissue per animal. Care was taken to guarantee that a cell is not scored twice. The slide score data from each experiment was processed using the Excel macro program provided in Comet IV. Comparisons between the vehicle control group response and that of the test item dose groups were made. The primary end-points are percentage tail DNA (%Tail intensity) and median percentage tail intensity.
Each slide was assessed for the incidence of ‘hedgehog’ cells to give an indication of cell integrity. Hedgehogs are cells that exhibit a microscopic image consisting of a small or non-existent head, and large diffuse tails and are considered to be heavily damaged cells, although the etiology of the hedgehogs is uncertain.
Individual slide data, animal data and grouped data are presented. The median percentage DNA for each slide was determined and the mean of the median value calculated for each animal. The mean of the individual animal means is calculated to give a group mean.
Statistics:
With no indication of any increase in percentage tail intensity or dose related increases in median percentage tail intensity in the test item groups statistical analysis was considered to be unnecessary.
Key result
Sex:
male
Genotoxicity:
negative
Toxicity:
no effects
Vehicle controls validity:
valid
Negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
The comet assay did not demonstrate any significant increases or dose related increases in the percentage tail intensity or median percentage tail intensity in the liver, glandular stomach or duodenum and was therefore considered to be non-genotoxic to these tissues. The positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in all tissues analyzed indicating that the test method itself was operating as expected. The vehicle control groups all had percentage tail intensity and median percentage tail intensity values within the expected range.

The 100 mg/kg dose group of the glandular stomach had one animal (animal 29) which had a very high percentage tail intensity in the A replicate slide only. This was considered to be an anomalous result for this slide replicate and may have been due to a few highly damaged cells being present as indicated by the high standard deviation compared to the other slides in the group. Overall the group did not demonstrate a significant increase in tail intensity over the control.

There was no marked increase in hedgehog frequency for any of the test item dose levels in any of the tissues investigated.

Summary Table of the Comet Data

Glandular Stomach

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Vehicle (Arachis oil)

5.01

3.92

2.01

MTD (200 mg/kg)

5.69

3.25

1.19

MTD/2 (100 mg/kg)

6.42

5.41

3.67

MTD/4 (50 mg/kg)

4.61

3.95

1.92

Positive
(N-Nitroso-N-Methylurea)

7.43

28.10

26.61

Liver

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Vehicle (Arachis oil)

0

0.17

0

MTD (200 mg/kg)

0

0.42

0.02

MTD/2 (100 mg/kg)

0

0.21

0.01

MTD/4 (50 mg/kg)

0

0.27

0.01

Positive
(N-Nitroso-N-Methylurea)

10.24

22.73

22.81

Duodenum

Dose Level

Group Mean % Hedgehogs

Group Mean % Tail Intensity

Group Mean of Median % Tail Intensity

Vehicle (Arachis oil)

15.10

5.04

2.64

MTD (200 mg/kg)

11.36

5.44

3.17

MTD/2 (100 mg/kg)

11.44

5.04

2.71

MTD/4 (50 mg/kg)

14.14

5.36

2.79

Positive
(N-Nitroso-N-Methylurea)

22.10

43.73

42.76


Conclusions:
In the Comet element of the test, the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non-genotoxic to the liver, glandular stomach and duodenum investigated in vivo.
Executive summary:

 Introduction

The Comet Assay is a standard follow-up in vivo study. 

The basic principle of the single cell gel electrophoresis assay is the migration of DNA in an agarose matrix under electrophorectic conditions. When viewed under a microscope, a cell nucleus after electrophoresis has the appearance of a ‘comet’, with a head (the nuclear region) and a tail containing DNA fragments or strands migrating in the direction of the anode. The tail of the comet will elongate and broaden with increasing levels of damage to the DNA

The primary target tissues of the comet phase assay are liver, glandular stomach, and duodenum. Comet slides were prepared from the testes and bone marrow but not scored. Bone Marrow was sampled for micronucleus slides.

Methods

A range-finding test was performed to find suitable dose levels of the test item. The Comet assay main test was conducted at the maximum tolerated dose (MTD) 200 mg/kg with 100 mg/kg and 50 mg/kg as the lower dose levels. Groups, each of seven rats, were dosed three times at 0, 24 and 44 hours with the test item. Animals were killed 4 hours after the third dose administration. 

 

The groups of rats from each dose level were killed by humane euthanasia (carbon dioxide asphyxiation) approximately 48 hours after the start of the test. The glandular stomach, duodenum, liver, bone marrow and testes were processed for comet slides. Samples of liver, duodenum, testes and glandular stomach were preserved in formalin for possible histopathology in the event of a positive response. Comet slides were not prepared for the positive control animals dosed with CP.

Results

 

In the Comet Assay, the positive control group induced a marked increase in percentage tail intensity and median percentage tail intensity in all tissues analyzed indicating that the test method itself was operating as expected. The vehicle control groups all had percentage tail intensity and median percentage tail intensity values within the expected range.

 

There was no statistically significant marked increase in percentage tail intensity or median percentage tail intensity for any of the test item dose groups in the liver, duodenum or glandular stomach when compared to the vehicle control, confirming that the test item did not induce DNA damage in the tissue investigated under the conditions of the test.

 

There was no evidence of any significant increases in the incidence of micronucleated polychromatic erythrocytes in animals dosed with the test item when compared to the vehicle control group.

 

The positive control group (cyclophosphamide) showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test.


 

1.4        Conclusion

In the Comet Test, the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 did not induce any increases in the percentage tail intensity values in the liver, duodenum or glandular stomach. The test item was considered to be non-genotoxic to the liver, duodenum or glandular stomachin vivounder the conditions of the test. 

In the Micronucleus Test the test item, Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 was considered to be non genotoxic under the conditions of the test

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

Mode of Action Analysis / Human Relevance Framework

The in-vitro testing indicated a potential for genotoxicity with in particular clastogenicity in the mammalian cell chromosome aberration test and mutagenicity in the presence of rat S9. There was also a weak response in only one strain indicating gene mutation in the bacteria in the Ames test.

To determine if this genotoxic potential in-vitro was relevant to human exposure to the test substance, in-vivo test were conducted.  The two appropriate tests were the in-vivo bone marrow micronucleus test in rats, this test was done to establish if the in-vitro clastogenicity was reproducible in vivo. The study showed that the test substance reached the bone marrow but there was no evidence of increased micronuclei so the clastogenicity was not confirmed in vivo. The second in vivo test was the Comet test, this test looks for DNA damage, which would indicate a mutagenic potential in vivo, this test did not find any evidence of such damage in the three tissues examined, the liver, duodenum or glandular stomach. So the mutagenicity was not confirmed in vivo. Based on the two negative in vivo tests the tets substance Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane, List No 940-783-4 isconsidered no to represent a mutagenic or clastogenic hazard to humans.

Additional information

Justification for classification or non-classification

The three in vitro assays showed a positive result, although the Ames result was only in one strain. To establish if a classification as a mutagen is required it was agreed with ECHA to do in-vivo micronucleus and Comet tests in rats. These tests were both clearly negative and showed no evidence in-vivo of clastogenicity or other DNA damage. Therefore the potential for genotoxicity seen in the in-vitro test was not seen in-vivo. Based on this there is no requirement to classify Bis(2-chloroethoxy)methane 1,15-dichloro-3,5,8,11,13-penta-oxa pentadecane 1-(2-chloroethoxy)-2-(2-chloroethoxymethoxy)ethane as a mutagen based on EU CLP(GHS) criteria.