Octylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Octylamine was not mutagenic in an in vitro mammalian cell assay (HPRT assay using mouse lymphoma TK+/- cells), with or without metabolic activation and was negative in an Ames test performed with several S. typhimurium strains (TA 1535, TA1537, TA98 and TA100) with and without metabolic activation.

Link to relevant study records

Reference

Reference 1

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

as of 1997

Principles of method if other than guideline:

HPRT assay for the detection of mutations at the hypoxanthine-guanine phosphoribosyl transferase (hprt) locus (6-thioguanine [6TG] resistance) in mouse lymphoma cells using a fluctuation protocol according to Cole et al. 1983.
Reference:
Cole J, Arlett C F, Green M H L, Lowe J and Muriel W 1983: A comparison of the agar cloning and microtitration techniques for assaying cell survival and mutation frequency in L5178Y mouse lymphoma cells. Mutation Research, 111, 317-386

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Target gene:

Induction of a forward mutation in the X-linked hprt locus: Resistance to the toxic analogue 6-thioguanine (6TG) results from lack of hypoxanthine-guanine phosphoribosyl transferase (HPRT) activity. Thus, the hprt-negative mutants are unable to use 6TG and survive in its presence.

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI media (containing antibiotics and varying concentrations of horse serum, heat-inactivated, from 0 - 20 %)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically "cleansed" against high spontaneous background: yes,
- Other procedures: purge of TK-negative mutants

Additional strain / cell type characteristics:

other: TK proficient (TK+)

Metabolic activation:

with and without

Metabolic activation system:

post-mitochondrial fraction from livers of male SD rats previously induced with Arochlor-1254

Test concentrations with justification for top dose:

Experiment 1: 0, 10, 20, 30, 40, 50, 60, 70, 75, 80, 90, 100 µg/mL (evaluated)
Experiment 2: 0, 10, 20, 30, 40, 50, 55, 60, 65, 70, 72.5, 75, 80, 90 µg/mL (evaluated)

Vehicle / solvent:

- Vehicle/solvent used: DMSO
- Justification for choice of solvent/vehicle: sufficient water solubility of the test substance

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO, diluted 100-fold in treatment medium

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitroquinoline-N-oxide (-S9); benzo(a)pyrene (+S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium, incubation in centrifuge tubes, culture flasks, and wells of microtiter plates,
depending on the operation step

DURATION
- Preincubation period: no data, pre-culture in RPMI 10 after thawing up to an appropriate cell density
- Exposure duration: 3 h
- Incubation temperature: 37 +- 1 °C
- Expression time (cells in growth medium): 7 d
- Selection time (if incubation with a selection agent): 12 to 13 d

SELECTION AGENT (mutation assays): 6-thioguanine (6-TG)

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: Selection of 6-TG resistance: 4 microtiter plates of 96 wells each; at 2 x10^4 cells per well each
(= 384 x2 x10^4 cells per test concentration)

DETERMINATION OF CYTOTOXICITY
- Method: Relative survival in the presence and absence of test substance (by visual counting of viable clones in microtiter plates)

DETERMINATION of VIABILITY (after expression period)
- Method: visual counting of viable clones in microtiter plates

DETERMINATION of 6-TG RESISTANCE (after expression period)
- Method: visual inspection of microtiter plates for the number wells containing clones.

Evaluation criteria:

ACCEPTANCE CRITERIA
The assay was considered valid if the following criteria were met:
1. the mutant frequencies in the negative (vehicle) control cultures fell within the normal range (not more than three times the historical mean value)
2. at least one concentration of each of the positive control chemicals induced a clear increase in mutant frequency (the difference between the positive and negative control mutant frequencies was greater than half the historical mean value).

EV ALUATION CRITERIA
For valid data, the test article was considered to induce forward mutation at the hprt locus in mouse lymphoma L5178Y cells if:
1. the mutant frequency at one or more concentrations was significantly greater than that of the negative control (p < 0.05)
2. there was a significant concentration﷓relationship as indicated by the linear trend analysis (p < 0.05)
3. the effects described above were reproducible.
Results that only partially satisfied the assessment criteria described above were considered on a case-by-case basis.

Statistics:

Statistical significance of mutant frequencies was carried out according to the UKEMS guidelines. Thus the control log mutant frequency (LMF) was compared with the LMF from each treatment concentration, and secondly the data were checked for a linear trend in mutant frequency with test article treatment. These tests require the calculation of the heterogeneity factor to obtain a modified estimate of variance.

Reference:
Robinson et al. 1990: Statistical evaluation of bacterial/mammalian fluctuation tests. In Statistical Evaluation of Mutagenicity Test Data (Ed D J Kirkland), Cambridge University Press, pp 102 – 140.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related: The highest concentration to reliably provide >10 % RS was 40.41 µg/mL in the absence and 80.81 µg/mL in the presence of S9.

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: marked increases of >= 1 pH unit at >= 1293 µg/mL
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: no

RANGE-FINDING/SCREENING STUDIES: for cytotoxicity testing up to 1293 µg/mL

COMPARISON WITH HISTORICAL CONTROL DATA: Acceptance criterion, Control/Historical ratio > 0.5, fulfilled for positive controls

Remarks on result:

other: strain/cell type: mouse lymphoma L5178Y cells

Remarks:

Migrated from field 'Test system'.

Summary of mutation data [table 8; means of two replicate cultures]

 

Experiment 1 (3 hour treatment in the absence and presence of S-9)

 

Treatment (µg/mL)	-S-9			Treatment (µg/mL)	+S-9
	% RS	MF§			% RS	MF§
0	100	5.84		0	100	3.74
20	86	6.48	NS	20	92	3.07	NS
30	68	5.98	NS	30	89	2.46	NS
40	46	7.90	NS	40	73	3.42	NS
50	43	7.15	NS	50	55	3.37	NS
60	18	5.93	NS	60	43	5.09	NS
				70	24	2.22	NS
				75	12	2.11	NS
Linear trend			NS	Linear trend			NS
NQO				B[a]P
0.1	63	19.94		2	63	48.97
0.15	52	30.99		3	19	163.83

 

Experiment 2 (3 hour treatment in the absence and presence of S-9)

 

Treatment (µg/mL)	-S-9			Treatment (µg/mL)	+S-9
	% RS	MF§			% RS	MF§
0	100	4.98		0	100	4.40
10	86	4.14	NS	20	93	4.88	NS
20	59	4.09	NS	40	65	3.14	NS
30	48	3.48	NS	50	52	3.26	NS
40	41	4.40	NS	60	42	1.82	NS
50	34	1.88	NS	65	40	2.42	NS
55	30	2.47	NS	70	35	3.63	NS
60	22	4.61	NS	80	38	2.57	NS
65	10	4.69	NS	90	22	2.76	NS
Linear trend			NS	Linear trend			NS
NQO				B[a]P
0.1	33	28.54		2	46	31.06
0.15	31	16.57		3	28	69.69

 

§       = 6TG resistant mutants/10⁶viable cells 7 days after treatment

% RS =Percent relative survival adjusted by post treatment cell counts

NS    = not significant

Conclusions:

Interpretation of results (migrated information):
negative

N-octylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under test conditions employed in this study. This included treatments up to highly toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system.

Executive summary:

N-octylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells in a valid study that was conducted in accordance with OECD test guideline No. 476 and under GLP conditions. This included treatments up to highly toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system. Vehicle controls and positive control substances performed as expected (Covance, 2010). The study is considered to be valid and acceptable for assessment.

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Non-GLP guideline study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix

Test concentrations with justification for top dose:

0, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment, SPT)
0, 4, 20, 100, 500 and1500 µg/plate (2nd experiment, SPT)
0, 0.8, 4, 20, 100 and 500 µg/plate (3rd experiment, PPT, with S-9 mix)
0, 4, 20, 100, 500, 1000 µg/plate (4th experiment, PPT, without S-9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: [DMSO]
- Justification for choice of solvent/vehicle: The test substance was completely soluble in DMSO

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

with S-9 mix for strains TA 100, TA 98, TA 1537, TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: N-methyl-N'-nitro-N-nitroso-guanidine

Remarks:

without S-9 mix for strains TA 100 and TA 1535

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylendiamine

Remarks:

without S-9 mix for strain TA 98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

other: 9-aminoacridine chloride monohydrate

Remarks:

without S-9 mix for strain TA 1537

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 20 minutes
- Exposure duration: 48 hours

DETERMINATION OF CYTOTOXICITY
- Method: relative total growth

Evaluation criteria:

In general, a substance to be characterized as positive in the Ames test has to fulfill the following requirements:
- doubling of the spontaneous mutation rate (control)
- dose-response relationship
- reproducibility of the results

Statistics:

no data

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

depending on strain and test conditions without S-9 mix as doses >= 500 µg/plate and with S-9 mix from about 1000-1500 µg/plate onward

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

ADDITIONAL INFORMATION ON CYTOTOXICITY:
A bacteriotoxic effect (reduced his- background growth, decrease in the number of his+ revertants) was observed depending an the strain and test
canditions without S-9 mix at doses >= 500 µg/plate and with S-9 mix form about 1000 µg - 1500 µg/plate onward.

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

A not dose dependent maximum increase of the mutant frequency of 1.6 was observed. The test substance is therefore not mutagenic in the Ames test.

Conclusions:

Under the conditions of the test octylamine was negative with and without metabolic activation.

Executive summary:

In a non-GLP study according to OECD TG 471 (Ames test) the Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with several concentrations of octylamine in four experiments (plate incorporation and preincubation test) with and without metabolic activation:

1. 0, 20, 100, 500, 2500 and 5000 μg/plate (1st experiment, SPT)

2. 0, 4, 20, 100, 500 and1500 μg/plate (2nd experiment, SPT)
3. 0, 0.8, 4, 20, 100 and 500 μg/plate (3rd experiment, PPT, with S-9 mix)
4. 0, 4, 20, 100, 500, 1000 μg/plate (4th experiment, PPT, without S-9 mix)

The preincubation time was 20 minutes. After 2 days revertant colonies were counted. A not dose dependent maximum increase of the mutant frequency of 1.6 was observed.

The test substance is therefore not mutagenic in the Ames test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

N-octylamine hydrochloride was not clastogenic in an in vivo micronucleus assay with bone marrow from exposed mice (study performed according to OECD TG 474 under GLP). Two in vitro tests support the negative results for genetic toxicity: Octylamine was not mutagenic in an in vitro mammalian cell assay (HPRT assay using mouse lymphoma TK+/- cells), with or without metabolic activation and was negative in an Ames test performed with several S. typhimurium strains (TA 1535, TA1537, TA98 and TA100) with and without metabolic activation.

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:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: GLP guideline study; but as read-across from supporting substance maximum reliability is 2. Read-across hypothesis: for details please see read-across report in IUCLID section 13.

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Germany
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: about 30 g
- Housing: Makrolon cages, type MI
- Diet (e.g. ad libitum): Standardized pelleted feed (Maus/Ratte Haltung "GLP", Provimi Kliba SA, Kaiseraugst, Switzerland)
- Water (e.g. ad libitum): ad libitum


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 30 - 70
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

purified water
- Vehicle(s)/solvent(s) used: purified water
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, purified water was selected as the vehicle.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:

The substance to be administered per kg body weight was dissolved in purified water. The pH values of all test substance preparations were between 4.0 - 6.0 prior to dosing without thoroghly.
To achieve a solution of the test substance in the vehicle, the test substance preparation was shaken thoroughly.
All test substance formulations wereprepared immediately before administration.

Duration of treatment / exposure:

The animals were treated once and samples of bone marrow were taken 24 hours and 48 hours after the treatment.

Frequency of treatment:

The animals were treated once.

Remarks:

Doses / Concentrations:
0, 75 , 150, 300 mg/kg
Basis:
no data

No. of animals per sex per dose:

5 male animals per dose

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPP) / Vincristine Sulphate (VCR)
- Route of administration: oral / i.p.
- Doses / concentrations: 20 mg / 0.15 mg

Tissues and cell types examined:

In general 2000 polychromatic erythrocytes (PCEs) from each of the male animals of every test group are evaluated and investigated for micronuclei.
The normochromatic erythrocytes (NCEs) which occur are also scored.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Pretest were performed

DETAILS OF SLIDE PREPARATION:

The bone marrow was prepared according to the method described by SCHMID, W. (7, 8) and SALAMONE, M. et al. (6).
• The two femora of the animals sacrificed by cervical dislocation were prepared by dissection and removing all soft tissues.
• After cutting off the epiphyses, the bone marrow was flushed out of the diaphysis into a centrifuge tube using a cannula filled with fetal calf serum (FCS) which was at 37°C (about 2 mL/femur).
• The suspension was mixed thoroughly with a pipette, centrifuged at 300 x g for 5 minutes, the supernatant was removed and the precipitate was resuspended in about 50 µL fresh FCS.
• 1 drop of this suspension was dropped onto clean microscopic slides, using a Pasteur pipette. Smears were prepared using slides with ground edges, the preparations were dried in the air and subsequently stained.

• The slides were stained in eosin and methylene blue (modified May-Grünwald solution or Wrights solution) for about 5 minutes.
• After having briefly been rinsed in purified water, the preparations were soaked in purified water for about 2 - 3 minutes.
• Subsequently, the slides were stained in Giemsa solution (15 mL Giemsa, 185 mL purified water) for about 15 minutes.
• After having been rinsed twice in purified water and clarified in xylene, the preparations were mounted in Corbit-Balsam.

Evaluation criteria:

A finding is considered positive if the following criteria are met:
• Significant and dose-related increase in the number of PCEs containing micronuclei.
• The number of PCEs containing micronuclei has to exceed both the concurrent negative control and the highest value of the historical control range.
A test substance is considered negative if the following criteria are met:
• The number of cells containing micronuclei in the dose groups is not significantly above the negative control and is within the historical control data.

Statistics:

The statistical evaluation of the data was carried out using the program system MUKERN (BASF Aktiengesellschaft).
The asymptotic U test according to MANN-WHITNEY (modified rank test according to WILCOXON) was carried out to clarify the question whether there were significant differences between the control group and dose groups with regard to the micronucleus rate in polychromatic erythrocytes. The relative frequencies of cells containing micronuclei of each animal were used as a criterion for the rank determination for the U test. Significances were identified as follows:
* p ≤ 0.05
** p ≤ 0.01

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Clinical signs of toxicity in test animals: no adverse signs or symptoms
- Mortality: none

The number of normochromatic erythrocytes containing micronuclei did not differ to any appreciable extent in the negative control or in the various dose groups at any of the sacrifice intervals.

No inhibition of erythropoiesis induced by the treatment of mice with Octylamine hydrochloride was detected; the ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all dose groups.

Read-across hypothesis: for details please see read-across report in IUCLID section 13.

 

Percent micronucleated PCE/200 PCE

dose	24 hours sacrifice interval	48 hours sacrifice interval
vehicle control	1.4 % mPCE	0.8 % mPCE
300 mg/kg b.w.	1.4 % mPCE	1.5 % mPCE
150 mg/kg b.w.	1.1 % mPCE
75 mg/kg b.w.	1.3 % mPCE
positive control (CCP)	15 % mPCE, mainly small micronuclei
positive control (VCR)	30.6 % mPCE, 8 % was lange micronuclei

Conclusions:

Interpretation of results (migrated information): negative
Thus, under the experimental conditions chosen here, the test substance Octylamine hydrochloride has no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Executive summary:

In this in vivo micronucleus assay (according to OECD TG 474, GLP) 5 male NMRI mice per dose group were exposed once orally via gavage against 0, 75 , 150 or 300 mg octylamine hydrochloride/kg bw. Samples of bone marrow were taken 24 hours and 48 hours after the treatment. 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group were evaluated and investigated for micronuclei. The normochromatic erythrocytes (NCEs) which occur are also scored. Positive controls and vehicle controls were examined as well.

Under the experimental conditions chosen here, the test substance Octylamine hydrochloride had no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Additional information from genetic toxicity in vivo:

Genetic toxicity in vivo (Micronucleus test):

In this in vivo micronucleus assay (according to OECD TG 474, GLP) 5 male NMRI mice per dose group were exposed once orally via gavage against 0, 75 , 150 or 300 mg octylamine hydrochloride/kg bw. Samples of bone marrow were taken 24 hours and 48 hours after the treatment. 2000 polychromatic erythrocytes (PCEs) from each of the animals of every test group were evaluated and investigated for micronuclei. The normochromatic erythrocytes (NCEs) which occur are also scored. Positive controls and vehicle controls were examined as well.

Under the experimental conditions chosen here, the test substance Octylamine hydrochloride had no chromosome-damaging (clastogenic) effect nor does it lead to any impairment of chromosome distribution in the course of mitosis (aneugenic activity) in bone marrow cells in vivo (BASF, 2007).

Genetic toxicity in vitro:

N-octylamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells in a valid study that was conducted in accordance with OECD test guideline No. 476 and under GLP conditions. This included treatments up to highly toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system. Vehicle controls and positive control substances performed as expected (Covance, 2010). The study is considered to be valid and acceptable for assessment.

In a non-GLP study according to OECD TG 471 (Ames test) the Salmonella typhimurium strains TA1535, TA1537, TA98 and TA100 were treated with several concentrations of octylamine in four experiments (plate incorporation and preincubation test) with and without metabolic activation:

1) 0, 20, 100, 500, 2500 and 5000 µg/plate (1st experiment, SPT)

2) 0, 4, 20, 100, 500 and1500 µg/plate (2nd experiment, SPT)

3) 0, 0.8, 4, 20, 100 and 500 µg/plate (3rd experiment, PPT, with S-9 mix)

4) 0, 4, 20, 100, 500, 1000 µg/plate (4th experiment, PPT, without S-9 mix)

The preincubation time was 20 minutes. After 2 days revertant colonies were counted. A not dose dependent maximum increase of the mutant frequency of 1.6 was observed. The test substance is therefore not mutagenic in the Ames test (BASF, 1989).

Justification for selection of genetic toxicity endpoint
This in vivo miclonucleus assay was performed according to OECD TG 474, under GLP. It is the only in vivo study available.

Justification for classification or non-classification

Based on the results of one in vivo study (micronucleus test, performed according to OECD TG under GLP) and two in vitro studies (Ames test and HPRT test, both performed according to respective OECD test guideline) no classification for the endpoint "genetic toxicity" is required for octylamine.