7-azatridecane-1,13-diamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In a bacterial reverse mutation test (AMES test, OECD TG 471) no mutagenic activity was observed when the registration substance was tested up to the limit concentration.

No data on cytogenicity or mutagenic activity in mammalian cells are available for the registration substance. However adequate and reliable studies performed with a surrogate substance (multi-constituent substance containing high levels of this registration substance) are at hand.

No in vitro cytogenicity studies are available, but results of an in vivo cytogenicity study (equivalent to OECD TG 475) testing a surrogate substance are reported in the in vivo section below.

A surrogate substance showed no HPRT gene mutation in CHO cells both in the absence and the presence of activation system (equivalent to OECD 476).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 3 FEB 1999 to 5 APR 1999

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

version of 1997

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL: the substance appeared to be stable under the conditions of the study, no evidence of instability was observed.

Species / strain / cell type:

S. typhimurium, other: TA 97a, TA 98, TA 100, and TA 1535

Species / strain / cell type:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with and without

Metabolic activation system:

aroclor 1254 induced male rat liver S9-mix

Test concentrations with justification for top dose:

0, 10, 50, 100, 500, 1000, 2500, and 5000 µg/plate (highest concentration is maximum concentration requiered according to guideline)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used:sterile water
- Justification for choice of solvent/vehicle: no evidence for instability was observed

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-acetylaminofluorene

9,10-dimethylbenzanthracene

2-nitrofluorene

sodium azide

N-ethyl-N-nitro-N-nitrosoguanidine

other: ICR-191 dihydrochloride (CASRN: 17070-45-0)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium; in agar (plate incorporation)

DURATION
- Preincubation period: no
- Exposure duration:incubation at 37°C for 48 hours

NUMBER OF REPLICATIONS: 3 plates per concentration

DETERMINATION OF CYTOTOXICITY
- yes

Rationale for test conditions:

AN individual trial must have included a negative and a positive control and at least 5 concentration levels of the test substance for each tester strain and condition.
A data point, concnetration level or trial was excluded from analysis when acceptability criteria were not met - these are as follows:
- all tester strains must have had their strain characteristic specifics and exhibit a characteristic number of revertants per plate in the absence of the test substance
- mean positive control values must exhibit at least a three-fold increase of revertants compared to solvent control
- a minimum of three non-toxic concentrations were requiered (non toxic, i.e. < 50% reduction in mean number of revertants per plate relative to the mean of the concurrent negative control)

Evaluation criteria:

POSITIVE, if the mean number of revertant colonies per plate in at least one strain with or without metabolic activation system was at least two times greater than the mean of concurrent vehicle control and there was a concentration-related increase over the range tested

NEGATIVE, if no two-foldincrease in number of revertant colonies or no concentration-related increase over the range tested

Results not meeting the criteria for positive or negavtive were evaluated using scientific judgment and may have been reported as EQUIVOCAL.

Statistics:

mean number of three plates/concentration level and condition as well as standard deviation was calculated

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

E. coli WP2 uvr A pKM 101

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 2500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 100, and TA 1535

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 1000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

not examined

Key result

Species / strain:

S. typhimurium, other: TA 97a, TA 98, TA 100, and TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

starting at 500 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of this study the test item showed no mutagenic activity in bacterial tester strains.

Executive summary:

In a guideline study according to OECD TG 471 under GLP conditions, mutagenic activity of the test item was investigated in a plate incorporation assay. Salmonella typhimurium strains TA 97a, TA1535, TA98 and TA100 as well as Escherichia coli strain WP2uvrA were exposed to concentrations of 0, 10, 50, 100, 500, 1000, 2500 or 5000 µg test item per plate either with or without metabolic activation system (aroclor induced male rat liver S9 -mix). Three plates at each concentration level were used. No precepitation of the test substance occurred. Cytotoxicity was observed as low as 500 µg/plate in strains tested without metabolic activation system, or as low as 1000 µg/plate in some strains tested with metabolic activation system. All positive controls induced marked increases in frequency of revertant colonies. All negative controls were found to be in an acceptable range. The test item showed no mutagenic activity.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

read-across source

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested concentrations (i.e. 1, 10, 100, 500, and 1000 µg TM/mL) resulted in 103, 94, 65, 51 and 22% of mean relative cell survival respectively. I.e. cytotoxicity is obvious at 1000 µg/ml (acc. to general def. of cytotoxicity which is 10-20% of control).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

tested concentrations (i.e.10, 50, 100, 175, 250 µg TM/mL) resulted in 100, 92, 66, 38 and 29% of mean relative cell survival respectively. I.e. cytotoxicity is obvious at ca. 1000 µg/ml (acc. to general def. of cytotoxicity which is 10-20% of control).

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other:

Remarks:

test material test concentrations correspond to 0.5-0.8, 5-8, 50-80, 250-400, 500-800 µg BHT/mL

Conclusions:

Under the conditions used in this study the test item was not mutagenic in the HGPRT-test with CHO cells both in the presence (5%(v/v)) and absence of rat liver S-9 mix (i.e. metabolic activation).

Executive summary:

The study used as source investigated Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine (EC 907 -605 -7, which contains relevant amounts of the submission substance,

7-azatridecane-1,13-diamine).

The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Results obtained in this in vivo rat bone marrow chromosome aberration test (equivalent to OECD TG 475) demonstrated that the test item is not a cytogenetic active agent under the conditions tested

(note: in this study a surrogate substance (i.e. reaction mass 7-azatridecane-1,13-diamine and hexamethylenediamine) was tested, however the dose tested was adjusted to 100% BHMT, which is the submission substance in this registration).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / bone marrow chromosome aberration

Type of information:

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

Adequacy of study:

key study

Justification for type of information:

For details on endpoint specific justification please see read-across report in section 13 or find a link in cross-reference “assessment report”.

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

read-across: supporting information

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

A variety of clinical signs of toxicity were observed in treated rats

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Results obtained in this in vivo rat bone marrow chromosome aberration test demonstrated that the test item is not a cytogenetic active agent under the conditions tested.

Executive summary:

The study used as source investigated Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine (EC 907 -605 -7, which contains relevant amounts of the submission substance,

7-azatridecane-1,13-diamine). In this study also the test material was adjusted to 100% of BHMT, which is the submission substance of this registration, thus the study investigated the submission substance up to the limit dose in a dose range finding study and up to the maxiumum tolerated dose (MTD) in the main test as required by the current guideline.

The study results of the source compound were considered applicable to the target compound. Justification and applicability of the read-across approach (structural analogue) is outlined in the read-across report in section 13 or find a link in cross reference “assessment report”.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In a guideline study according to OECD TG 471 under GLP conditions, mutagenic activity of the submission substance was investigated in a plate incorporation assay. Salmonella typhimurium strains TA 97a, TA1535, TA98 and TA100 as well as Escherichia coli strain WP2uvrA were exposed to concentrations of 0, 10, 50, 100, 500, 1000, 2500 or 5000 µg test item per plate either with or without metabolic activation system (aroclor induced male rat liver S9 -mix). Three plates at each concentration level were used. No precepitation of the test substance occurred. Cytotoxicity was observed as low as 500 µg/plate in strains tested without metabolic activation system, or as low as 1000 µg/plate in some strains tested with metabolic activation system. All positive controls induced marked increases in frequency of revertant colonies. All negative controls were found to be in an acceptable range. The test item showed no mutagenic activity.

No data on cytogenicity or mutagenic activity in mammalian cells are available for the registration substance. However adequate and reliable studies performed with a surrogate substance (multi-constituent substance containing high levels of this registration substance) are at hand.

One study investigated the potential of the test item (surrogate of the submission substance) to induce gene mutations at the HGPRT locus in Chinese hamster ovary cells in vitro following the principles of guideline OECD 476 and in compliance with GLP. The assay was performed in two independent experiments, using identical procedures, both with and without rat liver microsomal activation.

The test article was tested with the following concentrations:

without S9-mix: 10, 50, 100, 175, and 250 µg test material/ml (5 hours of treatment with test material; test material test concentrations correspond to 5 -8, 25 -40, 50-80, 87.5 -140, 125 -200 µg BHT/mL)

with S9-mix: 1, 10, 100, 500, and 1000 µg test material /ml (5 hours of treatment with test material; test material test concentrations correspond to 0.5-0.8, 5-8, 50-80, 250-400, 500-800 µg BHT/mL)

According to the preliminary experiment for toxicity the concentration ranges were selected. Starting from 100 µg test material/ml relative initial survival was slightly decreasing with increasing concentrations tested, reaching cytotoxicity effect level (i.e. only 10 -20% survival of control) at concentrations of 333 µg test material/ml in the test without metabolic activation and at 1000 µg test material/ml in thes tests with various concentrations of the S-9 mix. Nevertheless up to the highest concentration tested in the mutagenicity assay no distinct decrease of the cloning efficiency was observed.

Up to the highest investigated dose no increase in mutant colony numbers was obtained in two independent experiments.

Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies.

In conclusion, the test item does not induce gene mutations in the HGPRT-test with Chinese hamster cells, either in the presence or in the absence of a metabolic activation system, under the experimental conditions described. Therefore the test item is considered to be non-mutagenic in this HGPRT assay.

To investigate cytogenetic potential of the test material an in vivo rat bone marrow chromosomal aberration assay was performed comparable to OECD TG 475 (study pre-guideline) and according to GLP (US FDA requirement of 1978). The test material dosed at 500 mg/kg bw (note: in this study the dose tested was adjusted to 100% BHMT, which is the submission substance of this dossier) did not produce any evidence of chromosome damage as measured by increases in chromosome aberrations, altered mitotic index (as measure for cytotoxic effects), or chromosome number as compared to concurrent controls in this assay. The positive control substance, cyclophosphamide, produced significant increase in the percent of cells displaying chromosome aberrations, average number of aberrations and decreased the mitotic index and chromosome number, confirming senstitivity of the test system applied. Under the conditions of this assay, the test substance is not cytogenetically active.

In both studies used as source the test material investigated was Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine (EC 907 -605 -7, which contains relevant amounts of the submission substance, i.e. 7 -azatridecane-1,13 -diamine), however in slightly different compositions.

a) In the HPGRT study the test material contained between 50 to 80% of 7 -azatridecane-1,13 -diamine (i.e. BHMT, the submission substance), below 10% of another amine and water. Both of the amines in the reaction mass are classified as skin corrosive substances, thus for both constituents of the test material a relevant and comparable level of cytotoxicity is expected in a cell based assay (comparable due to structural similarity). Cytotoxicity was observed in the dose range finding study of this gene mutation study in mammalian cells, thus the highest tested doses were limited to the doses were 10 -20% of relative survival was seen. In theory, the concentrations which could have been tested with the submission substance, if pure test material would have been used, could have been slightly higher however no relevant influence is assumed, as the results obtained were clearly negative at the tested concentrations up to the appropriate level inducing cytotoxic effects. The study is therefore regarded adequate to cover the data requirements.

b) In the in vivo bone marrow chromosomal aberration study the test material dosing was adjusted by the study authors to 100% of 7 -azatridecane-1,13 -diamine (i.e. BHMT), which is the submission substance of this registration, thus the study investigated the submission substance up to the limit dose in a dose range finding study and up to the maximum tolerated dose (MTD) in the main test as required by the current guideline. The study is therefore regarded adequate to cover the data requirement.

Justification for classification or non-classification

In the adequate and reliable data presented above no mutagenic activity of the submission substance was observed, thus no classification for germ cell mutagenicity according to the criteria of Regulation (EC) No 1272/2008 is proposed.