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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: GLP guideline study

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 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
as of 1997
Principles of method if other than guideline:
HPRT assay for the detection of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol according to Cole et al. 1983.
Reference:
Cole J, Arlett C F, Green M H L, Lowe J and Muriel W 1983: A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Mutation Research, 111, 317-386
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Isopropylamine (MIPA)
- Substance type: organic
- Physical state: liquid
- Analytical purity: 99.91 % (GC) [see Report. Appendix 7: OXEA, Specification of 30 Oct. 2009]
- Impurities (identity and concentrations): Water (0.06 %), n-i-propylidene-i-propylamine (0.014%)
- Expiration date of the lot/batch: Sep. 2010
- Stability under test conditions: stable
- Storage condition of test material: stored at 2 - 8 °C under desiccant in the dark

Method

Target gene:
Induction of a forward mutation in the X-linked hprt locus: Resistance to the toxic analogue 6-thioguanine (6TG) results from lack of hypoxanthine-guanine phosphoribosyl transferase (HPRT) activity. Thus, the hprt-negative mutants are unable to use 6TG and survive in its presence.
Species / strain
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Type and identity of media: RPMI media (containing antibiotics and varying concentrations of horse serum, heat-inactivated, from 0 - 20 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes,
- Other procedures: purge of TK-negative mutants
Additional strain / cell type characteristics:
other: TK proficient (TK+)
Metabolic activation:
with and without
Metabolic activation system:
post-mitochondrial fraction from livers of male SD rats previously induced with Arochlor-1254
Test concentrations with justification for top dose:
Experiment 1: 0, 80, 120, 210, 240, and 270 µg/mL (evaluated)
Experiment 2: 0, 100, 150, 175, 200, 250, 300, and 400 µg/mL (evaluated)
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: sufficient water solubility of the test substance
Controls
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
water as vehicle
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: 4-nitroquinoline-N-oxide (-S9); benzo(a)pyrene (+S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium, incubation in centrifuge tubes, culture flasks, and wells of microtiter plates,
depending on the operation step

DURATION
- Preincubation period: no data, pre-culture in RPMI 10 after thawing up to an appropriate cell density
- Exposure duration: 3 h
- Incubation temperature: 37 +- 1 °C
- Expression time (cells in growth medium): 7 d
- Selection time (if incubation with a selection agent): 12 d

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Selection of 6-TG resistance: 4 microtiter plates of 90 wells at 2 x10^4 cells per well each
(= 384 x2 x10^4 cells per test concentration)

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival in the presence and absence of test substance (by visual counting of viable clones in microtiter plates)

DETERMINATION of VIABILITY (after expression period)
- Method: visual counting of viable clones in microtiter plates

DETERMINATION of 6-TG RESISTANCE (after expression period)
- Method: visual inspection of microtiter plates for the number wells containing clones.
Evaluation criteria:
ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1. the mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
2. at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

EV ALUATION CRITERIA
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05)
2. there was a significant concentration﷓relationship as indicated by the linear trend analysis (p < 0.05)
3. the effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:
Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Reference:
Robinson et al. 1990: Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed D J Kirkland), Cambridge University Press, pp 102 – 140.

Results and discussion

Test results
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
dose-related: The highest concentrations to provide >10 % RS were 148 µg/mL +S9 and 296 µg/mL -S9.
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: marked increases of >= 1 pH unit at >= 240 µg/mL
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: only at the highest concentration (which turned out to be too toxic)

RANGE-FINDING/SCREENING STUDIES: for cytotoxicity testing up to solublity limit of 500 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: Acceptance criterion, Control/Historical ratio > 0.5, fulfilled for positive controls
(See Appendices)

Remarks on result:
other: strain/cell type: mouse lymphoma L5178Y cells
Remarks:
Migrated from field 'Test system'.

Any other information on results incl. tables

Summary of mutation data [mean of two replicate cultures]:

Experiment 1 (3 hour treatment in the absence and presence of S-9)

[for individual replicates, see Appendix 2, Table 10 (-S9), and Appendix 3, Table 18 (+S9)]

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

% RS

MF§

% RS

MF§

0

100

1.28

0

100!

1.26

80

97

0.23

NS

40

85

1.79

NS

120

81

1.22

NS

200

66

2.11

NS

210

36

2.66

NS

240

58

0.51

NS

240

30

1.62

NS

280

45

1.94

NS

270

19

0.78

NS

300

33

2.20

NS

350

17

0.39

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.1

44

19.72

2

54

10.78

0.15

34

40.90

3

20

48.09

Experiment 2 (3 hour treatment in the absence and presence of S-9)

[for individual replicates, see Appendix 4, Table 26 (-S9), and Appendix 5, Table 34 (+S9)]

Treatment

(µg/mL)

-S-9

Treatment

(µg/mL)

+S-9

% RS

MF§

% RS

MF§

0

100

5.14

0

100

6.41

100

98

4.46

NS

200

90

7.21

NS

150

82

4.28

NS

250

93

3.43

NS

175

70

4.92

NS

300

60

4.95

NS

200

56

4.65

NS

325

53

5.67

NS

250

36

2.97

NS

350

32

4.64

NS

300

28

5.45

NS

375

20

5.70

NS

400

3

6.88

NS

Linear trend

NS

Linear trend

NS

NQO

B[a]P

0.1

59

54.25

2

66

80.64

0.15

58

66.29

3

39

104.52

§       6TG resistant mutants/106viable cells 7 days after treatment

% RS = Percent relative survival adjusted by post treatment cell counts

NS    = not significant

     !   = Based on one replicate only

         

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

Isopropylamine (MIPA) did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under test conditions employed in this study. Thisincluded treatments up to highly toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system.