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Genetic toxicity in vitro

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Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
1995.12.8-1997.7.8
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Structural similarities of the target substance to the source substances include the (bi)cyclic ring structure with a carboxylic acid anhydride group as the single reactive moiety. For the target substance the bicyclic ring structures contains a double bond at a specific location within the ring and also contains a substituted methyl group at a specific location on the ring structure whereas for the source substance neither the position of the double bond nor the methyl group are specified.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a specific isomer of the source substance, tetrahydromethylphthalic anhydride (MTHPA), in which neither the location of the double bond nor the methyl substitution are defined. MTHPA, has a stated composition comprising the target substance 1,2,3,6-tetrahydro-3-methylphthalic anhydride (CAS No. 5333-84-6, EC No. 226-247-6) together with tetrahydro-4-methylphthalic anhydride (CAS No. 34090-76-1, EC No. 251-823-9), 1,2,3,6-tetrahydro-4-methylphthalic anhydride (CAS No. 3425-89-6, CAS No. 222-323-8) and 3-cyclohexene-1,2-dicarboxylic anhydride, 4-methyl- (CAS No. 19438-64-3).


3. ANALOGUE APPROACH JUSTIFICATION
Generally, the physical and chemical properties do not show major differences. All the above mentioned cyclic anhydrides have comparable boiling temperatures, low vapour pressure and, from a physiological point of view, similar partition coefficients. Differences in melting point may be explained by the fact that the substances are representing multi-constituent or mono-constituent substances.
The cyclic anhydrides rapidly hydrolyse in contact with water and the methyl substituted cyclic anhydrides appear to show a low potential for biodegradation. Based on their physico-chemical properties the substances are expected to have low bioaccumulation potentials and comparable adsorption/desorption properties. The dicarboxylic acid degradation product arising from hydrolysis is the moiety of concern with respect to effects in the aquatic environment. The substances have a rather a low potential to cause toxicity to water based species including fish, daphnia, algae and microorganisms.
Acute toxicity is low. All of the substances are expected to be highly irritant to the eye and be both skin and respiratory sensitisers and a harmonised classification is in place for these properties (Index No. 607-240-000). The absence of a mutagenic potential has been demonstrated in various guideline in-vitro tests. The outcome of the available mammalian studies conducted by the oral route suggest local effects (irritation of the stomach mucosa) probably arising from pH effects of the di-acid degradation product to be significant.
Due to structural similarities, comparable physical/chemical properties such as the molecular weight, partition coefficient and vapour pressure, the toxicokinetic profile of the registered substance and the potential structural analogue substances are also expected to be comparable in terms of physiological absorption, distribution, metabolism and excretion processes. The carboxylic acid anhydride group which rapidly hydrolyses to form the di-acid once in contact with physiological liquid, mainly determine the fate of the substance within the body. Available toxicokinetic data demonstrate that cyclic anhydrides are commonly metabolised to the corresponding di-carboxylic acids within the body and are finally excreted in urine.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
JAPAN: Guidelines for Screening Mutagenicity Testing Of Chemicals
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
Principles of method if other than guideline:
Guidelines for screening Mutagenicity testing of Chemicals (Japan) and OECD Test Guideline 471 and 472
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
not indicated
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media: provided by Dr. A. N. Ames, California University, USA on 1975-10-31
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media: provided by Dr.Gada, National Institute of Genetics, Japan on 1979-5-9
- Properly maintained: yes, -80 °C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
S9 mix
Test concentrations with justification for top dose:
-S9 mix; 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate
+S9 mix; 0, 156, 313, 625, 1250, 2500, 5000 ug/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: well-known solvent for general purpose
Details on test system and experimental conditions:
Type: Ames test
METHOD OF APPLICATION: in medium; in agar (plate incorporation); preincubation; in suspension; as impregnation on paper disk


DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS:2

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other: The test substance is considered to be positive for mutagenic activity when assay plates with the test substance show a significant increase in revertant colony count as compared with that on negative control plates and when this effect is reasonably reproducible or dose dependent.
Evaluation criteria:
no details given
Statistics:
no details given
Species / strain:
other: Salmonella typhimurium, TA100, TA1535, TA98, TA1537, Escherichia coli Wp2 uvrA
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
other: without metabolic activation(-S9mix)| 500 ug/plate (TA1535),| 1000 ug/plate (TA100, TA98, TA1537),| 2500 ug/plate (WP2)|with metabolic activation(+S9mix)| 5000 ug/plate (TA100, TA1537)
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
This chemical did not induce mutations in the S. typhimurium and E. coli strains.

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: 7.4
Remarks on result:
other: other: Salmonella typhimurium, TA100, TA1535, TA98, TA1537, Escherichia coli Wp2 uvrA

no remarks

Conclusions:
Interpretation of results: negative

Tetrahydromethylphthalic anhydride (MTHPA) was not mutagenic in Salmonella typhimuriumTA100, TA1535, TA98, TA1537 and Escherichia coli WP2uvrA at concentrations up to 5 mg/plate or 2 mg/plate, with or without an exogenous metabolic activation system, respectively.
Executive summary:

In a reverse gene mutation assay in bacteria, strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA were exposed to tetrahydromethylphthalic anhydride in DMSO at concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of mammalian metabolic activation (S9 mix, Rat liver, induced with Phenobarbital and 5,6-benzoflavone).

Tetrahydromethylphthalic anhydride was tested up to limit concentration (5000 µg/plate). No cytotoxicity occurred. The test chemical did not induce mutations in the S. typhimurium and E. coli strains The positive controls induced the appropriate responses in the corresponding strains. There was no evidence of induced mutant colonies over background.

This study is classified as acceptable. This study satisfies the requirement for the Japanese Guidelines for Screening Mutagenicity Testing Of Chemicals Test Guideline and OECD 471 and 472 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint:
in vitro cytogenicity / chromosome aberration study in mammalian cells
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
2009-11-2 to 2010-1-4
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Structural similarities of the target substance to the source substances include the (bi)cyclic ring structure with a carboxylic acid anhydride group as the single reactive moiety. For the target substance the bicyclic ring structures contains a double bond at a specific location within the ring and also contains a substituted methyl group at a specific location on the ring structure whereas for the source substance neither the position of the double bond nor the methyl group are specified.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a specific isomer of the source substance, tetrahydromethylphthalic anhydride (MTHPA), in which neither the location of the double bond nor the methyl substitution are defined. MTHPA, has a stated composition comprising the target substance 1,2,3,6-tetrahydro-3-methylphthalic anhydride (CAS No. 5333-84-6, EC No. 226-247-6) together with tetrahydro-4-methylphthalic anhydride (CAS No. 34090-76-1, EC No. 251-823-9), 1,2,3,6-tetrahydro-4-methylphthalic anhydride (CAS No. 3425-89-6, CAS No. 222-323-8) and 3-cyclohexene-1,2-dicarboxylic anhydride, 4-methyl- (CAS No. 19438-64-3).


3. ANALOGUE APPROACH JUSTIFICATION
Generally, the physical and chemical properties do not show major differences. All the above mentioned cyclic anhydrides have comparable boiling temperatures, low vapour pressure and, from a physiological point of view, similar partition coefficients. Differences in melting point may be explained by the fact that the substances are representing multi-constituent or mono-constituent substances.
The cyclic anhydrides rapidly hydrolyse in contact with water and the methyl substituted cyclic anhydrides appear to show a low potential for biodegradation. Based on their physico-chemical properties the substances are expected to have low bioaccumulation potentials and comparable adsorption/desorption properties. The dicarboxylic acid degradation product arising from hydrolysis is the moiety of concern with respect to effects in the aquatic environment. The substances have a rather a low potential to cause toxicity to water based species including fish, daphnia, algae and microorganisms.
Acute toxicity is low. All of the substances are expected to be highly irritant to the eye and be both skin and respiratory sensitisers and a harmonised classification is in place for these properties (Index No. 607-240-000). The absence of a mutagenic potential has been demonstrated in various guideline in-vitro tests. The outcome of the available mammalian studies conducted by the oral route suggest local effects (irritation of the stomach mucosa) probably arising from pH effects of the di-acid degradation product to be significant.
Due to structural similarities, comparable physical/chemical properties such as the molecular weight, partition coefficient and vapour pressure, the toxicokinetic profile of the registered substance and the potential structural analogue substances are also expected to be comparable in terms of physiological absorption, distribution, metabolism and excretion processes. The carboxylic acid anhydride group which rapidly hydrolyses to form the di-acid once in contact with physiological liquid, mainly determine the fate of the substance within the body. Available toxicokinetic data demonstrate that cyclic anhydrides are commonly metabolised to the corresponding di-carboxylic acids within the body and are finally excreted in urine.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
in vitro mammalian chromosome aberration test
Target gene:
no details given
Species / strain / cell type:
Chinese hamster lung fibroblasts (V79)
Details on mammalian cell type (if applicable):
- Type and identity of media: provided by ECACC (European Collection of Cells Cultures)
- Properly maintained: yes, -80 +/- 10°C
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Type and composition of metabolic activation system:
- source of S9 : S9 fraction of Phenobarbital (PB) and β-naphthoflavone (BNF) induced rat liver
- method of preparation of S9 mix: Complete S9 mix was freshly prepared containing components with the following ratios:
S9 fraction - 3 mL
HEPES (N-2-Hydroxyethylpiperazine-N-2-Ethane Sulphonic Acid) 20 mM - 2 mL
KCl 330 mM - 1 mL
MgCl2 50 mM - 1 mL
NADP (β-Nicotinamide Adenine Dinucleotide Phosphate) 40 mM - 1 mL
Glucose-6-phosphate 50 mM - 1 mL
DME medium - 1 mL
Test concentrations with justification for top dose:
Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/mL test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/mL test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/mL test item.
Vehicle / solvent:
solvent: DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
ethylmethanesulphonate
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-Nitrosodimethylamine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
DURATION
Experiment A with 3/20 h treatment/sampling time without and with S9 mix: 19.53, 39.06, 78.12, and 156.25 μg/ml test item.
Experiment B with 20/20 h treatment/sampling time without S9 mix: 1.22, 2.44, 4.88, 9.76 and 19.53 μg/ml test item.
Experiment B with 20/28 h treatment/sampling time without S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.
Experiment B with 3/28 h treatment/sampling time with S9 mix: 4.88, 9.76, 19.53, 39.06 and 78.12 μg/ml test item.

Preparation of cultures
Cell cultures were treated with Colchicine (0.2 μg/ml) two hours prior to harvest. Following the selection time, cells were swollen with 0.075 M KCl hypotonic solution, then washed in fixative (approx. 10 min. in 3:1 mixture of methanol: acetic-acid until the preparation becomes plasma free) and dropped onto slides and air-dried. The preparation was stained with 5 % Giemsa for subsequent scoring of chromosome aberration frequencies. For control of bias, all slides were coded and scored blind.
Evaluation criteria:
The Chromosome Aberration Assay is considered valid if following criteria are met:
– the number of aberrations found in the negative and /or solvent controls falls within the range of historical laboratory control data.
– the positive control items produce biologically relevant increases in the number of cells with structural chromosome aberrations.
Statistics:
no details given
Species / strain:
Chinese hamster lung fibroblasts (V79)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Data on pH: No effects
- Data on osmolality: No effects
- Possibility of evaporation from medium: n/a
- Water solubility: n/a
- Precipitation and time of the determination: Not observed

RANGE-FINDING/SCREENING STUDIES
A dose selection cytotoxicity assay was performed to establish an appropriate concentration range for the Chromosome Aberration Assays. Toxicity was determined by cell counting and results noted as cell survival in the treatment group (in %) relative to the solvent control. These results were used to select concentrations ranging from little to maximum(< 50% survival) toxicity for evaluation in the main studies.

STUDY RESULTS
- Concurrent vehicle negative and positive control data : Valid

For all test methods and criteria for data analysis and interpretation:
- Concentration-response relationship where possible : Negative
- Statistical analysis; p-value if any: Negative

Chromosome aberration test (CA) in mammalian cells:
- Genotoxicity results (for both cell lines and lymphocytes)
o Definition for chromosome aberrations, including gaps : The nomenclature and classification of chromosome aberrations were based upon ISCN, 1985 (An International System for Human Cytogenetic Nomenclature, ed. D.G. Harnden et al. S. Karger, Switzerland) and Savage, 1976 (Annotation: Classification and relationships of induced chromosome structural change. Journal of Medical Genetics, 13: 103-122) and 1983 (Some practical notes on chromosomal aberration. Clinical Cytogenetics Bulletin, 1: 64-76).
o Number of cells scored for each culture and concentration, number of cells with chromosomal aberrations and type given separately for each treated and control culture, including and excludling gaps : At least 200 metaphase cells containing 2 N ± 2 centromeres were evaluated for structural aberrations from each experimental group. Chromatid and chromosome type aberrations (gaps, deletions and exchanges) were recorded separately. Additionally the number of polyploid and endoreduplicated cells were scored. No statistically significant differences were noted between treatment and negative control groups.
o Changes in ploidy (polyploidy cells and cells with endoreduplicated chromosomes): Not seen

HISTORICAL CONTROL DATA (with ranges, means and standard deviation, and 95% control limits for the distribution as well as the number of data)
- Positive historical control data:
3/20 hour treatment/sampling time –S9
Range: 20-33; mean: 26.00; standard deviation: 3.52; number of data: 8
3/20 hour treatment/sampling time +S9
Range: 17-22; mean: 21.50; standard deviation: 2.03; number of data: 8
20/28 hour treatment/sampling time –S9
Range: 22-35; mean: 27.94; standard deviation: 3.32; number of data: 8
3/28 hour treatment/sampling time +S9
Range: 18-26; mean: 20.75; standard deviation: 1.98; number of data: 8
- Negative (solvent/vehicle) historical control data:
3/20 hour treatment/sampling time –S9
Range: 2-6; mean: 3.31; standard deviation: 1.08; number of data: 8
3/20 hour treatment/sampling time +S9
Range: 2-5; mean: 3.00; standard deviation: 0.89; number of data: 8
20/28 hour treatment/sampling time –S9
Range: 2-5; mean: 3.38; standard deviation: 0.72; number of data: 8
3/28 hour treatment/sampling time +S9
Range: 2-5; mean: 3.38; standard deviation: 0.96; number of data: 8
Conclusions:
Interpretation of results: Negative

Tetrahydromethylphthalic anhydride (MTHPA) tested up to cytotoxic concentrations, both with and without metabolic activation, did not induce structural chromosome aberrations in this test in Chinese Hamster lung cells. Therefore, tetrahydromethylphthalic anhydride (MTHPA) is considered as not clastogenic in this system.
Executive summary:

Tetrahydromethylphthalic anhydride (MTHPA), of which 3 -MTHPA D4 is an isomer, has been tested in a Chromosome Aberration Assay in V79 cells using OECD test methods.

There were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted. There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases.

Tetrahydromethylphthalic anhydride (MTHPA) was therefore considered as not clastogenic in this test system. 

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From 2009-09-14 to 2009-12-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study without detailed documentation
Justification for type of information:
1. HYPOTHESIS FOR THE ANALOGUE APPROACH
Structural similarities of the target substance to the source substances include the (bi)cyclic ring structure with a carboxylic acid anhydride group as the single reactive moiety. For the target substance the bicyclic ring structures contains a double bond at a specific location within the ring and also contains a substituted methyl group at a specific location on the ring structure whereas for the source substance neither the position of the double bond nor the methyl group are specified.


2. SOURCE AND TARGET CHEMICAL(S) (INCLUDING INFORMATION ON PURITY AND IMPURITIES)
The target substance is a specific isomer of the source substance, tetrahydromethylphthalic anhydride (MTHPA), in which neither the location of the double bond nor the methyl substitution are defined. MTHPA, has a stated composition comprising the target substance 1,2,3,6-tetrahydro-3-methylphthalic anhydride (CAS No. 5333-84-6, EC No. 226-247-6) together with tetrahydro-4-methylphthalic anhydride (CAS No. 34090-76-1, EC No. 251-823-9), 1,2,3,6-tetrahydro-4-methylphthalic anhydride (CAS No. 3425-89-6, CAS No. 222-323-8) and 3-cyclohexene-1,2-dicarboxylic anhydride, 4-methyl- (CAS No. 19438-64-3).


3. ANALOGUE APPROACH JUSTIFICATION
Generally, the physical and chemical properties do not show major differences. All the above mentioned cyclic anhydrides have comparable boiling temperatures, low vapour pressure and, from a physiological point of view, similar partition coefficients. Differences in melting point may be explained by the fact that the substances are representing multi-constituent or mono-constituent substances.
The cyclic anhydrides rapidly hydrolyse in contact with water and the methyl substituted cyclic anhydrides appear to show a low potential for biodegradation. Based on their physico-chemical properties the substances are expected to have low bioaccumulation potentials and comparable adsorption/desorption properties. The dicarboxylic acid degradation product arising from hydrolysis is the moiety of concern with respect to effects in the aquatic environment. The substances have a rather a low potential to cause toxicity to water based species including fish, daphnia, algae and microorganisms.
Acute toxicity is low. All of the substances are expected to be highly irritant to the eye and be both skin and respiratory sensitisers and a harmonised classification is in place for these properties (Index No. 607-240-000). The absence of a mutagenic potential has been demonstrated in various guideline in-vitro tests. The outcome of the available mammalian studies conducted by the oral route suggest local effects (irritation of the stomach mucosa) probably arising from pH effects of the di-acid degradation product to be significant.
Due to structural similarities, comparable physical/chemical properties such as the molecular weight, partition coefficient and vapour pressure, the toxicokinetic profile of the registered substance and the potential structural analogue substances are also expected to be comparable in terms of physiological absorption, distribution, metabolism and excretion processes. The carboxylic acid anhydride group which rapidly hydrolyses to form the di-acid once in contact with physiological liquid, mainly determine the fate of the substance within the body. Available toxicokinetic data demonstrate that cyclic anhydrides are commonly metabolised to the corresponding di-carboxylic acids within the body and are finally excreted in urine.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Type of assay:
mammalian cell gene mutation assay
Target gene:
The mouse lymphoma assay (MLA), employing the tk+/- (thymidine kinase) locus in L5178Y cells has been used.
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
The original L5178Y TK+/- 3.7.2 C mouse lymphoma cell line was obtained from the American Type Culture Collection.
Cells are stored as frozen stocks in liquid nitrogen. Each batch of frozen cells was purged of TK-/--mutants and checked for the absence of mycoplasma.
For each experiment, one vial was thawed rapidly, the cells were diluted in RPMI 10 medium and incubated at 37 +/- 1 °C in a humidified atmosphere containing approximately 5 % CO2 in air. Subcultures were established in an appropriate number of flasks from well-growing cells.
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
The test item was investigated in the presence of an appropriate metabolic activation system, which is a cofactor-supplemented post-mitochondrial fraction (S9).
Test concentrations with justification for top dose:
Assay 1:
3-hour treatment, in absence of exogeneous metabolic activation (-S9):
5.12; 12.8; 32; 80; 140 and 200 μg/mL.
3-hour treatment, in presence of exogeneous metabolic activation (+S9):
38.4; 96; 240; 600; 1200 and 1800 μg/mL.
Assay 1 (additional test):
3-hour treatment (-S9 Mix): 80; 140; 200; 400; 800 and 1200 μg/mL.
Assay 2:
3-hour treatment (+S9 Mix): 96; 240; 600; 1200; 2000 and 3000 μg/mL.
24-hour treatment (-S9 Mix): 25.6; 64; 160; 280; 400 and 800 μg/mL.
Vehicle / solvent:
DMSO
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
Details on test system and experimental conditions:
Assay 1.:
A 3-hour treatment was performed in the presence and absence of S9 Mix.

Assay 2.:
A second assay was performed because of the verification and completion of the results obtained in the Assay 1 was required based on OECD Guideline 476.
In the Assay 2 a 24-h treatment (without S9) was performed according to the ICH recommendation.
A 24-hour treatment was performed without metabolic activation, and a 3-hour treatment with addition of metabolic activation.
Evaluation criteria:
The test item is considered to be mutagenic in this assay if all the following criteria are met (based on Moore et al.):
1. The assay is valid;
2. Statistically significant (p < 0.05) increases in mutation frequency are observed in treated cultures compared to the corresponding negative control values at one or more concentrations;
3. The increases are reproducible between replicate cultures and between tests (when treatment conditions were the same).
4. There is a significant dose-relationship as indicated by the adequate trend analysis;
5. The mutation frequency at the test concentration showing the largest increase is at least 126 mutants per 106 viable cells (GEF = the Global Evaluation Factor) higher than the corresponding negative control value.
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 data were checked for a linear trend in mutant frequency with treatment dose using the adequate regression analysis.
Species / strain:
mouse lymphoma L5178Y cells
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
Results of the Assay 1.:
The treatment duration was 3h and the treatment was performed in presence and also in absence of exogenous metabolic activation (S9 Mix).
In this assay the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with historical data. The parallel tested positive control chemicals induced a statistically significant increase in the mutant frequency (2 Sample t-Test, α = 0.01) additionally the IMF (Induced Mutation Frequency: number of mutants over the control value per 10exp6 viable cells) exceeded the GEF (Global Evaluation Factor, which is equal to 126).
In this assay no cytotoxicity [based on the harmonised relative survival (harmonised RS) and RTG data] was observed at the applied test item concentrations in the absence of S9 Mix, and 65.8 % toxicity (based on the harmonised RS data) was obtained at the concentration level of 1800 μg/mL in presence of S9 Mix.
The evaluated mutation frequencies obtained in the First Assay (Assay 1) did not show any significant differences from the mutation frequency of the corresponding vehicle control and did not show dose-response relationship at the applied concentrations (Dunnett’s Test, α = 0.05). Additionally, the MF values of test item treatments were far below the GEF criterion for positive call.
Precipitation of the test item was not observed during the treatments (either in the presence and absence of S9 Mix).

Results of the Assay 2.:
The treatment duration was 3h in presence of metabolic activation (S9 Mix) and 24h in absence of metabolic activation (S9 Mix).
In the Assay 2 the plating efficiencies of the negative and positive controls in the viability test (PEviability) as well as the mutation frequency of the current negative control were within the acceptable ranges and in accordance with historical data. The parallel tested positive control chemicals induced a statistically significant increase (high above the GEF) in the mutant frequency (2 Sample t-Test, α = 0.01).
3h treatment in presence of S9 Mix:
At the highest examined concentration level of 3000 μg/mL strong cytotoxic effect of the test item was observed.
In this part of the Assay 2 the obtained mutation frequencies were statistically evaluated at the concentration range of 96-2000 μg/mL. The mutation frequency of the concentration level of 2000 μg/mL was statistically significantly higher than the mutation frequency of the vehicle control (Dunnett’s Test, α = 0.05). A slight dose-relationship was also observed between the variances. The higher value remained below the GEF criterion for positive call (IMF: 98), furthermore at this concentration high level of toxicity (89.12 %) was observed.
The obtained statistical significance was not considered as a sign of the mutagenic effect of the test item.
24h treatment in absence of S9 Mix:
The concentration level of 400 μg/mL was very toxic, resulted 2.39 % relative survival (based on the harmonised RS). 12.81 % relative survival was achieved at the concentration level of 280 μg/mL. The relative survival data at the concentration range of 25.6- 160 μg/mL changed in the range of the biological variability of the test.
The mutation frequencies at the concentration levels of 280 and 160 μg/mL were statistically significantly higher than the mutation frequency of the vehicle control (Dunnett’s Test, α = 0.05). The mutation frequencies showed clear dose-related increase, that would confirm a putative potential mutagenic effect of the test item, but similarly to the results of the 3-hour treatments the mutation frequencies remained below the GEF criterion (IMF: 114 at the concentration level of 280 μg/mL and 40 at the concentration level of 160 μg/mL).

Summary of the overall study:
In all phases of the Assay 2 there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than the mutation frequencies of the corresponding vehicle control (Dunnett’s Test, α = 0.05). However, the changes of the mutation frequencies were not dose-related, and the GEF criterion for positive call was not attained in any case. In the case of the 3-hour treatments (in absence of S9 at the Completed Assay 1) the higher frequencies were observed in the repeated (partially completed) assays only. In the Assay 1 no statistical significant differences were detected between the treatment and vehicle control mutation rates. Thus, the observed statistical significances were not evaluated as biologically relevant.
Conclusions:
Interpretation of results: negative

Under the conditions of this study, the test item tetrahydromethylphthalic anhydride (MTHPA) does not induce gene mutations in presence and absence of metabolic activation in the cultured mammalian cells used.
Executive summary:

An in vitro mammalian cell assay was performed in mouse lymphoma L5178Y TK+/- 3.7.2 C cells at the tk locus to test the potential of tetrahydromethylphthalic anhydride (MTHPA), of which 3 -MTHPA D4 is an isomer, to cause gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (S9 Mix) and for 24 hours without metabolic activation following OECD/EU test methods.

In the first assay, mutation frequencies did not show any statistical or biological significant differences from controls. In a second assay there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than mutation frequencies of the vehicle control. However, while the changes in mutation frequencies were dose-related, the GEF criterion for these to be regarded as positive was not achieved and the obtained statistical significances were regarded as not biologically relevant.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Various studies have been undertaken on tetrahydromethylphthalic anhydride (MTHPA). This is a structural analogue of 3 -MTHPA D4 in which the methyl substitution is not defined or fixed in a specific position on the carbon cyclic structure, as opposed to 3 -MTHPA D4 where the methyl substitution is fixed at the 3C position. 3 -MTHPA D4 is regarded as a specific isomer of MTHPA.

Bacterial cell gene mutation assay:

A reverse gene mutation assay (Ames test) has been conducted using the pre-incubation method with strains TA100, TA1535, TA98, TA1537 of S. typhimurium and Escherichia coli Wp2 uvrA. These were exposed to concentrations of 0, 62.5, 125, 250, 500, 1000, 2000 ug/plate (-S9 mix) and 0, 156, 313, 625, 1250, 2500, 5000 ug/plate (+S9 mix) in the presence and absence of a mammalian metabolic activation system. No cytotoxicity occurred and the substance did not induce mutations in the S. typhimurium and E. coli strains examined.

Cytogenicity:

A chromosome aberration assay in V79 cells has been conducted using OECD test methods. There were no biologically significant increases in the number of cells showing structural chromosome aberrations, either in the absence or in the presence of metabolic activation, up to and including cytotoxic concentrations. There were no statistical differences between treatment and control groups and no dose-response relationships were noted.There were no biologically relevant increases in the rate of polyploid or endoreduplicated metaphases. The substance was therefore considered as not clastogenic in this test system

In a second study, CHL/IU cell cultures/primary lymphocyte cultures were exposed to concentrations of 0, 0.075, 0.15, 0.30 and 0.60 mg/mL with and without metabolic activation. Structural chromosomal aberrations were not induced up to 0.30 mg/mL (24 and 48 hours continuous treatment without S9). Polyploidy (1.13%) was increased at 0.30 mg/mL with 48 hours continuous treatment without metabolic activation. Polyploidy (1.25 -1.88 %) was statistically increased at 0.11 -0.43 mg/mL (all concentrations) with short-term treatment in the presence of S9, indicating a potential effect of the substance. Whereas this study showed no indication of clastogenic properties, a polyploidy inducing effect cannot be excluded. Therefore a second test was performed. However, the biological relevance of the observed variation was considered equivocal as the test design is of limited use to investigate such effects.

Mammalian cell gene mutation assay:

An in vitro mammalian cell assay has been performed in mouse lymphoma L5178Y TK+/- cells to test the potential for gene mutation and/or chromosome damage. Treatments were carried out for 3 hours with and without metabolic activation (S9 Mix) and for 24 hours without metabolic activation following OECD/EU test methods. In the first assay, mutation frequencies did not show any statistical or biological significant differences from controls. In a second assay there were one or two concentration levels where the obtained mutation frequencies were statistically significantly higher than mutation frequencies of the vehicle control. However, while the changes in mutation frequencies were dose-related, the GEF criterion for these to be regarded as positive was not achieved and the obtained statistical significances were regarded as not biologically relevant.


Short description of key information:
Genotoxicity in-vitro: Bacterial reverse mutation - Negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

Based on results of the three different in vitro genetic toxicity studies tetrahydromethylphthalic anhydride (MTHPA), of which 3 -MTHPA D4 is an isomer, was not classified and labelled as genotoxic according to Directive 67/548/EEC (DSD) and to Regulation 1272/2008/EC (CLP).