m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05-Nov-2012 to 24-Mar-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: Cultured peripheral human lymphocytes

Details on mammalian cell type (if applicable):

- Type and identity of media:
Blood samples
Blood samples were collected by venapuncture using the Venoject multiple sample blood collecting system with a suitable size sterile vessel containing sodium heparin. Immediately after blood collection lymphocyte cultures were started.
- Culture medium
Culture medium consisted of RPMI 1640 medium, supplemented with 20% (v/v) heat-inactivated (56°C; 30 min) foetal calf serum, L-glutamine (2 mM), penicillin/streptomycin (50 U/mL and 50 µg/mL respectively) and 30 U/mL heparin.
- Lymphocyte cultures
Whole blood (0.4 mL) treated with heparin was added to 5 mL or 4.8 mL culture medium (in the absence and presence of S9-mix, respectively). Per culture 0.1 ml (9 mg/mL) phytohaemagglutinin was added.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: not applicable, immediately after blood collection lymphocyte cultures were started.
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: not applicable, immediately after blood collection lymphocyte cultures were started.

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone.

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3hr exposure;: 10, 33, 100, 333 and 1000 µg/mL
With S9-mix, 24 and 48hr exposure; 24 and 48 hr fixation: 10, 33, 100, 333, 1000 and 2773 µg/mL
First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 0.1, 3 and 9 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 10, 30 and 50 µg/ mL
Second cytogenetic test:
Without S9-mix, 24 hr exposure; 24 hr fixation: 0.3, 1 and 3 µg/mL
Without S9-mix, 48 hr exposure; 48 hr fixation: 0.3, 1 and 3 µg mL
With S9-mix, 3 hr exposure; 48 hr fixation: 3, 30 and 50 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 0.5 µg/mL for a 3 h exposure period, 0.2 µg/mL for a 24 h exposure period and 0.1 µg/mL for a 48 h exposure period

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9 Migrated to IUCLID6: in Hank's Balanced Salt Solution: 10 µg/mL

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 48 hr
- Exposure duration: 3 hr (with and without S9-mix), 24 and 48 hr (without S9-mix)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 hr

SPINDLE INHIBITOR (cytogenetic assays): colchicine
STAIN (for cytogenetic assays): Giemsa

NUMBER OF REPLICATIONS: duplicates in two independent experiments

NUMBER OF CELLS EVALUATED: 100 metaphase chromosome spreads per culture

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index of each culture was determined by counting the number of metaphases per 1000 cells

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A test substance was considered positive (clastogenic) in the chromosome aberration test if:
a) It induced a dose-related statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.
b) A statistically significant and biologically relevant increase in the frequencies of the number of cells with chromosome aberrations was observed in the absence of a clear dose-response relationship.

A test substance was considered negative (not clastogenic) in the chromosome aberration test if none of the tested concentrations induced a statistically significant (Chi-square test, one-sided, p < 0.05) increase in the number of cells with chromosome aberrations.

Statistics:

The incidence of aberrant cells (cells with one or more chromosome aberrations, gaps included or excluded) for each exposure group outside the laboratory historical control data range was compared to that of the solvent control using Chi-square statistics.

Species / strain:

lymphocytes: human pheripheral blood

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTOR:
- Effects of pH: No
- Effects of osmolality: No

- Precipitation: Precipitation in the exposure medium was observed at dose levels of 1000 µg/ml and above

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 10 µg/ml and above in the absence of S9, 3 hr treatment/24 hr fixation; at dose levels of 10 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr and at dose levels of 33 µg/ml and above in the presence of S9, 3 hours treatment, 24 hours fixation



COMPARISON WITH HISTORICAL CONTROL DATA:
- The number of cells with chromosome aberrations found in the solvent and positive control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide induced appropriate responses.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Appropriate toxicity was reached at the dose levels selected for scoring.

Conclusions:

Interpretation of results (migrated information):
positive with metabolic activation
positive without metabolic activation

it is concluded that this test is valid and that m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is clastogenic in human lymphocytes.

Executive summary:

The number of cells with chromosome aberrations found in the solvent control cultures was within the laboratory historical control data range. Positive control chemicals, mitomycin C and cyclophosphamide, both produced a statistically significant increase in the incidence of cells with chromosome aberrations, indicating that the test conditions were adequate and that the metabolic activation system (S9-mix) functioned properly.

 

Both in the absence and presence of S9-mix, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced statistically significant, dose dependent increases in the number of cells with chromosome aberrations both when gaps were included and excluded in two independently repeated experiments.

 

No effects of m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline on the number of polyploid cells and cells with endoreduplicated chromosomes were observed both in the absence and presence of S9-mix. Therefore it can be concluded that m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline does not disturb mitotic processes and cell cycle progression and does not induce numerical chromosome aberrations

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

18-Mar-2013 to 30-Dec-2013

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)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-Guanine Phosphoribosyl Transferase (HPRT) in V79 cells

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media:
- Minimum Eagles Medium with Earle’s salts (MEME, Invitrogen Corporation), supplemented with 5 ml of MEM non-essential amino acids solution (Invitrogen Corporation), 0.11 g of sodium pyruvate (Invitrogen Corporation), 0.292 g of L-glutamine (Invitrogen Corporation) and 2.2 g of sodium bicarbonate (Merck) per litre. Final concentrations of streptomycin and penicillin G (Invitrogen Corporation) were 50 µg/mL and 50 U/ml, respectively.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: no
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Dose range finding test:
Without and with S9-mix, 3 hours treatment: 0.3, 1, 3, 10, 33, 100 and 1000 µg/mL
Without S9-mix, 24 hours treatment: 0.3, 1, 3, 10, 33, 100 and 1000 µg/ml
Experiment 1:
Without S9-mix, 3 hours treatment: 0.03, 0.1, 0.3, 1, 3, 6 and 10 µg/mL
With S9-mix , 3 hours treatment: 10, 33, 100, 150, 175, 200, 215 and 230 µg/ml
Experiment 2
Without S9-mix, 24 hours treatment: 0.01, 0.03, 0.1, 0.3, 1, 2, 3 and 5 µg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: dimethyl sulfoxide (DMSO) was used as solvent

Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

3-methylcholanthrene

7,12-dimethylbenzanthracene

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration:
Short-term treatment
With and without S9-mix: 3 hours
Prolonged treatment period
Without S9-mix: 24 hours
- Expression time (cells in growth medium): 6 days
- Selection time (if incubation with a selection agent): 7 days

SELECTION AGENT (mutation assays): 5 µg/mL thioguanine (6-TG)

NUMBER OF REPLICATIONS:
- Solvent controls: Duplicate cultures
- Treatment groups and positive control: Single cultures

NUMBER OF CELLS EVALUATED: 1.5 x 10E6 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiments)

Evaluation criteria:

ACCEPTABILITY OF THE ASSAY
A mutation assay was considered acceptable if it met the following criteria:
a) The absolute colony forming efficiency of the solvent controls should be between 60 and 120%
b) At least seven doses of the test substance should have an acceptable number of surviving cells (106) which could be analysed for expression of the HPRT mutation.
c) The spontaneous mutant frequency in the solvent-treated control will be < 6 per 10E5 clonable cells.
d) The positive control substances induced significant (at least three-fold) increases in the mutant frequency.
e) The selected dose range has to include a clearly toxic concentration (10 to 20%) of the average of solvent controls or should exhibit limited solubility or should extend to 5 mg/plate.


DATA EVALUATION
Any increase of the mutation frequency should be evaluated for its biological relevance including a comparison of the results with the historical control data range.

A test substance is considered positive (mutagenic) in the mutation assay if it induces at least a three-fold increase in the mutation frequency compared to the solvent control in a dose-dependent manner. An observed increase should be biologically relevant and will be compared with the historical control data range.

A test substance is considered equivocal (questionable) in the mutation assay if no clear conclusion for positive or negative result can be made after an additional confirmation study.

A test substance is considered negative (not mutagenic) in the mutation assay if:
a) None of the tested concentrations show a mutation frequency of at least three-fold compared to the solvent control.
b) The results are confirmed in an independently repeated test.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

without

Genotoxicity:

positive

Remarks:

The mutagenicity was confined only to incubations with a prolonged treatment period

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS:
- Effects of pH: No
- Effects of osmolality: No
- Precipitation: Precipitation in the exposure medium was observed at the dose level of 1000 µg/mL.

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 10 µg/ml and above in the absence of S9-mix after the 3 hour treatment period, at the dose level of 1000 µg/ml in the presence of S9-mix after the 3 hour treatment period and at dose levels of 3 µg/ml and above in the absence of S9-mix after the prolonged treatment period .

COMPARISON WITH HISTORICAL CONTROL DATA:
The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range and within the acceptability criteria of this assay.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- Toxicity was observed at dose levels of 1 µg/ml and above in the absence of S9-mix after the 3 hour treatment period, at dose levels of 150 µg/ml and above in the presence of S9-mix after the 3 hour treatment period and at dose levels of 3 µg/ml and above in the absence of S9-mix after the prolonged treatment period .

Remarks on result:

other: strain/cell type: CH-V79-4

Remarks:

Migrated from field 'Test system'.

Conclusions:

Interpretation of results (migrated information):
positive

It is concluded that m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is mutagenic in the gene mutation test with V79 Chinese hamster cells. The mutagenicity was confined only to incubations with a prolonged treatment period in the absence of a metabolic activation

Executive summary:

The spontaneous mutation frequencies in the solvent-treated control cultures were between the minimum and maximum value of the historical control data range (except in the absence of S9-mix), but the spontaneous mutation frequencies in the solvent-treated control cultures were within the acceptability criteria of this assay.

 

Mutation frequencies in cultures treated with positive control chemicals were increased by 8.5- and 87-fold for ethylmethanesulphonate in the absence of S9-mix and by 11- and 3.5-fold for7,12-dimethylbenz[a]anthracene and 3-methylcholanthrene, respectively in the presence of S9-mix. It was therefore concluded that the test conditions were appropriate and that the metabolic activation system (S9-mix) functioned properly.

 

In the absence of S9-mix, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not induce a significant increase in the mutation frequency in the first experiment after the short treatment period. The test substance induced an 11-fold dose related increase in the mutation frequency after the prolonged treatment period. Although the 11-fold increase was observed at the severe toxic highest dose level, increases above the historical control data range, up to 6.2-fold, were already seen at lower dose levels (from 0.03 µg/ml and above). Therefore, the test substance is considered to be mutagenic in the absence of S9-mix.

 

In the presence of S9-mix, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline did not induce a significant increase in the mutation frequency.

Reference 3

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28-Nov-2012 to 10-Dec-2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

- S. typhimurium: Histidine gene
- E. coli: Tryptophan gene

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:

Rat liver S9-mix induced by a combination of phenobarbital and ß-naphthoflavone

Test concentrations with justification for top dose:

Experiment 1
Main study:
Without and with S9-mix: 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate
Experiment 2:
Without and with S9-mix: 100, 333, 1000, 3330 and 5000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle:

Test compound was soluble in DMSO and DMSO has been accepted and approved by authorities and international guidelines

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 Migrated to IUCLID6: 650 µg/plate in DMSO for TA100

Positive control substance:

2-nitrofluorene

Remarks:

without S Migrated to IUCLID6: 10 µg/plate in DMSO for TA98 and 15 µg/plate for TA1537

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

without S9 Migrated to IUCLID6: 10 µg/plate in DMSO for WP2uvrA

Positive control substance:

sodium azide

Remarks:

without S9 Migrated to IUCLID6: 5 µg/plate in saline for TA1535

Positive control substance:

other: 2-aminoanthracene in DMSO for all tester strains

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hour

NUMBER OF REPLICATIONS:
- Doses of the test substance were tested in triplicate in each strain. Two independent experiments were conducted.

NUMBER OF CELLS EVALUATED: 10E8 per plate

DETERMINATION OF CYTOTOXICITY
- Method: The reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies.

OTHER EXAMINATIONS:
- The presence of precipitation of the test compound on the plates was determined.

Evaluation criteria:

A test substance is considered negative (not mutagenic) in the test if:
a) The total number of revertants in tester strain TA100 is not greater than two (2) times the concurrent control, and the total number of revertants in tester strains TA1535, TA1537, TA98 or WP2uvrA is not greater than three (3) times the concurrent control.
b) The negative response should be reproducible in at least one independently repeated experiment.

A test substance is considered positive if:
a) A two-fold (TA100) or more or a three-fold (TA1535, TA1537, TA98, WP2uvrA) or more increase above solvent control in the mean number of revertant colonies is observed in the test substance group.
b) In case a repeat experiment is performed when a positive response is observed in one of the tester strains, the positive response should be reproducible in at least one independently repeated experiment.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

with the exception of tester strain TA1537

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: No precipitation was observed up to and including the top dose of 5000 µg/plate

COMPARISON WITH HISTORICAL CONTROL DATA:
- The negative and strain-specific positive control values were within our laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
The bacterial background lawn was slightly reduced and the number of revertants was extremely reduced in TA1537 at the highest concentration tested. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed in all other strains.

Although the reduction in the number of revertant colonies in the tester strains TA1535 and TA100 in the absence and presence of S9-mix at the test substance concentration of 5000 µg/plate was not less than the minimal value of the historical control data range, a clear reduction in the number of revertant colonies was observed compared to the number of revertant colonies seen at 3330 µg/plate (mutagenic response).

Conclusions:

Interpretation of results (migrated information):
positive

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is mutagenic in the Salmonella typhimurium reverse mutation assay and in the Escherichia coli reverse mutation assay.

Executive summary:

The negative and strain-specific positive control values were within the laboratory background historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

 

In the absence of S9-mix, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced dose related increases in four tester strains (TA1535, TA98, TA100 and WP₂uvrA). The increases observed were abovethelaboratory historical control data range and 2.3- to 22-fold the concurrent vehicle controls. Although the increases observed in WP₂uvrA were not 3-fold, the increases were above the historical control data range and therefore considered to be biologically relevant.

 

In the presence of S9-mix, m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced dose related increases in four tester strains (TA1535, TA98, TA100 and WP₂uvrA). The increases observed were abovethelaboratory historical control data range and 2.6- to 90-fold the concurrent vehicle controls. Although the increases observed in WP₂uvrA in the first experiment was not 3-fold, the increases were above the historical control data range and therefore considered to be biologically relevant.

 

Since 2.3- to 90-fold, dose related increases were observed in four tester strains, both in the absence and presence of S9-mix, these increases are considered biologically relevant and m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is mutagenic in the absence and presence of S9-mix.

Genetic toxicity in vivo

Description of key information

Mutagenicity studies have been conducted in vitro which all turmed out to be positive, excepet for the point mutation assay (equivocal results and cell transformation assay were the substance is considered without an effect suggesting a cell transformation potential). Both key studies are positive. In the in vivo test, both supporting studies are positive but micronucleus test is negative (it is a read-across test with m-isomer)

A transgenic rodent assay was conducted to clarify the genetic toxicity of p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline.

Under the conditions of the OECD 488 Transgenic Rodent Assay study doses up to and including a top dose of 25 mg/kg/day were concluded to be negative for the induction of cII mutants in liver, but positive for the induction of cII mutants in glandular stomachat a dose of 12.5 mg/kg bw d and therefore based on the target organ toxicity presented in the OECD 407 and 408 studies this positive finding could be a result of site of contact epithelial cell cytotoxicity or an allergenic response associated with the strong sensitizing properties of the registered substance. The positive finding in the stomach at 25 mg/kg bw d is therefore unlikely to represent a true genotoxic mode of action. Given this uncertainty the registrant has still taken a conservative approach and classified the substance as Mutagen Cat 2 even though the STOT RE classification is expected to be protective of any genotoxic/none genotoxic hazard to the digestive tract.

Mutant Frequency Data Summary -Liver

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	52.1 +- 26.2
2 6.3 mg/kg/d	46.0 +-21.4
3 12.5 mg/kg/d	39.7 +-14.5
4 25 mg/kg/d	42.6 +-13.2
5 Positive Control	199.6 +-35.3*

* Statistically Significant (1 -way ANOVA, p<0.001) compared to G1

Mutant Frequency Data Summary -Glandular Stomach

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	27.2 +-4.7
2 6.3 mg/kg/d	39.6 +-15.2
3 12.5 mg/kg/d	33.1 +-8.8
4 25 mg/kg/d	54.1 +-15.2*
5 Positive Control	393.9 +-120.8*

* Statistically Significant (1 -way ANOVA, p<0.001) compared to G1

As per section 25 of OECD TG 488, DNA sequencing of mutants can be used to further understand weak or borderline negative responses and understand if the spectrum is different from the background. Sequencing of mutants would be useful for the present substance, to eliminate the potential for an irrelevant positive result and to further develop a mechanistic hypothesis.

Further investigations regarding the conduct of such studies are subject to review.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16-Sep-2013 to 22-Oct-2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld, Germany
- Age at study initiation: 6 weeks
- Weight at study initiation: 34.8 ± 1.3 g (range 33 – 38 g).
- Assigned to test groups randomly: yes
- Fasting period before study: Feed was withheld 3 - 4 h prior to dosing until administration
- Housing: In groups of 5 animals per sex per cage in polycarbonate cages containing sterilised sawdust as bedding material. Paper bedding was provided as cage-enrichment
- Diet (e.g. ad libitum): free access
- Water (e.g. ad libitum): free access
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.4 - 23.0 °C
- Humidity (%): 38 - 87%
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was suspended in propylene glycol . The specific gravity of propylene glycol is 1.036 g/ml. m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline concentrations were vortexed and treated with ultra-sonic waves to obtain a homogeneous suspension.

- Justification for choice of solvent/vehicle: A homogeneous suspension could be obtained in propylene glycol and propylene glycol is accepted and approved by authorities and international guidelines

- Concentration of test material in vehicle: 43.8, 87.5 and 175 mg/ml
- Amount of vehicle (if gavage or dermal): The dosing volume was 10 ml/kg body weight

Duration of treatment / exposure:

Treatment:
Solvent, positive control, low and mid dose level: 24 hours
Highest dose level: 24 and 48 hours

Frequency of treatment:

Once

Remarks:

Doses / Concentrations:
438, 875, and 1750 mg/kg body weight
Basis:
nominal conc.

No. of animals per sex per dose:

At least five animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s):
- Route of administration: Oral
- Doses / concentrations: 40 mg/kg body weight

Tissues and cell types examined:

Bone marrow smears

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
-The dose level selected should be ideally be the maximum tolerated dose level or that which produces some evidence of toxicity up to a maximum recommended dose of 2000 mg/kg.

DETAILS OF SLIDE PREPARATION:
- The smears are air-dried, fixed in methanol and stained using the "Wright-stain-procedure" in an "Ames" HEMA-tek slide stainer, allowed to air-dry and vover-slipped using mounting medium.

METHOD OF ANALYSIS:
- The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio of polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes.

Evaluation criteria:

A test substance is considered positive in the micronucleus test if:
-It induced a biologically as well as a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the frequency of micronucleated polychromatic erythrocytes (at any dose or at any sampling time) and the number of micronucleated polychromatic erythrocytes in the animals are above the historical control data range.

A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (Wilcoxon Rank Sum Test, one-sided, p < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes and the number of micronucleated polychromatic erythrocytes in the animals are within the historical control data range.

Statistics:

Wilcoxon Rank Sum Test, one-sided, p < 0.05

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 1750 and 2000 mg/kg BW
- Clinical signs of toxicity in test animals:
One male and one female were dosed with 2000 mg test substance per kilogram body weight. The female had a hunched posture, showed ataxia and was lethargic within 4 hours after dosing. The female died within 19 hours after dosing. The male animal had a hunched posture within 4 hours after dosing. The animal recovered from the treatment within 19 hours after dosing. Two additional males were dosed with 2000 mg/kg body weight. One of the animals died within 20 hours after dosing. Three males and and three females dosed with 1750 mg/kg body weight showed no treatment related clinical signs after dosing


RESULTS OF DEFINITIVE STUDY
- Dose range: 438, 875 and 1750 mg/kg BW
- Clinical signs of toxicity in test animals:
No treatment related clinical signs or mortality were noted in animals dosed with 438 and 875 mg
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight, or control animals receiving vehicle or cyclophosphamide.

The following clinical observations were made in the groups treated with 1750 mg
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight:

During the first 2 hours after treatment none of the animals showed treatment related clinical signs. Within 19 hours after dosing 3 animals had died, a third animal showed a hunched posture and was lethargic, a fourth animal was lethargic, showed ventral recumbency and had no reaction to a stimulus. This animal died within 23 hours after dosing. After replacement of the dead animals 4 animals were available for the 24 hour sampling time and 5 animals were available for the 48 hour sampling time.

The other animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight showed no treatment related clinical signs or mortality.

- Induction of micronuclei (for Micronucleus assay):
No increase in the mean frequency of micronucleated polychromatic erythrocytes above the historical control data range (0 – 5) was observed in the bone marrow of animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight. Two animals treated with 438 mg/kg body weight had a high incidence of 6 and 7 micronucleated polychromatic erythrocytes which is above the historical control data range.

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes compared to the corresponding vehicle control group in animals treated with 1750 mg/kg body weight at both sampling times.
One out of four animals dosed with 1750 mg/kg body weight sampled after 24 hours showed an increase above the historical control data range (0 – 5) and 3 out of 5 animals at the 48 hours sampling time.

- Ratio of PCE/NCE (for Micronucleus assay):
The animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight and the animals of the positive control group showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

- Appropriateness of dose levels and route: Adequate evidence of test material toxicity was demonstrated via the oral route administration.

Conclusions:

Interpretation of results (migrated information): negative
m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is not clastogenic or aneugenic in the bone marrow micronucleus test in male mice up to a dose of 875 mg/kg

Executive summary:

Micronucleated polychromatic erythrocytes

No increase in the mean frequency of micronucleated polychromatic erythrocytes above the historical control data range (0 – 5) was observed in the bone marrow of animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight. Two animals treated with

438 mg/kg body weight had a high incidence of 6 and 7 micronucleated polychromatic erythrocytes which is above the historical control data range.

 

m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the mean number of micronucleated polychromatic erythrocytes compared to the corresponding vehicle control group in animals treated with 1750 mg/kg body weight at both sampling times. One out of four animals dosed with 1750 mg/kg body weight sampled after 24 hours showed an increase above the historical control data range (0 – 5) and 3 out of 5 animals at the 48 hours sampling time.

Polychromatic to normochromatic ratio

The animals treated with 438 and 875 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which indicated a lack of toxic effects on the erythropoiesis.

 

The animals treated with 1750 mg m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline/kg body weight and the animals of the positive control group showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, demonstrating toxic effects on erythropoiesis.

Discussion and conclusion

A high level of mortality was observed in the animals dosed with 1750 mg/kg body weight in the main study (4 out of 13 animals died), which did not reflect the data obtained in the dose range finding study where no treatment related clinical signs or mortality was observed.

 

According to the guidelines, a substance should be tested up to the maximum of toxicity, which is defined as the dose producing signs of toxicity such that higher dose levels, based on the same dosing regimen, would be expected to produce lethality. When using this definition, the high dose of 1750 mg/kg where lethality was observed, is considered too high for evaluation of genotoxicity in this assay.

 

Due to excessive toxicity at 1750 mg/kg, the micronucleus scoring at this dose level has no toxicological value.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: gene mutation

Type of information:

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

Adequacy of study:

key study

Study period:

15th November 2019 to 6th March 2020

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

For further details please see Read Across Justification document enclosed in Chapter 13.

Reason / purpose for cross-reference:

read-across source

Qualifier:

according to guideline

Guideline:

OECD Guideline 488 (Transgenic Rodent Somatic and Germ Cell Gene Mutation Assays)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

transgenic rodent mutagenicity assay

Specific details on test material used for the study:

ID: p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline
Purity: 96.5% (per Certificate of Analysis)
Expiry Date: 25 August 2021
Description: Clear pale whitish yellow viscous liquid
Storage Conditions: stored at room temperature, protected from light upon receipt on 11 October 2019. Transferred to storage at 2 to 8ºC, protected from light the same day.

Species:

rat

Strain:

Fischer 344

Remarks:

Big Blue

Details on species / strain selection:

The Big Blue® in vivo mutation assay is a Transgenic Rodent (TGR) Mutation assay described in OECD Test Guideline 488 (OECD, 2013). TGR assays in general, and the Big Blue® assay in particular, have been reviewed by OECD (OECD, 2009 and 2011a) and identified in OECD Test Guideline 488 (OECD, 2011b and OECD, 2013) as being appropriate to investigate in vivo mutagenicity in any tissue of interest. In addition, the TGR assays are also recommended to investigate a potential mutagenic mode of action in the etiology of rodent tumors.

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BioReliance colony housed at Taconic Biosciences, Inc., Hudson, NY
- Age at study initiation: 8 weeks
- Assigned to test groups randomly: yes
- Fasting period before study: N/A
- Housing: Animals were housed in an environmentally
controlled room with continuous recording of room temperatures of 69 - 75°F and
relative humidity of 30 - 70%, with a 12-hour light/12-hour dark cycle. The animal rooms were supplied with at least 10 changes of fresh HEPA-filtered air every hour.
Animals were group housed during acclimation and following test animal selection,
in polycarbonate cages. Cages and feeders were changed at least once weekly.
Animals were transferred to clean racks at least once every other week.
Environmental enrichment (in the form of Nylabones®) was also provided. Access
to the Animal Facility was dictated by Standard Operating Procedures (SOPs) and
was restricted to authorized personnel only.
- Diet (e.g. ad libitum): All animals received TEKLAD Global Diet #2018C (Certified 18% Protein Rodent
Diet, Envigo, Madison, Wisconsin), in stainless steel rodent feeders, ad libitum from
arrival until termination.
- Water (e.g. ad libitum): Drinking water from an automatic watering system was provided ad libitum from
arrival until termination.
- Acclimation period: The animals were acclimated for 7 days (Groups 1-5) or between 5 and 21 days
(Groups 6 and 7), prior to the first dose administration. Animals were observed
daily for signs of illness or death. Prior to randomization, animals were examined
and approved for use by the Veterinarian.

Study Dates:
Animal arrival:
11th October 2019
Experimental Completion:
6th March 2020

Terminal Necropsy date:
19 October 2017
02 November 2017 (Groups 6 and 7)

Route of administration:

oral: gavage

Vehicle:

1% carboxymethylcellulose (medium viscosity) and 1% Polysorbate 80 in deionized water,

Component Supplier/Source Lot/Batch Number Expiration Date
Deionized (DI) water Milli-Q IQ 7000 Not applicable Day of use
Carboxymethylcellulose Sigma-Aldrich SLBW0425 31 May 2021
Polysorbate 80 Sigma-Aldrich BCBW3990 30 November 2021

Details on exposure:

Dose formulations were prepared weekly.
A suitably-sized amber glass vial with a PTFE stir bar was calibrated to the target batch size, and an appropriate amount of test substance was measured into it,. Approximately 70% of the total volume of vehicle was added to each vial and stirred magnetically. Additional vehicle was added until the final target volume was achieved. In the range finder assay, the mixtures were stirred magnetically for a total of 33 to 40 minutes and sonicated for 10 minutes until uniform. In the mutation assay, the mixtures were stirred magnetically for 50 to 143 minutes until uniform with no sonication. Once uniform in appearance, the formulations were aliquoted for daily use and were stored refrigerated (2 8°C), protected from light until delivered to in-life for dosing.
All formulations were removed from refrigerated storage and allowed to equilibrate at room temperature for at least 30 minutes while stirring prior to and during dosing.
Residual dose formulations were discarded after use.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Post exposure period:

3 days

Dose / conc.:

0 mg/kg bw/day

Remarks:

Vehicle Control

Dose / conc.:

6.3 mg/kg bw/day

Dose / conc.:

12.5 mg/kg bw/day

Dose / conc.:

25 mg/kg bw/day

No. of animals per sex per dose:

6 Main Cohort 4 TK Cohort

Control animals:

yes

Positive control(s):

20 mg/kg/day ENU on six non-consecutive days
Positive control animals (Group 5) were exposed by oral gavage to 20 mg/kg/dose of ethyl
nitrosourea (ENU) on Days 1, 2, 3, 12, 19 and 26.

Tissues and cell types examined:

For Groups 1-4, liver and glandular stomach were collected for mutant analysis. Tissues were weighed, flash frozen in liquid nitrogen and stored at or below -65°C, following applicable SOPs. Organ weight data were collected to predict number of DNA extractions possible from a tissue. Duodenum and testes were also collected and stored frozen as indicated above for possible mutation analysis, but ultimately, were not analyzed.

Details of tissue and slide preparation:

Liver and glandular stomach tissue samples from the first five surviving animals/group (Groups 1-4 only) were processed for DNA isolation and mutant analysis following BioReliance SOP based on Agilent RecoverEase Methods (Agilent, 2018). Tissues from the sixth animal/group were retained frozen as reserve samples, as applicable. DNA was extracted following BioReliance SOP. Isolated DNA samples were stored at 2 8°C.

DNA isolated from previous positive control animals (F344 transgenic male rats exposed to N-ethyl-N-nitrosourea (ENU) by oral gavage, at 20 mg/kg/day on six non-consecutive days) was used as a packaging positive control. These animals (previously dosed during BioReliance Study No. AF39AF.171.BTL) are collectively designated in the post life data as Group 5. This use of “packaging controls” is permitted by OECD TG 488 as a way to reduce unnecessary animal usage. The goal of the positive control in this case is to demonstrate the ability to recover induced mutants from the study target tissues. The study number and animal numbers were included in the raw data and the final mutation analysis report.

Statistics:

Dunnett’s test was conducted on body weight, body weight changes, and organ weight data. All required statistics were based on a significance value of p < 0.05.

Key result

Sex:

male

Genotoxicity:

positive

Toxicity:

not specified

Remarks:

Toxicity seen at similar dose levels in repeat dose studies

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 25 mg/kg bw/d

Remarks:

Glandular stomach

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 25 mg/kg bw/d

Remarks:

Liver

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 12.5 mg/kg bw/d

Remarks:

Liver and Glandular Stomach

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: 6.25 mg/kg bw/d

Remarks:

Liver and Glandular Stomach

Additional information on results:

RESULTS, 5-DAY RANGE-FINDING STUDY

Mortality and In-life Observations
Treatment with p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline resulted in no mortality at any dose level in the range-finding assay. Clinical signs of decreased motor activity and hunched posture were observed at the mid and high dose levels, while ruffled fur was also observed in one animal at the high dose level. During detailed hands-on observations, thinness was also observed at the mid and high dose levels, and diarrhea was also observed at the high dose level.

Body Weights and Body Weight Gains
Statistically significant differences were noted for group mean body weight at each dose level, with body weight gains decreasing with increasing dose level. The overall mean body weight gain (Day 1-5) was also significantly lower at each dose level compared to the vehicle control group.



RESULTS, 28-DAY STUDY

Mortality and In-life Observations
No mortality was observed following treatment with p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline at any dose level with the exception of one animal from the vehicle control group in the TK cohort, which was found dead on Day 15. Clinical signs of ruffled fur were observed in one animal at the low dose, while piloerection was observed with most animals at the high dose.

Body Weights and Body Weight Gains
Body weights did not appear to be significantly impacted at any dose level as compared to the vehicle control group in the main cohort. However, the overall mean body weight gain was significantly lower at the high dose level in the TK cohort compared to the vehicle control group. Any other statistically significant differences noted in body weights or body weight changes were considered incidental, as they were not dose-related, and were not toxicologically relevant.

Necropsy and Organ Weights
Organ weights were collected mainly for predicting the number of possible DNA extractions from a tissue (for the Big Blue® assay), rather than toxicity evaluation.

Mutant Frequency Analysis
Treatment with p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline in 1% carboxymethylcellulose (medium viscosity) and 1% Polysorbate 80 in deionized water did not cause a statistically elevated mutant frequency (MF) at the cII gene in the liver of Big Blue® rats, but did cause a statistically elevated mutant frequency (MF) at the cII gene in the glandular stomach of Big Blue® rats at 25 mg/kg/day. The positive control ENU treatment produced a statistically significant increase in mutant frequencies for both tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants, following exposure to a known direct acting mutagen.

Mutant Frequency Data Summary -Liver

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	52.1 +- 26.2
2 6.3 mg/kg/d	46.0 +-21.4
3 12.5 mg/kg/d	39.7 +-14.5
4 25 mg/kg/d	42.6 +-13.2
5 Positive Control	199.6 +-35.3*

* Statisticall Significant (1 -way ANOVA, p<0.001) compared to G1

Mutant Frequency Data Summary -Glandular Stomach

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	27.2 +-4.7
2 6.3 mg/kg/d	39.6 +-15.2
3 12.5 mg/kg/d	33.1 +-8.8
4 25 mg/kg/d	54.1 +-15.2*
5 Positive Control	393.9 +-120.8*

* Statisticall Significant (1 -way ANOVA, p<0.001) compared to G1

Conclusions:

Under the conditions of this study, the administration of p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline at doses up to and including a top dose of 25 mg/kg/day was concluded to be negative for the induction of cII mutants in liver, but positive for the induction of cII mutants in glandular stomach at only the highest dose tested (25 mg/kg/day). At 6.25 and 12.5 mg/kg bw d the glandular stomach was also negative for the induction of cII mutants.

As per section 25 of OECD TG 488, DNA sequencing of mutants can be used to further understand weak or borderline negative responses and understand if the spectrum is different from the background. Sequencing of mutants would be useful for the present substance, to eliminate the potential for an irrelevant positive result and to further develop a mechanistic hypothesis.
Further investigations regarding the conduct of such studies are subject to review.

Executive summary:

The purpose of this study was to determine the effect of p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)anilineon mutation frequency at thecIIgene in liver and glandular stomach from male transgenic Fischer 344 Big Blue^®rats. The Big BlueAssay is a Transgenic Rodent (TGR) mutation assay, described in OECD Technical Guideline 488 (OECD, 2013).

In therange-finding portion of thestudy,p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline was administered at the following dose levels: 0 (vehicle control), 25, 100, and 200 mg/kg/day (Groups 1-4, respectively), to five male rats per group;1% carboxymethylcellulose (medium viscosity) and 1% Polysorbate 80 in deionized waterwas used as the vehicle. Animals were dosedby oral gavage for up to 5 consecutive days, at a dose volume of 10 mL/kg. Animals were sacrificed by CO₂overdose on Day 5 after completion of data collection.

Treatment withp-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)anilineresulted in no mortality at any dose level in the range-finding assay. Clinical signs of decreased motor activity and hunched posture were observed at the mid and high dose levels, while ruffled fur was also observed in one animal at the high dose level. During detailed hands-on observations, thinness was also observed at the mid and high dose levels, and diarrhea was also observed at the high dose level.Statistically significant differences were noted for group mean body weight at each dose level, with body weight gains decreasing with increasing dose level. The overall mean body weight gain (Day 1-5) was also significantly lower at each dose level compared to the vehicle control group.

Based on the results of the range-finding assay,p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline was administered at the following dose levels in the transgenic mutation portion of the study: 0 (vehicle control), 6.3, 12.5, and 25 mg/kg/day (Groups 1-4, respectively), to six male rats per group in the main study cohort. In addition, these dose levels were administered to four male F344 wild type rats per group in the TK cohort. The vehicle was1% carboxymethylcellulose (medium viscosity) and 1% Polysorbate 80 in deionized water. Test substance and vehicle control formulations were administered once daily for 28 consecutive days, at a dose volume of 10 mL/kg. DNA isolated from previous positive control animals (F344 transgenic male rats exposed toN-ethyl-N-nitrosourea (ENU) by oral gavage, at 20 mg/kg/day on six non-consecutive days) was used as a packaging positive control. These animals (previously dosed during BioReliance Study No. AF39AF.171.BTL) are collectively designated in the post‑life data as Group 5.N‑ethyl-N-nitrosourea (ENU) is a potent direct acting mutagen, demonstrated to be mutagenic in the target tissues.

Animals in Groups 1-4weresacrificed by CO₂overdose on Day 28 (TK cohort) or Day 31 (Main cohort). At necropsy for the Main cohort animals, liver and glandular stomach tissues were collected, weighed, flash frozen and stored at -65°C or less for subsequent mutation analysis. The tissues from the first five surviving animals/group were processed for DNA isolation and analysis ofcIImutants following BioReliance SOPs. Duodenum and testes were also collected at necropsy for possible mutation analysis, but ultimately, were not analyzed.

In the transgenic mutation portion of the study, no mortality was observed following treatment withp-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)aniline at any dose level with the exception of one animal from the vehicle control group in the TK cohort, which was found dead on Day 15. Clinical signs of ruffled fur were observed in one animal at the low dose, while piloerection was observed with most animals at the high dose. Body weights did not appear to be significantly impacted at any dose level as compared to the vehicle control group in the main cohort. However, the overall mean body weight gain was significantly lower at the high dose level in the TK cohort compared to the vehicle control group. Any other statistically significant differences noted in body weights or body weight changes were considered incidental, as they were not dose-related, and were not toxicologically relevant.

Treatment with p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)anilinein 1% carboxymethylcellulose (medium viscosity) and 1% Polysorbate 80 in deionized water did not cause a statistically elevated mutant frequency (MF) at thecIIgene in the liver of Big Blue^®rats, but did cause a statistically elevated mutant frequency (MF) at the cII gene in the glandular stomach of Big Blue^®rats at 25 mg/kg/day. The positive control ENU treatment produced a statistically significant increase in mutant frequencies for both tissues tested, demonstrating the utility of the test system to detect and quantify induced mutants, following exposure to a known direct acting mutagen.

Under the conditions of this study, the administration of p-(2,3-epoxypropoxy)-N, N-bis(2,3-epoxypropyl)anilineat doses up to and including a top dose of 25 mg/kg/day was concluded to be negative for the inductionofcIImutants in liver, but positive for the induction ofcIImutants in glandular stomach.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Several mutagenicity studies have been conducted in in-vitro test systems which all turned out to be positive excepted for the point mutation assay (equivocal results and cell transformation assay were the substance is considered without an effect suggesting a cell transformation potential).

In the in-vivo mutagenicity assays (micronucleus test in the Mouse) performed with the structural analogue and used as read-across,no significant mutagenic potential in-vivo was noted.

Altogether, it was judgedthat p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline substance is mutagenic in vitro and not mutagenic in in-vivo experiment. The following table presents results of the transgenic rodent assay:

Mutant Frequency Data Summary -Liver

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	52.1 +- 26.2
2 6.3 mg/kg/d	46.0 +-21.4
3 12.5 mg/kg/d	39.7 +-14.5
4 25 mg/kg/d	42.6 +-13.2
5 Positive Control	199.6 +-35.3*

* Statistically Significant (1 -way ANOVA, p<0.001) compared to G1

Mutant Frequency Data Summary -Glandular Stomach

Group No.	Mutant Frequency +- StDv (x10 -6)
1 Vehicle	27.2 +-4.7
2 6.3 mg/kg/d	39.6 +-15.2
3 12.5 mg/kg/d	33.1 +-8.8
4 25 mg/kg/d	54.1 +-15.2*
5 Positive Control	393.9 +-120.8*

* Statistically Significant (1 -way ANOVA, p<0.001) compared to G1

Justification for selection of genetic toxicity endpoint
Concerning the genetic in vitro, two bacterial reverse mutation test (positive), a gene mutation assay on Saccharomyces cerevisiae (positive), a point mutation assay with mouse Lymphoma cells (ambiguous results) and two cell transformation studies (Without an effect suggesting a cell transformation potential) are available and are considered as supporting studies. As key studies, an in vitro Chromosome aberration in cultured peripheral Human lymphocytes (OEC 473) and an in vitro mammalian cell gene mutation test with L5178Y mouse Lymphoma test (OECD 476) were performed. Both key studies are positive.

Regarding the genetic in vivo, a sister chromatid exchange study (positive and a nucleus anomaly test in somatic interphase (positive), and a Micronucleus test in bone marrow cells of the mouse (Negative),

were performed and are considered as supporting studies. As key study, a transgenic Rodent assay was conducted (positive in glandular stomach at doses only above 25 mg/kg bw/d).

All of these studies (in vivo and in vitro) are taken into consideration.

Justification for classification or non-classification

 

 In vitro and in vivo studies have been conducted with the p-isomer and m-isomer substances in order to clarify its mutagenic potential. While both substances are considered to be clearly positive in the in vitro studies. An in vivo micronucleus study performed with the m-isomer was negative at the maximum tolerated dose level. The reason is likely the fast detoxification of the epoxy group by epoxide hydrolases in vivo. A recent transgenic rodent assay conducted using doses up to and including a top dose of 25 mg/kg/day was concluded to be negative for the induction of cII mutants in liver, but positive for the induction of cII mutants in glandular stomach

 

 Under the conditions of the OECD 488 Transgenic Rodent Assay studydoses up to and including a top dose of 25 mg/kg/day were concluded to be negative for the induction of cII mutants in liver, but positive for the induction of cII mutants in glandular stomach at a dose of 12.5 mg/kg bw d and therefore based on the target organ toxicity presented in the OECD 407 and 408 studies this positive finding could be a result of site of contact epithelial cell cytotoxicity or an allergenic response associated with the strong sensitizing properties of the registered substance. The positive finding in the stomach at 25 mg/kg bw d is therefore unlikely to represent a true genotoxic mode of action. Given this uncertainty the registrant has still taken a conservative approach and classified the substance as Mutagen Cat 2 even though the STOT RE classification is expected to be protective of any genotoxic/none genotoxic hazard to the digestive tract.

Further to the results from the transgenic rodent assay, as per section 25 of OECD TG 488, DNA sequencing of mutants can be used to further understand weak or borderline negative responses and understand if the spectrum is different from the background. Sequencing of mutants would be useful for the present substance, to eliminate the potential for an irrelevant positive result and to further develop a mechanistic hypothesis. 

     

Taking all results and uncertainties into consideration a conservative approach has been taken for the two isomers and the registrant has self-classified for mutagenicity (Muta 2, H341) according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).