Amines, N-(3-aminopropyl)-N-(C16-18 and C18-unsatd. alkyl)trimethylenedi-

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Data is based on read across from a structurally similar branched triamine C12, which was found to be non-mutagenic in a bacterial mutagenicity test (Ames) and in a mammalian mutagenicity test with mouse lymphoma cells. The branched triamine C12 was not clastogenic in an in vitro mammalian chromosome aberration test with human lymphocytes. Based on the negative results in in vitro studies, the performance of additional in vivo testing is not indicated.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 0.2 of IUCLID.

Species / strain:

S. typhimurium, other: TA 1535, TA 1537, TA100, TA98, and TA102.

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 50 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: Since the test item was toxic in the preliminary test, the choice of the highest dose-level for the main test was based on the level of toxicity, according to the criteria specified in the international guidelines.

ADDITIONAL INFORMATION ON CYTOTOXICITY: In the main study in the experiments without S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥ 50 μg/plate. In the experiments with S9 mix, a moderate to marked toxicity was induced generally at dose-levels ≥ 100 μg/plate in the first experiment and ≥ 50 μg/plate in the second experiment.
In the preliminary cytotoxicity test, with S9, toxicity was induced at dose levels ≥ 100 µg/plate in TA 98 strain and ≥ 500 µg/plate in other strains.

Conclusions:

Under these experimental conditions, the test item does not show mutagenic activity in the bacterial reverse mutation test with Salmonella typhimurium.

Executive summary:

A Bacterial Reverse Mutation Test was performed according to OECD No. 471and EEC B.13/14.

To assess the toxicity of the test item to the bacteria, six dose-levels (one plate/dose-level; 10, 100, 500, 1000, 2500 and 5000 µg/plate) were tested in the TA 98, TA 100 and TA 102 strains, with and without S9 mix. The evaluation of the toxicity was performed on the basis of the observation of the decrease in the number of revertant colonies and/or a thinning of the bacterial lawn.

With dose levels based on the results of the preliminary toxicity test two independent experiments (without S9-mix: First experiment 6.25, 12.5, 25, 50 and 100 µg/plate, for the TA 98 and TA 1537 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the remaining strains. Second experiment: 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains. With S9 mix First experiment: 12.5, 25, 50, 100 and 200 µg/plate for all tester strains. Second experiment (preincubation method): 3.125, 6.25, 12.5, 25 and 50 µg/plate, for the TA 98, TA 1537 and TA 1535 strains, 12.5, 25, 50, 100 and 200 µg/plate, for the TA 100 and TA 102 strains) using three plates/dose-level, each strain was tested, with and without S9 mix, with at least five dose-levels of the test item, the vehicle control and the appropriate positive control.

Preliminary toxicity and First test: The direct plate incorporation method was performed as follows: test item solution (0.1 mL), S9 mix when required (0.5 mL) and bacterial suspension (0.1 mL) were mixed with 2 mL of overlay agar (containing traces of the relevant aminoacid and biotin and maintained at 45°C).

After rapid homogenization, the mixture was overlaid onto a Petri plate containing minimum medium.

Second test: The preincubation method was performed as follows: test item solution (0.1 mL), S9 mix (0.5 mL) and the bacterial suspension (0.1 mL) were incubated for 60 minutes at 37°C before adding the overlay agar and pouring onto the surface of a minimum agar plate.

After 48 to 72 hours of incubation at 37°C, revertants were scored with an automatic counter (Cardinal counter, Perceptive Instruments, CB9 7 BN, ).

This study is considered valid if the following criteria are fully met:

. the number of revertants in the vehicle controls is consistent with laboratory historical data

. the number of revertants in the positive controls is higher than that of the vehicle controls and is consistent with laboratory historical data.

A reproducible two-fold increase in the number of revertants compared with the vehicle controls, in any strain at any dose-level and/or evidence of a dose-relationship was considered as a positive result. Reference to historical data, or other considerations of biological relevance may

also be taken into account in the evaluation of the data obtained.

Preliminary toxicity test:

No precipitate was observed in the Petri plates when scoring the revertants at all dose-levels. Both with and without S9 mix, the test item was strongly toxic at dose-levels ≥ 500 µg/plate. In the TA 98 strain, the test item was also toxic at 100 µg/plate.

Mutagenicity experiments:

Without S9-mix: A moderate to marked toxicity was induced generally at dose-levels ≥ 50 µg/plate. No increase in the number of revertants was noted in all tester strains, in both experiments.

With S9-mix: A moderate to marked toxicity was induced generally at dose-levels ≥ 100 µg/plate in the first experiment and ≥ 50 µg/plate in the second experiment. No increase in the number of revertants which could be considered as relevant was induced in any of the five tester strains.

Criteria for validity were met.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 0.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 0.2 of IUCLID.

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Without S9 mix: a slight to strong decrease in the mitotic index was induced at dose-levels ≥ 7.5 μg/mL. With S9 mix: a slight to strong toxicity was generally induced at dose-levels ≥ 5 μg/mL.

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: For the choice of the highest dose for treatment in the first experiment of the present study, significant variations in pH and strong toxicity induced in CIT Study No. 23283 MLY (In vitro mammalian cell gene mutation test performed on the same compound) were taken into account.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Without S9 mix: a slight to strong decrease in the mitotic index was induced at dose-levels ≥ 7.5 μg/mL. With S9 mix: a slight to strong toxicity was generally induced at dose-levels ≥ 5 μg/mL.

PRECIPITATION CONCENTRATION: No precipitation observed
MITOTIC INDEX: 
First experiment
-S9 mix:  (3 hour treatment, 20 hour harvest)
Doses	0      0.137  0.41  1.23  3.69  11.07 	
MI	7.70   5.90   6.35  7.30  6.95  0.90	   
OC 	100    77     82    95    90    12        

Doses   33.22  99.67  299   *Pos. control
MI      0.00   0.00   0.00   230 
OC      0      0      0      30 	

+ S9 mix: (3 hour treatment, 20 hour harvest)
Doses	0      0.137  0.41  1.23  3.69	11.07	
MI      6.55   5.45   5.9   6.25  5.65	0.00		
OC      100    83     90    95	  86	0		

Doses   33.22  99.67   299  *Pos. control
MI      0.00   0.00    0.00  315/1.75    
OC      0      0       0     48/27

MI = Mitotic index in % (mean)
OC = % of control
* -S9 mix =MCC  3 ug/mL, +S9-mix = CPA 25 ug/mL/ CPA 50
ug/mL

Second experiment
1 -S9 mix: 
Doses	0     0.31  0.63  1.25 2.5  5    7.5 *Pos. control
MI      4.10  4.75  4.60  5.25 5.20 4.50 4.05 3.65 
OC	100   116   112	  128  127  110	 99   98
2 - S9 mix:  
MI	5.00  4.95  5.65  4.75 4.25 5.10 2.90	-
OC	100   99    113	  95   85   102	 58	-
3 + S9 mix: 	
MI      4.50  3.75  3.95  3.65 3.30 2.70 0.00 1.50/0.75
OC      100   83    88	  81   73   60	 0    33/17
4 + S9 mix: 
MI      2.05  3.25  1.75  3.00  2.80 2.80 0.00	-
OC      100   159   85    146	137  137  0     -

MI = Mitotic index in % (mean)
OC = % of control
*-S9 mix =MCC  0.2 ug/mL, +S9-mix = CPA 25 ug/mL/ CPA 50
ug/mL 
1: 3 hour treatment, 20 hour harvest
2: 44 hour treatment, 44 hour harvest
3: 3 hour treatment, 20 hour harvest
4: 3 hour treatment, 44 hour harvest


CYTOTOXIC CONCENTRATION: 
- With metabolic activation: 
First and second experiment >= 5 µg/mL: 40 to 100% decrease in mitotic index.- Without metabolic activation: 
First experiment: after 3 hours treatment >= 11.07 µg/mL: 88 to 100% decrease in mitotic index.
Second experiment: after 44 hours treatment at 7.5 µg/mL:42% decrease in mitotic index.
TEST-SPECIFIC CONFOUNDING FACTORS: -

STATISTICAL RESULTS: 
Chromosomal aberration analysis: 
 
Experiments without S9 mix
The dose-levels selected for metaphase analysis were as  follows:   0.41, 1.23 and 3.69 µg/mL, for the 3-hour treatment (first experiment), higher dose-levels being strongly toxic,  2.5, 5 and 7.5 µg/mL, for the 20-hour treatment (second experiment),   7.5 µg/mL, for the 44-hour
treatment (second experiment), 7.5 µg/mL being the highest dose used for treatment in this second experiment.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 hours treatments.

Experiments with S9 mix
The dose-levels selected for metaphase analysis were as follows: 0.41, 1.23 and 3.69 µg/mL, for the 20-hour harvest time in the first experiment, 1.25, 2.5 and 5 µg/mL, for the 20-hour harvest time in the second
experiment, 5 µg/mL, for the 44-hour harvest time. The highest doses selected for metaphase analysis were the highest readable doses (higher dose-levels being strongly ntoxic). No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

Conclusions:

Under these experimental conditions, the test item did not induce chromosome aberrations in cultured human lymphocytes.

Executive summary:

According to OECD No. 473 and EEC B.10 a chromosome abrerration test was performed with Triameen Y12D in human lymphocytes.

Primary culture of human lymphocytes were prepared from whole blood samples obtained from two healthy donors.

Based on the results from CIT 23283 MLY  (the pH of the treated cultures showed a significant difference in comparison with the vehicle control culture, at dose-levels ≥ 2 mM (7.9 ≤ pH ≤ 8.5, versus pH 7.0 for the vehicle control), at dose-levels ≥ 0.2 mM, a strong toxicity was induced (all cells were found dead 3 hours following the treatment))  the following dose levels were selected:

First experiment both with and without S9 mix:

0.137, 0.41, 1.23, 3.69, 11.07, 33.22, 99.67 and 299 µg/mL

Second experiment both with and without S9 mix:

0.31, 0.63, 1.25, 2.5, 5 and 7.5 µg/mL.

In two independent experiments, using duplicate cultures, the cells were tested, with and without S9 mix, at the doses stated above with the vehicle control and the positive controls; mitomycin C (MMC, without S9 mix:), 3 µg/mL (3 hrs) or 0.2 µg/mL (continuous treatment) or Cyclophosphamide (CPA, with S9 mix) 50 µg/mL and 25 µg/mL (the dose-level which gave a satisfactory response in terms of quality and quantity of metaphases and extent of chromosomal damage was selected for analysis).

To prepare each culture, 0.5 mL of heparinised whole blood was added to 5 mL of RPMI 1640 medium containing 20% fetal calf serum, L-glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and phytohaemagglutinin (PHA: a mitogen to stimulate the lymphocytes to divide). The cultures were then placed at 37°C for 48 hours.

In the first experiment, lymphocyte cultures were then exposed to the test or control items, both in the absence and presence of S9 mix, for 3 hours then rinsed. One and a half hours before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest time was 20 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles. As this experiment gave negative results, a second experiment was performed as follows:

- without S9 mix, cells were exposed continuously to the test or control items, until harvest,

- with S9 mix, cells were exposed to the test or control items for 3 hours and then rinsed.

One and a half hour before harvest, each culture was treated with a colcemid solution (10 µg/mL) to block cells at the metaphase-stage of mitosis. Harvest times were 20 hours and 44 hours from the beginning of treatment, corresponding to approximately 1.5 normal cell cycles and 24 hours later.

After hypotonic treatment (KCl 0.075 M), the cells were fixed in a methanol/acetic acid mixture (3/1; v/v), spread on glass slides and stained with Giemsa.  All the slides were coded, so that the scorer is unaware of the treatment group of the slide under evaluation ("blind" scoring).

The cytotoxicity of the test item was evaluated using the mitotic index (number of cells in mitosis/1000 cells examined), which indicates whether a item induces mitotic inhibition. Mitotic index was determined without blind scoring.  Analysis of 200 metaphases/dose-level (with 44 to 46 chromosomes) was made, with 100 metaphases/culture whenever possible. Only 50 metaphases/culture were analysed when at least 10% cells with structural chromosome aberrations were observed.  All metaphase analyses were performed blind.

The following structural aberrations were recorded for each metaphase: gaps, chromatid and chromosome breaks and exchanges, and others (multiple aberrations and pulverizations). In addition, the following numerical aberrations were recorded when encountered: polyploidy and endoreduplication. The analysis of the slides was performed at Microptic, cytogenetic services (2 Langland Close Mumbles, , ), in compliance with GLP, and the Principal Investigator was Natalie Danford.

Treatment of results

All the data were presented in tabular form in which the type of structural chromosome aberrations, the total number of aberrations and the frequency of cells with structural chromosome aberrations, including and excluding gaps, are shown. A cell having one or more of the above-mentioned structural chromosome aberrations was recorded as a single cell with structural chromosome aberrations. Therefore the total frequency of cells with structural chromosome aberrations was not necessarily equivalent to the total number of aberrations.

Acceptance criteria

This study was considered valid when the following criteria were met:

- the frequency of cells with structural chromosome aberrations in the vehicle controls was consistent with our historical data,

- the frequency of cells with structural chromosome aberrations in the positive controls was significantly higher than that of the controls and consistent with our historical data.

Statistical analysis

For each test and for each harvest time, the frequency of cells with structural chromosome aberrations (excluding gaps) in treated cultures was compared to that of the vehicle control cultures. If necessary, the comparison was performed using the χ² test, in which p = 0.05 was used as the lowest level of significance.

Evaluation criteria

A reproducible and statistically significant increase in the frequency of cells with structural chromosome aberrations for at least one of the dose-levels and one of the two harvest times was considered as a positive result. Reference to historical data or other considerations of biological relevance, was also taken into account in the evaluation of the findings

Experiments without S9 mix:

Cytotoxicity: In the first experiment, after 3 hours treatment, a strong toxicity was induced at dose-levels ≥ 11.07 µg/mL (88-100% decrease in the mitotic index).

In the second experiment, after 44 hours treatment, a slight decrease in the mitotic index was noted at 7.5 µg/mL (42% decrease).

Chromosomal aberration analysis: The dose-levels selected for metaphase analysis were as follows: . 0.41, 1.23 and 3.69 µg/mL, for the 3-hour treatment (first experiment), higher dose-levels being strongly toxic, . 2.5, 5 and 7.5 µg/mL, for the 20-hour treatment (second experiment), . 7.5 µg/mL, for the 44-hour treatment (second experiment), 7.5 µg/mL being the highest dose used for treatment in this second experiment.

No significant increase in the frequency of cells with structural chromosomal aberrations was noted after 3, 20 as well as 44 hours treatments.

Experiments with S9 mix:

Cytotoxicity: A slight to strong toxicity was generally induced at dose-levels ≥ 5 µg/mL (40-100% decrease in the mitotic index).

Chromosomal aberration analysis: The dose-levels selected for metaphase analysis were as follows: . 0.41, 1.23 and 3.69 µg/mL, for the 20-hour harvest time in the first experiment, . 1.25, 2.5 and 5 µg/mL, for the 20-hour harvest time in the second experiment, . 5 µg/mL, for the 44-hour harvest time.

The highest doses selected for metaphase analysis were the highest readable doses (higher dose-levels being strongly toxic).

No significant increase in the frequency of cells with structural chromosomal aberrations was noted in both experiments and at both harvest times.

The frequencies of cells with structural chromosome aberrations of the vehicle and positive controls were as specified in acceptance criteria. The study was therefore considered valid.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 0.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

read-across based on grouping of substances (category approach)

Adequacy of study:

key study

Justification for type of information:

For details and justification of read-across please refer to the report attached in section 0.2 of IUCLID.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

with and without S9 mix, 3 hour treatment: >= 10 µM without S9 mix, 24 hour treatment: >= 0.75 µM

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: To assess the cytotoxicity of the test item, at least six dose-levels (one culture/dose-level) were tested both with and without metabolic activation.
A treatment of 3 hours was performed using a final concentration of 10E6 cells/mL. At the end of treatment, cells were washed and then cell concentration was adjusted in order to seed 1.6 cells per well in the 96-well microtiter plates. After at least 7 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- S9: a marked toxicity was noted mainly at 10 μM, after 3 hours of treatment. After 24 hours treatment, a marked to strong toxicity was noted at 0.75 μM. +S9: a marked to strong toxicity was induced at dose-levels ≥ 10 μM.

Conclusions:

Under these experimental conditions, the test item did not show mutagenic activity in this mouse lymphoma assay.

Executive summary:

According to OECD No 476 and EEC B.17 a gene mutation assay was performed with Triameen Y12D in L5178Y TK+/- Mouse Lymphoma Cells.

 

To assess the cytotoxicity of the test item, doses of  0.02, 0.2, 1, 2, 5 and 10 mM (one culture/dose-level) were tested both with and without metabolic activation.  A treatment of 3 hours was performed using a final concentration of 106 cells/mL. At the end of treatment, cells were washed and then cell concentration was adjusted in order to seed 1.6 cells per well in the 96-well microtiter plates.  After at least 7 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.

 

Two independent mutagenicity experiments were performed,

Without S9 mix:    

First experiment (3-hour treatment), 0.31, 0.63, 1.25, 2.5, 5 and 10 µM, 

Second experiment (24-hour treatment, because first experiment was negative), 0.063, 0.125, 0.25, 0.5, 0.75 and 1 µM

With S9 mix:

First experiment 0.63, 1.25, 2.5, 5, 10 and 20 µM

Second experiment 0.63, 1.25, 2.5, 5, 10 and 15 µM,

 

The following controls were included using duplicate cultures:

- vehicle controls: cultures treated with the vehicle,

- positive controls: cultures treated with:

- MMS, in the absence of S9 mix,

- CPA, in the presence of S9 mix

 

Cells were grown in RPMI 1640 medium containing L-Glutamine (2 mM), penicillin (100 U/mL), streptomycin (100 µg/mL) and sodium pyruvate (200 µg/mL), supplemented by heat inactivated horse serum at 5% (RPMI 5), 10% v/v (RPMI 10) or 20% v/v (RPMI 20)

 

For the 3-hour treatment, approximately 0.5*106cells/mL in 20 mL culture medium (RPMI 5) were exposed to the test or control items, at 37°C.  For the 24-hour treatment, approximately 0.15*106cells/mL in 20 mL culture medium (RPMI 5) were exposed to the test or control items, at 37°C using a gentle shaking. At the end of the treatment period, the cells were rinsed. After centrifugation and removing of the supernatant, the pellet were suspended in RPMI 10 and the cells were counted using an haemocytometer and seeded as follows

 

Survival plates

1.6 cells/well (one 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates) to determine cytotoxicity using cloning efficiency (CE0). After at least 7 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted. To enable the expression of the mutant phenotype, the remaining cells were incubated at a density of 2*105 cells/mL in RPMI 10 medium for 48 hours, at 37°C in a humidified atmosphere of 5% CO2/95% air. During this expression period, cultures were maintained at the density of approximately 2*105 cells/mL where possible. At the end of this expression period, the cell densities of the cultures were determined using an haemocytometer and seeded after serial dilutions as follows:

 

Viability plates

1.6 cells/well (one 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates) to define the number of viable cells (CE2). After at least 7 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air, the clones were counted.

 

Mutant plates

2000 cells/well (one 96-well plate/culture = two plates/dose-level, except for the vehicle control where two 96-well plates/culture were used = total of four plates) to select the TFTR (trifluorothymidine resistant) mutant cells (for the determination of CEMUTANT). After 11-12 days of incubation at 37°C in a humidified atmosphere of 5% CO2/95% air in the presence of 4 µg TFT/mL of culture medium, the clones were counted, differentiating small and large colonies:

- size of small colonies: 25% of the diameter of the well,

- size of large colonies: > 25% of the diameter of the well

For scoring of colonies in mutant plates, the following parameters were considered:

- well containing mutant colony (small or large),

- well not containing mutant colony,

- when both small and large colonies are present in the same well two mutant colonies were counted (one small and one large)

 

Treatment of results

Data from cytotoxicity plates (empty wells) are used to calculate CE0 (cloning efficiency after treatment) and RCE0 (survival relative to vehicle controls after treatment). These values give a measure of the toxicity of the treatment with the test item. CE0 is calculated from the zero term of the Poisson distribution:

 

CE0 = -Ln [empty wells/total wells]/number of cells/well (+/- 1.6)

RCE0 = [CE0 treated/ CE0 vehicle control]*100

 

Data from viability plates (empty wells) are used to calculate CE2 (cloning efficiency at the end of the expression period) and RCE2 (viability relative to vehicle controls at the end of the expression period). These values indicate the viability of the cell populations at the end of the expression period. CE2is calculated from the zero term of the Poisson distribution:

 

CE2 = -Ln [empty wells/total wells]/number of cells per well (+/- 1.6)

RCE2 = [CE2 treated/ CE2 vehicle control]*100

 

Data from the mutant plates (empty wells) are used to calculate CEmutant (cloning efficiency in selective medium). This value is an indicator of the absolute mutant frequency. CEmutant is calculated from the zero term of the Poisson distribution (only mutant clones are able to grow in TFT containing medium):

 

CEmutant  =  -Ln [empty wells/total wells]/number of cells per well (+/- 2000)

The relative mutant frequency (MF) = CEmutant*106/CE2

 

Daily growth on day 1 and on day 2, cell count factor, survival, RS (relative survival), SG (suspension growth), RSG (relative suspension growth) and RTG (relative total growth) are calculated as follows:

 

Daily cell growth on day 1 (or 2) = cell concentration on day 1 (or 2)/ adjusted concentration on day 0 (or 1) = 2 x 105#

# or appropriate cell concentration if lower

SG = Daily growth on day 1 x daily growth on day 2

RSG = [SG treated/ SG vehicle control]*100

RTG (%) = RSG*RCE2

Cell count factor = treated post-treatment cell count/ vehicle control post-treatment cell count

Survival = CE0*cell count factor

RS = (treated survival value/vehicle control survival value)*100

 

Evaluation criteria

A reproducible two-fold increase in the mutant frequency when compared with the vehicle controls, at any dose-level and/or evidence a dose-relationship were considered as a positive result. Reference to historical data, or other considerations of biological relevance were also taken into account in the evaluation of the data obtained. Positive response observed only at high levels of cytotoxicity (survival lower than 10%) were not considered.

 

This study was considered valid since the following criteria were fulfilled:

-the cloning efficiency of the vehicle controls were between 0.6-1.4 for

CE0 and between 0.7-1.3 for CE2 (see § 2.3),

-the mutation frequency of the vehicle controls were between 60-250*10-6,

-the mutation frequency of the positive controls were higher than that of the vehicle controls (more than two fold) and consistent with our historical data.

 

Preliminary toxicity test:

At the end of the treatment period, the pH of the treatment cultures showed a significant difference in comparison with the vehicle control culture, at dose-levels ≥ 2 mM (7.9 ≤ pH ≤ 8.5, versus pH 7.0 for the vehicle control). The test item was markedly to strongly toxic at dose-levels ≥ 0.02 mM, as shown by 99-100% (without S9 mix) or 87-100% (with S9 mix) decrease in the relative survival (RS).

 

Mutagenicity tests without S9-mix:

Cytotoxicity: After 3 hours treatment, a marked toxicity was noted mainly at 10 µM, as shown by 59% decrease in the CE0, 81% decrease in the RS, as well as 95% decrease in the relative total growth (RTG). After 24 hours treatment, a marked toxicity was noted at 0.75 µM (86% decrease in the RS) and all the cells were found dead following the treatment at 1 µM.

Mutagenicity: No noteworthy increase in the mutation frequency was induced, both after 3-hour and 24-hour treatments.

 

Mutagenicity tests with S9-mix:

Cytotoxicity: A marked to strong toxicity was induced at dose-levels ≥ 10 µM, as shown mainly by 77-100% decrease in the RTG. Mutagenicity: No noteworthy increase in the mutation frequency was induced, in both experiments.

This information is used in a read-across approach in the assessment of the target substance. For details and justification of read-across please refer to the read-across report attached to IUCLID section 0.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Data is based on read across from a structurally similar branched triamine C12. Branched triamine C16-18 only differs in the length of the alkyl chain, which is suspected to influence aspects related to bioavailability, but not aspects of chemical reactivity and route of metabolization, aspects that influence specific mechanisms of toxicity relevant for genotoxicity. For these reasons, many of the studies can best be performed on the substance with the shortest chain length within a group of structurally similar substances, as this is considered to represent the most sensitive species to show specific effects.

 

In vitro genotoxicity

The test substance was found non-mutagenic in a bacterial mutagenicity test (Ames test), performed in accordance with EU Method B.13/14 and under GLP, with Salmonella typhimurium strains TA 1535, TA 1537, TA100, TA98, and TA102 using test concentrations up to 200 μg/plate, both with and without metabolic activation (CIT, 2002d). The tests were performed up to cytotoxic levels. Two independent experiments were performed, using 3 plates per dose level. No increased number of revertants was observed in any experiment, indicating that the substance is not mutagenic in the Ames test.

 

Negative results were observed also in a mammalian mutagenicity test with mouse lymphoma L5178Y cells performed in accordance with EU Method B.17 and GLP (CIT, 2002e), both with and without metabolic activation. Two independent experiments were performed, using 3- and 24-hours exposure duration. Test concentrations up to 20 μM (with S9 mix) and 10 μM (without S9) were used and cytotoxicity was observed with and without S9 mix, 3-hour treatment, from 10 µM and without S9 mix, 24 hour treatment, from 0.75 µM.

 

The test substance was also not clastogenic in an in vitro mammalian chromosome aberration test with human lymphocytes, performed in accordance with EU Method B.10 and under GLP (CIT, 2002f), both in the presence and absence of metabolic activation. Two independent experiments were performed, using 3 hours exposure duration (experiment 1) or continuous exposure until harvest (experiment 2). The test concentrations used were up to and including 299 µg/mL (1 mM) in experiment 1 and 7.5 µg/mL in experiment 2. Without S9 mix, a slight to strong decrease in the mitotic index was induced at dose levels ≥ 7.5 μg/mL, while with S9 mix a slight to strong toxicity was generally induced at dose levels ≥ 5 μg/mL.

 

In vivo genotoxicity

In accordance with Column 2 of REACH Annex VIII, in vivo testing needs to be considered, if positive results have been observed in any of the available in vitro studies. As in the present case all available in vitro studies were negative, the performance of additional in vivo studies is not indicated.

Justification for classification or non-classification

Data is based on read across from astructurally similar branched triamine C12 which is considered to be predictive for branched triamine C16-18. Based on the results of the in vitro genetic toxicity studies, there is no need to classify for genotoxicity according to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.