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Genetic toxicity in vitro

Description of key information

In Vitro (Mutagenic effects - bacterial): NOAEL; OECD 471; Ames study; not mutagenic; Reliability=1

In Vitro (Cytogenicity - mammalian): NOAEL; OECD 487; GLP; mammalian cell micronucleus test (CHO cells); positive with and without metabolic activation; Reliability= 1.

In Vitro (Mutagenic effects-mammalian): NOAEL; OECD 476 GLP; 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:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
yes
Remarks:
A chemical reaction with dosing vehicle due to known chemical properties and reactivity of the test substance was observed. This deviaiton was not suspected to affect the conduct or outcome of the study.
Qualifier:
according to
Guideline:
EPA OPPTS 870.5100 - Bacterial Reverse Mutation Test (August 1998)
Deviations:
yes
Remarks:
A chemical reaction with dosing vehicle due to known chemical properties and reactivity of the test substance was observed. This deviaiton was not suspected to affect the conduct or outcome of the study.
Qualifier:
according to
Guideline:
EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)
Deviations:
yes
Remarks:
A chemical reaction with dosing vehicle due to known chemical properties and reactivity of the test substance was observed. This deviaiton was not suspected to affect the conduct or outcome of the study.
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus in the genome
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Species / strain / cell type:
E. coli WP2 uvr A
Metabolic activation:
with and without
Metabolic activation system:
Liver homogenate (S9) prepared from male Sprague-Dawley rats induced with Aroclor 1254
Test concentrations with justification for top dose:
Toxicity-Mutation test: 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate
Mutagenicity Test (TA98, TA100, TA1535, TA1537 without activation): 66.7, 100, 333, 667, 1000 µg/plate
Mutagenicity Test (TA98, TA100, TA1535 with activation): 33.3, 66.7, 100, 333, 667 µg/plate
Mutagenicity Test (TA1537 with activation): 33.3, 66.7, 100, 333, 667, 1000 µg/plate
Mutagenicity Test (WP2urvA with/without activation): 100, 333, 667, 1000, 2000 µg/plate
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance was soluble in DMSO at 50 mg/mL, the highest stock concentration that was prepared for use on this study.
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
4-nitroquinoline-N-oxide
2-nitrofluorene
sodium azide
benzo(a)pyrene
other: acridine mutagen ICR-191, 2-aminoanthracene
Remarks:
Positive controls dissolved in DMSO, except sodium azide and ICR-191(dissolved in sterile water); vehicle and positive controls purchased from reliable commercial vendors expected to be free of contaminants; visually examined for indication of instability
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation); In the non-activated assays, 0.5 mL of sham mix and 100 µL of vehicle, test substance dilution, or positive control were added to pre-heated (45–48ºC) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain.
In the S9-activated assays, 100 µL of the vehicle, test substance dilution, or positive control were added to pre-heated (45–48ºC) glass culture tubes containing 2 mL of selective top agar, followed by 100 µL of tester strain and 0.5 mL of S9 mix. All mixtures were vortexed and overlaid onto the surface of minimum glucose agar plates.

DURATION: After the overlay solidified, the plates were inverted and incubated for approximately 65 to 69 hours at 37 ± 2ºC. Plates that were not evaluated immediately following the incubation period were stored at approximately 4ºC.

NUMBER OF REPLICATIONS: The toxicity-mutation test used duplicate plates for each dose level and the mutagenicity test used triplicate plates.

DETERMINATION OF CYTOTOXICITY: The appearance of the bacterial background lawn was assessed microscopically for test substance toxicity and precipitation. Toxicity was scored relative to the concurrent tester strain specific negative control, and evaluated as a decrease in the mean number of revertant bacterial colonies per plate. In addition, the thinning or disappearance of the bacterial background lawn was considered a sign of toxicity. Plates with observation of background lawn toxicity or precipitation were assigned a toxicity or precipitation code. Background lawn toxicity and precipitation were documented by exception only; no codes were assigned to plates with normal background lawn and that were free of precipitation.

OTHER:Revertant colonies were counted with the Sorcerer automated colony counter using Ames Study Manager software. Plates that could not be accurately counted automatically were counted manually.
Evaluation criteria:
Strains TA1535 and TA1537:
Data were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 3.0-fold the mean concurrent negative control value. This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

Strains TA98, TA100 and WP2uvrA
Data sets were judged positive if the increase in mean revertants at the highest numerical dose response was ≥ 2.0-fold the mean concurrent negative control value. This increase in the mean number of revertants per plate must be accompanied by a dose response associated with increasing concentrations of the test substance unless observed at the top dose level only.

A data set may be judged equivocal if there was a biologically relevant increased response that only partially meets criteria for a positive response. A response was evaluated as negative if it was neither positive nor equivocal.
Statistics:
For all replicate plates, the results were presented as the mean revertants per plate and standard deviation.
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:
cytotoxicity
Remarks:
background lawn reduction and >50% reduction in revertent colonies at varying dose levels
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
background lawn reduction and >50% reduction in revertent colonies at varying dose levels
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
RANGE-FINDING/SCREENING STUDIES:
In the toxicity-mutation test, the maximum dose evaluated was 5000 µg/plate for tester strains TA98, TA100, TA1535, TA1537, and WP2uvrA in the absence and presence of S9 metabolic activation. This dose was achieved using a concentration of 50 mg/mL and a 100 µL plating aliquot. The dose levels used in this test were 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg/plate. The plate incorporation method was employed. No positive mutagenic responses were observed at any dose level in any tester strain in the absence or presence of S9 metabolic activation. No test substance precipitation was observed. Toxicity was observed as both background lawn reduction and as a >50% reduction in revertant colonies at varying dose levels (see Table below).

Additional tables containing revertant data for the toxicity-mutation and mutagenicity tests are in the attached pdf document.

Cytotoxicity in Toxicity-Mutation Test

Tester Strain

Metabolic Activation

Toxicity starting at (µg/plate)

>50% reduction in revertant colonies

Background lawn reduction

TA98

-

1000

667

+

667

333

TA100

-

1000

667

+

1000

667

TA1535

-

1000

667

+

667

1000

TA1537

-

667

667

+

1000

1000

WP2urvA

-

3333

3333

+

3333

1000

 

Cytotoxicity in Mutagenicity Test

Tester Strain

Metabolic Activation

Toxicity starting at (µg/plate)

>50% reduction in revertant colonies

Background lawn reduction

TA98

-

none

667

+

none

667

TA100

-

none

1000

+

none

667

TA1535

-

none

1000

+

none

667

TA1537

-

1000

667

+

1000

667

WP2urvA

-

none

2000

+

2000

1000

Conclusions:
The test substance was negative in this in vitro test.

Executive summary:

The test substance was evaluated for mutagenicity in the Bacterial Reverse Mutation Test using the plate incorporation method. Salmonella strains TA98, TA100, TA1535, and TA1537 and Escherichia coli strain WP2uvrA were tested in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). The test was performed in 2 phases. The first phase was the toxicity-mutation test, which established the dose range for the mutagenicity test, and provided a preliminary mutagenicity evaluation. The second phase was the mutagenicity test, which evaluated and confirmed the mutagenic potential of the test substance.

Based on the toxicity-mutation test, the dose levels evaluated in the mutagenicity test ranged from 33.3 to 2000 µg/plate. No positive mutagenic responses were observed at any dose level or with any tester strain in either the absence or presence of S9 metabolic activation. No test substance precipitation was observed. Toxicity was observed as both background lawn reduction and as a >50% reduction in revertant colonies at varying dose levels. All criteria for a valid study were met. Under the conditions of this study, the test item showed no evidence of mutagenicity in the Bacterial Reverse Mutation Test either in the absence or presence of Aroclor-induced rat liver S9. It was concluded that the test substance was negative in this in vitro test.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
other: OECD Guideline 487 (In Vitro Mammalian Cell Micronucleus Test, Guidelines for the Testing of Chemicals) (adopted 2014)
Deviations:
yes
Remarks:
A chemical reaction with dosing vehicle due to known chemical properties and reactivity of the test substance was observed. This deviaiton was not suspected to affect the conduct or outcome of the study.
Qualifier:
according to
Guideline:
other: EC Directive (640/2012/EC Method B.49)
Deviations:
yes
Remarks:
A chemical reaction with dosing vehicle due to known chemical properties and reactivity of the test substance was observed. This deviaiton was not suspected to affect the conduct or outcome of the study.
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
micronuclei in Chinese Hamster Ovary (CHO-K1) cells
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
Chinese Hamster Ovary (CHO-K1) cells
- Periodically checked for Mycoplasma contamination: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor-induced rat liver S9
Test concentrations with justification for top dose:
Preliminary Toxicity test: 5, 10, 25, 50, 75, 125, 250, 500, 1000, and 2000 µg/mL
Micronucleus Assay (4-hour activated and non-activated): 5, 10, 25, 50 and 75 µg/mL
Micronucleus Assay (24-hour non-activated): 10, 25, 30, 35, 40, 45, and 50 µg/mL
Repeat Micronucleus Assay (4-hour activated and non-activated): 5, 10, 25, 50, 100, 200, 300, 400, and 500 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Dimethyl sulfoxide (DMSO)
- Justification for choice of solvent/vehicle: Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance formed a transparent amber solution in DMSO at 200 mg/mL, the highest stock concentration used in the study.
Untreated negative controls:
yes
Remarks:
DMSO
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; The CHO-K1 cultures were initiated in labeled sterile, 48-well tissue culture treated plates by seeding 0.5 mL of appropriately concentrated seeding stock to required wells. Plates were incubated at 37ºC ± 2ºC in a humidified atmosphere of 5 ± 2% CO2 in air. Approximately 24 hours after seeding, the culture medium was aspirated and replaced with either 500 µL of test substance or vehicle prepared in treatment media, or 495 µL of either complete F12-K media, or S9 mix, containing 1% DMSO which was then inoculated with 5 µL of appropriate positive control for a final volume of 500 µL.
DURATION: In the non-activated test system, the treatment times were approximately 4 and 24 hours, and in the S9-activated test system, approximately 4 hours. The cells were harvested at approximately 24 hours from the initiation of the treatment, at a time point representing approximately a 1.5 normal cell cycle time.

STAIN (for cytogenetic assays): Trypan blue

NUMBER OF REPLICATIONS: Triplicate

NUMBER OF CELLS EVALUATED: 20000 nucleated cells per sample

DETERMINATION OF CYTOTOXICITY: As indicated by the frequency of beads among the total nucleated cells counted.

OTHER: The osmolality and pH of the vehicle control, as well as the 75 and 50 μg/mL test substance concentration in the culture media, were determined for the micronucleus assay. The pH of the treatment medium was evaluated both at the beginning and end of the treatment period by visual determination using the pH-sensitive color indicator present in the treatment medium. Precipitation was also evaluated both at the beginning and the end of the treatment period by visual determination. Relative survival was determined via a bead ration obtained during flow cytometric analysis of the micronuclei. Relative survival of ≤ 50% was not reached in the 4-hour non-activated and activated conditions; therefore the micronucleus assay repeated to include additional dose levels.
Evaluation criteria:
The following conditions were used as a guide to determine a positive response:
− A statistically significant increase (p < 0.05, Fisher’s exact test) in the percentage of cells with micronuclei was seen in one or more treatment groups relative to the vehicle control response.
− The observed increased frequencies were accompanied by a concentration-related increase when evaluated by trend test.
− Any of the results were outside the 95% control limit distribution of the historical negative control data. (Note: Statistically significant values that did not exceed the historical control range for the negative/vehicle control may be judged as not being biologically significant.)

The following condition was used as a guide to determine an equivocal response:
− The data did not allow a conclusion of positive or negative.

The test substance was judged negative if the following conditions were met:
− There was no statistically significant increase in the percentage of cells with micronuclei in any treatment group relative to the vehicle control group.
− There was no concentration-related increase when evaluated with an appropriate trend test.
− All results were within the 95% control limit distribution of the historical negative control data.
Statistics:
Statistical analysis consisted of a Fisher’s exact test to compare the percentage of cells with micronuclei in the test substance treated groups with the vehicle control response. A Cochran-Armitage test for dose responsiveness was conducted only if statistically significant values, based on the Fisher’s exact test, were found.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
positive
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Substantial toxicity observed at ≥45 µg/L in 24-hr non-activated condition (micronucleus assay). Substantial toxicity (≤50% survival relative to vehicle control) at 4-hr activated and non-activated conditions (repeat assay).
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 pH change was observed in any condition
- Effects of osmolality: Observed changes in osmolality were ≤20% and were, therefore, not considered significant.
- Evaporation from medium:
- Water solubility:
- Precipitation: Test substance precipitation was not observed in any condition.
- Other confounding effects:

RANGE-FINDING/SCREENING STUDIES: No test substance precipitation was observed during the preliminary toxicity test. Substantial toxicity (≤50% survival relative to the vehicle control) was observed at concentrations ≥75 µg/mL in the 4-hour S9-activated and non-activated test condition and at concentrations ≥50 µg/mL in the 24-hour non-activated test condition. Osmolality in the non-activated test system was 462, 459, and 462 mOsm/kg at 0, 2000, and 1000 µg/mL, respectively. Osmolality in the S9-activated test system was 471, 476, and 475 mOsm/kg at 0, 2000, and 1000 µg/mL, respectively. pH in the non-activated test system was 8.10, 7.91, and 7.56 at 0, 2000, and 1000 µg/mL, respectively. pH in the S9-activated test system was 7.41, 7.28, and 7.08 at 0, 2000, and 1000 µg/mL, respectively. Observed changes in osmolality were ≤20% and were, therefore, not considered significant.

COMPARISON WITH HISTORICAL CONTROL DATA:

Cell proliferation calculations were conducted to determine the population doubling time. Cells used in the initial micronucleus assay exhibited an average population doubling of 1.68 and in the repeat micronucleus assay a 1.82 average population doubling. 

 

The percentage of micronuclei was significantly increased in the 4-hour non-activated condition in respect to the vehicle control in a dose-responsive manner. The percentage of cells with micronuclei in the 4-hour non-activated condition at concentrations of ≥50 µg/mL exceeded the negative historical 95% control limits.

 

Since survival levels of ≤50% were not reached in the 4-hour non-activated test condition, a repeat assay was conducted. The percentage of micronuclei was significantly increased in respect to the vehicle control in a dose-responsive manner up to 100 µg/mL with a 27.64% survival. At the next higher concentration level, 200 µg/mL, a decrease in micronuclei was observed in conjunction with a decrease in relative survival. The percentage of cells with micronuclei at 50 and 100 µg/mL exceeded the negative historical 95% control limits.

 

The percentage of micronuclei was significantly increased in the 4-hour activated test condition with respect to the vehicle control. A significant dose response was observed in the 4-hour activated test condition. The percentage of cells with micronuclei in the 4-hour activated condition ≥50 µg/mL exceeded the negative historical 95% control limits. Relative survival levels of ≤50% were not reached resulting in a repeat micronucleus assay. The repeat micronucleus assay flow cytometric analysis was conducted on dose levels up to 300 µg/mL in the activated condition, vehicle and positive controls. Higher doses were not analyzed due to the excessive toxicity observed at the 300 µg/mL. The percentage of micronuclei was significantly increased in the 4-hour activated test conditions with respect to the vehicle control. The percentage of cells with micronuclei in the 4-hour activated condition at 50 µg/mL exceeded the negative historical 95% control limits. A significant dose response was observed in the 4-hour activated test condition. In the 4-hour activated condition, a dose-dependent increase in micronuclei was observed up to 50 µg/mL with a 108.6% survival. At the next dose level, 100 µg/mL, a dose-dependent decrease in micronuclei was observed in conjunction with a dose-dependent decrease in survival. The percentage of micronuclei was not significantly increased in the 24-hour activated test conditions with respect to the vehicle control. Relative survival levels of ≤50% were reached in the 24-hour non-activated condition. 

 

Micronucleus Summary – Micronucleus Test

Treatment µg/mL

S9 Activation

Treatment Time (hours)

Average Relative Survival (%)

Average MN Frequency (%)

Fold Increase in Micronuclei

Vehicle

-S9

4

100.00

1.18a

NA

5

-S9

4

138.12

1.10

0.94

10

-S9

4

106.02

1.30

1.11

25

-S9

4

152.92

1.27

1.08

50

-S9

4

91.21

3.04b,d

2.58

75

-S9

4

83.19

3.74b,d

3.18

0.4 MMC

-S9

4

62.37

8.03c,d

6.83

0.8 MMC

-S9

4

62.14

8.24c,d

7.00

Vehicle

+S9

4

100.00

3. 57a

NA

5

+S9

4

179.49

3.66

1.02

10

+S9

4

127.95

2.99

0.84

25

+S9

4

188.77

3.49

0.98

50

+S9

4

104.52

5.05b

1.41

75

+S9

4

84.86

7.15b

2.00

2 CP

+S9

4

116.43

5.97c,d

1.67

4 CP

+S9

4

129.11

5.65d

1.58

Vehicle

-S9

24

100.00

5.20a

NA

10

-S9

24

102.73

3.64

0.70

25

-S9

24

91.19

4.54

0.87

30

-S9

24

91.43

3.48

0.67

35

-S9

24

61.58

5.58b

1.07

40

-S9

24

80.17

5.96b

1.15

45

-S9

24

45.80

4.76

0.92

50

-S9

24

50.57

4.69

0.90

0.4 MMC

-S9

24

46.49

12.66c,d

2.44

0.8 MMC

-S9

24

45.07

9.70c,d

1.87

aWithin negative historical 95% control limits

b Above negative historical 95% control limits

cWithin historical positive control range

dStatistically significant increase relative to the vehicle

 

Micronucleus Summary – Repeat Micronucleus Test

Treatment µg/mL

S9 Activation

Treatment Time (hours)

Average Relative Survival (%)

Average MN Frequency (%)

Fold Increase in Micronuclei

Vehicle

-S9

4

100.00

1.63a

NA

5

-S9

4

63.47

0.76

0.46

10

-S9

4

79.77

0.76

0.47

25

-S9

4

57.77

1.13

0.69

50

-S9

4

43.70

3.94b,d

2.41

100

-S9

4

27.64

4.12b,d

2.52

200

-S9

4

19.77

0.38

0.23

0.4 MMC

-S9

4

73.04

4.53c,d

2.77

0.8 MMC

-S9

4

35.85

6.71c,d

4.11

Vehicle

+S9

4

100.00

2.26a

NA

5

+S9

4

164.02

2.81

1.24

10

+S9

4

152.35

3.12

1.38

25

+S9

4

177.17

3.54

1.57

50

+S9

4

108.06

6.18b,d

2.74

100

+S9

4

79.41

1.54

0.68

200

+S9

4

50.12

0.11

0.05

300

+S9

4

10.72

0.36

0.16

2 CP

+S9

4

105.91

4.49c,d

1.99

4 CP

+S9

4

85.7

6.68d

2.96

aWithin negative historical 95% control limits

b Above negative historical 95% control limits

cWithin historical positive control range

dStatistically significant increase relative to the vehicle

 

Additional tables containing individual cytotoxicity and micronuclei assessments are in the attached pdf document.

Conclusions:
The test substance was positive for induction of micronuclei in the in vitro mammalian micronucleus test in Chinese hamster ovary cells.
Executive summary:

The test substance was evaluated for its ability to induce micronuclei in Chinese hamster ovary (CHO-K1) cells in vitro in the absence and presence of an exogenous metabolic activation system (Aroclor-induced rat liver S9). Dimethyl sulfoxide (DMSO) was chosen as the dosing vehicle based on the solubility of the test substance and compatibility with the target cells. The test substance formed a transparent amber solution in DMOS at 200 mg/mL, the highest stock concentration used in the study. The test substance was observed to react chemically with the vehicle during formulation. This was due to the known chemical properties and reactivity of the test substance.

A preliminary toxicity test was conducted to establish the concentration range for the micronucleus assay. The cells were exposed to 10 concentrations of the test substance ranging from 5 to 2000 µg/mL, as well as the vehicle and positive controls. The top dose, 2000 µg/mL, was pH adjusted prior to the preparation of subsequent doses. No additional pH adjustments were required. In the preliminary toxicity test the cells were treated for 4 and 24 hours in the non-activated test condition and for 4 hours in the S9-activated test condition. All cells were harvested 24 hours after treatment initiation and relative cell survival was calculated. No test substance precipitation was observed. Substantial toxicity (≤50% survival relative to the vehicle control) was observed at concentrations ≥75 µg/mL in the 4-hour S9-activated and non-activated test condition and at concentrations ≥50 µg/mL in the 24-hour non-activated test condition.

The concentrations chosen for the micronucleus assay were 5, 10, 25, 50, and 75 µg/mL in the 4-hour activated and non-activated test conditions and 10, 25, 30, 35, 40, 45, and 50 µg/mL in the 24-hour non-activated test condition.

In the micronucleus assay the cells were exposed to 5-7 concentrations of the test substance ranging from 5 to 75 µg/mL, and to the vehicle and positive controls. No pH adjustment was required. In the micronucleus assay the cells were treated for 4 and 24 hours in the non-activated test condition and for 4 hours in the S9-activated test condition. All cells were harvested 24 hours after treatment and analysed for the presence of micronuclei by flow cytometry. No test substance precipitation was observed.

Analysis was conducted on all concentration levels, vehicle and positive controls for all test conditions in the micronucleus assay. Substantial toxicity was not observed at any concentration level in the 4-hour S9-activated and non-activated test condition. Substantial toxicity was observed at concentrations ≥45 µg/mL in the 24-hour non-activated test condition. The percentage of micronuclei was significantly increased in the 4-hour activated and non-activated test conditions as compared to the concurrent vehicle controls. A statistically significant dose response was observed in the 4-hour activated and non-activated test conditions. The percentage of cells with micronuclei in the 4-hour activated condition at concentrations ≥50 µg/mL and the 4-hour non-activated condition at concentrations ≥50 µg/mL exceeded the negative historical 95% control limits. The percentage of micronuclei was not significantly increased in the 24-hour non-activated test condition.

Since relative cell survival of ≤50% was not reached in the first trial in the 4-hour conditions, a repeat assay was conducted. The concentrations selected for the repeat assay were 5, 10, 25, 50, 100, 200, 300, 400, and 500 µg/mL.

In the repeat micronucleus assay the cells were exposed to 9 concentrations of the test substance ranging from 5 to 500 µg/mL, as well as the vehicle and positive controls. The formulation of the top concentration level, 500 µg/mL, was adjusted for pH prior to the preparation of subsequent doses. No additional pH adjustments were required. The cells were treated for 4 hours in the non-activated and activated test conditions. All cells were harvested 24 hours after the initiation of the treatment and analysed for the presence of micronuclei by flow cytometry. No test substance precipitation was observed.

The repeat micronucleus assay flow cytometric analysis was conducted on test substance concentrations ≤300 µg/mL in the activated condition and ≤200 µg/mL in the non-activated condition, and on the vehicle and positive controls. Relative cell survival was calculated during the flow cytometric analysis and cells treated with higher concentrations were not analysed due to the excessive decrease in relative survival observed at the 300 and 200 µg/mL concentration levels.

The percentage of micronuclei was significantly increased in the 4-hour activated and non-activated test conditions as compared to the concurrent vehicle control. The percentage of cells with micronuclei in the 4-hour activated condition at 50 µg/mL and the percentage of cells with micronuclei in the 4-hour non-activated condition at 50 and 100 µg/mL exceeded the negative historical 95% control limits. A significant dose response was observed in the 4-hour activated and non-activated test conditions. In the 4-hour activated condition a dose-dependent increase in micronuclei was observed at concentrations ≤50 µg/mL. In the 4-hour non-activated condition, a dose-dependent increase in micronuclei was observed at concentrations ≤100 µg/mL.

All criteria for a valid study were met. Under the conditions of this study, the test substance was found to induce micronuclei in the in vitro mammalian micronucleus test in Chinese hamster ovary cells in the non-activated and S9-activated test systems. It was concluded that the test substance was positive in this in vitro test.

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
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 isophthalic acid, has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labeling and/or risk assessment.
Justification for type of information:
The test substance rapidly hydrolyses to isophthalic acid (IPA). Therefore, the study with IPA is being used to fulfil this data requirement. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.
Reason / purpose:
read-across: supporting information
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Remarks:
The study was conducted according to the guideline in effect dated 1997.
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.

Additional tables containing cytotoxicity and mutagenicity data are in the attached pdf document.

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

In Vivo (Clastogenic effects - mammalian): NOAEL; OECD 474; GLP; In vivo mouse micronucleus study; no mammalian mutagenesis was observed at any exposure level; 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:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
The study, if used in support of terephthalic acid, has a reliability of 1 (reliable without restriction). This study was selected as the key study because the information provided for the hazard endpoint is sufficient for the purpose of classification and labelling and/or risk assessment. Guideline study performed under GLP requirements.
Justification for type of information:
The test substance rapidly hydrolyses to isophthalic acid (IPA) and IPA is structurally similar to terephthalic acid (TPA). Therefore, the study with TPA is being used to fulfil this data requirement. Additional documentation, provided within the IUCLID Assessment Reports section, supports the read-across approach.
Reason / purpose:
read-across: supporting information
Qualifier:
according to
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 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:
1000 mg/kg and above; 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.

Administration of the test substance did not induce any increase in the incidence of micronucleated PCEs in any of the treated groups.

Summary of Bone Marrow Micronucleus Study

Treatment

Sex

Time
(hr)

Number of Mice 

PCE/Total

Change from Control (%)

Micronucleated Polychromatic Erythrocytes

Erythrocytes
(Mean +1- SD)

Number per 1000 PCEs
(Mean +1- SD)
 

Number per PCEs Scored1

Corn oil

 

 

 

 

 

 

 

20 mL/kg

M

24

5

0.456 ± 0.03

0.3 ± 0.27

3 / 10000

 

F

24

5

0.477 ± 0.06

0.1 ± 0.22

1 I 10000

Terephthalic acid

 

 

 

 

 

 

 

200 mg/kg

M

24

5

0.455 ± 0.06

0

0.4 ± 0.22

4 / 10000

 

F

24

5

0.445 ± 0.03

-7

0.4 ± 0.22

4 / 10000

400 mg/kg

M

24

5

0.474 ± 0.06

4

0.3 ± 0.27

3 / 10000

 

F

24

5

0.435 ± 0.03

-9

0.2 ± 0.27

2 / 10000

800 mg/kg

M

24

5

0.465 ± 0.04

2

0.2 ± 0.27

2 / 10000

 

F

24

5

0.467 ± 0.07

-2

0.2 ± 0.27

2 / 10000

CP

 

 

 

 

 

 

 

50 mg/kg

M

24

5

0.342 ± 0.03

-25

16.7 ± 5.73

*167 / 10000

 

F

24

5

0.339 ± 0.02

-29

21.5 ± 3.04

*215 I 10000

Corn oil

 

 

 

 

 

 

 

20 mL/kg

M

48

5

0.484 ± 0.04

0.5 ± 0.00

5 / 10000

 

F

48

5

0.463 ± 0.03

0.3 ± 0.27

3 / 10000

Terephthalic acid

 

 

 

 

 

 

 

800 mg/kg

M

48

5

0.495±0.05

2

0.0 ± 0.00

0 / 10000

 

F

48

5

0.476±0.04

3

0.2 ± 0.27

2 / 10000

1*p≤0.05 (Kastenbaum-Bowman Tables)

 

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 24 Hours After a Single Dose of Terephthalic Acid

Treatment

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE
(Number/PCE scored)

Corn oil

 

 

 

 

20 mL/kg

M

101

0.446

0 / 2000

 

 

102

0.408

1 / 2000

 

 

103

0.464

1 / 2000

 

 

104

0.468

0 / 2000

 

 

105

0.493

1 / 2000

 

F

106

0.438

0 / 2000

 

 

107

0.561

1 / 2000

 

 

108

0.434

0 / 2000

 

 

109

0.438

0 / 2000

 

 

110

0.516

0 / 2000

Terephthalic acid

 

 

 

 

200 mg/kg

M

111

0.438

1 / 2000

 

 

112

0.419

1 / 2000

 

 

113

0.553

1 / 2000

 

 

114

0.428

0 / 2000

 

 

115

0.435

1 / 2000

 

F

116

0.456

0 / 2000

 

 

117

0.488

1 / 2000

 

 

118

0.441

1 / 2000

 

 

119

0.420

1 / 2000

 

 

120

0.420

1 / 2000

400 mg/kg

M

121

0.465

1 / 2000

 

 

122

0.434

0 / 2000

 

 

123

0.414

1 / 2000

 

 

124

0.566

0 / 2000

 

 

125

0.491

1 / 2000

 

F

126

0.423

0 / 2000

 

 

127

0.418

0 / 2000

 

 

128

0.413

0 / 2000

 

 

129

0.481

1 / 2000

 

 

130

0.439

1 / 2000

800 mg/kg

M

131

0.525

0 / 2000

 

 

132

0.456

0 / 2000

 

 

173*

0.408

1 / 2000

 

 

134

0.458

0 / 2000

 

 

135

0.478

1 / 2000

 

F

136

0.462

1 / 2000

 

 

137

0.388

0 / 2000

 

 

138

0.564

0 / 2000

 

 

139

0.484

1 / 2000

 

 

140

0.435

0 / 2000

CP

 

 

 

 

50 mg/kg

M

141

0.350

23 / 2000

 

 

142

0.322

30 / 2000

 

 

143

0.323

32 / 2000

 

 

144

0.333

29 / 2000

 

 

145

0.384

53 / 2000

 

F

146

0.366

34 / 2000

 

 

147

0.345

44 / 2000

 

 

148

0.315

51 / 2000

 

 

149

0.340

44 / 2000

 

 

150

0.329

42 / 2000

* Replacement animal

 

Induction of Micronucleated Polychromatic Erythrocytes in Bone Marrow Cells Collected 48 Hours After a Single Dose of Terephthalic Acid

Treatment

Sex

Animal Number

PCE/Total Erythrocytes

Micronucleated PCE
(Number/PCE scored)

Corn oil

 

 

 

 

20 mL/kg

M

151

0.472

1 / 2000

 

 

152

0.470

1 / 2000

 

 

153

0.447

1 / 2000

 

 

154

0.485

1 / 2000

 

 

155

0.544

1 / 2000

 

F

156

0.454

1 / 2000

 

 

157

0.466

1 / 2000

 

 

158

0.480

0 / 2000

 

 

159

0.425

0 / 2000

 

 

160

0.490

1 / 2000

Terephthalic acid

 

 

 

800 mg/kg

M

161

0.541

0 / 2000

 

 

162

0.454

0 / 2000

 

 

163

0.444

0 / 2000

 

 

164

0.494

0 / 2000

 

 

165

0.543

0 / 2000

 

F

166

0.439

1 / 2000

 

 

167

0.500

1 / 2000

 

 

168

0.530

0 / 2000

 

 

169

0.455

0 / 2000

 

 

170

0.458

0 / 2000

 

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

The test substance was negative in a bacterial reverse mutation assay in Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537 and E.coli WP2 uvrA. The test substance was positive in an in vitro micronucleus study in Chinese hamster ovary cells.

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.

Based on the above data, overall, the test substance is considered negative for genotoxicity.

Justification for classification or non-classification

Except for a positive response in an in vitro micronucleus assay in CHO cells (test substance) and a positive response in the chromosome aberration test in cultured human lymphocytes (read-across substance), the substance and the read-across substances did not produce mutagenicity when evaluated in cell culture or laboratory animals. Overall, the test substance is considered negative for genotoxicity and therefore 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.