Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Not mutagenic or genotoxic

Link to relevant study records

Referenceopen allclose all

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
Guideline study performed under 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
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus Holdings LLC, Batch 48654
- Expiration date of the lot/batch: 17 May 2020
- Purity test date: N/A

RADIOLABELLING INFORMATION (if applicable)
- Radiochemical purity: N/A
- Specific activity: N/A
- Locations of the label: N/A
- Expiration date of radiochemical substance: N/A

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room Temperature
- Stability under test conditions: Assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: 20 minute sonication at 37 degrees C required to dissolve, stable for at least 4 hours
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: N/A

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: N/A
- Preliminary purification step (if any): N/A
- Final dilution of a dissolved solid, stock liquid or gel: 1 mg/ml
- Final preparation of a solid: N/A

FORM AS APPLIED IN THE TEST (if different from that of starting material) : N/A

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable) : N/A

OTHER SPECIFICS:
Target gene:
N/A
Species / strain / cell type:
lymphocytes:
Remarks:
Human peripheral blood lymphocytes from healthy and non-smoking donors with no known recent exposure to genotoxic chemicals and radiation
Cytokinesis block (if used):
colcemid
Metabolic activation:
with and without
Metabolic activation system:
Phenobarbital/β-naphthoflavone induced male Wistar rat liver S9 mix containing MgCl2 (8 mM), KCl (33 mM), glucose-6-phosphate (5 mM) and NADP (4 mM) in sodium-ortho-phosphate-buffer (100 mM, pH 7.4)
Vehicle / solvent:
cell culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): 500 µL heparinized whole blood in 4.5 mL complete culture medium. Culture medium: RPMI 1640 supplemented with 15% FBS, 100 U/100 µg/ml penicillin/streptomycin solution, 0.24 g/ml PHA-L.

DURATION : See table

ANALYSIS OF METAPHASE CELLS: Evaluation of the cultures is performed [according to standard protocol of the "Arbeitsgruppe der Industrie, Cytogenetik"] using microscopes with 100x oil immersion objectives.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid

STAIN: BrdU

NUMBER OF METAPHASE SPREADS ANALYSED PER DOSE (if in vitro cytogenicity study in mammalian cells): At least 300

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED:
The cultures are harvested by centrifugation 24 h after beginning of treatment. The supernatant is discarded and the cells are resuspended in approximately 5 mL hypotonic solution (0.4 % KCl). The cell suspension is incubated at room temperature for 20 min. After removal of the hypotonic solution by centrifugation the cells are fixed with 3+1 methanol + glacial acetic acid. The fixation procedure is repeated twice. Slides are prepared by dropping the cell suspension onto a clean microscopic slide. The cells are stained with giemsa and according to the Fluorescent plus Giemsa technique, respectively. The slides are coverslipped using 2-3 drops of Eukitt(R). Afterwards they are air dried.

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index
- Any supplementary information relevant to cytotoxicity: The number of polyploid cells is scored

OTHER EXAMINATIONS:
- Determination of polyploidy: Near tetraploid karyotype
- Determination of endoreplication: N/A
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable): N/A

- OTHER:
Evaluation criteria:
All structural chromosomal aberrations including breaks, fragments, deletions, exchanges and chromosomal disintegration are recorded. Gaps are recorded as well but not included in the calculation of the aberration rates. The definition of a gap is as follows: an achromatic region (occurring in one or both chromatids) independent of its width. The remaining visible chromosome regions should not be dislocated either longitudinally or laterally.

Providing that all acceptability criteria are fulfilled, a test chemical is considered to be clearly positive if, in any of the experimental conditions examined:
a) at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) the increase is dose-related when evaluated with an appropriate trend test,
c) any of the results are outside the distribution of the historical negative control data
When all of these criteria are met, the test chemical is then considered able to induce chromosomal aberrations in cultured mammalian cells in this test system.

Providing that all acceptability criteria are fulfilled, a test chemical is considered clearly negative if, in all experimental conditions examined
a) none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
b) there is no concentration-related increase when evaluated with an appropriate trend test,
c) all results are inside the distribution of the historical negative control data.
The test chemical is then considered unable to induce chromosomal aberrations in cultured human peripheral blood lymphocyte cells in this test system.
Statistics:
Not reported
Key result
Species / strain:
lymphocytes:
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Remarks on result:
other: precipitation at highest dose(s)
Conclusions:
The test substance was evaluated for potential to cause chromosome aberrations using an in vitro Chromosome Aberration (CA) Test according to OECD 473 in human lymphocytes. The test substance was determined to be non-cytogenic according to the results of this test.
Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
2017-2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 490 (In Vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene)
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell gene mutation tests using the thymidine kinase gene
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Vertellus, 48654
- Expiration date of the lot/batch: 17 May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: assumed to be stable
- Solubility and stability of the test substance in the solvent/vehicle: the test substance was soluble in RPMI (+ 5% HS) cell culture medium in concentrations up to 2 mg/mL after treatment with ultrasound (for 20 minutes at 37 °C)

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Final dilution of a dissolved solid, stock liquid or gel: 2 mg/mL
Target gene:
Thymidine kinase (TK)
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
with and without
Metabolic activation system:
S-9 liver fraction (10% v/v), from male rats induced with phenobarbital and B-napthoflavone for three consecutive days by oral route.
Test concentrations with justification for top dose:
Doses are half-log intervals, with the high dose at the limit of 5000 µl/plate.
Vehicle / solvent:
RPMI + 5% HS cell culture medium
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
methylmethanesulfonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: plate incorporation (first test) and preincubation (second test)
- Cell density at seeding (if applicable): 0.1 ml of a 10-h bacterial culture (having a density of at least 10E7/11mL)

DURATION
- Preincubation period: 4 h
- Exposure duration: 48-72 h for plate incorporation; preincubation for 30 m before plating in top agar
- Expression time (cells in growth medium):
- Fixation time (start of exposure up to fixation or harvest of cells): cells not fixed

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: not applicable


DETERMINATION OF CYTOTOXICITY
- Method: The induced mutant frequence meets or exceeds the GEF of 126 mutants per 10E6 cells

Rationale for test conditions:
Pre-experiments were carried out in order to determine the toxicity of the test item. Several concentrations of the test item were tested. The maximum test concentration was 2 mg/mL, the maximum soluble concentration in cell culture medium. After a two day growth period the relative suspension growth (RSG) of the treated cell cultures was calculated according to the method of Clive and Spector.
Evaluation criteria:
For a test material to be classified as mutagenic, the test item must exceed the Global Evaluation factor (GEF) of 126 mutants per 10E6 cells and a dose-dependent increase in mutant frequency must be detected.

For a test to be considered valid, the mean of the vehicle control revertant colony numbers for each strain should lie within or close to the current historical control range of the laboratory unless otherwise justified by the Study Director.

The positive control compounds must produce an increase in mean revertant colony numbers of at least twice that of the concurrent vehicle controls.


A test item will be considered non-mutagenic (negative) in the test system if the above criteria are not met.
If the results obtained fail to satisfy the criteria for a clear “positive” or “negative” response, the test data should be evalueated by expert judgment and or further possible investigations.
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Key result
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid

Table 1: Main experiment WITHOUT enzymatic activation

 Test group Conc. [µg/mL]  RCE [%]  RTG [%]  MF [mutants/ 10^6 cells]   IMF [mutants/ 10^6 cells]  GEF exceeded Statistical Significant Increase  Precipitate
 C1  0     100    100      72.4     /  /  /  -
 C2  /  /  /  -
 2  .5  115.7   93.6   61.7   -10.7  -  -  -
 3  1.25  111.6   100.5   49.6  -22.8  -  -  -
 4  2.5  97.6  80.9    56.7 -15.7  -  -  -
 5  5 109.7  76.7  49.4 -23.1  -  -  -
 6  10  88.8  48.2  75.7 3.3  -  -  -
 10  40 86.1  15.7   77.7 5.3  -  -  -
 12  50 102.4  16.1  56.8 -15.6  -  -  -
 EMS  300 90.2  73.0  739.1 666.7  +  +  -
 MMS  10  81.1  68.7    978.5   906.0  +  +  -

Table 2: Main experiment WITH enzymatic activation

 Test group Conc. [µg/mL]  RCE [%]  RTG [%]  MF [mutants/ 10^6 cells]   IMF [mutants/ 10^6 cells]  GEF exceeded Statistical Significant Increase  Precipitate
 C1  0     100    100      80.8     /  /  /  -
 C2  /  /  /  -
 3  2.5  97.6    93.8    67.3   -13.5    -  -  -
 4  5.0  111.7   115.6   54.9  -26.0  -  -  -
 5  10  83.7

 74.7

  108.9

28.0

 -

 -

 -

 7

 30

 88.9

  58.6

62.3

-18.5

 -

 -

 -

 9

 40

78.8

45.2

55.9

-25.0

 -

 -

 -

 11

 50

102.5

   40.2

  47.1

-33.7

 -

 -

 -

 12

 100

 86.2

  21.2

  59.3

-21.5

 -

 -

 -

B[a]P

 2.5

71.2

35.7 

991.4

 910.6

 +

 +

 -

C: Negative Controls

a: Relative Cloning Efficiency, RCE = [(CE dose group / CE of corresponding controls) x 100]

Cloning Efficiency, CE = ((-LN (((96 - (mean P1,P2)) / 96)) / 1.6) x 100)

b: Relative Total Growth, RTG = (RSG x RCE)/100

c: Mutant Frequency, MF = {-ln [negative cultures/total wells (selective medium)] / -ln [negative cultures/total wells (non selective medium)]}x800

d: Induced Mutant Frequency, IMF = mutant frequency sample – mean value mutant frequency corresponding controls

e: Global Evaluation Factor, GEF (126); +: GEF exceeded, -: GEF not exceeded

f: statistical significant increase in mutant frequency compared to negative controls (Mann Whitney test, p<0.05). +: significant, -: not significant.

EMS: Ethylmethanesulfonate

MMS: Methylmethanesulfonate

B[a]P: Benzo[a]pyrene

Conclusions:
The test substance was evaluated for mutagenicity potential according to OECD Guideline Section 4, No. 490 and EC No 440/2008, L142, Annex Part B, B 17, "In vitro Mammalian Cell Gene Mutation Tests Using the Thymidine Kinase Gene." The result of the test was negative, indicating that the test substance is non-mutagenic.
Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2016-01-31 to 2016-02-17
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well documented GLP compliant study report
Justification for type of information:
The analogue approach is used for the hazard assessment of toxicological, eco-toxicological and environmental fate endpoints for the registration Pentamid™ KH (EC 934-047-1). The hypothesis is that data can be read-across between Pentamid™ KH and its structural analogues, based on structural similarity and common breakdown/metabolic products (Scenario 1 of the Read-Across Assessment Framework (RAAF, ECHA, 2015)).

For mammalian toxicity the “parent” substances show no significant systemic or dermal toxicity in mammals and vertebrates. One of the metabolic products of Pentamid™ KH, the well-studied terephthalic acid, has no significant systemic toxicity. The amine metabolite, however, shows diffuse mammalian systemic toxicity at moderate concentrations. This metabolite is employed in the risk assessment, in agreement with the European Union draft Risk Assessment Report of 2008.

The sole classification for Pentamid™ KH is the precautionary aquatic chronic 4 category, based on low water solubility and a log Kow higher than 4.

Read-across data, all evaluated as reliable according to Klimisch scores of 1 or 2, to estimate the toxicity of the registered substance is used for fulfilling the data requirements of the REACH registration and classifying potential hazards. This read-across approach is adequate for the purposes of risk assessment and classification and labeling.
Reason / purpose:
read-across source
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine for the S. typhimurium strains, and tryptophan for E. coli.
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:
Aroclor 1254 induced rat liver S9-mix
Test concentrations with justification for top dose:
For direct plate assay with the tester strains, TA1535, TA1537 and TA98 in the absence and presence of S9-mix: 5.4, 17, 52, 164 and 512 μg/plate.
For Pre incubation experiment: tested at the dose levels of 5.4, 17, 52, 164 and 512 μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA (in the absence and presence of S9-mix)
Vehicle / solvent:
Ethanol
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
9-aminoacridine
2-nitrofluorene
sodium azide
N-ethyl-N-nitro-N-nitrosoguanidine
benzo(a)pyrene
other: ICR-191 (Sigma); 2-aminoanthracene (2AA)
Evaluation criteria:
Data evaluation and statistical procedures:

In addition to the criteria stated below, any increase in the total number of revertants was
evaluated for its biological relevance including a comparison of the results with the historical
control data range.
A test item is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is not greater than two (2)
times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537
or TA98 is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one follow up experiment.

A test item is considered positive (mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 or WP2uvrA is greater than two (2) times
the concurrent control, or the total number of revertants in tester strains TA1535, TA1537 or TA98
is greater than three (3) times the concurrent control.
b) In case a repeat experiment is performed when a positive response is observed in one of the
tester strains, the positive response should be reproducible in at least one follow up experiment.
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 98
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A pKM 101
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
In the direct plate test as well as in the pre-incubation test, no increase in the number of revertants was observed upon treatment with HOSTAGEL HT 300 under all conditions tested.

No reduction of the bacterial background lawn and no biologically significant decrease in the number of revertants were observed. For strain TA1537 fluctuation of revertants below lab historical controls with no dose relation was observed at lowest dose.

Migrated from field 'Test system'.
Conclusions:
negative with metabolic activation
negative without metabolic activation
Based on the results obtained with the OECD 471 AMES test, the source test item is regarded as not mutagenic. The target substance is not classified for mutagenicity according to Regulation EC No. 1272/2008.
Executive summary:

The source substance was tested in the Salmonella typhimurium reverse mutation assay with four

histidine-requiring strains of Salmonella typhimurium (TA1535, TA1537, TA100 and TA98) and in

the Escherichia coli reverse mutation assay with a tryptophan-requiring strain of Escherichia coli

(WP2uvrA). The test was performed in two independent experiments, at first a direct plate assay

was performed and secondly a pre-incubation assay both in the absence and presence of S9-mix

(rat liver S9-mix induced Aroclor 1254).

The test item was tested in the first mutation assay at a concentration range of 5.4 to 512

μg/plate in the absence and presence of S9-mix in the tester strains TA1535, TA1537 and TA98.

The source material precipitated on the plates at the top dose of 512 μg/plate. The bacterial

background lawn was not reduced at any of the concentrations tested. No biologically relevant

devrease in the number of revertants was observed.

In the second mutation experiment, the source substance was tested up to concentrations of 512

μg/plate in the tester strains TA1535, TA1537, TA98, TA100 and WP2uvrA in the pre-incubation

assay. It precipitated on the plates at dose levels of 164 and 512 μg/plate. The

bacterial background lawn was not reduced at any of the concentrations tested and no

biologically relevant decrease in the number of revertants was observed.

The source substance did not induce a significant dose-related increase in the number of revertant

(His+) colonies in each of the four tester strains (TA1535, TA1537, TA98 and TA100) and in the

number of revertant (Trp+) colonies in tester strain WP2uvrA both in the absence and presence of

S9-metabolic activation. These results were confirmed in an independently repeated experiment.

Based on the results of this study it is concluded that the source material is not mutagenic in the

Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation

assay. The target substance is not classified for mutagenicity according to Regulation EC No. 1272/2008.

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Not applicable

Additional information

Justification for classification or non-classification

The registered substance was tested in an in vitro chromosomal aberrations assay (OECD 473) and in vitro mouse lymphoma assay (OECD 490), with findings of no clastogenicity or mutagenicity. A close structural analogue was tested in an Ames assay, with a finding of no mutagenicity. The criteria for classification for mutagenicity according to Regulation EC No. 1272/2008 are not met.