Dibutylamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

- HPRT (GLP-compliant study according to OECD 476): negative

- AMES (comparable to a NTP standard protocol): negative

- CA (similar to OECD 473): ambiguous

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 476 (In Vitro Mammalian Cell Gene Mutation Test using the Hprt and xprt genes)

GLP compliance:

yes

Type of assay:

in vitro mammalian cell gene mutation test using the Hprt and xprt genes

Target gene:

hypoxanthine-guanine phosphoribosyl transferase

Species / strain / cell type:

mouse lymphoma L5178Y cells

Details on mammalian cell type (if applicable):

- Type and identity of media: RPMI 1640 media
- Periodically checked for Mycoplasma contamination: yes

Metabolic activation:

with and without

Metabolic activation system:

rat S9 mix

Test concentrations with justification for top dose:

Range finder: 0; 40.41; 80.81; 161.6; 323.3; 646.5; 1293 µg/ml (with and without S9 mix)
Experiment 1: 0, 50, 100, 150, 240, 280, 320, 360 µg/ml (without S9 mix)
0, 80, 160, 240, 280, 400, 450, 525 µg/ml (with S9 mix)
Experiment 2: 0, 50, 100, 150, 250, 300, 320, 340, 360, 400, 450 µg/ml (without S9mix)
0, 100, 200, 300, 350, 400, 450, 500, 525, 550,600 µg/ml (with S9 mix)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Positive control substance:

other: see remarks

Remarks:

4-nitroquinoline 1-oxide 0.10/0.15 µg/ml without S9 mix; benzo[a]pyrene 2.00/3.00 µg/ml

Details on test system and experimental conditions:

METHOD OF APPLICATION:
- in medium

DURATION
- Exposure duration: 3 h
- Expression time (cells in growth medium): 7 days

NUMBER OF REPLICATIONS: 2

Evaluation 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. The control log mutant frequency (LMF) was copared with the LMF from each treatment concentration and 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.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of S-9 ranging from 40.41 to 1293 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested (1293 µg/mL) was not plated due to excessive toxicity and complete toxicity (0% RS) was observed at the highest concentration plated in the absence and presence of S-9 (646.5 µg/mL). The highest concentration to provide >10% RS was 323.3 µg/mL in the absence and presence of S-9, which gave 12% and 37% RS, respectively.
No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1293 µg/mL) as compared to the concurrent vehicle controls (individual data not reported). Marked changes in pH were observed at 1293 µg/mL as compared to the concurrent vehicle controls but no marked changes were observed up to 646.5 µg/mL (individual data not reported). As no concentration tested in the Mutation Experiments was greater than 600 µg/mL no further measurements were performed.

In Experiment 1 ten concentrations, ranging from 50 to 500 µg/mL in the absence of S‑9 and from 80 to 600 µg/mL in the presence of S‑9, were tested. Seven days after treatment, the highest two concentrations tested in the absence of S‑9 (400 and 500 µg/mL) and the highest concentration tested in the presence of S‑9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 200 µg/mL in the absence of S‑9 and 320 and 360 µg/mL in the presence of S‑9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of S‑9. The highest concentrations plated were 360 µg/mL in the absence of S‑9 and 525 µg/mL in the presence of S‑9, which gave 18% and 22% RS in the absence and presence of S-9, respectively. Although no concentration tested in the presence of S-9 gave the desired 10 to 20% RS the toxicity level observed at 525 µg/mL (22% RS) was sufficiently close to 10 to 20% RS to be considered acceptable and the data are therefore considered valid.

In Experiment 2 ten concentrations, ranging from 50 to 450 µg/mL in the absence of S‑9 and from 100 to 600  µg/mL in the presence of S‑9, were tested. Seven days after treatment all concentrations in the absence and presence of S-9 were selected to determine viability and 6TG resistance. The highest concentrations tested, 450 µg/mL in the absence of S‑9 and 600 µg/mL in the presence of S‑9, gave 26% and 20% RS in the absence, respectively (seeTable8). It may be noted that in the absence of S‑9 a concentration of 400 µg/mL gave 20% RS and the data are therefore considered valid.

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	1.71		0		100	2.62
50		94	3.38	NS	80		78	1.18	NS
100		73	2.49	NS	160		74	1.75	NS
150		64	2.19	NS	240		59	2.60	NS
240		58	1.63	NS	280		52	1.90	NS
280		54	2.28	NS	400		44	2.49	NS
320		30	1.69	NS	450		32	1.79	NS
360		18	1.81	NS	525		22	1.78	NS
Linear trend			NS		Linear trend			NS
NQO					B[a]P
0.1		62	24.79		2		54	32.03
0.15		73	16.36		3		39	58.99

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	2.19		0		100	1.18
50		97	1.55	NS	100		94	1.36	NS
100		90	1.55	NS	200		81	1.45	NS
150		82	2.76	NS	300		69	2.42	NS
250		51	3.65	NS	350		65	3.61	NS
300		54	2.46	NS	400		68	4.32	NS
320		47	5.65	NS	450		52	2.67	NS
340		58	0.60	NS	500		34	1.51	NS
360		45	4.60	NS	525		35	2.02	NS
400		20	3.06	NS	550		25	2.98	NS
450		26	2.54	NS	600		20	2.45	NS
Linear trend			NS		Linear trend			*
NQO					B[a]P
0.1		86	12.51		2		39	25.22
0.15		58	15.93		3		18	26.40

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

%RS  Percent relative survival adjusted by post treatment cell counts

NS    Not significant

*, **, ***       Test for linear trend: χ²(one-sided), significant at 5%, 1% and 0.1% level respectively

Conclusions:

A GLP-compliant study according to OECD 476 was performed. It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study.

Executive summary:

A GLP-compliant in vitro mammalian cell gene mutation test using the Hprt and xprt genes according to OECD 476 was performed. In the cytotoxicity Range-Finder Experiment, six concentrations were tested in the absence and presence of rat S9 ranging from 40.41 to 1293 µg/mL (equivalent to 10 mM at the highest concentration tested). The highest concentration tested (1293 µg/mL) was not plated due to excessive toxicity and complete toxicity (0% RS) was observed at the highest concentration plated in the absence and presence of rat S9 (646.5 µg/mL). The highest concentration to provide >10% RS was 323.3 µg/mL in the absence and presence of rat S9, which gave 12% and 37% RS, respectively.

No marked changes in osmolality were observed in the Range-Finder at the highest concentration tested (1293 µg/mL) as compared to the concurrent vehicle controls (individual data not reported). Marked changes in pH were observed at 1293 µg/mL as compared to the concurrent vehicle controls but no marked changes were observed up to 646.5 µg/mL (individual data not reported). As no concentration tested in the Mutation Experiments was greater than 600 µg/mL no further measurements were performed.

In Experiment 1 ten concentrations, ranging from 50 to 500 µg/mL in the absence of rat S9 and from 80 to 600 µg/mL in the presence of rat S9, were tested. Seven days after treatment, the highest two concentrations tested in the absence of rat S9 (400 and 500 µg/mL) and the highest concentration tested in the presence of rat S9 (600 µg/mL) were considered too toxic for selection to determine viability and 6TG resistance. In addition, concentrations of 200 µg/mL in the absence of rat S9 and 320 and 360 µg/mL in the presence of rat S9 were not selected as there were sufficient non-toxic concentrations. All other concentrations were selected in the absence and presence of rat S9. The highest concentrations plated were 360 µg/mL in the absence of rat S9 and 525 µg/mL in the presence of rat S9, which gave 18% and 22% RS in the absence and presence of rat S9, respectively. Although no concentration tested in the presence of rat S9 gave the desired 10 to 20% RS the toxicity level observed at 525 µg/mL (22% RS) was sufficiently close to 10 to 20% RS to be considered acceptable and the data are therefore considered valid.

In Experiment 2 ten concentrations, ranging from 50 to 450 µg/mL in the absence of rat S9 and from 100 to 600  µg/mL in the presence of rat S9, were tested. Seven days after treatment all concentrations in the absence and presence of rat S9 were selected to determine viability and 6TG resistance. The highest concentrations tested, 450 µg/mL in the absence of rat S9 and 600 µg/mL in the presence of rat S9, gave 26% and 20% RS in the absence, respectively (seeTable8). It may be noted that in the absence of rat S9 a concentration of 400 µg/mL gave 20% RS and the data are therefore considered valid.

It is concluded that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells when tested under the conditions employed in this study. These conditions included treatments up to toxic concentrations in two independent experiments in the absence and presence of a rat liver metabolic activation system (S9).

Reference 2

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

Comparable to NTP standard protocol

Principles of method if other than guideline:

according to Haworth et al. (1983): Environ. Mutagen. 5, Suppl. 1, 3-142

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine operon

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

Metabolic activation systems were derived from Arochlor-induced livers of male SD rats and male Syrian hamsters.

Test concentrations with justification for top dose:

0, 100, 333.3, 1000, 3333.3, 10000 µg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water

Untreated negative controls:

yes

Remarks:

water

Negative solvent / vehicle controls:

yes

Remarks:

sterility control

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

sodium azide

other: without S9: 4-nitro-o-phenylenediamine (TA98 ); with S9: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days

NUMBER OF REPLICATIONS: Each trial consisted of triplicate plates and was done in a replicate.

Evaluation criteria:

Positive if a reproducible dose related response over the solvent control was obtained .

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:

at 10000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

TA1535

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean±SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	11	2.2	29	4.6	12	2.4	14	1.8	6	1.5	15	2
100	17	2	22	4.5	10	1	16	1.5	9	1.7	20	3.4
333.3	16	1.7	26	0.6	12	2	19	1.2	10	2.2	16	2.4
1000	15	2.3	23	2.4	14	2	10	2.2	12	3	15	3.5
3333.3	17	0.7	20	0.7	11	2.3	17	1.8	8	2	15	1.5
10000	15s	2.8	27	2.2	10	3.8	10	1.9	15s	7.3	16	1.7
Positive Control	413	7.4	433	9.9	258	6.2	401	10.6	151	1.5	210	6

 

TA100

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	128	4.7	123	3.5	125	7.5	92	4.5	115	7.5	109	8.9
100	120	9.1	110	8	121	4.3	108	5.2	130	3.3	123	4.8
333.3	107	1.9	94	11.2	128	1	114	10.7	124	1.3	113	11
1000	119	7.7	103	10.5	129	3.8	96	1.8	126	12.2	109	7.8
3333.3	111	8	104	18.7	115	0.9	107	7.6	121	6.9	112	9.7
10000	117	7.7	110	8.8	115	5.7	100	8.1	116	5.6	118	10.7
Positive Control	484	9.6	520	8	1526	17.3	1511	44.1	839	28.5	498	19.2

 

TA98

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Negative)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	36	5.2	37	1.3	40	4.3	47	4.5	44	2.6	44	2.3
100	39	1.7	23	1.7	41	0.9	47	2.9	41	2.3	37	2
333.3	36	9.1	40	6.1	44	0.9	46	2.5	46	2.7	28	4.6
1000	35	3.1	24	2.9	44	2.3	38	0.6	38	0.7	36	3.5
3333.3	39	7.4	28	1.2	36	2	34	2.7	37	3.7	35	1.8
10000	30	4.8	34	3.3	32s	3.5	39	0.9	31	1	31	1.9
Positive Control	436	30.4	472	14.2	1208	12.7	1391	32.4	839	12.5	570	53.5

 

TA1537

Dose	No Activation (Negative)		No Activation (Negative)		10% HLI (Equivocal)		10% HLI (Negative)		10% RLI (Negative)		10% RLI (Negative)
ug/Plate	Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM		Mean ± SEM
0	9	0.3	9	1.5	7	1	20	0.6	10	2	16	1.8
100	7	1.2	8	0.6	10	1.2	14	1.5	7	0	18	2.7
333.3	8	1	11	1	16	1	20	3.2	7	0.9	12	1.7
1000	8	1.2	6	1.2	17	3.9	17	1.3	9	1.3	18	1.3
3333.3	11	1.7	8	0.3	11	1.9	14	2.5	7	2.5	12	2.1
10000	8t	2	12	2.3	16	3.6	12	2.7	7	2	14	4
Positive Control	178	18.6	286	38.5	308	21.4	281	16.3	313	37	157	4.2

 

Abbreviations:
RLI = induced male Sprague Dawley rat liver S9
HLI = induced male Syrian hamster liver S9
s = Slight Toxicity; p = Precipitate; x = Slight Toxicity and Precipitate; T = Toxic; c = Contamination

Under the conditions tested, no mutagenic activity was observed in any strain/activation combination in the bacterial AMES-Test. The positive controls showed the expected values.

Conclusions:

An Ames-test, comparable to a NTP standard protocol, was performed. Under the conditions tested, no mutagenic activity was observed in any strain/activation combination. The positive controls showed the expected values.

Executive summary:

An Ames-test, comparable to the NTP standard protocol, was performed. Four different strains (S. typhimurium TA 1535, TA 1537, TA 98 and TA 100) were tested with and without metabolic activation for 2 days (preincubation period: 20 min). The metabolic activation systems were derived from Arochlor-induced livers of male SD rats and male Syrian hamsters. Each trial consisted of triplicate plates and was done in a replicate. Under the conditions tested, no mutagenic activity was observed in any strain/activation combination. The positive controls showed the expected values.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

not specified

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Test concentrations with justification for top dose:

=< 0.2 mg/ml (max. effective concentration)

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: ethanol

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

1 % ethanol, 0.5 % DMSO, saline

True negative controls:

not specified

Positive controls:

not specified

Positive control substance:

not specified

Evaluation criteria:

Evaluation criteria (Ishidate and Odashima 1977): Spontaneous background aberrations of the solvent controls were about 3%.
Negative if =< 4.9 %
suspicious if 5 - 9.9 %
positive if >=10 %

Species / strain:

Chinese hamster lung (CHL/IU)

Metabolic activation:

without

Genotoxicity:

ambiguous

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

not specified

At 0.2 mg/ml, 6 % of the cells showed chromosomal aberrations (gaps, breaks, and translocations) after 48 h. 

Conclusions:

An in vitro mammalian chromosome aberration test similar to OECD 473 was performed in Chinese hamster lung (CHL/IU) cells without metabolic activation. At 0.2 mg/ml, 6 % of the cells showed chromosomal aberrations (gaps, breaks, and translocations) after 48 h. Therefore, results of this study were ambiguous concerning the genotoxicity of the test substance.

Executive summary:

An in vitro mammalian chromosome aberration test similar to OECD 473 was performed in Chinese hamster lung (CHL/IU) cells without metabolic activation. Three different doses were added to 3-day-old cultures. Chromosome preparations were made, as a rule at 24 and 48 h, or when necessary at 6 h, after the treatment. Cells were treated with colcemid (0.2 µg/ml) for 2 h, and after trypsinization, they were incubated in 0.075 M KCI hypotonic solution for 15 min at 37°C. The cells were fixed with ice-cold fixative (methanol:glacial acetic acid, 3:1 v/v) which was changed 3 times. A few drops of the suspension were then placed on clean dry slides which were held horizontally under an electric heater. The slides were stained with 1% Giemsa's buffered solution (pH 6.8) for 20 min. The number of cells with chromosomal aberrations was recorded on 100 well-spread metaphases at the magnification of 700.

At 0.2 mg/ml, 6 % of the cells showed chromosomal aberrations (gaps, breaks, and translocations) after 48 h. Therefore, results of this study were ambiguous concerning the genotoxicity of the test substance. The vehicle and untreated negative controls showed valid results.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

- Mammalian Erythrocyte Micronucleus Test (GLP-compliant study according to OECD 474): negative

Link to relevant study records

Reference

Endpoint:

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

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Remarks:

DRF included in same report

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

ICR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Sprague-Dawley, Inc., Frederick, MD
- Age at study initiation: 6-8weeks old
- Weight at study initiation: 27.4-35.8g (males) and 24.5-30.6g (females)
- Assigned to test groups randomly: yes
- Fasting period before study: no
- Housing: 5 of the same sex per cage
- Diet (e.g. ad libitum): Purina certified rodent chow 5002 ad libitum
- Water (e.g. ad libitum): tap water ad libitum
- Acclimation period: 5 days at least

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23.3°C (74°F)
- Humidity (%): 50+/-20
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: corn oil
- Supplier: Super G

Details on exposure:

gavage at constant volume of 20 ml/kg.

Duration of treatment / exposure:

one

Frequency of treatment:

once

Post exposure period:

5 mice/group were sacrified at 24, 48 and 72 hours (All 5 mice treated with Cyclophosphamide were sacrified at 24h).

Dose / conc.:

0 mg/kg bw/day (nominal)

Dose / conc.:

55 mg/kg bw/day (nominal)

Dose / conc.:

110 mg/kg bw/day (nominal)

Dose / conc.:

220 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Vehicle control n=15 per sex
55 mg/kg n=15 per sex
110 mg/kg n=15 per sex
220 mg/kg n=20 per sex
Cyclophosphamide 60 mg/kg n=5 per sex

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Justification for choice of positive control(s): commonly used
- Doses / concentrations: 60 mg/kg in distilled water
- Supplier: Sigma Chemical Company

Tissues and cell types examined:

bone marrow from femurs

Details of tissue and slide preparation:

Slides were fixed in methanol and colored with may-Grünwald-Giemsa.

Evaluation criteria:

1000 polychromatic erythrocytes were scored for the presence of micronuclei.

The mean incidence of micronucleated polychromatic erythrocytes must not exceed 5/1000 polychromatic erythrocytes (0.5%) in the vehicle control.
The incidence of micronucleated polychromatic erythrocytes in the positive control group must be significantly increased relative to the vehicle control group (p<0.05).

Statistics:

Kastenbaum-Bowman

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

- Mortality: 3/20 males and 1/20 females treated at 220 mg/kg died on the day of exposure.
- Clinical signs: noted on the days following dose administration, included lethargy in male and female mice at 55, 110, and 220 mg/kg.
- Reductions up to 19 % in the ration of polychromatic erythrocytes per total erythrocytes were observed. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in the dibutylamine treated grouop was not statistically increased relative to the respective negative controls.
Cyclophosphamide induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p<0.05).

Summary of bone marrow micronucleus study

Treatment	Sex	Time (h)	Number of mice	PCE/total erythrocytes	Micronucleated polychromatic erythrocytes
					Number per 1000 PCE	Number per PCE scored
corn oil 20ml/kg	M	24	5	0,53	0,8+/-0,84	4/5000
		48	5	0,58	1,4+/-1,14	7/5000
		72	5	0,57	0,8+/-0,84	4/5000
	F	24	5	0,7	2,0+/-1,00	10/5000
		48	5	0,59	1,0+/-0,71	5/5000
		72	5	0,61	1,2+/-0,84	6/5000
Bi-n-butylamine 55mg/kg	M	24	5	0,54	0,6+/-0,55	3/5000
		48	5	0,58	1,0+/-1,41	5/5000
		72	5	0,65	1,2+/-0,84	6/5000
	F	24	5	0,72	2,6+/-1,82	13/5000
		48	5	0,48	1,2+/-0,84	6/5000
		72	5	0,62	0,6+/-0,89	3/5000
Bi-n-butylamine 110mg/kg	M	24	5	0,52	0,2+/-0,45	1/5000
		48	5	0,65	1,0+/-0,71	5/5000
		72	5	0,56	1,4+/-2,07	7/5000
	F	24	5	0,66	0,0+/-0,00	0/5000
		48	5	0,51	1,8+/-0,84	9/5000
		72	5	0,66	0,8+/-0,84	4/5000
Bi-n-butylamine 220mg/kg	M	24	5	0,52	0,8+/-0,84	4/5000
		48	5	0,63	1,8+/-1,30	9/5000
		72	5	0,57	1,4+/-1,14	7/5000
	F	24	5	0,59	2,6+/-2,61	13/5000
		48	5	0,49	0,6+/-0,89	3/5000
		72	5	0,56	1,0+/-0,71	5/5000
Cyclophosphamide 60mg/kg	M	24	5	0,54	17,4+/-3,21	87/5000*
	F	24	5	0,46	34,4+/-2,41	172/5000*

p<0.05

Conclusions:

A GLP-compliant in vivo Mammalian Erythrocyte Micronucleus Test was performed according to OECD TG 474 in male/female ICR mice. The test substance was found to be negative regarding the endpoint genotoxicity.

Executive summary:

An in vivo Mammalian Erythrocyte Micronucleus Test was performed according to OECD TG 474 (GLP-compliant) in male/female ICR mice. Treatment with the test substance was once orally via gavage at constant volume of 20 ml/kg. 5 mice per group were sacrified at 24, 48 and 72 hours (All 5 mice treated with Cyclophosphamide were sacrified at 24h). Results show that 3/20 males and 1/20 females treated at 220 mg/kg died on the day of exposure. Clinical signs on the days following dose administration, included lethargy in male and female mice at 55, 110, and 220 mg/kg were noted. Reductions up to 19 % in the ration of polychromatic erythrocytes per total erythrocytes were observed. The number of micronucleated polychromatic erythrocytes per 1000 polychromatic erythrocytes in the test substance treated group was not statistically increased relative to the respective negative controls. Cyclophosphamide induced a significant increase in micronucleated polychromatic erythrocytes in both male and female mice (p<0.05).

However, the test substance was found to be negative regarding the endpoint genotoxicity.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro studies:

A GLP-compliant in vitro mammalian cell gene mutation test using the Hprt and xprt genes according to OECD 476 showed that the test substance did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells and is therefore negative in that assay (2010, K1).

The test substance was tested for mutagenicity using the Salmonella/microsome preincubation assay using the standard protocol approved by the National Toxicology Program. The test substance was tested at doses of 0.10, 0.33, 1.0, 3.3, and 10 mg/plate in as many as 5 Salmonella typhimurium strains (TA1535, TA1537, TA97, TA98, and TA100) in the presence and absence of rat or hamster liver S-9. The test substance was negative in these tests and the highest ineffective dose tested in any Salmonella typhimurium strain was 10.000 mg/plate (1986, K2). Further studies with limited information or significant

methodological deficiencies showed also negative results in the ames test (1958, K3; 1977, K3; 1977, K4; 1978, K4; 1977, K4).

 

In a publication an in vitro cytogenetics assay using Chinese hamster cells the test substance in ethanol (1% solution) caused chromatid gaps, chromatid or chromosomal breaks and translocations in 6% of cells and showed therefore ambiguous results (1977, K2). An increase in the frequency of chromosomal breaks was also observed in a further publication at a concentration of 1 mM. The number of breaks per cell was 0.12 vs. 0.03, 0.02, and 0.03 for the control with saline, ethanol, and acetone (1977, K3). In the same publication a slight increase in the SCE rate was also observed, but not considered substance-related (5.83 and 7.08, respectively, vs. 3.36, 3.68, and 3.41 in the controls) (1977, K4). Another study with significant methodological deficiencies showed also a positive result in an UDS test (1977, K3).

In vivo studies:

A GLP-compliant in vivo Mammalian Erythrocyte Micronucleus Test was performed according to OECD TG 474 in male/female ICR mice. 20 male/female animals per group were orally administered single doses of 0, 55, 110 and 220 mg/kg bw. Mortality occurred among high-dose males (3/20) and females (1/20), and lethargy was observed in both males and females of all treatment groups. While treated mice exhibited slight reductions in polychromatic erythrocytes to erythrocyte ratios (up to 19%) relative to vehicle control (corn oil) animals, there was no significant increase in micronucleated polychromatic erythrocytes at 24, 48 or 72-hour post-treatment examination in either male or female treated mice when compared to controls (1995, K1). Two further in vivo studies with limited information or significant methodological deficiencies showed negative test results (1981, K3; 1978, K4). The positive result in another in vivo study was considered false-positive by the author (1978, K4). In a further study, the test substance was only tested in combination with sodium nitrite. This combination was found to be positive in that in vivo assay (1996, K3).

Justification for classification or non-classification

Several Ames-tests (OECD 471), with or without metabolic activation through S9-Mix, a HPRT forward mutation assay (OECD Guideline 476), a chromosomal aberration assay (OECD Guideline 473), and a valid in vivo Test (OECD Guideline 475) were performed to test for a mutagenic effects. Therefore, the available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse findings on genotoxicity was observed in the valid in-vitro or in-vivo studies. As a result the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008,as amended for the thirteenth time in Regulation (EC) No 2018/1480.