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Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: OECD 471; Ames study; not mutagenic; Reliability = 2 [CAS# 100-21-0]

Cytogenicity - mammalian: OECD 473; chromosome aberration (human peripheral blood lymphocytes); positive with and without metabolic activation; Reliability = 2 [CAS# 100-21-0]

Mutagenic effects-mammalian: OECD 476; CHO/HGPRT study; not mutagenic; Reliability = 2 [CAS# 121-91-5]

Link to relevant study records

Referenceopen allclose all

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:
comparable to guideline study with acceptable restrictions
Remarks:
The test substance was concluded to be negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No indications of mutagenicity were observed at any dose level in any tester strain when tested up to 10000 μg/plate in the absence or presence of S9. The assay was conducted prior to the 1997 adaptation of the current OECD 471, and did not include an E.coli WP2 or S. typhimurium TA102 tester strain. These strains are known to specifically detect certain oxidising mutagens, cross-linking agents and hydrazines that other Salmonella tester strains may not be sensitive to. However, based on the physico-chemical characteristics of the test substance, the inclusion of a fifth tester strain would not have changed the clearly negative outcome of the Ames assay. In addition, there was no indication of in vitro or in vivo mutagenicity for any other genetic toxicology endpoint.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
Tested up to 10000 µg/plate with and without S9 metabolic activation in 4 Salmonella strains.
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate (S9)
Test concentrations with justification for top dose:
0, 500, 1000, 2500, 5000, 10000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Not reported
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: N-methyl-N'-nitro-N-nitrosoguanidine (TA1535 -S9 and TA100 -S9); 9-aminoacridine (TA1537 -S9); 2-nitrofluorene (TA98 -S9); 2-aminoanthracene (all strains +S9)
Details on test system and experimental conditions:
METHOD OF APPLICATION: Treatment without activation was conducted by adding 0.1 mL of overnight culture containing the test sample and 1e8 bacteria to top agar supplemented with L-histidine, NaCl, and biotin. The components were mixed and poured onto a plate containing Davis minimal agar. Treatments with activation were conducted as those without activation except that S9 mix was added to the bacteria/top agar mixture before it was poured onto a Davis minimal agar plate. The plates were incubated at approximately 37°C for approximately 48 hours.

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: 2 trials for all strains, except TA1537 which had 3 trials when tested with activation

DETERMINATION OF CYTOTOXICITY
- Method: Cytotoxicity of the test sample in the presence and absence of an activation system, as measured in strain TA1535, was the same as the method of application for mutagenesis except that 10e3 rather than 10e8 bacteria were used per plate and a non-limiting concentration of histidine was present.
Evaluation criteria:
A chemical is classified as nonmutagenic if the reversion frequency is less than two times the spontaneous frequency, and if less than 0.02 revertants/nmole are observed.
Statistics:
Data from replicate plates within a single experiments are averaged. The highest average number of revertants that is obtained is expressed as a multiple of the control value for the sensitive strain(s). When a test sample is active, the average numbers of revertants observed before activity plateaus or decreases at the various concentrations tested are submitted to linear regression analysis. The slope of the line thus obtained is used to determine the number of revertants/nmole or µg of test sample.
Species / strain:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Metabolic activation:
with and without
Genotoxicity:
negative
Remarks:
The hint of activity observed in strain TA1537 in Trial 2 in the presence of activation was not observed in other trials.
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Conclusions:
The test substance was not mutagenic in an Ames assay with Salmonella typhimurium strains TA 1535, TA 1537, TA 98 and TA 100, in the presence and absence of an exogenous activation system.
Executive summary:

The test substance was evaluated for mutagenicity in Salmonella typhimurium strains TA100, TA1535, TA98, and TA100 with and without an exogenous metabolic activation system (S9). The maximum concentration tested was 10000 µg/plate. The hint of activity observed in strain TA1537 in Trial 2 in the presence of activation was not observed in other trials. In this study, the test substance was negative.

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: see 'Remark'
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). The study, if used in support of terephthalic acid, has a reliability of 1 (reliable without restriction).
Qualifier:
according to guideline
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
yes
Remarks:
The culturing of cells, treatment with the test substance, harvesting, slide preparation and original cytogenetic analysis was conducted in SV1318. In this study, additional cytogenetic analysis was conducted
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.5375 - In vitro Mammalian Chromosome Aberration Test
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: Annex V to Council Directive 67/548/EEC on the approximation of law, regulations and administrative provisions relating to the classification, packaging and labelling of dangerous substances, published in the 26th Adaptation, Commission Directive 2000/32/
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: ICH Harmonised Tripartite Guidelines S2A: Guidance on Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (adopted at Step 4 of the ICH process 19 July 1995) and S2B: Genotoxicity: A Standard Battery for Genotoxicity Testing of Pharmaceu
GLP compliance:
yes
Type of assay:
in vitro mammalian chromosome aberration test
Species / strain / cell type:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Metabolic activation system:
(S9-mix)
Test concentrations with justification for top dose:
Two independent cytogenetic experiments using a range of concentrations were conducted initially to determine the limit concentration. The three concentrations tested in the chromosomal aberration analysis were 500, 250 and 50 µg/mL.
Vehicle / solvent:
The test substance was diluted in dried dimethylsulphoxide. Both positive control substances were prepared as solutions in sterile double deionised water.
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
other: Mitomycin C, cyclophosphamide
Details on test system and experimental conditions:
Duplicate human peripheral blood cultures were exposed to the solvent, test substance or positive control substance at appropriate concentrations in two experiments. A cytogenetic test was conducted using a sample of pooled blood. Cells were exposed to the test substance and control substances for a period of 3 hours both in the presence and absence of S9 mix. Solvent, untreated and positive control cultures were included. In a second, independent cytotoxic test, cells were exposed to the test substance and control substances for a period of 3 hours in the presence of S9-mix and 20 hours in the absence of S9 mix. Solvent, untreated and positive control cultures were included. Treatment of the cultures started approximately 48 hours after culture initiation. A single sampling time, 20 hours after the start of treatment (68 hours after culture initiation), was used. Cultures were established from blood samples from healthy volunteers. At 0 hours, cultures (10 mL) were established by the addition of 0.5 mL of whole blood to RPMI-1640 (Dutch modification) tissue culture medium supplemented with approximately 10% foetal bovine serum (FBS), 1.0 IU/mL heparin, L-glutamine (2 mM), 100 IU/mL penicillin and 100 μg/mL streptomycin. The lymphocytes were stimulated to enter cell division by addition of phytohaemagglutinin (PHA; at 5% v/v) and the cultures were maintained at approximately 37°C for 48 hours with gentle daily mixing where possible. Prior to treatment, the cultures which were used in the 20 hour study in experiment 2 were centrifuged and the culture medium changed with fresh supplemented RPMI-1640 culture medium. After approximately 48 hours from culture establishment, aliquots of the test substance, solvent control or positive control were administered to duplicate cultures as appropriate. In addition, 200 μL of a 1:1 mix of S9 and co-factor solution was added to each culture to be treated in the presence of S9-mix. In experiment, all cultures were kept at 37°C for 3 hours after which the culture medium as replaced with fresh RPMI01640 culture medium and then the cells were reincubated at 37°C for the remainder of the 68 hour period. Cultures from experiment 2 in the absence of S9-mix were treated for a period of 20 hours until the end of the 68 hour growth period. At 68 hours, the cells were harvested which involved centrifugation, removal of the supernatant and suspension in 0.075M KCl. The cells were fixed using a methanol/glacial acetic acid fixative (3:1 v/v). After at least two subsequent changes of fixative the cell suspension was mounted onto a microscope slide. The slides were examined and the mitotic index determined by examining 1000 lymphocytes per culture and calculating the percentage of cells in metaphase. For each experiment, both in the presence and absence of S9-mix, duplicate cultures treated with terephthalic acid at three concentrations were selected for chromosomal aberration analysis along with the appropriate solvent and positive control cultures.
Evaluation criteria:
The percentages of aberrant metaphases and the number of aberrations per cell were calculated for each treatment scored, both including and excluding cells with only gap-type aberrations
Statistics:
The Fisher Exact Probability Test (one-sided) is used to evaluate statistically the percentage of metaphases showing aberrations (excluding cells with only gap-type aberrations).
Data from each treatment group, in the presence and absence of S9-mix, was compared with the respective solvent control group value. The data have been interpreted as follows:
a) No statistically significant increase in the percentage of aberrant cells (at any concentration) above concurrent solvent control values indicates the study to be negative (non-clastogenic).
b) A statistically significant increase in the percentage of aberrant cells above concurrent solvent control values, which falls within the laboratory solvent control range indicates the study to be negative (non-clastogenic).
c) An increase in the percentage of aberrant cells, at least at one concentration, which is substantially greater than the laboratory historical solvent control values indicates the study to be positive (clastogenic).
d) A statistically significant increase in the percentage of aberrant cells which is above concurrent solvent values and which is above the historical solvent control range upper value but below that described in (c) may require further evaluation.
Species / strain:
lymphocytes: human peripheral blood
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
It was discovered that the pH limited the concentration at which the chromosomal aberration could be performed. The concentration of test substance above 500 µg/mL decreased the pH of the culture medium by 1.1 pH units. No significant effect on osmolality was observed.

A reduction in the mean mitotic activity, compared to the control, was observed in cultures from both experiment 1 (34% +S9-mix; 26% -S9 mix) and Experiment 2 (35% +S9-mix, 59% -S9 mix) treated with the highest concentrations of terephthalic acid selected for chromosomal aberration analysis.

CHROMOSOMAL ABERRATION ANALYSES
Statistically and biologically significant increases in the percentage of aberrant cells were observed in Experiment 2 following 20 hour treated in the absence of S9-mix. Small increases in the percentage of aberrant cells were observed following 3 hour treatment in Experiment 1 in the presence and absence of S9-mix and in Experiment 2 in the presence of S9-mix. The positive control materials, mitomycin C and cyclophosphamide, induced statistically and biologically significant increases in the percentage of aberrant cells, compared to the solvent control cultures.

Table 1 : Mean Chromosomal Aberrations and Mitotic Indices in the Absence of  Metabolic activation

Treatment

Mean % Aberrant Cells Excluding Gaps

Mean% Mitotic Index

Experiment 1

Solvent Control           10 μL/mL

Mitomycin                  0.5 μg/mL

Terephthalic acid

                                  500 μg/mL 

                                   250 μg/mL

                                   50 μg/mL

 

3.50

36.00**

 

7.00

4.00

3.00

 

10.7

10.4Δ

 

7.9

9.1

9.2

Experiment 2

Solvent Control         10 μL/mL

Mitomycin                 0.2 μg/mL

Terephthalic acid

                                  500 μg/mL 

                                   250 μg/mL

                                   50 μg/mL

 

4.0

20.00**

 

23.43**

12.00**

4.00

 

6.8

2.5Δ

 

2.8

5.0

6.4

** Statistically significant increase in the percentage of aberrant cells at p<0.01 using

Fisher's Exact Test (one-sided).

ΔPositive control mitotic index and % aberrant cells are determined from a single culture.

 

 Table 2 : Mean Chromosomal Aberrations and Mitotic Indices in the Presence of  Metabolic activation

Treatment

Mean % Aberrant Cells Excluding Gaps

Mean% Mitotic Index

Experiment 1

Solvent Control           10 μL/mL

Cyclophosphamide 50 μg/mL

Terephthalic acid

                                  500 μg/mL 

                                  250 μg/mL

                                  50 μg/mL

 

1.00

44.00**

 

4.50*

2.00

2.50

 

13.9

8.6Δ

 

9.2

9.1

10.6

Experiment 2-3 hour Treatment

Solvent Control           10 μL/mL

Cyclophosphamide 50 μg/mL

Terephthalic acid

                                  500 μg/mL 

                                  250 μg/mL

                                  50 μg/mL

 

4.5

38.00**

 

7.00

2.0

3.50

 

7.8

2.9Δ

 

5.1

5.1

7.4

* Statistically significant increase in the percentage of aberrant cells at p<0.05 using

Fisher's Exact Test (one-sided).

** Statistically significant increase in the percentage of aberrant cells at p<0.01 using

     Fisher's Exact Test (one-sided).

ΔPositive control mitotic index and % aberrant cells are determined from a single culture.

Conclusions:
Under the conditions of the assay, terephthalic acid is clastogenic to cultured human lymphocytes treated in vitro in the absence of S9-mix.
Executive summary:

In this study report, the results of the chromosomal aberration analysis from the previous studies SV1318 and SV1380 were combined. A small but statistically significant increase in the percentage of aberrant cells, compared to the solvent control values were recorded in cultures in experiment 1 in the absence of S9 mix and experiment 2 in the presence and absence of S9 mix. These increases were small, well within the historical control range and not all concentration related. They are therefore considered to be of no biological importance. The sensitivity of the test system and the metabolic activity of the S9 mix employed were clearly demonstrated by the increases in the percentage of aberrant cells induced by the positive control agents, mitomycin C and cyclophosphamide.

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). The study, if used in support of isophthalic acid, has a reliability of 1 (reliable without restriction).
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
GLP compliance:
yes
Type of assay:
mammalian cell gene mutation assay
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
- Type and identity of media: agar culture
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: not reported
- Periodically "cleansed" against high spontaneous background: no reported
Additional strain / cell type characteristics:
other: hypoxanthine-guanine phospho ribosyl transferase (HGPRT)
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S-9
Test concentrations with justification for top dose:
initial assay - non-activated: 3000, 2000, 1500, 500, 250 and 125 μg/mL
initial assay - presence of S-9: 3000, 2000, 1500, 1000, 500 and 250 μg/mL
confirmatory assay - in the absence and presence of S-9: 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: dimethylsulfoxide (DMSO)
- Justification for choice of solvent/vehicle: not reported
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
other: Ethyl methanesulfonate (EMS) was used as the positive control in the non-activated study at a final concentration of 0.2 μg/mL. Benzo(a)pyrene (B(a)P) was used as the positive control in the S-9 activated study at a final concentration of 4 μg/mL.
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)

The testing procedure is summarized as follows.
Cultures of CHO cells were grown in the absence of antibiotics for several passages. Mycoplasma agar plates and broth tubes were inoculated directly with the culture material and separate sets of cultures were inoculated aerobically and anaerobically. The broth tubes were subcultured three times
onto agar plates. The agar plates were examined no sooner than 14 days postinoculation for the presence of mycoplasma colonies. For the Hoechst staining procedure, the culture material was directly inoculated onto Vero cell cultures. After incubation for 3-5 days, the cell cultures were stained with the DNA--=binding fluorochrome and were evaluated microscopically by epifluorescence for the presence of mycoplasma.
SELECTION AGENT (mutation assays):
Biological Reagents:
Ham's F-12 medium without hypoxanthine supplemented with 5% dialyzed FBS, 1% penicillin-streptomycin and 1% L-glutamine (F12FBS5-Hx)
Hank's Balanced Salt Solution (HBSS), Ca + + and Mg+ + -free (CMF-HBSS)
Trypsin, 0.05%
6-Thioguanine (TG, 2-amino-6-mercaptopurine), 20 mM in 0.1 N NaOH
Cofactor pool (final concentration in S-9 mix): 4 mM nicotinamide adenine dinucleotide phosphate (NADP), 5 mM glucose-6-phosphate, 30 mM potassium chloride (KCl), 10 mM calcium chloride (CaCl2), 10 mM magnesium chloride (MgCl2) and 50 mM sodium phosphate buffer, pH 8.0
S-9, 9000 x g supernatant of an Aroclor-1254 induced Sprague-Dawley rat liver
homogenate, lot R-413
Giemsa stain, 10% aqueous
Methanol, 95%

NUMBER OF REPLICATIONS: initial treatment - duplicates; for evaluation of cytotoxicity, the replicates from each treatment condition were subcultured independently in F12FBS5-Hx, in triplicate, at a density of 100 cells/60 mm dish; For expression of the mutant phenotype, the replicates from each treatment condition were subcultured independently in F12FBS5-Hx, in duplicate, at a density of approximately 1,000,000 cells/100 mm dish; For selection of the TG-resistant phenotype, the replicates from each treatment condition were pooled and replated, in quintuplicate, at a density of 2 x 10e5 cells/100 mm dish in F12FBS5-Hx containing 10 μM TG.

NUMBER OF CELLS EVALUATED: The mutant frequency (MF) for each treatment condition was calculated by dividing the total number of mutant colonies by the number of cells selected (10e6 cells: 10 plates at 2 x 10e5 cells/plate), corrected for the cloning efficiency of cells prior to mutant selection. and is expressed as TG-resistant mutants per 10e6 clonable cells.

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth
Evaluation criteria:
The cloning efficiency of the solvent and untreated controls must be greater than 50%. The spontaneous mutant frequency in the solvent and untreated controls must fall within the range of 0-25 mutants per 10e6 clonable cells. The positive control must induce a mutant frequency at least three times that of the solvent control.
Statistics:
not reported
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:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no data
- Effects of osmolality: no data
- Evaporation from medium: no data
- Water solubility: no data
- Precipitation: no data
- Other confounding effects: no data

RANGE-FINDING/SCREENING STUDIES:
An initial and confirmatory mutagenicity assay were performed. Dose levels for the CHO/HGPRT mutation assay were selected following a preliminary toxicity test. Toxicity was based upon cloning efficiency after treatment relative to the solvent control. CHO cells were exposed to solvent alone and to nine concentrations of test article ranging from 5000 to 0.5 μg/mL in the toxicity test in the absence and presence of an S-9 reaction mixture. The osmolality of the highest concentration in treatment medium tested, 5000 μg/mL, was 417 mOsm/kg with a measured pH of 4.4. The pH of each treatment condition was measured and adjusted, as needed, to approximately 6.8-7.0 using 1 N NaOH. The highest soluble concentration in culture medium was approximately 1250 μg/mL, therefore, one-half of the doses selected for each assay were usually partially to completely insoluble in culture medium. The initial cytotoxicity study demonstrated large cytotoxic effects at 5000 μg/mL. This cytotoxic effect most likely resulted from the large amount of precipitate in the dosing flask since the limit of solubility in culture medium was achieved at 1250 μg/mL. All doses were prepared by combining sufficient treatment medium for the replicate flasks at each treatment condition in an appropriately sized container with the pH adjusted as necessary. The highest concentrations tested in the study required several pH adjustments at intervals during dose preparation. Multiple pH adjustments were necessary because precipitation in the treatment media caused the pH to again decline. The pH of the dosing solutions were adjusted prior to the addition of the dosing solutions to flasks containing the target cells. Dose levels of 4500, 3500, 2000, 1500, 1000 and 500 μg/mL for the non-activated study and 5000,4000,3000, 2000 and 1000 μg/mL for the activated study were selected. This study was terminated prior to selection due to excessive toxicity exhibited at most of the treatment levels tested. New doses were selected for the initial mutagenesis assay. These dose levels were 3000, 2000, 1500, 500, 250 and 125 μg/mL in the absence of S-9 and 3000, 2000, 1500, 1000, 500 and 250 μg/mL in the presence of S-9. The doses selected for the confirmatory mutagenesis assay were 4000, 3500, 3000, 2000, 1500 and 1000 μg/mL in the absence and presence of S-9. Inconsistencies in the cytotoxicity results were noted between replicate treatments. The results from this study were not reported and an additional study was performed using the dose levels of 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL in the absence and presence of S-9. Due to inconsistent results between the replicate flasks of the 4000 μg/mL treatment in the non-activated study, the plate counts for the "B" treatment were not included in any of the calculations performed for concurrent cytotoxicity or mutation frequency for this treatment condition.
Conclusions:
Under the conditions of these mutagenicity tests, test substance was negative in both the absence and presence of exogenous metabolic activation.
Executive summary:

The test substance was tested in the CHO/HGPRT mutation assay in the absence and presence of metabolic activation with Aroclor-induced rat liver S-9. The initial assay was conducted at dose levels of 3000, 2000, 1500, 500, 250 and 125 μg/mL in the non-activated study and at 3000, 2000, 1500, 1000, 500 and 250 μg/mL, in the presence of S-9. The confirmatory assay was conducted at dose levels of 4000, 3500, 3200, 3000, 2000, 1000 and 500 μg/mL in both the absence and presence of S-9. Under the conditions of these mutagenicity tests, test substance was negative in both the absence and presence of exogenous metabolic activation.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Clastogenic effects - mammalian: OECDE 474; in vivo mouse micronucleus study; negative; Reliability = 2 [CAS# 100-21-0]

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
This study is used for read-across and therefore has been assigned a reliability of 2 (reliable with restrictions). The study, if used in support of terephthalic acid, has a reliability of 1 (reliable without restriction). Guideline study performed under GLP requirements.
Qualifier:
according to guideline
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
GLP compliance:
yes
Type of assay:
micronucleus assay
Species:
mouse
Strain:
ICR
Sex:
male/female
Details on test animals or test system and environmental conditions:
Male and female ICR mice were obtained from Harlan Sprague Dawley, Inc. in two batches. At study initiation mice were 6-8 weeks old. Pilot study mouse weights were: males 29.5-34.1 g; females 25.2-27.4 g. Micronucleus assay mouse weights were: males 25.6-30.4 g; females 23.9-28.1 g.
Mice were quarantined for 5 days. The animal room was maintained at a temperature of 72±3°F, 50±20% relative humidity and a 12 hour light/dark cycle. Mice were housed in same sex groups of 5 in polycarbonate cages on racks. Hardwood chips were used as bedding. Food (Harlan TEKLAD certified Rodent 7012C) and tap water (Washington Suburban Sanitary Commission) were provided ad libitum.
Mice were numbered and identified by ear tag.
Route of administration:
intraperitoneal
Vehicle:
Corn oil. The vehicle was chosen as it permitted preparation of the highest soluble or workable stock solution, up to 100 mg/ml (compared to 0.5% CMC in water).
Details on exposure:
Mice were given a single intraperitoneal injection of the test substance, or vehicle alone. All mice were weighed immediately prior to dose administration and the dose volume based on individual body weights. Injections were kept to a constant volume of 20 ml/kg body weight. Additional animals (5/sex/group) were treated with vehicle or 800 mg/kg TPA and sacrificed at 48 hours. An additional group of 5 males and 5 females were dosed with 800 mg/kg TPA as a replacement group in case of mortality.
Duration of treatment / exposure:
Pilot study & Toxicity study: mice were observed for signs of toxicity for 3 days after administration.
Micronucleus study: single injection, mice were sacrificed 24 hours later. Additional mice from the vehicle control and high dose groups were sacrificed 48 hours after dose administration.
Frequency of treatment:
Single injection
Post exposure period:
Pilot study & Toxicity study: 3 days
Micronucleus study: 24 or 48 hours.
Remarks:
Pilot Study: 1, 10, 100, 1000 and 2000 mg/kg (nominal)
Remarks:
Toxicity Study: 1200, 1400, 1600 and 1800 mg/kg (nominal)
Remarks:
Main Micronucleus Study: 200, 400, and 800 mg/kg (nominal)
No. of animals per sex per dose:
Pilot study: 5 mice/sex received 2000 mg/kg, and 2 males/dose received either 1, 10, 100 or 1000 mg/kg
Toxicity study: 5 mice/sex/dose
Micronucleus study: controls - 10 mice/sex; 200 and 400 mg/kg - 5 mice/sex/dose, 800 mg/kg - 15 mice/sex/per dose, positive controls - 5 mice/sex/dose
Control animals:
yes, concurrent vehicle
Positive control(s):
In the micronucleus study, 5 males and 5 females were injected with cyclophosphamide dissolved in distilled water at a dose of 50 mg/kg.
Tissues and cell types examined:
Bone marrow cells obtained from the femurs.
Details of tissue and slide preparation:
Immediately follow sacrifice, the femurs were exposed, cut just above the knee, and the bone marrow was aspirated into a syringe containing foetal bovine serum. The cells were transferred to a capped centrifuge tube containing approximately 1 ml foetal bovine serum. The cells were pelleted by centrifugation at 100 x g for 5 minutes and the supernatant drawn off. The cells were resuspended and a small drop of bone marrow suspension was spread onto a clean glass slide. Two slides were prepared from each mouse. The slides were fixed in methanol and stained with May-Gruenwald-Giemsa and permanently mounted. Slides were coded prior to analysis.
Evaluation criteria:
2000 polychromatic erythrocytes were scored per slide for the presence of micronuclei. The number of micronucleated normochromatic erythrocytes per 2000 polychromatic erythrocytes was recorded, and the proportion of polychromatic erythrocytes to total erythrocytes over 1000 was recorded.
The test article was considered to induce a positive response if a dose responsive increase in micronucleated polychromatic erythrocytes was observed, and one or more doses were statistically elevated relative to the vehicle control.
Statistics:
Kastenbaum-Bowman tables were used to determine statistical significance at the 5% level.
Sex:
male/female
Genotoxicity:
negative
Toxicity:
yes
Remarks:
lethargy and piloerection at all doses
Vehicle controls validity:
valid
Negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
Pilot study: 3/5 males and 5/5 females died within 2 days administration of 2000 mg/kg. Lethargy and piloerection were noted in males at 1000 mg/kg and males and females at 2000 mg/kg. Tremors were observed in the males at 2000 mg/kg, and convulsions, prostation and crusty eyes were observed in females at this dose. All other animals appeared normal.

Toxicity study: mortality occurred within 3 days of dosing as follows: 2/5 males and 1/5 females at 1200 mg/kg; 4/5 males and 3/5 males at 1400 mg/kg; 2/5 males and 4/5 females at 1600 mg/kg; and 4/5 males and 2/5 females at 1800 mg/kg. Lethargy, piloerection and crusty eyes were seen in all mice at all doses. Tremors were seen in females at 1200 mg/kg, and in males and females at 1400 and 1800 mg/kg. Convulsions were seen in males and females at 1600 and 1800 mg/kg, and prostration in males at 1200 and 1400 mg/kg and males and females at 1600 and 1800 mg/kg. The maximum tolerated dose chosen for the micronucleus assay was 800 mg/kg.

Micronucleus study: 1 male mouse in the 800 mg/kg group was found dead the day after administration, but was replaced. Lethargy and piloerection were seen at all 3 doses in both sexes. Mice treated with vehicle alone and the positive control substance appeared normal throughout the study. Reductions of 2% to 9% in the ratio of polychromatic erythrocytes to total erythrocytes were observed in some of the treated groups compared to controls suggesting that erythropoiesis was not inhibited. There were no significant increases in the number of micronucleated polychromatic erythrocytes per 2000 polychromatic erythrocytes in treated groups compared to controls, irrespective of sex and time of bone marrow collection. The positive control substance induced a significant increase.
Conclusions:
Under the conditions of this study, the test substance did not induce a significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow and was concluded to be negative in the mouse micronucleus test.
Executive summary:

The test substance was tested in the mouse micronucleus assay. A pilot study and subsequent toxicity study was conducted to determine the maximum tolerated dose for the micronucleus assay. The test substance was administered in corn oil by a single intraperitoneal injection, at a constant injection volume of 20 ml/kg. Based on mortality and clinical signs observed during the pilot and toxicity studies, the maximum tolerated dose set for the micronucleus study was 800 mg/kg. Clinical signs included lethargy, piloerection, tremors and crusty eyes. In the micronucleus assay, the test substance was administered i.p. at doses of 0, 200, 400 and 800 mg/kg. Bone marrow cells were harvested 24 or 48 hours later. The positive control was cyclophosphamide. No significant increase in micronucleated polychromatic erythrocytes in test article treated groups relative to vehicle controls was observed. Under the conditions of this study, the test substance was concluded to be negative in the mouse micronucleus test.

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

Additional information

Terephthaloyl dichloride rapidly hydrolyses to terephthalic acid (TPA). TPA is structurally similar to isophthalic acid (IPA). Therefore, genetic toxicity studies with TPA and IPA are being used to meet this data requirement.

TPA was tested in a bacterial reverse mutation (Ames) assay, and was concluded to be negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No indications of mutagenicity were observed at any dose level in any tester strain when tested up to 10000 μg/plate in the absence or presence of S9. The assay was conducted prior to the 1997 adaptation of the current OECD 471, and did not include an E.coli WP2 or S. typhimurium TA102 tester strain. These strains are known to specifically detect certain oxidising mutagens, cross-linking agents and hydrazines that other Salmonella tester strains may not be sensitive to. However, based on the physico-chemical characteristics of the test substance, the inclusion of a fifth tester strain would not have changed the clearly negative outcome of the Ames assay.

IPA was negative in a CHO/HPRT mutation assay. TPA did induce chromosome aberration without metabolic activation in vitro in human peripheral blood lymphocytes, but was negative in an in vivo mouse micronucleus assay.

The test substance is considered negative for genotoxicity.

Justification for classification or non-classification

Except for a positive response in a chromosome aberration test in cultured human lymphocytes, the substance did not produce mutagenicity when evaluated in cell culture or laboratory animals. The substance does not need to be classified for mutagenicity according to EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.