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Genetic toxicity in vitro

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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:
29-Jul-2011 to 29-Aug-2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)
Deviations:
no
Principles of method if other than guideline:
The recommendations of the “International Workshop on Genotoxicity Tests Workgroup” (the IWGT), published in the literature (Clive et al., 1995, Moore et al., 1999, 2000, 2002, 2003, 2006 and 2007).
GLP compliance:
yes (incl. certificate)
Type of assay:
mammalian cell gene mutation assay
Target gene:
Thymidine kinase (TK) locus in L5178Y mouse lymphoma cells
Species / strain / cell type:
mouse lymphoma L5178Y cells
Details on mammalian cell type (if applicable):
- Source American Type Culture Collection, (ATCC, Manassas, USA) (2001).
- Type and identity of media:
- RPMI 1640 Hepes buffered medium (Dutch modification) containing penicillin/streptomycin (50 U/ml and 50 μg/ml, respectively), 1 mM sodium pyruvate and 2 mM L-glutamin supplemented with 10% (v/v) heat-inactivated horse serum (=R10 medium).
- 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 1:
Without and with S9-mix, 3 hours treatment: 33, 100, 333, 1000 and 2773 µg/mL
Dose range finding test 2:
Without S9-mix, 3 hours treatment: 0.01, 0.1, 1, 10 and 33 µg/mL
Mutation Experiment:
Without S9-mix, 3 hours treatment: 0.001, 0.01, 0.03, 0.1, 0.6, 1, 3.3 and 6.6 µg/mL
With S9-mix, 3 hours treatment: 0.3, 1, 3.3, 10, 33, 66, 100 and 125 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
Remarks:
dimethyl sulfoxide.
Positive controls:
yes
Positive control substance:
methylmethanesulfonate
Remarks:
without S9 Migrated to IUCLID6: 15 µg/mL
Negative solvent / vehicle controls:
yes
Positive controls:
yes
Positive control substance:
cyclophosphamide
Remarks:
with S9 Migrated to IUCLID6: 7.5 µg/mL
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
- Expression time (cells in growth medium): 2 days
- Selection time (if incubation with a selection agent): 11 days

SELECTION AGENT (mutation assays): 5 µg/mL trifluorothymidine (TFT)

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

NUMBER OF CELLS EVALUATED: 9.6 x 10E5 cells plated/concentration

DETERMINATION OF CYTOTOXICITY
- Method: relative suspension growth (dose range finding test) and relative total growth (mutation experiment)
Evaluation criteria:
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.

The global evaluation factor (GEF) has been defined by the IWTGP as the mean of the negative/solvent MF distribution plus one standard deviation. For the micro well version of the assay the GEF is 126.

A test substance is considered positive (mutagenic) in the mutation assay if it induces a MF of more than MF(controls) + 126 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 reaches a mutation frequency of MF(controls) + 126.
b) The results are confirmed in an independently repeated test.
Species / strain:
mouse lymphoma L5178Y cells
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 FACTORS
- Effects of pH: No
Solvent control: 7.2
1000 µg/ml: 7.2
- Effects of osmolality: No
Solvent control: 0.417 mOsm/kg
1000 µg/ml: 0.582 mOsm/kg
- Precipitation: Precipitation in the exposure medium was observed at the dose level of 2773 µg/mL (= 0.01 M)

RANGE-FINDING/SCREENING STUDIES:
- Toxicity was observed at dose levels of 0.01 µg/mL in the absence of S9, 3 hours treatment; at dose levels of 100 µg/mL in the presence of S9, 3 hours treatment.

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:
The relative total growth of the highest test substance concentration was reduced by 67 and 90% compared to the total growth of the solvent controls in the absence and presence of S9-mix, respectively.
Remarks on result:
other: strain/cell type: Test system L5178Y/TK+/-3.7.2C
Remarks:
Migrated from field 'Test system'.

P-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was tested up to concentrations of 6.6 and 125 µg/ml in the absence and presence of S9-mix, respectively. The incubation time was 3 hours. Since the relative suspension growth (after 48 hours cell culture) was 13% at the test substance concentration of 6.6 μg/ml in the absence of S9-mix and 16% at the test substance concentration of 125 μg/ml in the presence of S9-mix, these dose levels were selected as highest dose level to be tested. After 11 days of culturing the CEday2 appeared very low in the 2 highest dose groups. High toxicity was observed resulting in a relative total growth of 1 and 0. Consequently only 6 dose levels were analysed for expression of the TK mutation.

Conclusions:
Interpretation of results (migrated information):
positive

The mouse lymphoma assay was conducted according to OECD 476 guideline and GLP principles.
It is concluded that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is mutagenic in the mouse lymphoma L5178Y test system.
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.

Positive control chemicals, methyl methane sulfonate and cyclophosphamide induced appropriate responses.

In the absence of S9-mix, p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced an up to 15-fold increase in the mutation frequency.The increases observed were above the GEF + MF(controls)(203 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner. Therefore, these increases are considered relevant and p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is considered mutagenic in the absence of S9-mix.

 

In the presence of S9-mix, p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced an up to 33-fold increase in the mutation frequency.The increases observed were above the GEF + MF(controls)(182 per 106survivors), more than three-fold, outside the historical control data range and in a dose dependent manner. Therefore, these increases are considered relevant and p-(2,3 -epoxypropoxy)-N,N-bis(2,3 -epoxypropyl)aniline is considered mutagenic in the presence of S9 -mix.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April 14-July 10, 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-guideline study and pre-GLP study performed according to study protocol similar to OECD 471.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Version / remarks:
acc. Ames, B.N., Mc.Cann, J. and Yamasaki, E. (1975) Methods for detecting carcinogens and mutagens with the Salmonella/mammalian-microsome mutagenicity test. Mutation Research 31, pp. 347-364
Deviations:
yes
Remarks:
no independent repeat experiment
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
histidine locus of S.typhimurium
Species / strain / cell type:
other: Strains used: S.typhimurium TA98, TA100, TA1535, TA1537,TA1538 and S.cerevisia D4 [reported separately]
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate with S9 co-factors
Test concentrations with justification for top dose:
0.01, 0.1, 1, 5 and 10 µl per plate plus solvent and positive controls, with and without activation
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
EXPOSURE PERIOD: 48 hours
SELECTION AGENT (mutation assays): histidine deficient agar
NUMBER OF REPLICATIONS: 2 plates per assay
NUMBER OF CELLS EVALUATED: about 100'000'000 per plate

DETERMINATION OF CYTOTOXICITY
- visual evaluation of the transparence of the agar dish
Evaluation criteria:
Positive and negative results: Low cytotoxicity.
Positive: Increase of mutant colonies in comparison with solvent control by a factor of >2 (TA1535, TA1537, TA1538) or >2-3 (TA98, TA100) at 3 concentratons.
Negative result: Absence of mutagenicity acc. above critera and positive result in the positive control.
Statistics:
not performed
Species / strain:
S. typhimurium, other: TA 98, TA100, TA1535, TA1537, TA1538
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
in strains TA 100 and TA1535 with and without S9
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
valid
Remarks on result:
other: all strains/cell types tested
Remarks:
Migrated from field 'Test system'. Remarks: TA 98, TA100, TA1535, TA1537, TA1538

TA 1535 TA 1537 TA 1539 TA98 TA100
Solvent control -S9 12 14 8 29 89
Positive control -S9 2613 408 1764 1295 1249
0.01 µl / plate -S9 19 18 6 20 124
0.1 µl / plate -S9 38 16 7 20 199
1.0 µl / plate -S9 147 14 5 36 613
5.0 µl / plate -S9 56 3 6 25 494
10 µl / plate -S9 12 2 5 0 459
TA 1535 TA 1537 TA 1539 TA98 TA100
Solvent control +S9 21 23 19 40 120
Positive control +S9 429 456 1139 1357 784
0.01 µl / plate +S9 51 24 16 38 135
0.1 µl / plate +S9 181 24 10 42 313
1.0 µl / plate +S9 674 15 16 48 814
5.0 µl / plate +S9 457 9 10 32 982
10 µl / plate +S9 140 10 2 45 347
Conclusions:
Interpretation of results (migrated information):
positive

Positve with and without microsomal activation in S.typhimurium strains TA100 and TA1535. Negative with and without microsomal activation in strains TA98, TA1537 and TA1538.
Executive summary:

According to a pre-guideline and pre-GLP study performed according to study protocol similar to OECD 471, the substance was proved to be mutagenic in Salmonella typhimurium strains TA100 and TA1535 with and without metabolic activation.

Endpoint:
in vitro gene mutation study in bacteria
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
Nov. 17 - Nov. 19, 1981
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-GLP, pre-guideline study similar to the later OECD guideline 471
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Deviations:
no
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 472 (Genetic Toxicology: Escherichia coli, Reverse Mutation Assay)
Deviations:
no
GLP compliance:
no
Type of assay:
bacterial reverse mutation assay
Target gene:
his
Species / strain / cell type:
S. typhimurium TA 1538
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for identity with reference strain: yes
- Periodically "cleansed" against high spontaneous background: yes
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for identity with reference strains: yes
Species / strain / cell type:
E. coli WP2 uvr A
Details on mammalian cell type (if applicable):
- Type and identity of media:
- Properly maintained: yes
- Periodically checked for identity with reference strain: yes
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver S9 and cofactors
Test concentrations with justification for top dose:
5, 10, 50, 100, 500, 1000 and 5000 µg/plate
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in the agar (plate incorporation)

DURATION
- Preincubation period: none
- Exposure duration: 48 hours

SELECTION AGENT (mutation assays): his auxotrophy

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: estimation of plate background transparency
Evaluation criteria:
Doubling of the colony number with respect to the negative/solvent control at any concentration is evidence for mutagenicity
Statistics:
none
Additional information on results:
In the experiments performed without microsomal activation, treatment with the test material led to an increase in the number of backmutant colonies of strains TA 98, TA 100, TA 1535 and WP2 uvrA. This effect was observed at the concentrations of 50 or 100 µg/ 0.1 ml and above (strains TA 1535 and TA 100 respectively) and at the concentrations of 500 and 1000 µg/0.1 ml (strains TA 98 and WP2 uvrA).
In the experiments performed with microsomal activation this effect was observed at the concentrations of 50 or 100 µg/0.1 ml and above (strains TA 1535 and TA 100 respectively) and at the concentrations of 1000 and 5000 µg/0.1 ml (strain WP2 uvrA).
TA100 TA1535 WP2UVrA TA98 TA1537 TA1538
Solvent Control 158 12 17 19 4 11 without S9
Test material 5 µg/plate) 160 11 20 16 6 10
10 µg/plate) 179 13 17 20 5 10
50 µg/plate) 200 27 17 24 6 10
100 µg/plate) 479 39 25 20 4 9
500 µg/plate) 242 82 32 41 4 12
1000 µg/plate) 770 111 32 63 6 11
5000 µg/plate) 1393 55 11 26 2 1
Solvent Control 149 8 14 37 6 22 with S9
Test material 5 µg/plate) 162 10 17 26 7 21
10 µg/plate) 162 12 17 32 5 21
50 µg/plate) 197 22 14 34 4 24
100 µg/plate) 235 43 18 41 5 30
500 µg/plate) 569 268 21 34 5 20
1000 µg/plate) 980 609 33 38 7 28
5000 µg/plate) 2041 1156 59 56 3 6
Conclusions:
Interpretation of results (migrated information):
positive

The test substance showed a positive effect in strains TA100, TA 1535, TA98 in the experiment without S9 and a positive effect in strains TA100, TA1535, E.coli WP2uvrA in the experiment with S9.
Executive summary:

The test material proved mutagenic in the bacterial mutagenicity test in Salmonella strains TA100, TA1535 and E.Coli WP2uvrA and gave an equivocal result in strain TA98.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981-1982
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Non-GLP and non-guideline study performed in a laboratory which used to operate according to GLP. The test protocol was not validated and later evaluations (OECD SERIES ON TESTING AND ASSESSMENT Number 31, DETAILED REVIEW PAPER ON CELL TRANSFORMATION ASSAYS FOR DETECTION OF CHEMICAL CARCINOGENS [2007]) showed a high % of false positive and false negative assays.
GLP compliance:
no
Type of assay:
in vitro mammalian cell transformation assay
Test concentrations with justification for top dose:
0.000065 mg/ml
0.0000325 mg/ml
0.00001625 mg/ml
0.000008125 mg/ml
0.0000040625 mg/ml
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
The test substance was tested for cell-transformation inducing effects on mouse Balb/3T3 fibroblasts in vitro. The concentrations of the test material were determined on the basis of a cytotoxicity test. 5000 cells per dish were exposed to the test substance 48 hours after plating for an exposure period of 72 hours. Thereafter the cultures were washed and incubated with growth medium for four weeks. Culture medium was replaced on the 10th and 17th day. At the end of the incubation phase the cultures were fixed and stained with Giemsa. Cultures were evaluated under the microscope for colonies of transformed cells.
Statistics:
not required in this study
Additional information on results:
No colonies of transformed cells were detected. The substance is therefore considered without an effect suggesting of a cell transformation potential. The positive control cultures treated with methylcholanthrene (1.5 and 3.0 µg/ml) showed a transformation rate of 10.3 and 78 per 10'000 surviving cells. Overall assessment: negative.
Conclusions:
Interpretation of results (migrated information):
other: The substance is considered without an effect suggesting of a cell transformation potential.

No colonies of transformed cells were detected. The substance is considered without an effect suggesting of a cell transformation potential.
Executive summary:

The test substance was tested for cell-transformation inducing effects on mouse Balb/3T3 fibroblasts in vitro. The concentrations of the test material were determined on the basis of a cytotoxicity test. Cells were exposed to the test substance 48 hours after plating for an exposure period of 72 hours. Thereafter the cultures were washed and incubated with growth medium for four weeks. Culture medium was replaced on the 10th and 17th day. At the end of the incubation phase the cultures were fixed and stained with Giemsa. Cultures were evaluated under the microscope for colonies of transformed cells. No colonies of transformed cells were detected. The substance is considered without an effect suggesting of a cell transformation potential. The positive control cultures treated with methylcholanthrene (1.5 and 3.0 µg/ml) showed a transformation rate of 10.3 and 78 per 10'000 surviving cells.

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: genome mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
February 10 - July 27, 1987
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP and non-guideline study. The test protocol was according to test protocol published in the scientific literature. The test system has not been validated in a ring study and later evaluations (OECD SERIES ON TESTING AND ASSESSMENT Number 31, DETAILED REVIEW PAPER ON CELL TRANSFORMATION ASSAYS FOR DETECTION OF CHEMICAL CARCINOGENS [2007]) showed a high % of false positive and false negative assays.
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
GLP compliance:
yes (incl. certificate)
Type of assay:
in vitro mammalian cell transformation assay
Species / strain / cell type:
other: Mouse embryo fibroblasts BALB/3T3, clone A31-1-1
Metabolic activation:
with
Metabolic activation system:
rat liver microsomal fraction S9 from Aroclor 1254 induced livers of male Tif:RAIf (SPF) rats
Test concentrations with justification for top dose:
60 µg/ml
30 µg/ml
15 µg/ml
7.5 µg/ml
3.75 µg/ml
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
The test substance was tested for cell-transformation inducing effects on mouse Balb/3T3 fibroblasts in vitro in the presence of a metabolic activation system. The concentrations of the test material were determined on the basis of a cytotoxicity test. Cells in exponential phase of growth (48 hours after plating) were exposed to the test substance in the presence of S9 for an exposure period of 24 hours. Thereafter the cultures were washed and incubated in growth medium for four weeks.

Parallel to the transformation assay a cell viability control was done. 300 cells from the treated cultures were seeded and evaluated for colony formation 5 days thereafter.

Culture medium was replaced twice per week. At the end of the incubation phase the cultures were fixed and stained with Giemsa. Cultures were evaluated under the microscope for colonies of transformed cells. Transformed cells in this assay are so-called type III foci, showing prominent spindle shaped cells that are randomly oriented and exhibit criss-cross arrays at the periphery of the foci.
Evaluation criteria:
Statistical significance of differences between treated and control cultures according to the binomial distribution model.
Statistics:
Statistical significance in a test according to Tarone, R.E. and Gart, J.J. (1980): On the robustness of combined tests for trends in proportions. Journal of the American Statistical Association 75, pp. 110-116.

 

Transformation frequency

 

per plated cell

per surviving cell

 

x 104

x 104

 

 

 

Solvent control

0.67

1.53

Untreated control

0.31

0.78

2-AAF,  50 µg/ml

2.00

11.92

2-AAF, 100 µg/ml

2.14

29.22

 

 

 

3.75 µg/ml

1.07

2.74

7.5 µg/ml

0.43

1.01

15 µg/ml

0.67

1.76

30 µg/ml

1.23

3.93

60 µg/ml

0.67

16.22

The statistical comparison of the results from the solvent control and the various concentrations showing colonies of transformed cells revealed no significant differences at the confidence limit of 5%.

Conclusions:
Interpretation of results (migrated information):
other: The substance is considered without an effect suggesting of a cell transformation potential.

The substance is considered without an effect suggesting of a cell transformation potential.
Executive summary:

The test substance was tested for cell-transformation inducing effects on mouse Balb /3T3 fibroblasts in vitro. The concentrations of the test material were determined on the basis of a cytotoxicity test. Cells were exposed to the test substance 48 hours after plating for an exposure period of 24 hours in the presence of an S9 -based activation system. Thereafter the cultures were washed and incubated with growth medium for four weeks. Culture medium was replaced twice per week. At the end of the incubation phase the cultures were fixed and stained with Giemsa. Cultures were evaluated under the microscope for colonies of transformed cells. Acccording to the report, the substance is considered without an effect suggesting of a cell transformation potential.

Endpoint:
in vitro gene mutation study in mammalian cells
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1981
Reliability:
3 (not reliable)
Rationale for reliability incl. deficiencies:
other: Non-GLP study and pre-guideline study. Several concentrations treated, however, only 2 concentrations evaluated. Activation with S9 has not been used.
Qualifier:
no guideline followed
GLP compliance:
no
Type of assay:
mammalian cell gene mutation assay
Target gene:
Resistances to methotrexate, cytosine arabinoside or thymidine
Species / strain / cell type:
mouse lymphoma L5178Y cells
Additional strain / cell type characteristics:
not specified
Metabolic activation:
without
Test concentrations with justification for top dose:
0.0003 to 10000 µg/ml in the cytotoxicity test. The concentrations of 0.51 µg/ml (for 4 hours exposure experiment) and 0.21 µg/ml (for 18 hour exposure experiment) have been evaluated for mutagenicity.
Vehicle / solvent:
- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: no justification given.
Details on test system and experimental conditions:
METHOD OF APPLICATION: Treatment and expression in suspension cultures, selection in solid agar cultures

DURATION
- Preincubation period: continous culture in exponential growth phase
- Exposure duration: 4 hours and 18 hours
- Expression time (cells in growth medium): 72 hours
- Selection time (if incubation with a selection agent): 14 days for the 18-h test, 15 days for 4-h test
- Fixation time (start of exposure up to fixation or harvest of cells): no fixation, colony counting

SELECTION AGENT (mutation assays): 3 different agents: methotrexate, cytosine arabinoside or thymidine

NUMBER OF REPLICATIONS: not available

NUMBER OF CELLS EVALUATED: 4x10^5 cells in 5 ml tubes

DETERMINATION OF CYTOTOXICITY: cloning efficiency and relative total growth
Evaluation criteria:
Mutant frequency > 2.5 x higher than control
Statistics:
none
Duration cytosine arabinoside methotrexate thymidine
Mutant frequency (mutants/10^5 cells)
Control 1.08 17.72 23.56

0.51 µg/ml of test material

4 hours 0.64 29.39 37.92
Mutation factor (MF(treated / control)
0.59 1.66 1.61
Mutant frequency (mutants/10^5 cells)
Control 1.02 6.92 5.20
0.21 µg/ml of test material 18 hours 0.60 8.21 14.88
Mutation factor (MF(treated / control)
0.59 1.19 2.86
Conclusions:
Interpretation of results (migrated information):
ambiguous

Equivocal. The results of the two experiments demonstrate a high variability of the mutant frequencies between the controls. The results obtained with the treated cultures do not show a clear result.
Executive summary:

The study is not suited to evaluate mutagenicity in mammalian cells

Endpoint:
genetic toxicity in vitro
Remarks:
Type of genotoxicity: gene mutation
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
April 14-July 10, 1978
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Pre-guideline study and pre-GLP study performed according to study protocol similar to OECD 480.
Reason / purpose:
reference to same study
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)
Deviations:
yes
Remarks:
no independent repeat experiment
GLP compliance:
no
Type of assay:
other: Gene mutation assay in yeast
Target gene:
histidine locus of Saccharomyces cerevisiae
Species / strain / cell type:
Saccharomyces cerevisiae
Metabolic activation:
with and without
Metabolic activation system:
Aroclor 1254-induced rat liver homogenate with S9 co-factors
Test concentrations with justification for top dose:
0.01, 0.1, 1, 5 and 10 µl per plate plus solvent and positive controls, with and without activation
Vehicle / solvent:
DMSO
Details on test system and experimental conditions:
METHOD OF APPLICATION: in agar (plate incorporation)
EXPOSURE PERIOD: 48 hours
SELECTION AGENT (mutation assays): histidine deficient agar
NUMBER OF REPLICATIONS: 2 plates per assay
NUMBER OF CELLS EVALUATED: about 100'000'000 per plate

DETERMINATION OF CYTOTOXICITY
- visual evaluation of the transparence of the agar dish
Evaluation criteria:
Positive and negative results: Low cytotoxicity.
Positive: Increase of mutant colonies in comparison with solvent control by a factor of >2 at 3 concentratons.
Negative result: Absence of mutagenicity acc. above critera and positive result in the positive control.
Statistics:
not performed
Metabolic activation:
with and without
Genotoxicity:
positive
Remarks:
with and without S9
Cytotoxicity / choice of top concentrations:
no cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not examined
Positive controls validity:
other: a positive control (+S9) was not known in 1978
Additional information on results:
The result is considered valid because a positive response was obtained (dose-dependent, biological significant increase in mutant colonies)
Remarks on result:
other: strain/cell type: S. cerevisia, Strain D4
Remarks:
Migrated from field 'Test system'.
D4
Solvent control -S9 28
Positive control -S9 409
0.01 µl / plate -S9 34
0.1 µl / plate -S9 43
1.0 µl / plate -S9 64
5.0 µl / plate -S9 126
10 µl / plate -S9 140
D4
Solvent control +S9 33
Positive control +S9 31
0.01 µl / plate +S9 41
0.1 µl / plate +S9 42
1.0 µl / plate +S9 43
5.0 µl / plate +S9 127
10 µl / plate +S9 111
Conclusions:
Interpretation of results (migrated information):
positive

Positve with and without microsomal activation in S. cerevisiae.
Executive summary:

According to a pre-guideline and pre-GLP study performed according to study protocol similar to OECD 480, the substance was proved to be mutagenic in S.cerevisiae D4 with and without metabolic activation.

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:
August 01, 2011 - October 23, 2011
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.10 (Mutagenicity - In Vitro Mammalian Chromosome Aberration Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 from healthy adult, non-smoking, male volunteers, 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 : 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 S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 2773 µg/mL
Without S9-mix, 24 and 48hr exposure; 24/48 hr fixation: 33, 100, 333, 1000 and 2773 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 33, 100, 333, 1000 and 2773 µg/mL

Second dose range finding test:
Without S9-mix, 3hr exposure; 24 hr fixation: 0.3, 1, 3, 10 and 30 µg/mL
Without S9-mix, 24/48hr exposure; 24/48 hr fixation: 0.3, 1, 3, 10 and 30 µg/mL
With S9-mix, 3hr exposure; 24 hr fixation: 3, 10, 30, 50 and 75 µg/mL

First cytogenetic test:
Without S9-mix, 3 h exposure time, 24 h fixation time: 0.3, 5 and 7 µg/mL
With S9-mix, 3 h exposure, 24 h fixation time: 3, 40 and 45 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
Remarks:
dimethyl sulfoxide
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
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)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 hr

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

NUMBER OF REPLICATIONS: duplicates in one experiment

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: Cultured peripheral human lymphocytes
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 FACTORS
- Effects of pH: No
Solvent control: 7.40
1000 µg/ml: 7.47
- Effects of osmolality: No
Solvent control: 424 mOsm/kg
1000 µg/ml: 405 mOsm/kg
- Precipitation: Precipitation in the exposure medium was observed at the dose level of 2773 µg/ml in the first dose range finding test.

RANGE-FINDING/SCREENING STUDIES:
- Due to severe toxicity a second dose range finding test was performed.
- Second dose range finding: 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 3 µg/ml and above in the absence of S9 for the continuous treatment of 24 and 48 hr and at dose levels of 50 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.

LIST OF PROTOCOL DEVIATIONS:
- The slides were automatically embedded in a 1:10 mixture of xylene /pertex and mounted with a coverslip in an automated coverslipper.
Evaluation: Embedding slides and mounting the coverslip automatically instead of manually does not influence the study results.
- In the first dose range finding test the humidity was once below the range of 80-100% which was mentioned in the protocol, with a minimum of 54% for approximately 30 minutes. In addition the temperature in the dose range finding tests and the cytogenetic assay was several times below the range of 37.0 ± 1.0°C which was mentioned in the protocol, with a minimum of 34.3°C for approximately 1.5 hour.
Evaluation: These deviations were only for a short time and were caused by opening and closing of the incubator door during handling of the cell cultures. The number of cells with chromosome aberrations found in the negative control cultures were within the laboratory historical negative control data range; therefore these deviations of the temperature and temperature have no effect on the results of the study.
The study integrity was not adversely affected by the deviations.

Tables see the attached backgroud material.

Both in the absence and presence of S9-mix p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the number of cells with chromosome aberrations at the two highest concentrations tested, both when gaps were included and excluded. The number of cells with chromosome aberrations is high above the historical control data range and therefore biologically relevant.

 

No effects of p-(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 p-(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 under the experimental conditions described in this report.

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

A chromosome aberration study with p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline was performed according to OECD 473 guideline and GLP principles, in cultured peripheral human lymphocytes in one experiment. It is concluded that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is clastogenic in human lymphocytes.
Executive summary:

No effects of p-(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 p-(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 under the experimental conditions described in this report.

But both in the absence and presence of S9-mix p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline induced a statistically significant increase in the number of cells with chromosome aberrations at the two highest concentrations tested, both when gaps were included and excluded. The number of cells with chromosome aberrations is high above the historical control data range and therefore biologically relevant.

It is concluded that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline is clastogenic in human lymphocytes.

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)

Link to relevant study records
Reference
Endpoint:
in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus
Remarks:
Type of genotoxicity: chromosome aberration
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
16-Sep-2013 to 22-Oct-2013
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: The study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)
Deviations:
no
GLP compliance:
yes (incl. certificate)
Type of assay:
micronucleus assay
Species:
mouse
Strain:
NMRI
Sex:
male
Details on test animals 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.

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 judged that p-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline substance is mutagenic in vitro and not mutagenic in in-vivo experiment.


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), were performed and are considered as supporting studies. As key study, a Micronucleus test in bone marrow cells of the mouse was performed Negative), on m-(2,3-epoxypropoxy)-N,N-bis(2,3-epoxypropyl)aniline which is a read-across from a supporting substance (structural analogue or surrogate).

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, the results are not clear in the in vivo test performed with the p-isomer. A recent 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. Taken all results into consideration the two substances are classified for mutagenicity (Muta 2, H341)according to CLP regulation (Regulation EC No. 1272/2008) and DSD (Directive 67/548/EEC).