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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:
experimental study
Adequacy of study:
key study
Study period:
From December 09,2009 to March 27, 2010
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Well described GLP compliant study conducted to recognized international test guidelines.

Data source

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

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
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:
- Name of test material : 1, 2, 3, 6 TETRAHYDROPHTHALIC ANHYDRIDE
- Molecular formula : C8H8O3
- Molecular weight : 152.1473 g/mol
- Smiles notation : O=C1OC(=O)C2C1C/C=C\C2
- InChl : 1/C8H8O3/c9-7-5-3-1-2-4-6(5)8(10)11-7/h1-2,5-6H,3-4H2
- Physical state: solid, white scales
- Analytical purity: 99.92% (w/w)
- Lot/batch No.: SAT4209215
- Expiration date of the lot/batch: 03/06/2010
- Storage condition of test material: room temperature, protected from humidity.
- Container: opaque plastic bottle
- Amount received: 9.99 Kg

Method

Species / strain
Species / strain / cell type:
lymphocytes: Human
Metabolic activation:
with and without
Metabolic activation system:
S9
Test concentrations with justification for top dose:
1520, 760, 380, 190, 95.0, 47.5, 23.8, 11.9 µg/ml
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO batch nos. 1364641 54207P08 and 1395037 52208P07, obtained from Fluka AG;
Controlsopen allclose all
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
mitomycin C
Remarks:
without S9
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk

DURATION
- Exposure duration:
the treatment time was 3 hours after which the cells were allowed to recover prior to harvesting.
- Fixation time :
the harvest time of 24 hours, corresponding to approximately 1.5 cell cycle, was used.



Statistics:
For the statistical analysis, Fisher's Exact Test was is used to compare the number of cells bearing aberrations (assumed to be Poisson distributed) in control and treated cultures. The analysis was is performed using sets of data either including or excluding gaps. Following treatment with the test item, no statistically significant increase in the incidence of cells bearing aberrations including or excluding gaps over the control values, was observed in any experiment both in the absence or presence of S9 metabolism.

Results and discussion

Test results
Species / strain:
lymphocytes: Human.
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Moderate
Vehicle controls validity:
not valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: Slight dose-related reductions of the pH values over the controls (minimum value 6.50) were observed at the three higher dose levels.
- Effects of osmolality: No remarkable variations were observed of osmolality values over the control.


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

Any other information on results incl. tables

ASSAY RESULTS

One hundred metaphase spreads were scored for chromosomal aberrations from each culture with the exception of one replicate culture treated wi th Mitomycin-C from the first experiment where, due to the high incidence of aberrant cells (excluding gaps), scoring was terminated at 50 met apha ses. Following treatment with the test item, no relevant increase in the incidence of cells bearing aberrations including or excluding gaps ov er the c ontrol value was observed in any experiment in the absence or presence of S9 metabolism. Marked increases in the frequency of cells bearing aberra tions (including and excluding gaps) were seen in the cultures treated with the positive control substances, Mitomycin-C and Cyclophosphamide, in dicating the correct functioning of the assay system.

  MAIN ASSAY: 1

 

SOLVENT: DMSO

 

TREATMENT TIME: 3 hours

 

SAMPLING TIME: 24 hours

 

Treatment

Dose

Presence of S9 metabolism

Absence of S9 metabolism

 

µg/ml

%CA

Rel.MI

%CA

Rel.MI

Untreated

-

0.0

102

0.0

125

Solvent

1%

0.0

100

0.0

100

Test

380

0.0 (NS)

76

0.0 (NS)

118

Test

760

0.0 (NS)

69

0.0 (NS)

111

Test

1520

0.5 (NS)

65

0.0 (NS)

105

Mitomycin-C

0.50

-

-

31.3 ***

70

Cyclosphosphamide

18.0

16.5 ***

37

-

-

 

 Key:

 % CA  : Percentage of cells bearing aberrations (excluding gaps)

 Rel.MI : Mitotic Index relative to solvent controls (percent)

 -     : Not tested or not selected for the scoring of aberrations

 *     : Statistically significant at P<0.05

 **    : Statistically significant at P<0.01

 ***   : Statistically significant at P<0.001

 

 

MAIN ASSAY: 2

 

SOLVENT: DMSO

 

TREATMENT TIME: 24 hours

 

SAMPLING TIME: 24 hours

 

Treatment

Dose

Presence of S9 metabolism

Absence of S9 metabolism

 

µg/ml

%CA

Rel.MI

%CA

Rel.MI

Untreated

-

-

-

0.0

117

Solvent

1%

-

-

1.5

100

Test

190

-

-

0.0 (NS)

118

Test

380

-

-

0.0 (NS)

79

Test

760

-

-

1.5 (NS)

49

Mitomycin-C

0.30

-

-

29.0 ***

92

 

 Key:

 % CA  : Percentage of cells bearing aberrations (excluding gaps)

 Rel.MI : Mitotic Index relative to solvent controls (percent)

 -     : Not tested or not selected for the scoring of aberrations

 *     : Statistically significant at P<0.05

 **    : Statistically significant at P<0.01

 ***   : Statistically significant at P<0.001

 

Applicant's summary and conclusion

Conclusions:
Interpretation of results (migrated information):
negative

TETRAHYDROPHTHALIC ANHYDRIDE (THPA) does not induce chromosomal aberrations in human lymphocytes after in vitro treatment
Executive summary:

Tetrahydrophthalic anhydride (THPA) has been assayed for the ability to cause chromosomal damage in cultured human lymphocytes followingin vitrotreatment in the absence and presence of S9 metabolic activation. Methods used were in accordance with OECD/EU test methods. The substance does not induce chromosomal aberrations in human lymphocytes.