Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- negative: Ames test with Salmonella typhimurium TA 1535, TA 1537, TA 98, TA 100 and Escherichia coli WP2 uvrA (met. Act.: with and without) (OECD TG 471 (21 Jul 1997; GLP); REL1; cytotoxicity: yes

- negative: mammalian cell gene mutation assay, CHO cells (met. Act.: with and without) (OECD TG 476 (26 September 2014; GLP); REL1; cytotoxicity: yes

- negative: micronucleus assay, human peripheral blood lymphocytes (met. Act.: with and without) (OECD TG 487 (29 July 2016; GLP); REL1; cytotoxicity: yes

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
Study period:
2017-07-28 to 2017-09-15
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
(adopted July 21, 1997)
Deviations:
no
GLP compliance:
yes
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
SOURCE OF TEST MATERIAL
- provided by the sponsor

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Room temperature, protected from light and under nitrogen
- Solubility of the test substance in the solvent/vehicle:
test substance formed a clear solution in ethanol at a concentration of approximately 500 mg/mL with sonication at 37.0ºC for 20 minutes
- Stability of the test substance in the solvent/vehicle:
stable in ethanol, at concentrations of 0.0314 and 59.0 mg/mL, at room temperature for at least 3.5 hours, and at a concentration of 99.1 mg/mL for at least 3.1 hours.


Target gene:
histidine locus of several Salmonella typhimurium strains
tryptophan locus of Escherichia coli strain WP2 uvrA
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 mix
Test concentrations with justification for top dose:
preliminary toxicity assay: 6.67, 10.0, 33.3, 66.7, 100, 333, 667, 1000, 3333, and 5000 µg per plate
initial mutagenicity assay 5.00, 15.0, 50.0, 150, 500, 1500 and 3000 µg per plate (tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, and TA100 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150, 333 and 500 µg per plate (tester strains TA100 in the absence of S9 activation, and TA1535 in the presence and absence of S9 activation).
retest of mutagenicity assay: 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation
protocol criteria for top dose or toxicity were met
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: ethanol
- Justification for choice of solvent/vehicle: solubility pretest
Untreated negative controls:
yes
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
sodium azide
methylmethanesulfonate
other: 2 aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DETERMINATION OF CYTOTOXICITY
- Method: thinning of the microcolony lawn

METHOD OF APPLICATION: in agar (plate incorporation)

NUMBER OF REPLICATIONS: main test was performed in triplicates

DETERMINATION OF CYTOTOXICITY
- Method: thinning of the microcolony lawn
Rationale for test conditions:
The study was concluded to be negative without conducting a confirmatory (independent repeat) assay because the results were clearly negative; hence, no further testing was warranted.
Evaluation criteria:
For each replicate plating, the mean and standard deviation of the number of revertants per plate were calculated and are reported.
For the test substance to be evaluated positive, it must cause a dose-related increase in the mean revertants per plate of at least one tester strain over a minimum of two consecutive increasing concentrations of test substance as specified below:

Data sets were judged positive if the increase in mean revertants at the peak of the dose-response was equal to or greater than 3.0-times (Strains TA1535 and TA1537) respectively equal to or greater than 2.0-times (Strains TA98, TA100 and WP2 uvrA) the mean vehicle control value and above the corresponding acceptable vehicle control range.

Key result
Species / strain:
S. typhimurium, other: TA98, TA100, TA1535, TA1537
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
valid
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no precipitation observed up to the limit dose
- Other confounding effects: no
- Sterility Results: No contaminant colonies were observed on the sterility plates for the vehicle control, the test substance dilutions or the S9 and Sham mixes.


RANGE-FINDING/SCREENING STUDIES:
A range-finding study was conducted in all tester strains. Toxicity was observed beginning at 100, 333, 667, 1000, and 3333 µg per plate with all conditions.

Main test:
Toxicity was observed beginning at 150, 333, 1500, and 3000 µg per plate with all conditions except tester strain TA1535 in the presence of S9 activation
No precipitate was observed.

TA1535:
In the retest of mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation. No precipitate was observed. Toxicity was observed beginning at 1500 µg per plate. No positive mutagenic responses were observed for the test substance.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data: given in report
- Negative (solvent/vehicle) historical control data: given in report

Preliminary Toxicity Assay

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 1000

≥ 3333

TA100

≥ 333

≥ 1000

TA1535

≥ 100a

≥ 667

TA1537

≥ 667

≥ 3333

WP2uvrA

≥ 1000

≥ 3333

aToxicity was observed as a reduction in revertant counts.

Toxicity levels in main test

Tester Strains

Without metabolic activation

(µg per plate)

With metabolic activation

(µg per plate)

Toxicity

Toxicity

TA98

≥ 1500

3000

TA100

≥ 150

≥ 1500

TA1535

≥ 333

-

TA1537

≥ 1500

3000

WP2uvrA

≥ 1500

3000

In order to achieve the protocol criteria for top dose or toxicity, the test condition TA1535 in the presence of S9 activation was repeated. 

Conclusions:
Under the conditions of this study, CGE-PMDA adduct did not cause a positive mutagenic response with any of the tester strains in either the presence or absence of Aroclor induced rat liver S9. All criteria for a valid study were met as described in the protocol.
Executive summary:

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrAwere exposed to CGE-PMDA adduct in ethanol in concentrations of 0 (control), 5.00, 15.0, 50.0, 150, 500, 1500 and 3000 µg per plate (tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, and TA100 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150, 333 and 500 µg per plate (tester strains TA100 in the absence of S9 activation, and TA1535 in the presence and absence of S9 activation). In the retest of mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation.The assay was performed using the plate incorporation method.

 

The test substance was tested up to cytotoxic concentrations.Toxicity was observed beginning at 150, 333, 1500 and 3000 µg per plate with all conditions except tester strain TA1535 in the presence of S9 activation. In order to achieve the protocol criteria for top dose or toxicity, the test condition TA1535 in the presence of S9 activation was repeated.Toxicity was then observed beginning at 1500 µg per plate.Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

 

There was no evidence of an increase in the number of revertant colonies that was equal to or greater than 3.0-times the mean vehicle control value in strains TA1535 and TA 1537 or exceeded twice the background in the tester strains TA98, TA100 and WP2 uvrA examined at the peak of the dose-response. Therefore, test substance was considered to be non-genotoxic (nonmutagenic) in Salmonella tester strains TA98, TA100, TA1535, TA1537 andEscherichia coli strain WP2 uvrA under the conditions employed (plate incorporation assay).

 

Under the conditions of the study, the test substance was negative for mutagenic potential.

Endpoint:
in vitro cytogenicity / micronucleus study
Type of information:
experimental study
Adequacy of study:
key study
Study period:
20 July 2017 - 12 October 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)
Version / remarks:
updated and adopted 26 September 2014
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell micronucleus test
Target gene:
not applicable
Species / strain / cell type:
lymphocytes: human peripheral blood lymphocytes
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: Peripheral blood lymphocytes were obtained from a healthy non-smoking individual (no recent history of radiotherapy, viral infection, or the administration of drugs)

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: RPMI 1640 containing 15% fetal bovine serum, 2 mM L glutamine, 100 units penicillin, 100 µg/mL streptomycin, 37 ± 1C in a humidified atmosphere of 5 ± 1% CO2 in air
- Properly maintained: yes
Cytokinesis block (if used):
cytochalasin B
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
preliminary test: 0.5, 1.5, 5, 15, 50, 150, 500, 1500, 5000 µg/mL

concentrations in definitive tests, see section "Any other information on materials and methods"
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: solubility of the test substance, compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
cyclophosphamide
mitomycin C
other: vinblastine
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 4-24 hours
- Expression time (cells in growth medium): 24 hours

SPINDLE INHIBITOR (cytogenetic assays): cytochalasin B, 24 hours (± 30 minutes), 1.5 to 2 normal cell cycle

STAIN (for cytogenetic assays): acridine orange

NUMBER OF REPLICATIONS: two

METHODS OF SLIDE PREPARATION AND STAINING TECHNIQUE USED: cells were collected by centrifugation, and the suspension of fixed cells was applied to glass microscope slides and air-dried; stained with acridine orange

NUMBER OF CELLS EVALUATED: A minimum of 2000 binucleated cells from each concentration (if possible, 1000 binucleated cells from each culture) were examined and scored for the presence of micronuclei.

CRITERIA FOR MICRONUCLEUS IDENTIFICATION:
• the micronucleus should have the same staining characteristics as the main nucleus
• the micronuclei should be separate from the main nuclei or just touching (no cytoplasmic bridges)
• the micronuclei should be of regular shape and approximately 1/3 or less than the diameter of the main nucleus.


DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cloning efficiency; relative total growth; other:
- Any supplementary information relevant to cytotoxicity:

OTHER EXAMINATIONS:
- Determination of polyploidy:
- Determination of endoreplication:
- Methods, such as kinetochore antibody binding, to characterize whether micronuclei contain whole or fragmented chromosomes (if applicable):

- OTHER:
Evaluation criteria:
The test substance was considered to have induced a positive response if all of the following three criteria are met:
• at least one of the test concentrations exhibited a statistically significant increase when compared with the concurrent negative control (p ≤ 0.05), and
• the increase was concentration-related (p ≤ 0.05), and
• results were outside the 95% control limit of the historical negative control data.

The test substance was considered to have induced a clear negative response if none of the criteria for a positive response were met.
Statistics:
Statistical analysis was performed using the Fisher's exact test (p  0.05) for a pairwise comparison of the percentage of micronucleated cells in each treatment group with that of the vehicle control. The Cochran-Armitage trend test was used to assess dose-responsiveness.
Species / strain:
lymphocytes: human peripheral blood lymphocytes
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the highest dose of test substance in treatment medium was 9.5. The pH was adjusted in order to maintain neutral pH.
- Effects of osmolality: The osmolality of the test substance doses in treatment medium was considered acceptable
- Precipitation: >/=1500 µg/mL
- highest soluble concentration: 500 µg/mL

RANGE-FINDING/SCREENING STUDIES:
Cytotoxicity [>/= 50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >/= 150 µg/mL in the non activated and S9-activated 4-hour exposure groups, and at doses >/= 50 µg/mL in the non activated 24-hour exposure group. Based upon the results of the preliminary toxicity assay, the doses selected for the micronucleus assay were as follows:
Treatment Recovery
Time Time
non-activated: 4 hr 20 hr 25, 50, 75, 100, 125, 150 µg/mL
24 hr 0 hr 5, 10, 25, 37.5, 50, 75 µg/mL
S9-activated 4 hr 20 hr 25, 50, 75, 100, 125, 150 µg/mL

Conclusions:
Under the conditions of the assay described in this report, CGE-PMDA adduct was concluded to be negative for the induction of micronuclei in the non-activated and S9-activated test systems in the in vitro mammalian micronucleus test using human peripheral blood lymphocytes.
Executive summary:

CGE-PMDA adduct, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 in accordance with OECD Guideline 487. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay (OECD guidelines for complex reaction products). Cytotoxicity [>/=50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >/=150 µg/mL in the non‑activated and S9-activated 4-hour exposure groups, and at doses >/=50 µg/mL in the non‑activated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses >/=500 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 25 to 150 µg/mL for the non‑activated and S9-activated 4-hour exposure groups, and from 5 to 75 µg/mL for the non-activated 24-hour exposure group.

In the initial micronucleus assay, cytotoxicity (>/=50% CBPI relative to the vehicle control), was observed at doses >/=75 µg/mL in the S9‑activated 4-hour exposure group. In the non-activated 4 and 24-hour exposure groups, due to lack of requisite cytotoxicity, the micronucleus assay was repeated at doses ranging from 5 to 75 µg/mL for the non-activated 4-hour exposure group and from 5 to 50 µg/mL for the non-activated 24-hour exposure group. 

In the repeat micronucleus assay, cytotoxicity (>/=50% CBPI relative to the vehicle control), was observed at doses >/=50 µg/mL in the non-activated 4-hour exposure group and at doses >/= 36 µg/mL for the non-activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 5, 25, and 50 µg/mL for the non-activated 4-hour exposure group and 5, 15, and 36 µg/mL for the non-activated 24-hour exposure group.

No significant or dose‑dependent increases in micronuclei induction were observed in any treatment groups, with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate CGE-PMDA adduct was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. 

Endpoint:
in vitro gene mutation study in mammalian cells
Type of information:
experimental study
Adequacy of study:
key study
Study period:
29 November 2017 to 22 February 2018
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Version / remarks:
updated and adopted 29 July 2016
Deviations:
no
GLP compliance:
yes
Type of assay:
in vitro mammalian cell gene mutation test using the Hprt and xprt genes
Target gene:
HPRT
Species / strain / cell type:
Chinese hamster Ovary (CHO)
Details on mammalian cell type (if applicable):
CELLS USED
- Source of cells: CHO-K1-BH4, obtained from Dr. Abraham W. Hsie, Biology Division, Oak Ridge National Laboratory, Oak Ridge, TN
- Cell cycle length, doubling time or proliferation index: population doubling time of 12-14 hours, cloning efficiency generally greater than 80%
- Modal number of chromosomes: 20

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: Ham's F12 medium supplemented with 3 mM L glutamine and 5% (v/v) heat-inactivated and dialyzed fetal bovine serum (Complete Ham’s F12), 37 ± 1°C in a humidified atmosphere of 5 ± 1% CO2 in air
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9
Test concentrations with justification for top dose:
preliminary toxicity test: 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, and 5000 µg/mL, with and without S9
Visible precipitate was observed at >/=78.1 µg/mL without S9 and >/= 313 µg/mL with S9

definitive test:
Non-activated 5 ± 0.5 hours 5, 10, 20, 40, 55, and 75 µg/mL
S9-activated 5 ± 0.5 hours 40, 55, 75, 120, 165, and 225 µg/mL

retest:
Non-activated 5 ± 0.5 hours 2.5, 5, 10, 20, 30, and 40 µg/mL
S9-activated 5 ± 0.5 hours 20, 40, 55, 75, 100, and 120 µg/mL
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: Ethanol
- Justification for choice of solvent/vehicle: based on the solubility of the test article and compatibility with the target cells
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
True negative controls:
no
Positive controls:
yes
Positive control substance:
benzo(a)pyrene
ethylmethanesulphonate
Details on test system and experimental conditions:
METHOD OF APPLICATION: in medium
- Cell density at seeding (if applicable): approx. 5E06 in 10 mL Complete Ham’s F12 medium

DURATION
- Exposure duration: 5 ± 0.5 hours
- Expression time (cells in growth medium): 7 d (subcultured at 2- to 3-day intervals)
- Selection time (if incubation with a selection agent): 7 d
- Fixation time (start of exposure up to fixation or harvest of cells): 14 d

SELECTION AGENT (mutation assays): 6-thioguanine

NUMBER OF REPLICATIONS: duplicate + independet repeat

DETERMINATION OF CYTOTOXICITY
- Method: cloning efficiency; relative total growth

Evaluation criteria:
The test article was considered to have produced a positive response if it induced a statistically significant and dose-dependent increase in mutant frequency (p≤ 0.01) that exceeded the 95% confidence limit of the historical vehicle control data from this laboratory. If only one criterion was met (a statistically significant or dose-dependent increase or an increase exceeding the historical control 95% confidence interval), the results were considered equivocal. If none of these criteria were met, the results were considered to be negative.
Statistics:
Statistical analyses were performed using the method of Snee and Irr (1981), with significance established at the 0.05 level.
Species / strain:
Chinese hamster Ovary (CHO)
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Additional information on results:
TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH of the cultures was adjusted at concentrations ≥156 µg/mL with and without S9 to maintain neutral pH.
- Effects of osmolality: The osmolality of the cultures was acceptable as it did not exceed the osmolality of the vehicle control by more than 120%.
- Precipitation: Visible precipitate was observed at all concentrations at the beginning of treatment and at concentrations ≥120 µg/mL by the end of treatment with S9 and ≥40 µg/mL by the end of treatment without S9 in the definitive test and at concentrations ≥40 µg/mL at the beginning of treatment and at concentrations ≥100 µg/mL by the end of treatment with S9 and ≥30 µg/mL by the end of treatment without S9 in the independent repeat.

RANGE-FINDING/SCREENING STUDIES:
Adjusted relative survival was 41.64 and 37.17% at a concentration of 156 and 39.1 µg/mL with and without S9, respectively. Adjusted relative survival was or approximated 0% at all higher concentrations with and without S9.

HISTORICAL CONTROL DATA (with ranges, means and standard deviation and confidence interval (e.g. 95%)
- Positive historical control data:
- Negative (solvent/vehicle) historical control data:
Conclusions:
Under the conditions reoported in this test CGE-PMDA adduct was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system in a valid test.
Executive summary:

CGE-PMDA adductwas evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) in accordance with OECD Guideline 476. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, and 5000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at >/=78.1 µg/mL without S9 and >/= 313 µg/mL with S9

The concentrations 156 and 39.1 µg/mL could not be assessed for precipitate at the end of treatment due to volume loss in the flask. Adjusted relative survival was 41.64 and 37.17% at a concentration of 156 and 39.1 µg/mL, with and without S9, respectively. Adjusted relative survival was or approximated 0% at all higher concentrations, both with and without S9. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 40, 55, 75, 120, 165, and 225 µg/mL with S9 and 5, 10, 20, 40, 55, and 75 µg/mL without S9.

In the initial definitive mutagenicity assay (B1), visible precipitate was observed at all concentrations at the beginning of treatment and at concentrations ≥120 µg/mL by the end of treatment with S9, and ≥40 µg/mL by the end of treatment without S9. This trail was terminated as four acceptable concentrations were not available to evaluate a valid assay. A few dose concentrations did not express any colonies in the treatment condition without S9. The average absolute cloning efficiency of vehicle controls was below 60% in the treatment condition with S9.

In the retest of definitive mutagenicity assay (B2) the concentrations tested were 20, 40, 55, 75, 100, and 120 µg/mL with S9, and 2.5, 5, 10, 20, 30, and 40 µg/mL without S9. Visible precipitate was observed at concentrations ≥40 µg/mL at the beginning of treatment and at concentrations ≥100 µg/mL by the end of treatment with S9, and ≥30 µg/mL by the end of treatment without S9. The average adjusted relative survival was 93.27 and 27.345% at a concentration of 75 µg/mL with S9 and 30 µg/mL without S9, respectively. Cultures treated at concentrations of 20, 40, 55, and 75 µg/mL with S9, and 2.5, 5, 10, 20, and 30 without S9 were chosen for mutant selection. Although for the 10 and 2.5 µg/mL concentrations without S9 the mutant frequency values were outside of historical range (not statistically significant), higher dose concentrations were within the 95% control limits. Additionally, there was no dose-related trend observed. Based upon this the results for the absence of S9 are considered negative. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed with or without S9 (p > 0.01) with all other dose levels. The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate CGE-PMDA adduct was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system in a valid test.

 

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

Genetic toxicity in vivo

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Bacterial Reverse Mutation Assay

In a reverse gene mutation assay in bacteria according to OECD guideline 471 (adopted 21 July 1997), Salmonella typhimurium strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA were exposed to CGE-PMDA adduct in ethanol in concentrations of 0 (control), 5.00, 15.0, 50.0, 150, 500, 1500 and 3000 µg per plate (tester strains TA98, TA1537 and WP2 uvrA in the presence and absence of S9 activation, and TA100 in the presence of S9 activation) and 1.50, 5.00, 15.0, 50.0, 150, 333 and 500 µg per plate (tester strains TA100 in the absence of S9 activation, and TA1535 in the presence and absence of S9 activation). In the retest of mutagenicity assay, the dose levels tested were 5.00, 15.0, 50.0, 150, 500, 1500, 3000, and 5000 µg per plate with tester strain TA1535 in the presence of S9 activation. The assay was performed using the plate incorporation method.

The test substance was tested up to cytotoxic concentrations. Toxicity was observed beginning at 150, 333, 1500 and 3000 µg per plate with all conditions except tester strain TA1535 in the presence of S9 activation. In order to achieve the protocol criteria for top dose or toxicity, the test condition TA1535 in the presence of S9 activation was repeated. Toxicity was then observed beginning at 1500 µg per plate. Precipitation was not observed. The positive controls induced the appropriate responses in the corresponding strains. The mean numbers of revertant colonies in the negative controls were within the ranges of the historical control data.

There was no evidence of an increase in the number of revertant colonies that was equal to or greater than 3.0-times the mean vehicle control value in strains TA1535 and TA 1537 or exceeded twice the background in the tester strains TA98, TA100 and WP2 uvrA examined at the peak of the dose-response. Therefore, test substance was considered to be non-genotoxic (nonmutagenic) in Salmonella tester strains TA98, TA100, TA1535, TA1537 and Escherichia coli strain WP2 uvrA under the conditions employed (plate incorporation assay).

Under the conditions of the study, the test substance was negative for mutagenic potential.

 

Mammalian Cell Gene Mutation Assay

CGE-PMDA adduct was evaluated for its ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system, as assayed by colony growth in the presence of 6-thioguanine (TG resistance, TGr) in accordance with OECD Guideline 476. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the concentrations tested were 9.77, 19.5, 39.1, 78.1, 156, 313, 625, 1250, 2500, and 5000 µg/mL. The maximum concentration evaluated approximated the limit dose for this assay. Visible precipitate was observed at >/=78.1 µg/mL without S9 and >/= 313 µg/mL with S9

The concentrations 156 and 39.1 µg/mL could not be assessed for precipitate at the end of treatment due to volume loss in the flask. Adjusted relative survival was 41.64 and 37.17% at a concentration of 156 and 39.1 µg/mL, with and without S9, respectively. Adjusted relative survival was or approximated 0% at all higher concentrations, both with and without S9. Based upon these results, the concentrations chosen for the definitive mutagenicity assay were 40, 55, 75, 120, 165, and 225 µg/mL with S9 and 5, 10, 20, 40, 55, and 75 µg/mL without S9.

In the initial definitive mutagenicity assay (B1), visible precipitate was observed at all concentrations at the beginning of treatment and at concentrations ≥120 µg/mL by the end of treatment with S9, and ≥40 µg/mL by the end of treatment without S9. This trail was terminated as four acceptable concentrations were not available to evaluate a valid assay. A few dose concentrations did not express any colonies in the treatment condition without S9. The average absolute cloning efficiency of vehicle controls was below 60% in the treatment condition with S9.

In the retest of definitive mutagenicity assay (B2) the concentrations tested were 20, 40, 55, 75, 100, and 120 µg/mL with S9, and 2.5, 5, 10, 20, 30, and 40 µg/mL without S9. Visible precipitate was observed at concentrations ≥40 µg/mL at the beginning of treatment and at concentrations ≥100 µg/mL by the end of treatment with S9, and ≥30 µg/mL by the end of treatment without S9. The average adjusted relative survival was 93.27 and 27.345% at a concentration of 75 µg/mL with S9 and 30 µg/mL without S9, respectively. Cultures treated at concentrations of 20, 40, 55, and 75 µg/mL with S9, and 2.5, 5, 10, 20, and 30 without S9 were chosen for mutant selection. Although for the 10 and 2.5 µg/mL concentrations without S9 the mutant frequency values were outside of historical range (not statistically significant), higher dose concentrations were within the 95% control limits. Additionally, there was no dose-related trend observed. Based upon this the results for the absence of S9 are considered negative. No significant increases in mutant frequency, as compared to the concurrent vehicle controls, were observed with or without S9 (p > 0.01) with all other dose levels. The positive controls induced significant increases in mutant frequency (p < 0.01).

These results indicate CGE-PMDA adduct was negative for the ability to induce forward mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus (hprt) of Chinese hamster ovary (CHO) cells, in the presence and absence of an exogenous metabolic activation system in a valid test.

 

Micronucleus Test

CGE-PMDA adduct, was tested to evaluate the potential to induce micronuclei in human peripheral blood lymphocytes (HPBL) in both the absence and presence of an exogenous metabolic activation system. HPBL cells were treated for 4 hours in the absence and presence of S9, and for 24 hours in the absence of S9 in accordance with OECD Guideline 487. Ethanol was used as the vehicle.

In the preliminary toxicity assay, the doses tested ranged from 0.5 to 5000 µg/mL, which was the limit dose for this assay (OECD guidelines for complex reaction products). Cytotoxicity [>/=50% cytokinesis-blocked proliferation index (CBPI) relative to the vehicle control] was observed at doses >/=150 µg/mL in the nonactivated and S9-activated 4-hour exposure groups, and at doses >/=50 µg/mL in the nonactivated 24-hour exposure group. At the conclusion of the treatment period, visible precipitate was observed at doses >/=500 µg/mL in all three exposure groups. Based upon these results, the doses chosen for the micronucleus assay ranged from 25 to 150 µg/mL for the nonactivated and S9-activated 4-hour exposure groups, and from 5 to 75 µg/mL for the non-activated 24-hour exposure group.

In the initial micronucleus assay, cytotoxicity (>/=50% CBPI relative to the vehicle control), was observed at doses >/=75 µg/mL in the S9activated 4-hour exposure group. In the non-activated 4 and 24-hour exposure groups, due to lack of requisite cytotoxicity, the micronucleus assay was repeated at doses ranging from 5 to 75 µg/mL for the non-activated 4-hour exposure group and from 5 to 50 µg/mL for the non-activated 24-hour exposure group. 

In the repeat micronucleus assay, cytotoxicity (>/=50% CBPI relative to the vehicle control), was observed at doses >/=50 µg/mL in the non-activated 4-hour exposure group and at doses >/= 36 µg/mL for the non-activated 24-hour exposure group. The doses selected for evaluation of micronuclei were 5, 25, and 50 µg/mL for the non-activated 4-hour exposure group and 5, 15, and 36 µg/mL for the non-activated 24-hour exposure group.

No significant or dosedependent increases in micronuclei induction were observed in any treatment groups, with or without S9 (p > 0.05; Fisher’s Exact and Cochran-Armitage tests).

These results indicate CGE-PMDA adduct was negative for the induction of micronuclei in the presence and absence of the exogenous metabolic activation system. 

Justification for classification or non-classification

Based on reliable, relevant and adequate data on the test substance is considered to be not mutagenic and not clastogenic.

According to Regulation EC No. 1272/2008, no classification and labelling for mutagenicity is required.