Phthalic acid

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Phthalic acid was negative in several Ames Tests and in a mammalian cell Chromosomal Aberration assay. The hydrolysis product phthalic anhydride was shown to be no inducer of gene mutations in mammalian cells (HPRT Test).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline or GLP defined

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Principles of method if other than guideline:

The Ames Salmonella mutation test was used according to the plate incorporation procedure described by Maron and Ames (1983). Five Salmonella typhimurium strains were used: TA98, TA100, TA102; TA1535, and TA1537. This assay was performed with/without metabolic activation as an S9 mixture (Aroclor-1254 -induced rat liver homogenate). Negative and positive controls were used for each strain.

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Supplier: Sigma, thus, commercial grade is suggested

Target gene:

Ames Assay

Species / strain / cell type:

other: TA98, TA100, TA102, TA1535, TA 1537

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor-1254 induced rat liver homogenate)

Test concentrations with justification for top dose:

0; 20; 100; 500; 2500; 12500 µM

Untreated negative controls:

yes

Negative solvent / vehicle controls:

not specified

True negative controls:

yes

Positive controls:

yes

Positive control substance:

other: Without metabolical activation: 2-nitrofluorene (1µg per plate) for TA98, sodium azide (1.5 µg per plate) for TA100 and TA1535, mitomycin (1 µg per plate] for TA102, and acridine mutagen (1 µg per plate) for TA1537. With metabolical activation: 2-aminoant

Species / strain:

other: TA98, TA100, TA102, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Reverse Mutation Test Results for Phthalic Acid in Salmonella typhimurium:

Compound	Concentration (µM)	S9	TA98	TA100	TA102	TA1535	TA1537
Control	0	+	26.8 +1.6	101.2 +10.6	261.3 +14.3	8 +0.6	4 +0.5
	20	+	36.2 +1.8	158.6 +11.6	296.7 +15.4	13.6 +1.6	11.3 +1.6
	100	+	32.4 +1.5	148.6 +12.3	302.2 +16.8	16.2 +1.9	14.3 +1.4
	500	+	36.4 +2.1	152.9 +11.6	295.3 +12.5	14.4 +1.1	14.9 +1.1
	2500	+	29.7 +1.8	139.7 +10.3	297.6 +11.8	14.9 +1.1	13.7 +1.2
	12500	+	32.9 +1.5	154.2 +11.2	296.3 +10.8	15.3 +1.2	14.1 +0.9
2 -AA^a	1.0 µg/plate	+	402.3 +14.2	1278.5 +9.7	1358.2 +55.7	97.8 +2.8	75.5 +2.1
Control	0	-	22.6 +2.5	114.3 +10.2	257.6 +9.8	9.2 +0.6	5.2 +0.6
	20	-	29.3 +0.9	162.3 +14.3	286.3 +10.8	16.8 +1.2	13.5 +1.2
	100	-	35.2 +2.4	156.8 +11.6	294.5 +9.3	18.5 +2.1	16.2 +2.1
	500	-	31.8 +1.5	165.4 +13.6	300.5 +11.7	17.4 +1.5	15.8 +1.4
	2500	-	29.8 +2.4	159.6 +9.7	298.8 +10.6	17.6 +1.8	15.2 +1.6
	12500	-	33.5 +1.9	160.8 +10.2	297.6 +12.1	15.9 +1.3	13.4 +1.6
SA^b	1.5 µg/plate	-		1172 +52.3		103.5 +11
MMC^c	1.0 µg/plate	-			1256.9 +75.4	0
ICR-191^d	1.0 µg/plate	-					88.5 +3.2
2 -NF^e	1.0 µg/plate	-	386.7 +15.9

^a2 -Aminoanthracene (2 -AA); ^bSodium azide (SA); ^cMitomycin C (MMC); ^dAcridine (ICR-191); ^e2 -Nitrofluorene (2 -NF).

Executive summary:

The Ames Salmonella mutation test was used according to the plate incorporation procedure described by Maron and Ames (1983). Five Salmonella typhimurium strains were used: TA98, TA100, TA102; TA1535, and TA1537. This assay was performed with/without metabolic activation as an S9 mixture (Aroclor-1254 -induced rat liver homogenate). Negative and positive controls were used for each strain. The positive controls used in the tests performed without metabolic activation were as follows: 2 -nitrofluorene (1µg/plate) for TA98, sodium azide (1.5 µg/plate) for TA100 and TA1535, mitomycin (1µg/plate) for TA102, and acridine mutagen (1µg per plate) for TA 1537. The positive control used in the test performed with metabolic activation was 2 -aminoanthracene (1 µg per plate) for all strains. Five concentrations of the test substance were examined: 0; 20; 100; 500; 2500; 12500 µM using five bacterial strains and triplicate plates per dose. A significant increase in the number of revertants was observed in the presence of the positive control compounds. Negative and strain-specific positive control values were within historical lab range, demonstrating that the test conditions were effective and that the metabolic activation system functioned properly. The test substance did not produce mutagenic activity in any of the five bacterial strains tested under any of the activation conditions examined.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

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 gene mutation study in mammalian cells

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Justification for type of information:

REPORTING FORMAT FOR THE ANALOGUE APPROACH
[Please provide information for all of the points below. Indicate if further information is included as attachment to the same record, or elsewhere in the dataset (insert links in 'Cross-reference' table)]

1. HYPOTHESIS FOR THE ANALOGUE APPROACH
“The read-across hypothesis is that different substances give rise to (the same) common compounds to which the organism is exposed.”

Phthalic acid is the hydrolysis product of phthalic anhydride. In contact with water, phthalic anhydride is rapidly hydrolyzed to phthalic acid. Unconjugated phthalic acid was found in the urine of humans exposed to phthalic anhydride by the inhalation route, demonstrating systemic absorption and elimination via the urine and the existence of phthalic acid as the only hydrolysis product in vivo. Therefore, a read-across of systemic toxicity data obtained with the hydrolysis product phthalic acid is considered adequate for phthalic anhydride.
For further information see attached document:
Justification for a read-across between phthalic acid and phthalic anhydride

3. ANALOGUE APPROACH JUSTIFICATION
For further information see attached document:
Justification for a read-across between phthalic acid and phthalic anhydride

Reason / purpose for cross-reference:

read-across source

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.

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

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline or GLP defined

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Principles of method if other than guideline:

The Chinese hamster ovary (CHO) cell line was used for the CA test.

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

Supplier: Sigma; commercial grade can be suggested

Target gene:

Chromosome Aberration Test in CHO (Chinese hamster ovary) Cells

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Cytokinesis block (if used):

colcemid

Metabolic activation:

with and without

Metabolic activation system:

S9 (Aroclor-1254 induced rat liver homogenate)

Test concentrations with justification for top dose:

20; 100; 500; 2500; 12500 µM

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: Benzo[a]pyrene in the presence of S9 mixture, mitomycin C (MMC) in the absence of S9.

Details on test system and experimental conditions:

Cells were maintained in monolayer conditions in Eagle´s minimum essential medium supplemented with 10% fetal bovine serum , L-glutamine, and antibiotic at 37°C in a 5% CO2 atmosphere. For each treatment, 3x105 cells were cultured in duplicate in 5 ml of culture medium in a 25 ml flask, and treated with the test compounds in 10-µl reaction volumes for 24h. A 5 -h pulse treatment was then carried out with/without S9 mixture (metabolic activation) at a final concentration of 10% in the medium. Benzu[a]pyrene was used as a positive control substance in the presence of S9 mixture, and mitomycin C was used as a positive control in the absence of S9. After removing media, cells were rinsed in Dubecco´s phosphate-buffered saline and fresh medium was added to each flask. Colcemid was then added to each flask during the last 2h of incubation and cells were harvested after treatment with trypsin ethylenediamine tetraacetic acid (EDTA). Chromosome preparation was performed using standard methods, and cells were incubated in a hypotonic KCl (75 mM) solution at 37°C for 30 min and fixed in methanol:acetic acid (3:1, v/v) 3 times. Slides, maintained at 4°C, were prepared and 2 drops of fixed cell suspensions were spread on glass slides and air-dried. Cells were then stained with a 5% Giemsa solution, and slides were dried. At least 100 metaphases were then counted at 1000 X under an microscope.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

Positive controls validity:

valid

Chromosome Aberration Frequencies for Phthalic Acid:

With (+) or without (-) S9 mix	Concentrations	Observed cells	ctg^a	ctb^b	cte^c	csg^d	csb^e	cse^f	frg^g	Total
	DW^h	100	0	0	1	0	1	0	0	2
	MMC^i	100	5	6	4	2	5	4	6	32
	20	100	1	1	0	1	1	0	0	4
S9-	100	100	0	0	1	0	1	0	0	2
	500	100	1	0	0	0	0	0	1	2
	2500	100	0	1	0	0	0	2	0	3
	12500	100	1	0	1	1	0	0	2	5
S9+	DMSO^j	100	1	1	0	1	0	0	0	3
	Bap^k	100	9	4	7	8	10	11	8	57
	20	100	0	1	1	0	0	2	0	4
	100	100	2	0	0	0	2	0	0	4
	500	100	0	1	2	1	0	0	2	6
	2500	100	2	0	0	1	0	1	0	4
	12500	100	0	2	0	2	1	0	1	6

^aChromatic and isochromatic gap, ^bChromativ breakage, ^cChromatic exchange, ^dChromosome gap, ^eChromosome breakage, ^fChromosome exchange, ^gFragment, ^hDistilled water, ⁱMitomycin C, ^jDimethyl sulfoxide, ^kbenzol[a]pyrene.

Executive summary:

In a Chromosome Aberration test a Chinese hamster ovary (CHO) cell line was used. The test was performed with and without S9 mixture. Benzu[a]pyrene was used as a positive control substance in the presence of S9 mixture, and mitomycin C was used as a positive control in the absence of S9. Chromosomal aberration (CA) including gaps, breakages and exchanges were induced by the test substance at concentrations ranging from 20 µM/ml to 12500 µM/ml versus the untreated controls. Cells were exposed for 6h and harvested 18 h after treatment initiation. The test substance did not produce CA in CHO cells and was not cytotoxic under the experimental conditions.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In a micronucleus assay with intraperitoneal injection of phthalic acid to ICR mice no increased micronucleus frequency was reported.

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: No OECD guideline or GLP defined; short description

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Principles of method if other than guideline:

Seven- to 8 -wk-old male ICR strain mice were subjected to the Micronucleus (MN) test. Animals were administered the test substance ip and were sacrificed 24 h after treatment. Bone marrow was extracted and numbers of micronucleated cells were determined by counting numbers of polychromatic erythrocytes (PCE) from among at least 1000 PCE per animal. Micronucleated polychromatic erythrocytes MNPCE) that contained micronuclei were counted from among at least 1000 PCE.

GLP compliance:

not specified

Type of assay:

micronucleus assay

Specific details on test material used for the study:

Supplier: Sigma; commercial grade can be suggested

Species:

mouse

Strain:

ICR

Sex:

male

Route of administration:

intraperitoneal

Duration of treatment / exposure:

Animals were administered the test substance i.p. and were sacrificed 24h after treatment.

Frequency of treatment:

single dose

Post exposure period:

24h

Remarks:

Doses / Concentrations:
20, 100, 500, 2500, 12500 µM/kg)
Basis:
no data

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Tissues and cell types examined:

Bone marrow was extracted with fetal bovine serum (0.5 ml), and cell suspensions were centrifuges at 1000xg for 5 min. Number of micronucleated cells were determined by counting numbers of polychromatic erythrocytes (PCE) from among at least 1000 PCE per animal.Micronucleated polychromatic erythrocytes (MNPCE) that contained micronuclei were counted from among at least 1000 PCE.

Details of tissue and slide preparation:

MMC (positive control, 2 mg/kg) dissolved in sterile deionized water

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

valid

Negative controls validity:

other: Vehicle control= negative control

Positive controls validity:

valid

Micronucleus Induction in Mouse Bone Marrow Cells Treated with Phthalic Acid:

Samples	Treatments	Concentrations	Route	Number of mice tested	Exposure time (h)	MNPCE%^a(mean+ SD)	PCE/(PCE+NCE)^b (mean+SD)
Phthalic Acid	Control^c	0	i.p.	5	24	0.24 +0.05	0.11 +0.02
		20 µM/kg	i.p.	5	24	0.32 +0.34	0.15 +0.04
		100  µM/kg	i.p.	5	24	0.38 +0.35	0.13 +0.03
		500  µM/kg	i.p.	5	24	0.41 +0.25	0.16 +0.05
		2500  µM/kg	i.p.	5	24	0.31 +0.55	0.14 +0.02
		12500  µM/kg	i.p.	5	24	0.35 +0.26	0.18 +0.01
	MMC^d	2 mg/kg	i.p.	5	24	6.47 +0.54	0.62 +0.02

^aMicronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes, ^bPolychromatic erythrocytes/1000 erythrocytes, ^cSample dilution buffer, ^dMitomycin C.

Executive summary:

In a Micronucleus Assay in ICR strain mice the animals were injected i.p.with suspensions of test compound, which were treated as follows: group 1, vehicle alone (DMSO, negative control); groups 2, 3, 4, and 5 were treated with Phthalic Acid at 5 concentration levels (20, 100, 500, 2500, or 12500 µM/kg); and group 6 was treated with MMC (positive control, 2 mg/kg) dissolved in sterile deionized water. The animals were sacrificed 24 h after i.p. administration of the test substances. Micronuclei formations due to Phthalic Acid were evaluated using mouse bone marrow at concentrations up to 12500 µM/kg after 24 h of treatment. No mortalities were observed at these levels. Frequencies of MNPCE (micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes) in the vehicle control groups were 0.14% (PA). In general, MNPCE% was little higher for mouse bone-marrow cells treated with the test agent, 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 the mutagenic test used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro tests:

The Ames Salmonella mutation test was used according to the plate incorporation procedure described by Maron and Ames (1983). Five Salmonella typhimurium strains were used: TA98, TA100, TA102; TA1535, and TA1537. This assay was performed with/without metabolic activation. as an S9 mixture (Aroclor-1254 -induced rat liver homogenate). Negative and positive controls were used for each strain. Five concentrations of the test substance were examined: 0; 20; 100; 500; 2500; 12500 µM using five bacterial strains and triplicate plates per dose. A significant increase in the number of revertants was observed in the presence of the positive control compounds. Negative and strain-specific positive control values were within historical lab range, demonstrating that the test conditions were effective and that the metabolic activation system functioned properly. The test substance did not produce mutagenic activity in any of the five bacterial strains tested under any of the activation conditions examined (Lee, 2007).

This finding was confirmed by several other Ames assays.

In a Chromosome Aberration test a Chinese hamster ovary (CHO) cell line was used. The test was performed with and without S9 mixture. The test substance did not produce Chromosome Aberration in CHO cells and was not cytotoxic under the experimental conditions (Lee, 2007).

There is no mutation assay in mammalian cells in Phthalic acid available, therefore 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 concentrations of 10mM. The result was negative, with and without metabolic activation (BSL Bioservice, 2010).

In vivo tests:

In a Micronucleus Assay in ICR strain mice the animals were injected i.p.with suspensions of test compound, which were treated as follows: group 1, vehicle alone (DMSO, negative control); groups 2, 3, 4, and 5 were treated with Phthalic Acid at 5 concentration levels (20, 100, 500, 2500, or 12500 µM/kg); and group 6 was treated with MMC (positive control, 2 mg/kg) dissolved in sterile deionized water. The animals were sacrificed 24 h after i.p. administration of the test substances. Micronuclei formations due to Phthalic Acid were evaluated using mouse bone marrow at concentrations up to 12500 µM/kg after 24 h of treatment. No mortalities were observed at these levels. Frequencies of MNPCE (micronucleated polychromatic erythrocytes/1000 polychromatic erythrocytes) in the vehicle control groups were 0.14% (PA). In general, MNPCE% was little higher for mouse bone-marrow cells treated with the test agent, 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 the mutagenic test used.

A dominant lethal assay and a sperm head abnormality assay are reported in the literature. Both assays suffer from experimental limitations and can not be used for a hazard assessment. In a dominant lethal study in which male Swiss albino mice were treated for 5 consecutive days with a single i. p. injection of phthalic acid (either 40 or 80 mg/kg bw.) and then mated for 28 days with females of the same stock the test substance induced dominant lethal mutations in meiotic as well as post meiotic stages of spermatogenesis (Jha, 1998). The same authors report additional data in the same publication in a Sperm head abnormality assay in 5 male Swiss albino mice per dose group. The animals were injected with a single i. p. injection of phthalic acid. The doses injected were 0, 50, 100, 150, 200 and 300 mg/kg bw. Phthalic acid treatment resulted in statistically significant increase in the incidence of sperm head abnormalities (except at the lowest dose, i. e., 50 mg/kg bw), when animals were killed 1 and 3 weeks after exposure representing the spermatoa and spermacid stages of spermatogenesis, respectively. Various forms of abnormal sperm head shapes were recorded. The most common types were amorphous, elongate, without hook, giant amorphous. Statistically significant positive correlations (P<0.01) between dose and numbers of abnormal sperm were observed. There was a positive dose-response effect over the entire dose range tested (Jha, 1998). These studies are not reliable for hazard assessment because: 1) the test compound was injected i.p. and the vehicle contained 10% DMSO, 2) the authors indicate that phosphate buffered saline was used as a vehicle but did not indicate the pH of the injection solution; i.p. injection of a irritating compound is not relevant for human risk assessment, 3) the animals were dosed on 5 consecutative days in the dominant lethal assay (the corresponding guideline indicates single dosing and additional dosing should be justified by the investigator); 4) the number of implants per female were low in this experiment in all groups including control (ca. 6) compared to the typical number of implants for this strain (13-15); 5) no positive control compound was included in the assays to demonstrate the biological variability and/or sensitivity of the test systems.

Justification for classification or non-classification

Based on the available studies the test substance needs no classification for genetic toxicity according to EU guidelines.