Phthalic anhydride

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Phthalic anhydride was shown to be not mutagenic in the Ames test (OECD 471) and in the mammalian cell gene mutation assay (OECD 476), both performed with and without metabolic activation. In a reliable in vitro chromosomal aberration tests phthalic anhydride was found negative. In another study chromosomal aberrations were induced in mammalian cells in vitro at the highest phthalic anhydride concentrations (10 mM) only in the absence of S9 mix with concomitant marked cytotoxicity and compound precipitate.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

Preincubation method according to OECD TG 471.

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

No data

Species / strain / cell type:

other: Salmonella typhimurium TA100, TA 102, TA1535, TA98, TA1537, E. coli WP2uvrA

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

Preparation of S9 fraction:
Male Sprague-Dawley rats were used for the preparation of liver fractions. Sodium phenobarbital and 5,6-benzoflavone were used as an inducer of the rat metabolic activation system. Sodium phenobarbital was injected intraperitoneally into the rats 4 days before killing and 1, 2 and 3 days before killing 5,6 benzoflavone was injected intraperitoneally. From these rats liver S9 fraction was prepared according to Ames et al. (1975) Methods for detecting carcinogens and mutagens in the Salmonella /mammalian microsome mutagenicity test. Mutat. Res. 31, 347-364. S9 was dispensed into freezing ampules and stored at -80° C. Once the stock S9 had been thawed, remained S9 was not reused.

Test concentrations with justification for top dose:

0, 20, 39, 78, 156, 313, 625, 1250, 2500, 5000 ug/plate dissolved in DMSO

Vehicle / solvent:

solvent: DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: see 'Any other information on materials and methods incl. tables'

Details on test system and experimental conditions:

Ames test

Evaluation criteria:

The chemicals are considered to be mutagenic when dose-related increase in revertant colony count is observed and the number of revertant colonies per plate with the test substance is more than twice that of the negative control (solvent control) and when a reproducibility of the test result is observed.

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98 and TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: 2500 ug/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: negative

Phthalic anhydride was tested up to 5000 µg/plate. Cytotoxic effects were observed in the absence or in the presence of metabolic activator at concentrations equal or higher than 5000 or 313 µg/plate, respectively. Phthalic anhydride did not induce mutations in the bacterial mutation test, neither in the absence, nor in the presence of metabolic activator.

Positive and negative controls gave the expected values.

Executive summary:

Ames test preincubation method according to OECD TG 471.

Result: negative, phthalic anhydride did not induce mutations in the bacterial mutation test, neither in the absence or presence of metabolic activator.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Version / remarks:

1998

Principles of method if other than guideline:

The test item Phthalic Anhydride was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster.

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

no data

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix
The S9 liver microsomal fraction was prepared at BSL BIOSERVICE GmbH. Male Wistar rats were induced with Phenobarbital (80 mglkg bw) and B-Naphthoflavone (l00 mg/kg bw) for three consecutive days by oral route.

Test concentrations with justification for top dose:

The selection of the concentrations was based on data from the pre-experiments. Seven concentrations [0.0107, 0.034, 0.107, 0.34, 1.07,3.4, 10.7 roM] were tested with and without metabolic activation. The experimental conditions in these preexperiments were the same as described below for the main experiment.

In all main experiments the limit concentration of 10 mM was selected as the highest concentration.
Experiment I
with metabolic activation: 0.10,0.25,0.5,1.0,2.5,5.0,7.5 and 10 mM
and without metabolic activation: 0.025,0.05,0.5, 1.0,2.5,5.0, 7.5 and 10 mM
Experiment II
without metabolic activation: 0.10,0.25,0.5,1.0,2.5,5.0,7.5 and 10 mM
and with metabolic activation: 1.0,1.75,2.5,4.0,5.5,7.0,8.5 and 10 mM

Vehicle / solvent:

The test item was dissolved in cell culture medium (MEM + 0% FBS 4h treatment; MEM + 10% FBS 20h treatment) and diluted prior to treatment. The solvent was compatible with the survival of the cells and the S9 activity.

Additional Information: Phthalic Anhydride hydrolyses rapidly in the presence of water forming phthalic acid. The kinetic of the hydrolysis of phthalic anhydride was studied in buffered media. A half-life for phthalic anhydride of 30.5 seconds at pH
7.24 at 25°C was determined (Andres GO, Granados AM, Rossi RH (2001). Kinetic study on the hydrolysis of phthalate anhydride and aryl hydrogen phthalate. J. Org. Chern. 66, 7653-7657).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Ethylmethanesulfonate; 7,12-Dimethylbenz(a)anthracene

Details on test system and experimental conditions:

Mammalian cell culture systems are used to detect mutations induced by chemical substances. This in vitro experiment was performed to assess the potential of the test item to induce gene mutations by means of a HPRT (hypoxanthine-guaninephosphoribosyl-
transferase) assay using the Chinese Hamster V79 cell line. The HPRT system detects base pair mutations, frameshift mutations and small deletions. These are considered to be an initial step in the process leading to carcinogenesis. These cells are exposed to different concentrations of the test item, both with and without metabolic activation for a suitable period of time and subcultured to determine cytotoxicity and to allow phenotypic expression prior to mutant selection.
HPRT catalyses the conversion of the non-toxic 6-TO (6-thioguanine) to its toxic phosphorylated derivative. Cells deficient in HPRT are selected by resistance to 6-thioguanine. The deficiency of the "salvage" enzyme HPRT means that antimetabolites such as 6-TO are not incorporated into cellular nucleotids and the nucleotids needed for cellular metabolism are obtained solely from de novo synthesis. However, in the presence of the HPRT-enzyme, 6-TO is incorporated into cellular nucleotides, resulting in inhibition of cellular metabolism and cytotoxicity. Thus mutant cells are able to proliferate in the presence of 6-TO, whereas normal cells, which contain HPRT, are not.

Cells as monolayer cultures are exposed to the test item for a defined period of time (4h for short time exposure or 20 h for long time exposure). Cytotoxicity is determined by measuring the growth rate of the cultures.
The treated cultures are maintained in growth medium for 5-8 days to allow nearoptimal phenotypic expression of induced mutations. Mutant frequency is determined by seeding defined numbers of cells in medium containing the selective agent (6-TO) to detect mutant cells and in medium without selective medium to determine the cloning efficiency. After a suitable incubation time, cell colonies are counted. The number of mutant colonies in selective medium is adjusted by the number of colonies in non-selective medium to derive the mutant frequency.

There is no requirement for the verification of positive results. Negative or equivocal results should be clarified by further testing using modified experimental conditions. Study parameters which might be changed are concentrations, treatment time or metabolic activation conditions.
To establish a concentration response of the test item at least eight concentrations (single cultures) are tested. These concentration levels should yield a concentration related toxic effect. The highest concentration should induce a reduced level of survival of approximately 10-20% relative survival. The lowest concentration should be in the range of the negative control with respect to cell viability and proliferation.
For soluble, non-toxic test compounds the recommended maximum test concentration will be 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. Solvent or negative controls will be tested in duplicate.
Reference mutagens are tested in parallel to the test item in order to demonstrate the sensitivity of the test system.

Rationale for test conditions:

The selection of the concentrations was based on data from the pre-experiments. In all experiments 10 mM was selected as the highest concentration.
The test item was investigated at the following concentrations:
Main Experiment I
with metabolic activation: 0.10,0.25,0.5,1.0,2.5,5.0,7.5 and 10 mM
and without metabolic activation: 0.025,0.05,0.5, 1.0,2.5,5.0, 7.5 and 10 mM
Main Experiment II
without metabolic activation: 0.10,0.25,0.5,1.0,2.5,5.0,7.5 and 10 mM
and with metabolic activation: 1.0,1.75,2.5,4.0,5.5,7.0,8.5 and 10 mM

Evaluation criteria:

Evaluation of results:
A test is considered to be negative if there is no biological relevant increase in the number of mutants.
There are several criteria for determining a positive result:
- a reproducible three times higher mutation frequency than the solvent control for at least one of the concentrations;
- a concentration related increase of the mutation frequency; such an evaluation may be considered also in the case that a three-fold increase of the mutant frequency is not observed;
According to the OECD guidelines, the biological relevance of the results is the criterion for the interpretation of results, a statistical evaluation of the results is not regarded as necessary.

A mutation assay is considered acceptable if it meets the following criteria:
- Negative and/or solvent controls fall within the performing laboratories historical control data range: 4-47 mutants/10 EE6 cells
- The absolute cloning efficiency: ([number of positive cultures x 100] I total number of seeded cultures) of the negative and lor solvent controls is > 50%
- The spontaneous mutant frequency in the negative and/or solvent controls is in the range of BSL BIOSERVICE historical control data
- The positive controls (EMS and DMBA) induce significant increases (at least 3-fold increase of mutant frequencies related to the comparable negative control values and higher than the historical range of negative controls) in the mutant frequencies.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity nor precipitates, but tested up to recommended limit concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks:

DMBA and EMS

No precipitation of the test item was noted in the experiments.

No biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation with one exception. In experiment II without metabolic activation for the highest concentration evaluated (l0 mM) the relative growth was 17.6%.

In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose-response relationship was observed.

In detail:

In experiment I without metabolic activation mutant values of the negative controls and all test item concentrations found were within the historical control data of the test facility BSL BIOSERVICE (about 4-47 mutants per 10 EE6 cells). Mutation frequencies with the negative control without metabolic activation were found to be 5.56 and 11.43 mutants per 10 EE6 cells and in the range of 8.50 to 22.56 mutants per 10 EE6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.66 was found at a concentration of 5 mM with a relative growth of 123.9. No dose-response relationship could be observed.

With metabolic activation all mutant values of the negative controls and all test item concentrations were found within the historical control data of the test facility BSL BIOSERVICE (about 4-46 mutants per 10 EE6 cells). The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the solvent controls. No dose-response relationship could be observed. Mutation frequencies of the negative control were found to be 5.92 and 7.10 mutants per 10 EE6 cells and in the range of 6.14 to 13.71 mutants per 10 EE6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.11 was found at a concentration of 0.25 mM with a relative growth of 96.7%.

In experiment II without metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 4-47 mutants per 106 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies with the negative control were found to be 14.95 and 27.67 mutantsll06 cells and in the range of 7.89 to 26.75 mutantsll06 cells with the test item, respectively. The highest mutation rate (compared to the negative controls values) of 1.25 was found at a concentration of 5.0 mM with a relative growth of 107.2%.

In experiment II with metabolic activation all mutant values found were within the historical control data of the test facility BSL BIOSERVICE (about 4-46 mutants per 10 EE6 cells). No dose-response relationship could be observed. The mutation frequencies found in the groups treated with the test item did not show a biologically relevant increase as compared to the negative controls. Mutation frequencies of the negative control were found to be 4.67 and 6.97 mutants per 10 EE6 cells and in the range of 6.15 to 11.93 mutants/10 EE6 cells with the test item, respectively. The highest mutation rate (compared to the negative control values) of 2.05 was found at a concentration of 1.0 mM with a relative growth of 83.2%.

DMBA (1.5 µg/mL) and EMS (300 µg/mL) were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Conclusions:

Negative

Executive summary:

The test item Phthalic Anhydride was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster up to the limit concentration of 10 mM. No precipitation ofthe test item was noted in the experiments. No biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation with one exception. In experiment II without metabolic activation for the highest concentration evaluated (l0 mM) the relative growth was 17.6%. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Phthalic Anhydride is considered to be non-mutagenic in the HPRT locus using V79 cells.

Reference 3

Endpoint:

in vitro DNA damage and/or repair study

Remarks:

Type of genotoxicity: DNA damage and/or repair

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No data on purity of the compound

Principles of method if other than guideline:

CHO cells were incubated with test compound, positive control or solvent (Dimethylsulfoxide=DMSO).
A) Cells were incubated for 2 hours at 37 °C in the absence of S9-mix. BrdU was added and incubation was continued for 23.5 hours. Cells were washed, fresh medium containing BrdU and colcemid was added and incubation was continued for 2 to 3 hours. Cells were then collected by mitotic shake-off, fixed, air-dried and stained.
B)Cells were incubated for 2 hours at 37°C in the presence of S9-mix and 12 hours in the absence. Cells were washed and medium containing BrdU was added. Cells wee incubated for further 25,5 hours with colcemid present for the final 2 to 3 hours. Cells were collected by mitotic shake-off, fixed, air-dried and stained.

GLP compliance:

yes

Type of assay:

sister chromatid exchange assay in mammalian cells

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix
S-9 mix, consisting of 15 µL/mL liver homogenate (from male SD rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 5.4 mg/mL isocitric acid in serum-free medium

Test concentrations with justification for top dose:

0, 10, 30, 100, 300 ug/ml in DMSO

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: mitomycin c, cyclophosphamide

Details on test system and experimental conditions:

Treatments with the test item:
BrdUrd (10 µM) was added 2 hr after addtion of the test chemical (without S9) or immediately after the S9 mix plus chemical had been removed. The chemical treatment periods were approximately 25 hr without S9 and 2 hr with S9. The total incubation time with BrdUrd was 25-26 hr, with colcemid (0.1 µg/mL) present during the final 2-3 hr. Immediately before the cells were harvested, the cell monolayers were examined, and the degree of confluence and availability of mintoic cells were noted.
Cells were collected by mitotic shake-off, fixed, air-dried and stained.

Rationale for test conditions:

Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hours after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations.

Evaluation criteria:

For individual doses, absolute increases in SCEs per chromosome of 20% or more over the solvent control were considered significant.

Statistics:

Significance of relative SCEs/chromosome tested by linar regression versus log of the dose.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: dose selection was based on preliminary growth inhibition test (no details reported)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: negative

The highest concentration was selected as a cytotoxic level on the basis  of a preliminary study.

Without S9-mix:

Dose   total chromos.  Total SCE per µg/ml    SCE cell    
       no.                
0      2098               1012                 10,13
10     1047                472                  9,47
30     1047                518                 10,39
100    1051                495                  9,89
300    1049                497                  9,95

Positive control: TEM
0.015  1049               1607                 32,14

With S9-mix:

Dose   total chromos.  Total SCE per µg/ml    SCE cell
       no. 
0      1042                470                  9,47
10     1044                459                  9,23
30     1048                472                  9,46
100    1048                458                  9,18
300    1043                501                 10,09

Positive control: CP
1      1047               1043                 20,86

No significant increase in sister chromatid exchanges was observed at any  concentration investigated.

Executive summary:

In a Sister Chromatide Exchange (SCE) assay CHO cells were incubated with the test compound, positive control or solvent (DMSO) for 2 hours with S9 mix and for 25 hours without S9 mix. 2 -Bromodexoxyuridine is used for SCE staining and colcemid for mitotic arrest. Absolute increases in SCEs per chromosome were counted. Phthalic anhydride was negative in this SCE assay.

Reference 4

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no data on compound purity, only 100 metaphases per concentration scored

Principles of method if other than guideline:

Method according Galloway, Environ. Mutagen. 7, 1-51 (1985):
Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured. Test chemicals were supplied under code by the National Toxicology Program chemical repository (Radian Corp., Austin, TX) and were dissolved immediately before use in water, dimethyl sulfoxide (DMSO), ethanol, or acetone, in that order of preference.
Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured. Test chemicals were dissolved immediately before use. Chromosome Aberration Test: Cells were exposed to phthalic anhydride for 2 hours in the presence of metabolic activation (S9), and further incubated for 8-12 hours. In the tests without metabolic activation, the test chemical was left in culture until colcemid addition. This treatment yielded cells in their first mitosis. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix, consisting of 15 µL/mL liver homogenate (from male SD rats, induced with Aroclor 1254), 2.4 mg/mL NADP, and 5.4 mg/mL isocitric acid in serum-free medium

Test concentrations with justification for top dose:

0, 30, 100, 300 µg/ml DMSO
Dose selection was based on a preliminary growth inhibition test in which cells that excluded trypan blue were counted 24 hours after treatment. The top doses selected for the cytogenetics assays were those estimated to reduce growth by 50%. This approach was subsequently modified such that toxicity estimates were made from observations of cell monolayer confluence and mitotic activity in the same cultures used for analysis of SCEs or aberrations.

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: mitomycin-c, cyclophosphamide

Details on test system and experimental conditions:

Chromosome Aberration Test
Cells were exposed to phthalic anhydride for 2 hours in the presence of metabolic activation (S9), and further incubated for 8-12 hours. In the tests without metabolic activation, the cells were exposed to phthalic anhydride throughout the incubation period. This treatment yielded cells in their first mitosis. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis and from positive (triethylenemelamine, mitomycinC, or cyclophosphamid) and solvent control. All types of aberrations were recorded separately, but for data analysis they were grouped into categories of "simple" (breaks and terminal deletions), "complex" (exchanges and rearrangements), "other" (includes pulverized chromosomes), and "total." Gaps and endoreduplications were recorded but were not included in the totals.

Positive Controls
without S9-mix: Mitomycin-C;
with S9-mix: Cyclophosphamide

Statistics:

Armitrage test: Significance of percent cells with aberrations tested by linear regression trend test versus log of the dose.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

other: dose selection was based on preliminary growth inhibition test (no details reported)

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Remarks on result:

other: negative

Cloned

The highest concentration was selected as a cytotoxic level on the basis of a preliminary study.
Phthalic anhydride was negative in the chromosomal aberration assay at any dose tested (10 - 300 µg/ml).

Results without S9 mix:

Dose (µg/mL)	no. of cells	% aberrations total	% aberrations simple	% aberrations complex
0 (solvent control)	100	4	3	1
30	100	5	4	1
100	100	4	4	0
300	100	5	3	2
positive control TEM, 0.15	100	27	21	7

TEM: triethylenemelamine

Results with S9 mix:

Dose (µg/mL)	no. of cells	% aberrations total	% aberrations simple	% aberrations complex
0 (solvent control)	100	3	2	1
30	100	4	2	1
100	100	6	5	1
300	100	5	4	1
positive control CP, 0.15	100	25	15	12

CP: cyclophosphamide

Executive summary:

Cloned Chinese hamster ovary cells (CHO-W-B1) were cultured. Test chemicals were dissolved immediately before use. Chromosome Aberration Test Cells were exposed to phthalic anhydride for 2 hours in the presence of metabolic activation (S9), and further incubated for 8-12 hours. In the tests without metabolic activation, the cells were exposed to phthalic anhydride throughout the incubation period. This treatment yielded cells in their first mitosis. Cells were collected by mitotic shake-off. Slides were stained with Giemsa and coded, and 100 cells were scored from each of the three highest dose groups having sufficient metaphases for analysis. All types of aberrations were recorded.

Phthalic anhydride was negative in the chromosomal aberration assay at any dose tested (10 - 300 µg/ml).

Reference 5

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: no specific positive control included

Principles of method if other than guideline:

CHO cells and test compounds, e.g. phthalic anhydride were incubated at 37° C for 3 h in the presence or absence of S9. Cells were then washed, further incubated and harvested at 20 hours from the beginning of the treatment. Colcemid (0.1 µg/ml) was added 2-3 hours before chromosome aberration and cytotoxicity was tested.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

not applicable

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

6, 8, 10 mM in DMSO

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

other: positve only at the highest compound concentration which was "very toxic" (remaining cell counts 29%) and gave precipitate. Only a small, not statistically significant, increase in aberration was observed at a slightly lower concentration

Cytotoxicity / choice of top concentrations:

other: at the highest compound concentration (remaining cell counts 29%)

Vehicle controls validity:

not specified

Untreated negative controls validity:

not specified

Positive controls validity:

not specified

Additional information on results:

No specific positive controls were used, but there were positive effects with the following test items:
Dithiocarb gave clearly positive results without S9 activation with 16.5% cells with aberrations at 0.1 mM and 18.2% (25/137 cells) at 0.25 mM.
2,4-Dichlorophenol was positive with S9 mix at 0.6 mM (14.5% aberrant cells).

Phthalic anhydride (PA) caused a decrease in pH when added to culture medium and was immediately neutralized with 1N NaOH. In the range-finding experiment with PA, there was no statistically significant increase in aberrations at 10 mM with and without S-9 activation. Without S-9, cell counts were reduced to 59 % of controls at 10 mM. With S-9 there was little toxicity, and precipitate was visible at 8 and 10 mM. (no further data).

In a second experiment more toxicity was seen and there was a marked increase in aberrations, to 18.5 % compared with a control of 3 %, at the top dose of 10 mM without S-9, and only a borderline, nonsignificant increase at 10 mM with S-9.
The positive result with phthalic anhydride was found at a concentration that caused visible precipitate.

The study is of limited reliability because:
Effects were observed only at the highest compound concentration which was "very toxic" (remaining cell counts 29%) and gave precipitate. Only a small, not statistically sigificant, increase in aberration was observed at a slightly lower concentration (8 mM compared to 10 mM) which showed lower cytotoxicity (remaining cell counts 54%) and no precipitate. The authors stated in the discussion: "Although the results we present here are for only two compounds, phthalic anhydride and ethion-amide, the data clearly show not only that precipitate can be present in medium yet not visible even under darkfield microscopy, but also that aberrations induced above the precipitate level can be false-positives, i.e., found with nonmutagenic, noncarcinogens."

Limited documentation e.g.
- no data on precise number of cells scored
- no data on precise number and type of aberrations detected
- no positive control included into the experiment, but positive effects of two test items

Control values in the phthalic anhydride experiment (3%) mentioned in the Results section of the publication were out of the control values described in the Materials and Methods section. "The control levels of aberrations for CHO cells ranged from 0.00-2.25% cells with aberrations, with a mean of 1.50%". No explanation for these increased control values in the phthalic anhydride experiment is given.

Executive summary:

CHO cells and test compounds, e.g. phthalic anhydride were incubated at 37°C for 3 h in the presence or absence of S9. Cells were then washed, further incubated and harvested at 20 hours from the beginning of the treatment. Colcemid (0.1 µg/ml) was added 2-3 hours before chromosome aberration and cytotoxicity was tested.

Effects were observed only at the highest compound concentration which was "very toxic" (remaining cell counts 29%) and gave precipitate. Only a small, not statistically significant, increase in aberration was observed at a slightly lower concentration (8 mM compared to 10 mM) which showed lower cytotoxicity (remaining cell counts 54%) and no precipitate. Therefore the result is regarded as false positive.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a Micronucleus Assay mice were injected i.p.with suspensions of Phthalic Acid at 5 concentration levels (20, 100, 500, 2500, or 12500 µM/kg). The animals were sacrificed 24 h after i.p. administration of the test substances. Phthalic acid, the hydrolysis product of phthalic anhydride was not genotoxic according to the test utilized.

The study results obtained with phthalic acid as source fit to phthalic anhydride as target.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Phthalic anhydride was tested in the Ames Test preincubation method according to OECD TG 471. The test item did not induce mutations in the bacterial mutation test, neither in the absence nor presence of S9 rat liver metabolic activation. This negative result was confirmed by other negative Ames tests with the test item.

Phthalic Anhydride was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster up to the limit concentration of 10 mM. No precipitation ofthe test item was noted in the experiments. No biologically relevant growth inhibition was observed in experiment I and II with and without metabolic activation with one exception. In experiment II without metabolic activation for the highest concentration evaluated (l0 mM) the relative growth was 17.6%. In both experiments no biologically relevant increase of mutants was found after treatment with the test item (with and without metabolic activation). No dose response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test item Phthalic Anhydride is considered to be non-mutagenic in the HPRT locus using V79 cells.

In a Sister Chromatide Exchange (SCE) assay CHO cells were incubated with the test compound, positive control or solvent (DMSO) for 2 hours with S9 mix and for 25 hours without S9 mix. 2 -Bromodexoxyuridine is used for SCE staining and colcemid for mitotic arrest. Absolute increases in SCEs per chromosome were counted. Phthalic anhydride was negative in this SCE assay.

In one chomosomal aberration assay the registered substance showed negative results at concentrations of up to and including 300 µg/mL (Galloway, 1987). In a further chromosomal aberration test (Hilliard, 1988) a positive results was observed at the highest tested concentration (10 mM). This concentration was "very toxic" (remaining cell counts 29%) and showed precipitation. Only a small, not statistically significant, increase in aberration was observed at a slightly lower concentration (8 mM compared to 10 mM) which showed lower cytotoxicity (remaining cell counts 54%) and no precipitate. Hilliard (1988) discuss the discrepancy in the results compared to that of Galloway (1987) in 'the reason for the positive result in the present study probably include the use of a shorter treatment time, later harvest time (20 hr instead of 14 hr) and higher dose level (10 mM), which was very toxic and gave precipitate'.

In a Micronucleus Assay mice were injected i.p.with suspensions of Phthalic Acid, the hydrolysis product of phthalic anhydride, at 5 dose levels (20, 100, 500, 2500, or 12500 µM/kg) versus the positive control MMC (2 mg/kg). The animals were sacrificed 24 h after i.p. administration of the test substances. No mortalities were observed at these levels. Frequencies of MNPCE (micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes) in the treated goups were in general little higher than those treated with the vehicle, but no concentration-response relationship was observed. PCE/(PCE+NCE) values were also elevated in cells treated with the test agents, but again no concentration-response relationship was found.

In conclusion, the findings of the present study suggest that phthalic acid is not genotoxic in mutagenicity test utilized.

The study results obtained with phthalic acid as source fit to phthalic anhydride as target.

It can be concluded that phthalic anhydride is no inducer of gene mutations in bacteria and mammalian cells. However, it can induce chromosomal aberrations in vitro in extremely high, cytotoxic concentrations, and only in the absence of a metabolic activation system. This effect was not seen under in vivo conditions, where phthalic anhydride is rapidly hydrolyzed to the non-genotoxic phthalic acid.

Justification for classification or non-classification

Based on the available studies a non-classification is justified