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Genetic toxicity: in vitro

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Administrative data

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From October 4, 2018 to March 14, 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2019
Report date:
2019

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Test material

Constituent 1
Chemical structure
Reference substance name:
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
EC Number:
942-810-5
Molecular formula:
C43H24ClCuN15O16S6.xLi.yNa, (x + y) = 4; 0 < (x,y) < 4 with 1341.9 < MW < 1374 g/mol (UVCB substance), and traces of NaCl and NaSO4
IUPAC Name:
Reaction products of cuprate(4-), [C-(aminosulfonyl)-C-[[[2-[[4-chloro-6-[(2,5-disulfophenyl)amino]-1,3,5-triazin-2-yl]amino]ethyl]amino]sulfonyl]-29H,31H-phthalocyanine-C,C-disulfonato(6-)-N29,N30,N31,N32]-, tetrasodium and lithium chloride
Test material form:
solid: particulate/powder

Method

Species / strain
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Metabolic activation system:
S9 Mix
Test concentrations with justification for top dose:
Based on the preliminary test result of cytotoxicity, the highest dose tested in this assay was 5000 ug/mL.
Vehicle / solvent:
Dimethylsulfoxide (DMSO)
Controls
Untreated negative controls:
yes
Remarks:
Ultrapure water
Positive controls:
yes
Positive control substance:
cyclophosphamide
methylmethanesulfonate
Details on test system and experimental conditions:
Based on the preliminary test result of cytotoxicity, the definite test were performed at 5000, 2000, 8000 and 320 µg/mL, and two replicates were prepared for each level. The groups were treated for 4 hours with metabolic activation, 4 hour without metabolic activation and 24 hours without metabolic activation.
Evaluation criteria:
1. Test Validity
When the PE0 of negative control = 60%-140%, PE2 of negative control = 40%-140%; MF of the positive control ≥ 2-fold the negative control value, this study will be considered valid.
2. Result Evaluation
A test item is consider positive if it meets both of the following criteria:
(1) At least one or more concentration produces a concentration-related increase in mutant frequency.
(2) MF of the test item ≥ 2-fold the negative control value.

Results and discussion

Test results
Species / strain:
Chinese hamster Ovary (CHO)
Remarks:
CHO-K1
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
At the beginning and the end of treatment, no precipitate was observed in other dose.

Any other information on results incl. tables

Table 1. The Cytotoxicity Effect of CJ303 on L5178Y Cell

Treatment Dose (µg/mL) PE0 (%) RS0 (%) RSG (%) RS2 (%) RTG (%)
S9-
24h
Negative control  63.3 100.0 100.0 100.0 100.0
Solvent control  60.7 95.9 132.5 96.9 128.4
5000 50.3 79.5 43.3 91.5 39.6
2000 58.0 91.6 102.4 105.1 107.6
800 63.3 100.0 111.9 95.1 106.5
320 65.8 104.0 118.4 91.3 108.2
MMS 64.8 102.3 80.7 97.2 78.5
S9-
4h
Negative control  68.9 100.0 100.0 100.0 100.0
Solvent control  62.2 90.4 75.6 104.2 78.8
5000 63.2 91.7 94.1 89.6 84.3
2000 69.7 101.2 159.8 76.5 122.2
800 61.7 89.6 163.2 73.0 119.1
320 67.7 98.3 122.9 94.5 116.1
MMS 53.2 77.3 147.9 81.2 120.1
S9+
4h
Negative control  62.0 100.0 100.0 100.0 100.0
Solvent control  61.5 99.2 126.7 99.4 126.0
5000 50.9 82.1 82.2 99.4 81.8
2000 58.0 93.6 88.8 112.2 99.6
800 63.1 101.7 96.5 113.9 109.9
320 67.9 109.5 85.7 112.2 96.2
CP 60.7 97.9 134.6 98.6 132.7

Table 2. The mutant frequencies of CJ303 on L5178Y Cell

Treatment Dose (µg/mL) PE2 (%) MF (10^-6) SC (%)
S9-
24h
Negative control  62.7 83.4 58.3
Solvent control  60.7 120.0 -
5000 57.4 86.0 -
2000 65.9 94.9 -
800 59.6 92.5 -
320 57.2 119.3 -
MMS 60.9 488.4 72.9
S9-
4h
Negative control  67.8 77.2 67.5
Solvent control  70.6 93.0 -
5000 60.7 74.3 -
2000 51.8 92.6 -
800 49.5 97.1 -
320 64.0 116.0 -
MMS 55.0 524.3 78.2
S9+
4h
Negative control  60.5 80.3 69.3
Solvent control  60.2 82.0 -
5000 60.2 77.3 -
2000 67.9 85.5 -
800 68.9 94.8 -
320 67.9 72.7 -
CP 59.7 522.8 72.5

Table 3-1. The count result of PE0 and PE2 (S9- 24h)

Dose (µg/mL) Cell number PE0 PE2
EW TW EW TW
Negative control  011 33 96 36 96
012 32 96 33 96
021 40 96 35 96
022 35 96 37 96
Solvent control  D1 33 96 33 96
D2 40 96 40 96
5000 41 46 96 35 96
42 40 96 42 96
2000 31 40 96 35 96
32 36 96 32 96
800 21 32 96 38 96
22 38 96 36 96
320 11 43 96 41 96
12 33 96 36 96
MMS M1 40 96 32 96
M2 29 96 41 96

Table 3-2. The count result of PE0 and PE2 (S9- 4h)

Dose (µg/mL) Cell number PE0 PE2
EW TW EW TW
Negative control  011 30 96 31 96
012 29 96 33 96
021 34 96 35 96
022 35 96 31 96
Solvent control  D1 34 96 31 96
D2 37 96 31 96
5000 41 37 96 40 96
42 33 96 33 96
2000 31 30 96 45 96
32 33 96 39 96
800 21 40 96 44 96
22 32 96 43 96
320 11 33 96 33 96
12 32 96 36 96
MMS M1 39 96 36 96
M2 43 96 44 96

Table 3-3. The count result of PE0 and PE2 (S9+ 4h)

Dose (µg/mL) Cell number PE0 PE2
EW TW EW TW
Negative control  011 35 96 37 96
012 37 96 34 96
021 31 96 36 96
022 40 96 39 96
Solvent control  D1 39 96 38 96
D2 33 96 35 96
5000 41 43 96 42 96
42 42 96 32 96
2000 31 40 96 35 96
32 36 96 30 96
800 21 35 96 35 96
22 35 96 29 96
320 11 35 96 35 96
12 30 96 30 96
CP C1 40 96 39 96
C2 33 96 35 96

Applicant's summary and conclusion

Conclusions:
It was concluded that when tested up to 5000 µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.
Executive summary:

The mutagenic potential of CJ303 was assessed in the Mouse Lymphoma Cells (L5178Y), which can detect induced gene mutation.

 

L5178Y cells were exposed to CJ303 at the doses of 320µg/mL, 800µg/mL, 2000µg/mL and 5000µg/mL with metabolic activation for 4 hours, without metabolic activation for 4 hours and without metabolic activation for 24 hours, respectively. Dimethylsulfoxide (DMSO) was used as solvent control substances. Ultrapure water was used as negative control substances. Methyl methanesulfonate (MMS) and Cyclophosphamide (CP) were used as positive control substances for experiments with and without metabolic activation, respectively. After the treatment period, cytotoxicity was evaluated by the relative survival (relative to the negative control). All the culture were treated by trifluorothymidine (TF) to detect gene mutations.

 

When tested up to 5000µg/mL with or without metabolic activation, CJ303 did not induce any Gene Mutation in the L5178Y cell. The result of CJ303 in vitro Mammalian Cell Gene Mutation test was negative.