3,3'-[butane-1,4-diylbis(oxy)]bispropanamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: negative (BASF, 1999; OECD471)

Read-across: In vitro micronucleus assay: negative (BASF, 2012; OECD487); QSAR chromosomal aberration (+/- metabolic activation): negative (BASF, 2020; OASIS TIMES v2.27.19.13)

Read-across: HPRT locus assay: negative; (BASF, 2012; OECD476)

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Analytical purity: 99.4 g/100 g (GC)

Target gene:

The tester strains TA 1535, TA 1537, TA 98 and TA 100 selected by Ames and coworkers are derivatives of Salmonella typhimurium LT2 and have GC base pairs at the primary reversion site. All strains have a defective excision repair system (uvrB), which prevents the repair of lesions which are induced in the DNA, and this deficiency results in greatly enhanced sensitivity of same mutagens. Furthermore, all strains show a considerably reduced hydrophilic polysaccharide layer (rfa), which leads to an increase in permeability to lipophilic substances.
The strains TA 1535 and TA 100 are derived from histidine-prototrophic Salmonella strains by the substitution mutation his G 46 and are used to detect base pair substitutions. TA 1537 and TA 98 are strains for the detection of frameshift mutagens.
Escherichia coli WP2 uvrA which has an AT base pair at the primary reversion site is a derivative of E. coli WP2 with a deficient excision repair and is
used to detect substances which induce base pair substitutions.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not applicable

Species / strain / cell type:

E. coli WP2 uvr A

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

rat liver S9 mix

Test concentrations with justification for top dose:

1st experiment: 0; 20; 100; 500; 2,500 and 5,000 µg/plate
2nd experiment: 0; 4; 20; 100; 500 and 2,500 µg/plate

Vehicle / solvent:

water was used as vehicle

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

with S9 mix

Positive control substance:

2-acetylaminofluorene

Remarks:

all strains

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

other: N-methyl-N'-nitro-N-nitrosoguanidine

Remarks:

strains: TA 1535, TA 100

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

other: 4-nitro-o-phenylenidiamine

Remarks:

TA98

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

9-aminoacridine

Remarks:

TA1537

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Remarks:

without S9 mix

Positive control substance:

N-ethyl-N-nitro-N-nitrosoguanidine

Remarks:

E. coli WP2 uvrA

Details on test system and experimental conditions:

METHOD OF APPLICATION: standard plate and preincubation test

DURATION
- Preincubation period (for preincubation test): 20 min; subsequently, 2 ml of saft agar is added and, after mixing, the samples are poured onto the agar plates; for standard plate test, bacteria and test item were mixed and immediately poured onto agar plates
- Exposure duration: 48-72 h


DETERMINATION OF CYTOTOXICITY
- Method: count of bacterial colonies

Evaluation criteria:

The test chemical is considered positive in this assay if the following criteria are met:
- A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S-9 mix or after adding a metabolizing system.
A test substance is generally considered nonmutagenic in this test if:
- The nurnber of revertants for all tester strains were within the historical negative control range under all experimental conditions in two experiments carried out independently of each other.

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:

determined at 500-2500 µg/plate depending on strain

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No test substance precipitation was found.

Conclusions:

According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Executive summary:

The test substance was tested for its mutagenic potential based on the ability to induce point mutations in selected loci of several bacterial strais, i.e. Salmonella typhimurium and Escherichia coli, in a reverse mutation assay.

- Strains: TA 1535, Ta 100, TA 1537, TA 98 and E.coli WP2 uvrA

- Dose Range: 20 µg - 5000 µg/plate (SPT), 4 µg - 2500 µg/plate (PIT)

- Test Conditions: Standard plate test (SPT) and preincubation test (PIT) both with and without metabolic activation (Aroclor-induced rat liver S9 mix).

- Solubility: No precipitation of the test substance was found.

- Toxicity: A bacteriotoxic effect observed depending on the strain and test conditions from about 500 µg - 2500 µg/plate onward.

- Mutagenicity: An increase in the number of his+ or trp+ revertants was not observed in the standard plate test or in the preincubation test either without S9 mix or after the addition of a metabolizing system.

- Conclusion: According to the results of the present study, the test substance is not mutagenic in the Salmonella typhimurium / Escherichia coli reverse mutation assay under the experimental conditions chosen here.

Reference 2

Endpoint:

in vitro cytogenicity / micronucleus study

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study performed with another substance: Genotoxicity of 3,3'-[butane-1,4-diylbis(oxy)]bispropanamine can be reliably predicted based on read-across to 3,3'-Oxybis(ethyleneoxy)bis(propylamine) (CAS 4246-51-9). For justification of the read-across please refer to IUCLID section 13.

Qualifier:

according to guideline

Guideline:

other: OECD Guideline 487 (In vitro Mammalian Cell Micronucleus Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian cell micronucleus test

Specific details on test material used for the study:

- Name of test substance: 3,3’-Oxybis(ethyleneoxy)bis(propylamine)
- Batch identification: O 2895
- CAS No.: 4246-51-9
- Purity/composition: 99.7 corr. area-%
- Homogeneity: The homogeneity of the test substance was guaranteed by mixing before preparation of the test substance preparations
- Storage stability: The stability of the test substance under storage conditions is guaranteed until 15 Jun 2013 as indicated by the sponsor
- Date of production: 15 Jun 2011
- Molecular weight: 220.3092 g/mol
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: Room temperature

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: MEM (minimal essential medium with Earle's salts) containing a L-glutamine source supplemented with 10% (v/v) fetal calf serum (FCS), 1% (v/v) penicillin/streptomycin (10000 IU/10 000 µg/mL), and 1% (v/v) amphotericine B (250 µg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/ß-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment: 4 hours exposure; 24 hours harvest time; with and without S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 µg/mL;
2nd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 137.5; 275.0; 550.0; 1100.0; 2200.0 µg/mL; 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 62.5; 125.0; 250.0; 500.0; 1000.0 µg/mL
3rd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 3.1; 6.3; 12.5; 25.0; 50.0; 100.0; 200.0 µg/mL
4th Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.6; 1.3; 2.5; 5.0; 10.0; 20.0 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: culture medium (MEM)
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, culture medium (Minimal Essential Medium: MEM) was selected as vehicle.

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

without metabolic activation: 500 µg/mL ethyl methanesulfonate (EMS); with metabolic activation: 2.5 µg/mL cyclophosphamide (CPP)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Preincubation period: 6 h (attachment period)
- Exposure duration: 4 h / 24 h
- Recovery time: 20 h / 0 h

STAIN: Wrights solution (modified May-Grünwald solution); counterstained with 2.6% (v/v) Giemsa/Titrisol solution pH 7.2

NUMBER OF REPLICATIONS: 4 experiments, two test cultures per dose group = four culture preparations and thus of four slides per dose group

NUMBER OF CELLS EVALUATED: at least 1000 cells per culture, means at least 2000 cells per dose group

DETERMINATION OF CYTOTOXICITY
- Method: relative increase in cell count (RICC), proliferation index

OTHER:
- pH value: Changes in the pH were recorded by a change in the color of the indicator in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative control with and without S9 mix.
- Osmolarity: Osmolarity was measured, at least for the top dose and for the negative control with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after start of treatment of the test cultures (macroscopically) and at the end of treatment (macroscopically/ microscopically).

Evaluation criteria:

Acceptance criteria: The in vitro micronucleus assay is considered valid if the following criteria are met:
- The quality of the slides allowed the identification and evaluation of a sufficient number of analyzable cells.
- The number of cells containing micronuclei in the negative control was within the range of the historical negative control data.
- The positive control substances both with and without S9 mix induced a significant increase in the number of micronucleated cells.

Assessment criteria: A test substance is considered "positive" if the following criteria are met:
- A significant, dose-related and reproducible increase in the number of cells containing micronuclei.
- The number of micronucleated cells exceeds both the value of the concurrent negative control and the range of the historical negative control data.
A test substance generally is considered "negative" if the following criteria are met:
- The number of micronucleated cells in the dose groups is not significant increased above the concurrent negative control value and is within the range of the historical negative control data.

Statistics:

The statistical evaluation of the data was carried out using the MUVIKE program system (BASF SE). The proportion of cells containing micronuclei was calculated for each group. A comparison of each dose group with the concurrent negative control group was carried out using Fisher's exact test for the hypothesis of equal proportions. This test is Bonferroni-Holm corrected versus the dose groups separately for each time and was performed one sided.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH / osmolality: Osmolarity and pH values were not influenced by test substance treatment.
- Precipitation: No precipitation of the test substance in culture medium was observed.

RANGE-FINDING/SCREENING STUDIES: In the pretest for toxicity based on the purity and the molecular weight of the test substance 2200 µg/mL (approx. 10 mM) of the test material was used as top concentration. The cells were prepared at a harvest time of 24 hours (about 2 cell cycles) after 4 and 24 hours exposure time without S9 mix and after 4 hours exposure time with S9 mix. The pretest was performed following the method described for the main experiment. As indication of test substance toxicity relative increase in cell count (RICC) and cell attachment (morphology) were determined for dose selection. In the pretest various additional parameters were checked or determined for all or at least some selected doses. The following parameters are available: pH, osmolarity, and solubility. Following the requirements of the current guideline a test substance should be tested up to a maximum concentration of 5 mg/mL, 5 µL/mL or 10 mM, whichever is the lowest. In case of toxicity, the top concentration should produce 55% ± 5% cytotoxicity (relative increase in cell count [RICC] and/or proliferation index [PI]) compared to the respective vehicle control. For relatively insoluble test substances at least one concentration should be scored showing no precipitation in culture medium at the end of exposure period.
In the pretest the parameters pH value and osmolarity were not relevant influenced by the addition of the test substance preparation to the culture medium at the concentrations measured. However, a slight pH shift was observed at the highest required concentration prior to testing. Therefore, the pH of the stock solution was adjusted to a physiological value prior to application using small amounts of 32% (w/v) HCl.
In addition, no test substance precipitation was observed up to the highest required concentration of 2200 µg/mL.
After treatment with the test substance, either after 4 hours treatment in the absence and presence of S9 mix or after 24 hours continuous treatment in the absence of S9 mix no cytotoxicity indicated by reduced RICC of about or below 40 - 50% was observed.

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) was observed after 24 hours continuous treatment without S9 mix at least at the highest applied test substance concentration in all corresponding experimental parts of this study.

Summary table - experimental parts without S9 mix

Exp.	Exposure	Test groups	S9	Prec.*	Genotoxicity	Cytotoxicity
	period		mix		Micronucleated cells**	Proliferation index (PI)	RICC***
					[%]		[%]
1	4 hrs	Vehicle control^1	-	n.d.	0.5	2.33	100.0
		68.8 µg/mL	-	-	n.d.	n.d.	122.9
		137.5 µg/mL	-	-	n.d.	n.d.	119.7
		275.0 µg/mL	-	-	0.3	2.33	132.0
		550.0 µg/mL	-	-	0.6	2.41	121.6
		1100.0 µg/mL	-	-	0.6	2.45	108.2
		2200.0 µg/mL	-	-	n.s.	n.s.	106.6
		Positive control^1	-	n.d.	3.1^s	2.54	n.t.
2	24 hrs	Vehicle control^1	-	n.d.	n.d.	n.d.	100.0
		137.5 µg/mL	-	-	n.s.	n.s.	16.2
		275.0 µg/mL	-	-	n.s.	n.s.	11.4
		550.0 µg/mL	-	-	n.s.	n.s.	12.2
		1100.0 µg/mL	-	-	n.s.	n.s.	12.7
		2200.0 µg/mL	-	-	n.s.	n.s.	15.7
		Positive control^1	-	n.d.	n.d.	n.d.	n.t.
3	4 hrs	Vehicle control^1	-	n.d.	n.d.	n.d.	100.0
		3.1 µg/mL	-	-	n.d.	n.d.	105.2
		6.3 µg/mL	-	-	n.s.	n.s.	105.2
		12.5 µg/mL	-	-	n.s.	n.s.	97.6
		25.0 µg/mL	-	-	n.s.	n.s.	55.0
		50.0 µg/mL	-	-	n.s.	n.s.	6.3
		100.0 µg/mL	-	-	n.s.	n.s.	-6.2
		200.0 µg/mL	-	-	n.s.	n.s.	-6.3
		Positive control^1	-	n.d.	n.d.	n.d.	n.t.
4	24 hrs	Vehicle control^1	-	n.d.	0.7	2.48	100.0
		0.6µg/mL	-	-	1.0	2.62	103.1
		1.3µg/mL	-	-	0.7	2.39	102.8
		2.5µg/mL	-	-	0.7	2.59	88.0
		5.0µg/mL	-	-	n.s.	n.s.	81.5
		10.0µg/mL	-	-	n.s.	n.s.	38.3
		20.0µg/mL	-	-	n.s.	n.s.	44.4
		Positive control^1	-	n.d.	7.5^s	2.46	n.t.

* Precipitation in culture medium at the end of exposure period

^** Relative number of micronucleated cells per 1000 cells scored per test group

^*** Relative increase in cell count (RICC)

^S Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity

n.t. Not tested

¹ EMS 500 µg/mL

 

Summary table - experimental parts with S9 mix

Exp.	Exposure	Test groups	S9	Prec.*	Genotoxicity	Cytotoxicity
	period		mix		Micronucleated cells**	Proliferation index (PI)	RICC***
					[%]		[%]
1	4 hrs	Negative control	+	n.d.	0.8	2.37	100.0
		68.8µg/mL	+	-	n.d.	n.d.	104.4
		137.5µg/mL	+	-	1.3	2.36	109.6
		275.0µg/mL	+	-	0.9	2.17	105.8
		550.0µg/mL	+	-	1.4	2.38	64.5
		1 100.0µg/mL	+	-	n.s.	n.s.	90.7
		2 200.0µg/mL	+	-	n.s.	n.s.	79.9
		Positive control^1	+	n.d.	5.9^s	2.27	n.t.
2	4 hrs	Negative control	+	n.d.	0.7	2.46	100.0
		62.5µg/mL	+	-	n.d.	n.d.	97.0
		125.0µg/mL	+	-	0.6	2.44	108.0
		250.0µg/mL	+	-	0.5	2.46	109.5
		500.0µg/mL	+	-	1.1	2.48	101.2
		1 000.0µg/mL	+	-	n.s.	n.s.	109.9
		Positive control2	+	n.d.	7.4^s	2.56	n.t.

* Precipitation in culture medium at the end of exposure period

^** Relative number of micronucleated cells per 1000 cells scored per test group

^*** Relative increase in cell count (RICC)

^S Frequency statistically significant higher than corresponding control values

n.d. Not determined

n.s. Not scorable due to strong cytotoxicity

n.t. Not tested

¹ CPP 2.5 µg/mL

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

GLP guideline study performed with another substance. Genotoxicity of 3,3'-[butane-1,4-diylbis(oxy)]bispropanamine can be reliably predicted based on read-across to 3,3'-Oxybis(ethyleneoxy)bis(propylamine) (CAS 4246-51-9). For justification of the read-across please refer to IUCLID section 13.

Qualifier:

according to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

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

Specific details on test material used for the study:

- Name of test substance: 3,3’-Oxybis(ethyleneoxy)bis(propylamine)
- Batch identification: O 2895
- CAS No.: 4246-51-9
- Purity/composition: 99.7 corr. area-%
- Homogeneity: The homogeneity of the test substance was guaranteed on account of the high purity and was ensured by mixing before preparation of the test substance solutions
- Storage stability: The stability of the test substance under storage conditions is guaranteed until 15 Jun 2013 as indicated by the sponsor
- Date of production: 15 Jun 2011
- Molecular weight: 220.3092 g/mol
- Physical state, appearance: Liquid, colorless, clear
- Storage conditions: Room temperature

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

- Type and identity of media: Treatment medium (4-hour exposure period): Ham's F12 medium containing stable glutamine and hypoxanthine; Culture medium and Treatment medium (24-hour exposure): Ham's F12 medium containing stable glutamine and hypoxanthine supplemented with 10% (v/v) fetal calf serum (FCS); Pretreatment medium ("HAT" medium): Ham's F12 medium supplemented with hypoxanthine (13.6 x 10E-3 mg/mL), aminopterin (0.18 x 10E-3 mg/mL), thymidine (3.88 x 10E-3 mg/mL), 10% (v/v) fetal calf serum (FCS); Selection medium ("TG" medium): Hypoxanthine-free Ham's F12 medium supplemented with 6-thioguanine (10 μg/mL), 1% (v/v) stable glutamine (200 mM), 10% (v/v) fetal calf serum (FCS). All media were supplemented with 1% (v/v) penicillin/streptomycin (stock solution: 10000 IU / 10000 μg/mL) and 1% (v/v) amphotericine B (stock solution: 250 μg/mL)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (Each batch used for mutagenicity testing was checked for mycoplasma contamination)
- Periodically "cleansed" against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital/β-naphthoflavone-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment: 4 hours exposure; 24 hours harvest time; with and without S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 µg/mL;
2nd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 34.4; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 µg/mL; 4 hours exposure, 24 hours harvest time, with S9 mix: 0; 187.5; 375.0; 750.0; 1500.0; 2200.0 µg/mL
3rd Experiment: 24 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 µg/mL

Vehicle / solvent:

- Vehicle/solvent used: culture medium
- Justification for choice of solvent/vehicle: Due to the good solubility of the test substance in water, the aqueous culture medium (Ham's F12) was selected as vehicle

Negative solvent / vehicle controls:

yes

Positive controls:

yes

Remarks:

Without metabolic activation S9: 300 μg/mL ethyl methanesulfonate (EMS); with metabolic activation: 20 μg/mL methylcholanthrene (MCA)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

EXPERIMENTAL PROCEDURE
- Preparation of test cultures: The stocks of cells (1.0-mL portions) were thawed at 37°C in a water bath. 0.5 mL were pipetted into 25 cm² plastic flasks containing 5 mL Ham's F12 medium (incl. 10% [v/v] FCS). The medium was replaced after 24 hours to remove any dead cells. After at least 2 passages, cells were taken for the experiment, and for these there was another passage to prepare test cultures.
- Pretreatment of cells with "HAT" medium: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5x10E+05 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. After that, a passage in Ham's F12 medium incl. 10% (v/v) FCS was followed with a subsequent incubation for a further 3 - 4 days.
- Attachment period: For each test group, about 1x10E+06 logarithmically growing cells per flask (175 cm²) were seeded into about 20 mL Ham's F12 medium supplemented with 10% (v/v) FCS and incubated for about 20 - 24 hours. Two flasks (one flask referred to as A and one flask referred to as B) were used for each test group.
- Exposure period: After the attachment period, the medium was removed from the flasks and the treatment medium was added. The cultures were incubated for the respective exposure period at 37°C, 5% (v/v) CO2 and ≥ 90% humidity.
- Expression period: The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with at least 20 mL Ham's F12 medium incl. 10% (v/v) FCS and left to stand in the incubator for about 3 days (4-hour treatment) or 2 days (24-hour treatment). This was followed by the 1st passage. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection period: For selection of the mutants, six 75 cm² flasks with 3x10E+05 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF REPLICATIONS: Three independent experiments

DETERMINATION OF CYTOTOXICITY
- Cloning efficiency 1 (CE1; survival): For the determination of the influence of the test substance directly after the exposure period, per dose group about 200 cells were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10% (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours or 24 hours. The exposure periods were completed by rinsing several times with HBSS. Then the flasks were topped up with 5 mL Ham's F12 medium incl. 10% (v/v) FCS.
- Cloning efficiency 2 (CE2; viability): For the determination of the mutation rate after the expression period, about 200 cells were separated during the transfer into selection medium (after 7 – 9 days) and seeded in two flasks (25 cm²) containing 5 mL Ham's F12 medium incl. 10% (v/v) FCS each. In all cases, after seeding of the cells the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted. The absolute and relative cloning efficiencies (%) were calculated for each test group.

EVALUATION
- Cytotoxicity (CE, CE1, CE2): The cloning efficiency (CE, %) was calculated for each test group as follows:
CE[absolute] = (total number of colonies in the test group)/(total number of seeded cells in the test group) x 100
CE[relative] = (CE[absolute of the test group])/(CE[absolute of the vehicle/negative control]) x 100
The number of colonies in every flask was counted and recorded. Using the formula above the values of absolute cloning efficiencies (CE[absolute], CE1[absolute] and/or CE2[absolute]) were calculated. Based on these values the relative cloning efficiencies (CE[relative], CE1[relative] and/or CE2[relative]) of the test groups were calculated and given in percentage compared with the respective CE[absolute] value of the corresponding vehicle/negative control (vehicle/negative control = 100%).
- Mutant frequency: The number of colonies in every flask was counted and recorded. The sum of the mutant colony counts within each test group was subsequently normalized to 10E+06 cells seeded. The uncorrected mutant frequency (MF[uncorr.]) per 10E+06 cells was calculated for each test group as follows:
MF[uncorr.] = (total number of mutant colonies)/(number of seeded cells) x 10E+06
The uncorrected mutant frequency was corrected with the absolute cloning efficiency 2 for each test group to get the corrected mutant frequency (MF[corr.]):
MF[corr.] = (MF[uncorr.])/(CE2[absolute]) x 100

CHECK OR DETERMINATION OF FURTHER PARAMETERS
- pH: Changes in the pH were recorded by a change in the indicator color in the culture medium (phenol red: no color change from pH 6.7 - 8.3). The pH was measured, at least for the two top doses and for the negative controls with and without S9 mix.
- Osmolarity: Osmolarity was measured, at least for the top dose and for the negative controls with and without S9 mix.
- Solubility: Test substance precipitation was checked immediately after treatment of the test cultures and at the end of treatment.
- Cell morphology: The test cultures of all test groups were examined microscopically at the end of exposure period with regard to cell morphology, which allows conclusions to be drawn about the attachment of the cells.

Evaluation criteria:

ACCEPTANCE CRITERIA:
The HPRT assay is considered valid if the following criteria are met:
- The absolute cloning efficiencies of the negative/vehicle controls should not be less than 50% (with and without S9 mix).
- The background mutant frequency in the negative/vehicle controls should fall within our historical negative control data range of 0.00 – 16.43 mutants per 10E+06 clonable cells.
- The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data).
- At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

ASSESSMENT CRITERIA:
A finding is assessed as positive if the following criteria are met:
- Increase of the corrected mutation frequencies (MF[corr.]) both above the concurrent negative control values and our historical negative control data range.
- Evidence of reproducibility of any increase in mutant frequencies.
- A statistically significant increase in mutant frequencies and the evidence of a dose-response relationship.
Isolated increases of mutant frequencies above our historical negative control range (i.e. 15 mutants per 10E+06 clonable cells) or isolated statistically significant increases without a dose-response relationship may indicate a biological effect but are not regarded as sufficient evidence of mutagenicity.
The test substance is considered non-mutagenic according to the following criteria:
- The corrected mutation frequency (MF[corr.]) in the dose groups is not statistically significant increased above the concurrent negative control and is within our historical negative control data range.

Statistics:

An appropriate statistical trend test was performed to assess a dose-related increase of mutant frequencies. The number of mutant colonies obtained for the test substance treated groups was compared with that of the respective negative control groups. A trend is judged as statistically significant whenever the p-value (probability value) is below 0.10 and the slope is greater than 0. However, both, biological and statistical significance will be considered together.

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: The pH value of the test substance preparation was adjusted by adding small amounts of HCl.
- Precipitation: In this study, in the absence and the presence of S9 mix no precipitation in culture medium was observed up to the highest applied test substance concentration.

COMPARISON WITH HISTORICAL CONTROL DATA: In this study, no relevant increase in the number of mutant colonies was observed either without S9 mix or after the addition of a metabolizing system. In all experiments after 4 and 24 hours treatment with the test substance the values for the corrected mutation frequencies (MF[corr.]: 0.00 – 3.03 per 10+06 cells) were close to the respective vehicle control values (MF[corr.]: 0.26 – 3.52 per 10E+06 cells) and clearly within the range of our historical negative control data (without S9 mix: MF[corr.]: 0.00 – 16.43 per 10+06 cells; with S9 mix: MF[corr.]: 0.00 – 15.83 per 10+06 cells). The positive control substances EMS (without S9 mix; 300 μg/mL) and MCA (with S9 mix; 20 μg/mL) induced clearly increased mutant frequencies as expected. The values of the corrected mutant frequencies (without S9 mix: MF[corr.]: 75.04 – 452.96 per 10+06 cells; with S9 mix: MF[corr.]: 55.07 – 88.91 per 10E+06 cells) were clearly within our historical positive control data range (without S9 mix: MF[corr.]: 47.35 – 1 338.10 per 10E+06 cells; with S9 mix: MF[corr.]:
26.29 – 413.54 per 10E+06 cells).

ADDITIONAL INFORMATION ON CYTOTOXICITY: Cytotoxic effects indicated by clearly reduced cloning efficiencies of below 20% of control were observed in the 1st Experiment in the absence of S9 mix after 4 hours treatment and in the 2nd Experiment in the presence and absence of S9 mix. In the 3rd Experiment in the absence of S9 mix after 24hours treatment there was a decrease in the number of colonies in the highest applied concentrations. In detail, in the 1st Experiment in the absence of S9 mix, there was a decrease in the number of colonies at the highest applied concentration of 2 200.0 μg/mL (CE1 relative: 0.0%) after an exposure period of 4 hours. No cytotoxicity was observed in the presence of S9 mix. The cell densities were not distinctly reduced. In the 2nd Experiment in the absence of S9 mix after 24 hours treatment strong cytotoxicity was observed from the lowest concentration onward. Thus, with no remaining test group this experimental part was discontinued. The cell densities were distinctly reduced. In addition, in the presence of S9 mix, there was a decrease in the number of colonies at the highest applied concentration of 2200.0 μg/mL (CE1 relative: 0.0%) after an exposure period of 4 hours. In this experimental part the cell densities were not distinctly reduced. In the 3rd Experiment in the absence of metabolic activation after 24 hours treatment a decrease in the number of colonies was observed at the concentration of 6.25 μg/mL and above (CE1 relative: 0.0%). The cell densities were distinctly reduced.

CELL MORPHOLOGY: There were no adverse observations on cell morphology (cell attachment) in the absence and presence of S9 mix in the 1st Experiment and in the presence of S9 mix in the 2nd Experiment. After 24 hours treatment in the absence of S9 mix, in the 2nd and 3rd Experiment the morphology and attachment of the cells was adversely influenced at the highest applied concentrations.

Summary of results - experimental parts without S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Negative control	-	-	3.52	100.0	100.0
		68.8	-	-	n.c.^1	96.4	n.c.^1
		137.5	-	-	0.27	90.9	103.8
		275.0	-	-	1.32	96.9	100.5
		550.0	-	-	1.37	98.1	99.3
		1100.0	-	-	0.81	91.6	103.7
		2200.0	-	-	n.c.^2	0.0	n.c.^2
		Positive control^1	-	-	75.04	95.6	92.1
2	24	Negative control	-	-	n.c.^3	100.0	n.c.^3
		34.4	-	-		0.0	n.c.^3
		68.8	-	-		0.0	n.c.^3
		137.5	-	-		0.0	n.c.^3
		275.0	-	-		0.0	n.c.^3
		550.0	-	-		0.0	n.c.^3
		1100.0	-	-		0.0	n.c.^3
		2200.0	-	-		0.0	n.c.^3
		Positive control^1	-	-		69.4	n.c.^3
3	24	Negative control	-	-	0.26	100.0	100.0
		0.39	-	-	2.04	112.9	103.0
		0.78	-	-	3.03	113.3	94.2
		1.56	-	-	2.97	117.7	97.0
		3.13	-	-	0.54	64.9	103.0
		6.25	-	-	n.c.^2	0.0	n.c.^2
		12.50	-	-	n.c.^2	0.0	n.c.^2
		25.00	-	-	n.c.^2	0.0	n.c.^2
		50.00	-	-	n.c.^2	0.0	n.c.^2
		Positive control^1	-	-	452.96	102.9	66.6

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶ cells corrected with the CE₂ value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations are required

n.c.² Culture was not continued due to strong cytotoxicity

n.c.³ Culture was not continued due to missing the OECD recommendations

¹ EMS 300 µg/mL

 

Summary of results - experimental parts with S9 mix

Exp.	Exposure period [h]	Test groups [µg/mL]	S9 mix	Prec.*	Genotoxicity** MFcorr. [per 10^6cells]	Cytotoxicity***
						CE1 [%]	CE2 [%]
1	4	Negative control	+	-	2.63	100.0	100.0
		68.8	+	-	n.c.^1	104.1	n.c.^1
		137.5	+	-	n.c.^1	103.0	n.c.^1
		275.0	+	-	1.36	101.7	103.7
		550.0	+	-	2.53	106.8	98.2
		1100.0	+	-	0.56	86.4	105.9
		2200.0	+	-	1.58	39.1	110.8
		Positive control^2	+	-	55.07	99.7	105.9
2	24	Negative control	+	-	1.58	100.0	100.0
		187.5	+	-	0.00	108.8	^93.0
		375.0	+	-	2.10	105.4	96.1
		750.0	+	-	1.22	80.2	100.4
		1500.0	+	-	2.33	102.7	104.7
		2200.0	+	-	0.00	8.4	98.1
		Positive control^2	+	-	88.91	103.3	102.5

* Precipitation in culture medium at the end of exposure period

** Mutant frequency MF_corr.: mutant colonies per 10⁶ cellscorrected with the CE₂ value

*** Cloning efficiency related to the respective vehicle control

n.c.¹ Culture was not continued since a minimum of only four analysable concentrations are required

² MCA 20 µg/mL

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

In-vitro study: Bacterial systems

The mutagenic ability of the test item to induce point mutations in selected loci of several bacteria strains was assessed in an Ames test (BASF, 1999) according to OECD471 and GLP. S. typhimurium TA 1535, TA 1537, TA 98 and TA 100 and E. coli WP2 uvr A bacteria were incubated with increasing concentrations of the test item in the presence or absence of rat liver S9 mix (standard plate test of pre-incubation test). Even though a bacteriotoxic effect was observed depending on the strain and test conditions from about 500 µg – 2.500 µg/plate onward, no concentration-dependent increase of the numbers of revertants was observed in all strains following treatment with the test item. Thus, the test item is non-mutagenic under the experimental conditions chosen.

A mammalian cell gene mutation test is not available for the test substance. However, genotoxicity can be reliably predicted based on read-across to 3,3'-Oxybis(ethyleneoxy)bis(propylamine) (CAS 4246-51-9). For justification of the read-across please refer to the read-across justification document in IUCLID section 13.

 

In-vitro study: Mammalian cell gene mutation test (read-across CAS 4246-51-9)

An in vitro mammalian cell assay (BASF, 2012) was performed according to OECD guideline 476 in compliance with GLP. The test item was assessed for its potential to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, all with and without the addition of liver S9 mix from induced rats (exogenous metabolic activation). Based on the observations the following concentrations were tested and the test groups in bold type evaluated:

1st Experiment

without S9 mix (4-hour exposure period): 0; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 μg/mL

with S9 mix (4-hour exposure period): 0; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 μg/mL

2nd Experiment

without S9 mix (24-hour exposure period): 0; 34.4; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0 μg/mL

with S9 mix (4-hour exposure period): 0; 187.5; 375.0; 750.0; 1500.0; 2200.0 μg/mL

3rd Experiment without S9 mix (24-hour exposure period): 0; 0.39; 0.78; 1.56; 3.13; 6.25; 12.50; 25.00; 50.00 μg/mL

After an attachment period of 20 - 24 hours and a treatment period of 4 hours both with and without metabolic activation and 24 hours without metabolic activation, an expression phase of about 6 - 8 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted. The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both positive control items, EMS and MCA, led to the expected increase in the frequencies of forward mutations. In this study, in the 1st, 2nd and 3rd Experiment in the absence and in the second Experiment in the presence of metabolic activation the highest concentrations evaluated for gene mutations were clearly cytotoxic. On the basis from the results of the present study, the test item did not cause any relevant and dose-dependent increase in the mutant frequencies both without S9 mix and after adding a metabolizing system in three experiments performed independently of each other. Thus, under the experimental conditions of this study, the test item is not mutagenic in the HPRT locus assay under in vitro conditions.

A mammalian cell micronucleus test is not available for the test substance. However, genotoxicity can be reliably predicted based on read-across to 3,3'-Oxybis(ethyleneoxy)bis(propylamine) (CAS 4246-51-9). For justification of the read-across please refer to the read-across justification document in IUCLID section 13. Furthermore, the QSAR program OASIS TIMES v.2.27.19.13 with its module Chromosomal Aberrations S9 activated v.12.12 predicted no potential of the test substance to cause chromosomal aberrations.

In-vitro study: Mammalian cell micronucleus test(read-across CAS 4246-51-9)

An in vitro mammalian cell micronucleus test (BASF, 2012) was performed according to OECD TG 487 in compliance with GLP. The test item was assessed for its potential to induce micronuclei in V79 cells in vitro (clastogenic or aneugenic activity) both in the absence and the presence of a metabolizing system.

According to an initial range-finding cytotoxicity test for the determination of the experimental doses, the following doses were tested and the test groups in bold type were evaluated (plus asterisk: not scorable due to cytotoxicity):

1st Experiment:

4 hours exposure; 24 hours harvest time; without S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0; 2200.0* μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix: 0; 68.8; 137.5; 275.0; 550.0; 1100.0*; 2200.0* μg/mL

2nd Experiment:

24 hours exposure, 24 hours harvest time, without S9 mix: 0; 137.5*; 275.0*; 550.0*; 1100.0*; 2200.0* μg/mL

4 hours exposure, 24 hours harvest time, with S9 mix: 0; 62.5; 125.0; 250.0; 500.0; 1000.0* μg/mL

3rd Experiment:

24 hours exposure, 24 hours harvest time, without S9 mix: 0; 3.1; 6.3*; 12.5*; 25.0*; 50.0*; 100.0*; 200.0* μg/mL

4th Experiment:

24 hours exposure, 24 hours harvest time, without S9 mix: 0; 0.6; 1.3; 2.5; 5.0*; 10.0*; 20.0* μg/mL

A sample of at least 1000 cells for each culture was analyzed for micronuclei, i.e. 2000 cells for each test group. The vehicle controls gave frequencies of micronucleated cells within the historical negative control data range for V79 cells. Both positive control items, EMS and cyclophosphamide, led to the expected increase in the number of cells containing micronuclei. Cytotoxicity indicated by clearly reduced relative increase in cell count (RICC) was observed at the 24 hours harvest time with/without S9 mix at least at the highest applied test item concentration in all corresponding experimental parts of this study. On the basis of the results of the present study, the test item did not cause any biologically relevant increase in the number of cells containing micronuclei either without S9 mix or after adding a metabolizing system in four experiments carried out independently of each other. Thus, under the experimental conditions described, the test substance is considered not to have a chromosome-damaging (clastogenic) effect nor to induce numerical chromosomal aberrations (aneugenic activity) under in vitro conditions in V79 cells in the absence and the presence of metabolic activation.

Justification for classification or non-classification

Based on the available results of an in vitro genetic toxicity study in bacteria (Ames test) and consolidated by read-across to the negative results of a mammalian cell gene mutation test and a mammalian cell micronucleus test performed with a comparable compound (CAS 4246-51-9) and a QSAR prediction indicating no potential of the test substance to cause chromosomal aberrations, the registered substance has not to be classified and labelled as genotoxic according to Regulation (EC) No 1272/2008 (CLP).