Registration Dossier

Administrative data

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test similar to OECD 471: weak positive responses observed in three tester strains in presence of metabolic activation

Link to relevant study records
Reference
Endpoint:
in vitro gene mutation study in bacteria
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1980
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 471 (Bacterial Reverse Mutation Assay)
Qualifier:
according to
Guideline:
other: Industrial Safety and Health Department, Labor Standard Bureau, Ministry of Labor, Japan
GLP compliance:
not specified
Type of assay:
bacterial reverse mutation assay
Specific details on test material used for the study:
Purity: 75 wt%
Name cited in study report: NNN’N’-tetraglycidyl metaxylenediamine or PGA-X
Description at room temperatur: liquid
Target gene:
- S. typhimurium: Histidine gene
- Escherichia coli: Tryptophan gene
Species / strain / cell type:
S. typhimurium TA 1535, TA 1537, TA 98 and TA 100
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
S. typhimurium TA 1538
Additional strain / cell type characteristics:
not applicable
Species / strain / cell type:
E. coli WP2 uvr A
Additional strain / cell type characteristics:
not applicable
Metabolic activation:
with and without
Metabolic activation system:
Polychlorinated biphenyl (PCB, KC-500) induced S.D. rat S9
Test concentrations with justification for top dose:
10, 50, 100, 500, 1000 and 5000 µg/ plate. 5000 µg is the recommended maximum test concentration according to the OECD guideline.
Vehicle / solvent:
DMSO
Justification for choice of solvent: The substance is readily soluble in DMSO
Untreated negative controls:
no
Negative solvent / vehicle controls:
yes
Remarks:
DMSO
True negative controls:
no
Positive controls:
yes
Positive control substance:
9-aminoacridine
2-nitrofluorene
N-ethyl-N-nitro-N-nitrosoguanidine
other: 2-Aminoanthracene
Details on test system and experimental conditions:
METHOD OF APPLICATION:
preincubation

DURATION
- Preincubation period: 20 minutes at 37°C
- Exposure duration: 48 hours at 37 °C

NUMBER OF REPLICATIONS:
duplicates

DETERMINATION OF CYTOTOXICITY
Not specified
Evaluation criteria:
Test results were judged to be positive (+) if mutant colony counts were at least twice as high as natural mutant colony counts. Test results were judged to be negative (-) if mutant colony counts were lower.
Species / strain:
other: TA 98, TA 1537 and TA 1538
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Remarks:
Top dose, only in absence of S9
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 100
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
5000 µg/ plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
S. typhimurium TA 1535
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
at 1000 and 5000 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Key result
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with
Genotoxicity:
positive
Remarks:
1000 to 5000 µg/plate
Cytotoxicity / choice of top concentrations:
no cytotoxicity nor precipitates, but tested up to recommended limit concentrations
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
other: TA 100 and 1535
Metabolic activation:
without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Species / strain:
E. coli WP2 uvr A
Metabolic activation:
with and without
Genotoxicity:
negative
Cytotoxicity / choice of top concentrations:
cytotoxicity
Vehicle controls validity:
valid
Untreated negative controls validity:
not applicable
Positive controls validity:
valid
Additional information on results:
ADDITIONAL INFORMATION ON CYTOTOXICITY:
In absence of S9 inhibition of bacterial growth is observed at the top dose for all tester strains
Remarks on result:
other: mutagenic potential was fairly weak compared to that of existing mutagens.
Conclusions:
The substance shows weak mutagenic potential in the Salmonella Typhimurium TA 100, TA 1535 and Escherichia Coli WP2 uvra in presence of metabolic activation.
Executive summary:

The mutagenic activity of the test substance was evaluated in a study similar to OECD TG 471. A pre-incubation assay was performed in the tester strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2 uvr A both in absence and presence of Polychlorinated biphenyl (PCB), induced rat S9 as metabolic system. The substance was tested at concentrations ranging from 10 to 5000 µg/ plate (the maximum recommended dose level). Cytotoxicity was only observed at the top-dose in absence of S9, details on the method for cytotoxicity determination are not reported. Concurrent solvent and negative controls were included and gave appropriate responses. An increase in mutant frequencies was only observed at concentrations between 1000 to 5000 µg/plate in the strains TA 100 and TA 1535 and E. coli WP2 uvr A in presence of metabolic activation. The mutagenic potential was however fairly weak compared to that of existing mutagens. Although a weak positive response in the Salmonella typhimurium and Escherichia coli reverse mutation assays was obtained, insufficient data is available to concluded if the test substance is an actual mutagen.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

Testing proposal in vivo: combined OECD 474 (micronucleus test) and OECD 486 (Comet assay)

Link to relevant study records
Reference
Endpoint:
genetic toxicity in vivo, other
Remarks:
Combined gene mutation (comet assay) and micronucleus assay
Type of information:
experimental study planned
Study period:
according to decision by ECHA
Justification for type of information:
TESTING PROPOSAL ON VERTEBRATE ANIMALS

NON-CONFIDENTIAL NAME OF SUBSTANCE:
N, N, N’, N’-tetrakis(2, 3-epoxypropyl)-m-xylene-a, a’-diamine
CONSIDERATIONS THAT THE GENERAL ADAPTATION POSSIBILITIES OF ANNEX XI OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- Available GLP studies: There are no GLP studies available covering genetic toxicity information requirements.
- Available non-GLP studies: There is one well performed Ames test available, in which a weak positive response was observed in three of the tester strains in presence of metabolic activation. Since this data is insufficient for C&L, the need for additional information is triggered (ECHA Guidance in Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance).
- Historical human data: There are no historical human data available on genetic toxicity for the substance.
- (Q)SAR: At present there is no valid (Q)SAR model available which can fulfill the data requirements for vivo genetic toxicity of substances. (ECHA Guidance in Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance)
- In vitro methods: Because of the (weak) positive gene mutation test in bacteria, independent on the outcome of in vitro assays (in vitro micronucleus, chromosome aberration and in vitro mammalian cell gene mutation test) in vivo testing has to be performed for at least mutagenicity (ECHA Guidance in Information Requirements and Chemical Safety Assessment Chapter R 7a: Endpoint specific guidance). Therefore, the in vitro alternatives are not of added value for the assessment of the mutagenic potential of the substance nor form an alternative for the in vivo tests. An in vitro micronucleus test is only of limited value since a positive response will trigger an additional in vivo study as well. The proposed simultaneous assessment of mutagenicity (Comet assay) and clastogenicity (micronucleus test) in vivo is therefore the most efficient strategy in terms of animal use.
- Weight of evidence: There are no data available which are sufficient for a weight of evidence approach.
- Grouping and read-across: No substances or a category of substances are known which apply for read across addressing the genetic toxicity of the substance.
- Substance-tailored exposure driven testing: not applicable
- Approaches in addition to above: not applicable
- Other reasons: not applicable

CONSIDERATIONS THAT THE SPECIFIC ADAPTATION POSSIBILITIES OF ANNEXES VI TO X (AND COLUMN 2 THEREOF) OF THE REACH REGULATION ARE NOT ADEQUATE TO GENERATE THE NECESSARY INFORMATION:
- According to Column 2 Annex VII Further mutagenicity studies shall be considered in case of a positive result in the in vitro gene mutation study in bacteria.
According to Column 1 Annex VIII there are two data requirements: 1. In vitro cytogenicity study in mammalian cells or in vitro micronucleus study. 2. In vitro gene mutation study in mammalian cells, if a negative result in Annex VII and Annex VIII, 1.
- According to Column 2 Annex VIII: Appropriate in vivo mutagenicity studies shall be considered in case of a positive result in any of the genotoxicity studies in Annex VII or VIII.
- According to column 2 Annex IX: If there is a positive result in any of the in vitro genotoxicity studies in Annex VII or VIII and there are no results available from an in vivo study already, an appropriate in vivo somatic cell genotoxicity study shall be proposed by the registrant.
The points outlined above apply for the test substance and make in vivo testing unavoidable. Thus, in order to fulfill the information requirements a combined in vivo Comet assay (OECDE 489) and micronucleus test (OECD 474) is proposed.

FURTHER INFORMATION ON TESTING PROPOSAL IN ADDITION TO INFORMATION PROVIDED IN THE MATERIALS AND METHODS SECTION:
An in vivo test in mice is proposed in which the OECD guidelines 474 and 489 are combined, in order to fully cover the information requirements on mutagenic and clastogenic properties of the substance in one study, in order to reduce the use of test animals to a minimum.
Qualifier:
according to
Guideline:
OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)
Qualifier:
according to
Guideline:
OECD Guideline 489 (In vivo Mammalian Alkaline Comet Assay)
Endpoint conclusion
Endpoint conclusion:
no study available (further information necessary)

Additional information

Ames test (similar to OECD 471)

The mutagenic activity of the test substance was evaluated in a study similar to OECD TG 471. A pre-incubation assay was performed in the tester strains S. typhimurium TA 1535, TA 1537, TA 1538, TA 98 and TA 100 and E. coli WP2 uvr A both in absence and presence of Polychlorinated biphenyl (PCB), induced rat S9 as metabolic system. The substance was tested at concentrations ranging from 10 to 5000 µg/ plate (the maximum recommended dose level). Cytotoxicity was only observed at the top-dose in absence of S9, details on the method for cytotoxicity determination are not reported. Concurrent solvent and negative controls were included and gave appropriate responses. An increase in mutant frequencies was only observed at concentrations between 1000 to 5000 µg/plate in the strains TA 100 and TA 1535 and E. coli WP2 uvr A in presence of metabolic activation. The mutagenic potential was however fairly weak compared to that of existing mutagens. Although a weak positive response in the Salmonella typhimurium and Escherichia coli reverse mutation assays was obtained, insufficient data is available to concluded if the test substance is an actual mutagen (Mitsubishi Gas Chemical Company, 1980).

Testing proposal in vivo study

An additional in vivo test in mice, which combines the OECD guideline 474 and 489, is proposed.

Justification for classification or non-classification

The available data is insufficient for C&L of the substance for genetic toxicity in accordance with EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation No. 1272/2008 and its amendments. Therefore additional in vivo testing, more specifically a combined in vivo Comet assay and micronucleus test, is proposed.