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Toxicological information

Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to OECD test guideline under GLP conditions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012
Report Date:
2012

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): C.I. Pigment Rubine 264
- Lot/batch No.: H11601
- Expiration date of the lot/batch: 31 July, 2020

Method

Target gene:
tk locus
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI 10 medium
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no data
- Periodically "cleansed" against high spontaneous background: no data
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
50, 100, 250, 500, 1000 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: medium
- Justification for choice of solvent/vehicle: none required
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
RPMI 5 medium
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: without S9: 4-nitroquinoline-N-oxide; with S9: Cyclophosphamide
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: Test1: 3h; Test2: 24h
- Expression time (cells in growth medium): 2 days

NUMBER OF REPLICATIONS: duplicate

DETERMINATION OF CYTOTOXICITY
- Method: other: Harmonized relative survival
Evaluation criteria:
Determination of Survival or Viability
From the zero term of the Poisson distribution the probable number of clones/well (P) on microtiter plates in which there are empty wells (EW, without clones) out of a total of wells (TW) is given by:
P = -ln (EW/TW)
The plating efficiency (PE) in any given culture is therefore:
PE = P/1.6
The percentage relative survival (%RS) in each test culture was determined by comparing plating efficiencies in test and vehicle control cultures thus:
% RS = [PE (test)/PE (control)] x 100
To take into account any loss of cells during the 3 hour treatment period, percentage relative survival value for each dose of test item was adjusted as follows:
Harmonised %RS= %RS x (Post treatment cell concentration for dose/Post treatment cell concentration for vehicle control)
All percentage relative survival (%RS) values were adjusted as described above.
Additionally the suspension growth (SG), the relative viability (RV) and relative total growth (RTG) was calculated as follows:
SG = DCG1 x DCG2 (DCG: daily cell growth)
DCG (1 or 2) = cell concentration on day 1 or 2/ 2 x 10^5 (the initial cell concentration)
The RSG (relative suspension growth) was calculated as follows:
% RSG = [SG (test)/SG (control)] x 100
The percentage relative viability (%RV) in each test culture was determined by comparing plating efficiencies in test and vehicle control cultures thus:
% RV = [PE (test)/PE (control)] x 100
The relative total growth was calculated as follows:
RTG (%) = RV x RSG (%)

Determination of Mutant Frequency
It is usual to express mutant frequency (MF) as "mutants per 10^6 viable cells". In order to calculate this, the plating efficiencies of both mutant and viable cells in the same culture were calculated:
MF = [P (mutant)/2 x 103] x [1.6/P (viable)] x 10^6
= {-ln [EW/TW (mutant)]/-ln [EW/TW (viable)]} x 800
The small and large colony mutant frequencies were not calculated.
Statistics:
The heterogeneity of the obtained data was tested. The statistical significance of mutant frequencies (total wells with clones) was carried out using Dunnett’s Test, using TOXSTAT statistical software. The positive control data were compared with the respective vehicle control or untreated control data with 2 Sample t-Test, using TOXSTAT statistical software. The data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Water solubility: highly water insoluble
- Precipitation: yes

RANGE-FINDING/SCREENING STUDIES:
The concentrations applied in the assays (at the 3-hour and 24-hour treatments) were chosen according to the solubility and cytotoxicity results of the pre-experiments. The test item was considered as relatively insoluble therefore it was investigated beyond its limit of solubility under culture conditions.

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequency of the negative (vehicle) control cultures were within the expected normal range (50-170 mutants per 10^6 viable cells) in the performed experiments in line with the historical controls.
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'.

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative without and with metabolic activation

In a study according to OECD Test Guideline 476 (under GLP conditions), the structural analogue PR264 of the registered substance PR254 did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line). This result is read-across to the registered substance PR254.
Executive summary:

The genetic toxicity of the structural analogue PR264 of the registered substance PR254 was investigated in a Mouse Lymphoma assay using L5178Y cells according to OECD Test Guideline 476 under GLP conditions with and without metabolic activation (±S9 Mix). Based on the results of the preliminary Solubility and Toxicity Tests and regarding the practical difficulties with the handling, PR 264 was suspended and diluted in RPMI 5 Medium and the RPMI 5 Medium was parallel investigated as vehicle control.

The following concentrations were investigated in the Assay 1: 3-hour treatment (±S9 Mix): 50; 100; 250; 500 and 1000 μg/mL; The following concentrations were investigated in the Assay 2: 24-hour treatment (-S9 Mix): 50; 100; 250; 500 and 1000 μg/mL; 3-hour treatment (+S9 Mix): 50; 100; 250; 500 and 1000 μg/mL.

The performed Assays fulfilled the validity criteria regarding the negative control and positive controls as well as in connection with the number of analysable concentration levels (at least four). In the examined concentration range noticeable cytotoxicity did not occur at the 3-hour and 24-hour treatments.

In the performed assays the obtained mutation frequencies (in absence and also in presence of exogenous metabolic activation) did not show dose-related tendencies, did not exceed the relevant GEF thresholds for positive call and remained within the validity criterion range of the negative vehicle control cultures. The obtained statistically significantly differences from that of the corresponding vehicle control (Dunnett’s Test, α = 0.05) were considered to reflect the biological variability of the applied test system and were regarded as not biologically relevant. Under the conditions of this study, the PR 264 did not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells (L5178Y TK+/- 3.7.2 C mouse lymphoma cell line) used. This result is read-across to the registered substance PR254.