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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
15/04/2010 until 01/12/2010
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: This study was conduced following an accepted OECD guideline and in compliance with the Good Laboratory Practice.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2010
Report Date:
2010

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian chromosome aberration test

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
TEST ITEM LCE10031
IPL REGISTRATION NUMBER 100319
BATCH NUMBER T92235
EXPIRY DATE 27/05/2011 (i.e. retest date)
APPEARANCE pearls appearance
PURITY 99.45% (taking into account the water content but considered as
100 % as mentioned in the Study Plan)
SALT / BASE RATIO not applicable
WATER CONTENT 0.55 %
CORRECTION FACTOR none, at the sponsor’s request
QUANTITY SUPPLIED not determined
STORAGE CONDITIONS room temperature, protected from light
STABILITY UNDER
STORAGE CONDITIONS
up to expiry date (i.e. 27/05/2011)

Method

Target gene:
Aroclor1254
Species / strain
Species / strain / cell type:
human lymphoblastoid cells (TK6)
Details on mammalian cell type (if applicable):
Human lymphocytes were taken from healthy non-smoker subjects receiving no medication and who have
not suffered any recent viral infection. The blood was drawn onto lithium-heparin in a sterile Venoject
tube. The genotoxicity test was carried out using cells taken from two different donors (a man and a
woman)
Metabolic activation:
with and without
Metabolic activation system:
S9 fraction
Test concentrations with justification for top dose:
CYTOTOXICITY ASSAY:
PRELIMINARY CYTOTOXICITY ASSAY
Carried out both without and with metabolic activation using S9 from rat liver induced by Aroclor.
-Treatment durations:
• Without S9-mix : 4 h + 20 h recovery period (short treatment)
20 and 44 h without recovery period (continuous treatment)
• With S9-mix : 4 h + 20 h recovery period, with 5% or 10 % S9-mix
- Concentrations tested expressed as μg/mL LCE10031
• Without S9-mix : 162.5 – 81.25 – 40.63 – 20.31 – 10.16
(short and continuous treatments)
• With S9-mix : 162.5 – 81.25 – 40.63 – 20.31 – 10.16
(with either 5 or 10% S9-mix)
- Factor limiting the maximum
concentration tested : solubility

MUTAGENICITY ASSAYS:
Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers (S9-
mix)
- Number of assays : 2
- Number of replicate cultures : 2 per concentration
- Factor limiting the maximum
concentration tested : solubility
-Treatment durations:
• Without S9-mix : 4 h + 20 h recovery period (short treatment)
20 and 44 h without recovery period (continuous treatment)
• With S9-mix : 4 h + 20 h recovery period, with 5% or 10 % S9-mix
- Concentrations tested expressed as μg/mL LCE10031:
• Without S9 mix : 162.5 – 108.33 – 72.22 – 48.15 (assay 1: 4-hour treatment)
162.5 – 108.33 – 72.22 – 48.15 (assay 2: 20-hour treatment)
162.5 – 108.33 – 72.22 – 48.15 (assay 2: 44-hour treatment)
• With S9 mix : 162.5 – 108.33 – 72.22 – 48.15 (assay 1: with 5% S9-mix)
162.5 – 108.33 – 72.22 – 48.15 (assay 2: with 10% S9-mix)
Concentrations in bold were actually assessed
- Positive controls
- without S9-mix : mitomycin C 0.25 μg/mL, Sigma, batch 117K1683
(4 and 20-hour treatments)
(No positive controls are used for the 44-h treatment without metabolic activation)
- with S9-mix : cyclophosphamide 10 μg/mL, Sigma, batch 068K1131
In-Life Phase
Initiated - completed : 19/04/2010 – 30/07/2010
Vehicle / solvent:
Solvent used ethanol (Merck, Batch K40366283 938)
Stability in solvent unknown (dilutions were prepared extemporaneously)
S9 batch number IPL 09-C
Controls
Untreated negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C

Results and discussion

Test results
Species / strain:
human lymphoblastoid cells (TK6)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not specified
Positive controls validity:
valid
Remarks on result:
other: other:
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Under these experimental conditions, the test item induced no clastogenic activity in human lymphocytes both in presence and in
absence of metabolic activation, in the in vitro human lymphocyte metaphase analysis test.
Executive summary:

In the assays without metabolic activation (4, 20 or 44-hour treatment), strong to moderate cytotoxicity was noted at the highest concentration tested of 162.5 μg/mL, with 39.3 to 65.4% of mitotic index compared to the control. In the assays with metabolic activation with either 5 or 10% of S9-mix, moderate and no cytotoxicity was noted at the highest concentration tested of 162.5 μg/mL, with 106.7 and 65.6% of mitotic index compared to the control, respectively. Under these conditions, the concentration of 162.5 μg/mL was retained as the maximum concentration to be tested in each assay either with or without metabolic activation.

GENOTOXICITY ASSAY Carried out both without and with metabolic activation using Aroclor1254-induced S9 from rat livers (S9-mix) - Number of assays : 2 - Number of replicate cultures : 2 per concentration - Factor limiting the maximum concentration tested : solubility

-Treatment durations:

• Without S9-mix : 4 h + 20 h recovery period (short treatment) 20 and 44 h without recovery period (continuous treatment)

• With S9-mix : 4 h + 20 h recovery period, with 5% or 10 % S9-mix - Concentrations tested expressed as μg/mL LCE10031:

• Without S9 mix : 162.5 – 108.33 – 72.22 – 48.15 (assay 1: 4-hour treatment) 162.5 – 108.33 – 72.22 – 48.15 (assay 2: 20-hour treatment) 162.5 – 108.33 – 72.22 – 48.15 (assay 2: 44-hour treatment)

• With S9 mix : 162.5 – 108.33 – 72.22 – 48.15 (assay 1: with 5% S9-mix) 162.5 – 108.33 – 72.22 – 48.15 (assay 2: with 10% S9-mix)

The test item induced no statistically significant increase in the number of breaks per cell and in the frequency of aberrant cells excluding or including gaps in the treatments with or without metabolic activation. It is to be noted that during the 1st reading, an exchange was noted during the 2nd assay following a 44-hour treatment without S9-mix. As this event could constitute an alert for clastogenesis, a complementary reading was performed. In this new reading, no other specific event of clastogenesis was noted. Therefore, this effect was not considered as biologically significant. Furthermore, no statistically significant increase in the number of cells with numerical aberrations was noted in any treatment.

The acceptance criteria for the results were fulfilled. The current study was considered as valid. Under these experimental conditions, the test item induced no clastogenic activity in human lymphocytes both in presence and in absence of metabolic activation, in the in vitro human lymphocyte metaphase analysis test.