Benzenesulfonic acid, 4-C10-13-sec-alkyl derivs., compds. with 2-propanamine

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

LAS IPA:

The only study in which LAS IPA was tested is in the Ames reverse mutation assay (GLP, Guideline study) using S. typhimurium and E. coli strains up to cytotoxic concentrations, both with and without metabolic activation. No significant revertant colonies were observed. No other tests are available with the substance per se addressing the genotoxicity endpoint; hence, the endpoint was addressed further with information from read-across substances LAS Na and IPA.

IPA:

IPA was not mutagenic in the Ames test (Zeiger, 1987).

In a GLP, guideline study (Covance, 2010) the ability of isopropylamine (IPA) to cause gene mutations on mouse lymphoma L5178Y cells was examined. The results showed that IPA does not induce mutation at the hprt locus of L5178Y mouse lymphoma cells under the specific conditions employed. This included treatments up to highly toxic concentrations in two independent experiments, in the absence and presence of a rat liver metabolic activation system.

In another GLP, guideline study (Molinier, 1994), IPA did not cause chromosomal aberrations in human lymphocytes, when tested up to cytotoxic concentrations, both with and without metabolic activation. IPA is not clastogenic.

LAS Na:

LAS Na was not mutagenic in the Ames test (Schoeberl, 1993).

The potential of LAS Na to cause chromosomal aberrations in mammalian cells was examined with the use of Chinese hamster ovary cells, exposed to concentrations of 0.32 to 78 ug/ml with S9, and 1.25 to 156 ug/ml without S9. Positive responses were seen at cytotoxic concentrations only in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. Chromosomal aberrations seen at cytotoxicity levels can be considered as a secondary effect. The result suggests that LAS is not clastogenic (Murrie & Innes, 1997).

Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/ml without S9, and 0, 6, 10, 18, 30, and 60 ug/ml with S9. Preliminary tests show the test substance was cytogenic at concentrations of 50 µg/ml or greater with metabolic activation, and 100 µg/ml or above without metabolic activation. There was no biologically significant increase in mutation frequency in the treated groups and hence, LAS is considered not mutagenic to CHO cells both in the presence and absence of S9 (AVON, 1995).

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:

weight of evidence

Study period:

1995-05-16 to 1995-08-31

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

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

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

0, 0.6, 1, 1.8, 3, 6 µg/mL without S9
0, 6, 10, 18, 30, 60 µg/mL with S9

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

H0 medium

Positive controls:

yes

Positive control substance:

other: ethyl methane sulfonate; 3-(20-)methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 1 week
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 6 days at 37 degree C for cloning efficiency study, 9 days for mutation assay


STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test substance was considered mutagenic if a statistically significant dose-related increase in mutant frequency was found in concentrations with greater than 20% survival rate. The mean mutant frequency must also be significantly above the maximum spontaneous mutant frequency.

Statistics:

Statistical significance was determined by the t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test showed cytotoxicity at >= 50 µg/mL without S9, and >= 100 µg/mL with S9.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
In both the studies with and without S9, the mutant frequencies in the treated groups were statistically significantly higher than in the concurrent negative controls.However, the mutant frequencies in the treated groups were not significantly increased when compared to historical negative controls.There was also no dose-response relationship.The increased mutant frequency in treated groups was therefore not considered to be biologically significant.

Results of Test 1: Without S9 Mix            

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	82	3 ± 2
0.6	86	7 ± 1
1	85	3 ± 2
1.8	78	5 ± 2
3	86	1 ± 1
6	83	0 ± 1
EMS	83	277 ± 17

Results of Test 1: With S9 Mix     

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	88	1 ± 1
10	84	9 ± 4
18	78	5 ± 3
30	89	3 ± 2
60	89	7 ± 2
MCA	81	91 ± 9

Results of Test 2: Without S9 Mix

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	96	1 ± 1
0.6	92	2 ± 3
1	95	1 ± 1
1.8	93	5 ± 2
3	90	2 ± 1
6	91	6 ± 6
EMS	90	309 ± 20

Results of Test 2: With S9 Mix     

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	92	7 ± 3
10	88	9 ± 2
18	94	2 ± 1
30	93	2 ± 2
60	90	5 ± 1
MCA	95	89 ± 6

Conclusions:

The test substance is not mutagenic in either the presence or absence of metabolic activation.

Executive summary:

This study examined the potential of the test substance to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/mL without S9, and 0, 6, 10, 18, 30, and 60 µg/mL with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances.

Preliminary tests show the test substance was cytotoxic at concentrations of 50 µg/mL or greater with metabolic activation, and 100 µg/mL or above without metabolic activation.

There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unavailable - orginal study report date 2010

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)

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, 80, 120, 210, 240, and 270 µg/mL (evaluated)
Experiment 2: 0, 100, 150, 175, 200, 250, 300, and 400 µg/mL (evaluated)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

-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 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 90 wells 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related: The highest concentrations to provide >10 % RS were 148 µg/mL +S9 and 296 µg/mL -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 >= 240 µg/mL
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: only at the highest concentration (which turned out to be too toxic)

RANGE-FINDING/SCREENING STUDIES: for cytotoxicity testing up to solublity limit of 500 µg/mL

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

Summary of mutation data [mean of two replicate cultures]:

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

[for individual replicates, see Appendix 2, Table 10 (-S9), and Appendix 3, Table 18 (+S9)]

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	1.28			0		100!	1.26
80		97	0.23		NS	40		85	1.79		NS
120		81	1.22		NS	200		66	2.11		NS
210		36	2.66		NS	240		58	0.51		NS
240		30	1.62		NS	280		45	1.94		NS
270		19	0.78		NS	300		33	2.20		NS
						350		17	0.39		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		44	19.72			2		54	10.78
0.15		34	40.90			3		20	48.09

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

[for individual replicates, see Appendix 4, Table 26 (-S9), and Appendix 5, Table 34 (+S9)]

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	5.14			0		100	6.41
100		98	4.46		NS	200		90	7.21		NS
150		82	4.28		NS	250		93	3.43		NS
175		70	4.92		NS	300		60	4.95		NS
200		56	4.65		NS	325		53	5.67		NS
250		36	2.97		NS	350		32	4.64		NS
300		28	5.45		NS	375		20	5.70		NS
400		3	6.88		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		59	54.25			2		66	80.64
0.15		58	66.29			3		39	104.52

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

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

NS  = not significant

!   = Based on one replicate only

         

Conclusions:

In a mammalian cell gene mutation assay conducted according to OECD 476, the test item 2-propanamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells with and without metabolic activation under test conditions employed in this study at any tested concentrations. The test item can therefore be considered as non-mutagenic.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed 3 hours to 2-propanamine (99.91% purity) at concentrations of 0, 80, 120, 210, 240, and 270 µg/mL with and without S9 metabolic activation in the first experiment and at concentrations of 0, 100, 150, 175, 200, 250, 300, and 400 µg/mL with and without S9 metabolic activation in the second experiment.

Test item was tested up to solubility limit of 500 µg/mL. The positive controls induce the appropriate response.

Cytotoxicity was observed at the highest concentrations of 148 µg/mL and 296 µg/mL with and without S9 metabolic activation, respectively. There was no evidence of induced mutation at the hprt locus of L5178Y mouse lymphoma cells with and without metabolic activation under test conditions employed in this study at any tested concentrations. The test item can therefore be considered as non-mutagenic.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unavailable - orginal study report date 1994

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

Remarks:

However, in the presence of S9-mix, selection of the dose levels was based on pH and not on the mitotic index, since the use of non-physiological pH was not recommended.

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Donators: two healthy humans with no recent X-ray exposure and believed to be free of viral infection
- Time: on day of of culture
- Culture: whole blood in sterile tubes containing heparin

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9) of male SD rats induced with Arochlor 1254

Test concentrations with justification for top dose:

-S9 mix: 30, 100, and 300 µg/mL
+S9 mix: 250, 500, and 1000 µg/mL

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 h (-S9); 2 h (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h(+/-S9)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, applied 2 h before harvest

NUMBER OF REPLICATIONS: 2 per dose and interval

NUMBER OF CELLS EVALUATED: 200 metaphases per dose and interval

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, evaluated on 1000 cells each

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: gaps, chromatid / chromosomal breaks and exchanges, multiple aberrations and pulverisation

Evaluation criteria:

Validity:
- Aberrant cell frequencies (excluding gaps) in the controls fell within the range of historical data;
- The positive controls induced statistically significant increases in the aberrent cell frequencies that fall within the historical contrl ranges.

Evaluation criteria:
- a statistically significant increase in the aberrant cell frequency for at least one of the doses, for any harvest time.

Statistics:

X2 test (significance level of P = 0.05) to compare mean aberration frequencies with the controls.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >=300µg/mL (-S9), no cytotoxicity (+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: cytotoxicity

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- At 300 and 1000 µg/ml, the pH values were 8 and 10, respectively.
- 5000 µg/ml were hemolytic to the cells.

MITOTIC INDEX:
- With metabolic activation (Report, Table 3 and 4):
1000 µg/ml (2 treatment / 24 h harvest): 87 % of the control
1000 µg/ml (2 treatment / 48 h harvest): 93 % of the control

- Without metabolic activation (Report, Table 1 and 2):
300 µg/ml (24 h treatment/24 h harvest): 66 % of the control
1000 µg/ml (24 h treatment/24 h harvest): 0 % of the control

300 µg/ml (48 treatment/48 h harvest): 76 % of the control
1000 µg/ml (48 h treatment/48 h harvest): 45 % of the control

The aberrant cell frequencies in the negative controls were within the range of the historical data (0.4 +/-0.5 %), gaps excluded.

The aberrant cell frequencies in the positive controls were significantly higher than that of the negative controls (P 0.001).

Table1: Overview of aberrant cell frequency (from Report, Table 1 and 2):

	Number of aberrations				% aberrant cells
Doses [µg/mL]	+ gaps		- gaps		+ gaps		- gaps
Without metabolic activation: 24 / 24 h or 48 / 48 h exposure / harvest time
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
0	3	3	1	2	1.5	1.5	1.5	1.0
30	4	6	2	2	2.0	3.0	1.0	1.0
100	4	1	0	1	2.0	0.5	0.0	0.5
300	1	3	1	2	0.5	1.5	0.5	1.0
MMC [0.2]	42	-	36	-	19	-	16	-

MMC = mitomycin

Table 2: Overview of aberrant cell frequency (from Report, Table 3 and 4):

	Number of aberrations				% aberrant cells
Doses [µg/mL]	+ gaps		- gaps		+ gaps		- gaps
With metabolic activation: 2 / 24 h or 2 / 48 h exposure / harvest time
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
0	2	2	1	2	1.0	1.0	0.5	1.0
250	5	2	3	1	2.5	1.0	1.5	0.5
500	4	5	2	1	2.0	2.5	1.0	0.5
1000	1	2	0	1	0.5	1.0	0.0	0.5
CPA [50]	60	-	50	-	21.5	-	19.5	-

CPA = cyclophosphamide

Conclusions:

Under the conditions of this test 2-propanamine did not induce chromosomal aberrations in human lymphocytes when tested up to cytotoxic concentrations, both with and without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay according to OECD Guideline 473), human lymphocyte cultures were exposed to 2-propanamine (99.7% purity) in water at concentrations of 0, 250,500 and 1000 µg/mL with metabolic activation and 0, 30, 100 and 300 µg/mL without metabolic activation. S9 microsomal liver fraction, taken from Arochlor 1254-induced livers of male SD rats was used as metabolic activation system in the assay.

2-propanamine was tested up to cytotoxic concentrations. Positive controls induced the appropriate responses. Negative control responses were within the range of the historical data.

There was no evidence of chromosome aberrations in human lymphocytes, both with and without metabolic activation. Based on the results, the test item is therefore considered to be non-clastogenic in the chromosome aberration assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro mammalian cytogenetics (chromosome aberration test) OPPTS 870.5375; OECD 473.

Reference 4

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

1995-05-25 to 1996-02-07

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

However, this study does not adequately address the results obtained at mildly cytotoxic concentrations.

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

All concentrations in micrograms/mL

Test 1 with S9: 0.32, 0.63, 1.25, 2.5, 5, 10, 20, 39, 78 µg/mL
Test 1 without S9: 1.25, 2.5, 5, 10, 20, 39, 58,78, 156 µg/mL

Test 2 with S9: 2.5, 5, 10, 20, 26, 33, 39 µg/mL
Test 2 without S9: 20, 39, 58, 78, 130, 156 µg/mL

An additional test was done with S9 at the following dose levels:
2.5, 5, 7.5, 10, 15, 20, 25, and 30 µg/mL

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 6 hrs with S9, 22 hrs without S9
- Expression time (cells in growth medium): 16-40 hrs with S9, 40 hrs without S9
- Selection time (if incubation with a selection agent): 2 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24-48 hrs


SELECTION AGENT (mutation assays): Colcemid

NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 100 metaphases


DETERMINATION OF CYTOTOXICITY
- Method: number of cells per culture


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A dosage was considered toxic if cell count was less then 60% of cell cultures. A test substance was considered clastogenic if a single dose caused the percentage of aberrant cells to be consistently greater than the 99% confidence limits of negative controls and there was also an increase at another dose level.

Statistics:

95% and 99% confidence limits

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15 microgram/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=58 microgram/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9, only one culture (Test 2, 24 hr harvest, 20 ug/ml) showed a suspicious result. This single result was considered sporadic, as other cultures at this concentration, or at higher concentrations did not show a positive response. In Test 1, in the absence of S9, cytoxicity was seen at 78 micrograms/mL and above. In Test 2, in the absence of S9, cytoxicity was seen at concentrations of 58 micrograms/mL and above.

In Test 1, in the presence of S9, no positive results were seen at concentrations of up to 20 micrograms/mL. Metaphases could not be analyzed due to severe cytotoxicity at the 39 and 78 microgram/mL concentrations. In Test 2, in the presence of S9, one of the cultures at the 5 microgram/mL concentration gave a suspicious result, and both cultures at the 10 microgram/mL concentrations gave positive responses. Mild cytotoxity was also seen at the 10 microgram/mL concentration. At concentrations at and above 20 micrograms/mL, metaphases could not be analyzed due to severe cytotoxicity. No positive results were seen in the Test 2, 48 hr harvest cultures grown in the presence of S9, though moderate cytotoxicity was seen in one of the 20 microgram/mL cultures, and severe cytotoxicity was seen in all cultures above this concentration.

A third test was done in the presence of S9, which showed positive results at the 15 micrograms/mL concentration. However, this concentration was also moderately cytotoxic with only 26% of cells survival. However, due to the low survival of cells, these results are not definitive for determining clastogenicity. Higher concentrations were completely cytotoxic. An additional assessment was then performed at 10 micrograms/ml in the presence of S9, with negative results.

Abbreviations used in tables:

T - Toxicity evident from morphological changes

TT- Toxicity evident from reduced cell count (<60% of vehicle)

TTT- Too toxic for metaphase assessment

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F10 medium	0.01	1	0	Nil
Hams F10 medium	0.02	1	1	Nil
0.32	-	-	-	Nil
0.32	-	-	-	Nil
0.63	-	-	-	Nil
0.63	-	-	-	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	0.01	1	0	Nil
2.5	0.00	0	0	Nil
5	0.00	0	0	Nil
5	0.05	5	0	Nil
10	0.01	1	0	Nil
10	0.01	1	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	-	-	-	TTT
39	-	-	-	TTT
78	-	-	-	TTT
78	-	-	-	TTT
Cyclophosphamide (20 micrograms/ml)	0.14	8	4	-
Cyclophosphamide (30 micrograms/ml)	0.06	4	4	-
Cyclophosphamide (40 micrograms/ml)	0.33	20	19	-

Test 1: Without S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F10 medium	0.00	0	0	Nil
Hams F10 medium	0.00	0	0	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
20	-	-	-	Nil
20	-	-	-	Nil
39	0.01	1	0	Nil
39	0.00	0	0	Nil
58	0.01	1	0	Nil
58	0.00	0	0	Nil
78	0.00	0	0	T
78	0.00	0	0	T
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.03	3	1	-
Cyclophosphamide (20 micrograms/ml)	0.16	14	10	-

Test 2 With S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.01	1	0	Nil
Hams F-10 medium	0.02	2	1	Nil
2.5	0.07	2	1	Nil
2.5	0.04	3	1	Nil
5	0.04	3	2	Nil
5	0.06	6	4	Nil
10	0.12	8	6	T
10	0.19	13	5	T
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.38	20	17	-
Cyclophosphamide (50 micrograms/ml)	0.31	18	11	-

Test 2: With S9 Mix, 48 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.00	0	0	Nil
Hams F-10 medium	0.00	0	0	Nil
2.5	0.01	1	0	Nil
2.5	0.01	1	1	Nil
5	0.00	0	0	Nil
5	0.02	2	2	Nil
10	0.03	2	1	Nil
10	0.02	2	1	TT
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.03	3	2	-
Cyclophosphamide (50 micrograms/ml)	0.10	8	7	-

Test 2: Without S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.02	2	2	Nil
Hams F-10 medium	0.03	3	0	Nil
20	0.02	2	0	Nil
20	0.05	5	3	Nil
39	0.02	2	1	Nil
39	0.04	4	0	Nil
58	0.01	1	1	Nil
58	0.06	6	1	Nil
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.30	21	14	-
Methyl methane-sulphonate (20 micrograms/ml)	0.71	33	28	-

Test 2: Without S9 Mix, 48 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.01	1	1	Nil
Hams F-10 medium	0.00	0	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	0.01	1	1	Nil
39	0.00	0	0	Nil
58	0.00	0	0	T
58	0.01	1	0	T
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (20 micrograms/ml)	0.21	11	8	-
Methyl methane- sulphonate (40 micrograms/ml)	3.20	60	60	-

Test 3: With S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.04	4	0	Nil
Ham¿s F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

 

Test 3: With S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.04	4	0	Nil
Ham¿s F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

Test 3: With S9 Mix, 24 hr Harvest - Additional Assessment

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.02	2	0	Nil
10	0.01	1	0	Nil
10	0.02	2	0	Nil
Cyclophosphamide (30 micrograms/ml)	0.22	14	10	-

Conclusions:

The test substance is not clastogenic in the absence of metabolic activation.The test substance is also not clastogenic in the presence of metabolic activation at non-cytotoxic concentrations. Chromosomal abberations were detected only at cytotoxic concentrations, with metabolic activation.

Executive summary:

This study examined the potential of the test substance Marlon A 350 to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 µg/mL with S9, and 1.25 to 156 µg/mL without S9. Methyl methanesuflphonate and cyclophosphamide were used as positive controls.

No biologically significant results were seen in treated cultures in the absence of metabolic activation. Positive responses were seen at cytotoxic concentrations in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. These results indicate that LAS induces chromosomal aberrations at cytotoxic concentrations. However, this effect can be considered as secondary due to cytotoxicity.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Unavailable - orginal study report date 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Principles of method if other than guideline:

Directive 84/449/EEC, B.14 Mutagenicity (Salmonella typhimurium - reverse mutation assay)" 1984; equivalent to OECD 471

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced S9 fraction

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

Water solution at 50 g/L

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Positive controls:

yes

Remarks:

aminoanthracene

Positive control substance:

other: nitrofluorene, sodium azide and aminoacridine

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 µg/plate. No precipitation of the product was observed at any concentration tested.

Conclusions:

The substance was not mutagenic in Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537 and TA1538) in the presence and absence of metabolic activation in bacterial reverse mutation assay.

Executive summary:

The potential of the substance to induce reverse mutations in several strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) was evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The concentrations of the substance employed were 8, 40, 200, 1000 and 5000 µg/plate.

 

During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 µg/plate. No precipitate was observed at the maximum concentration tested. No positive responses were induced in any of the strains. Based on the results of this study, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 6

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

Unavailable - orginal publication date 1987

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

preincubation assay, two different sources of a microsomal metabolic system; 4 instead of 5 tester strains.

Principles of method if other than guideline:

Ames-Test modified acc. 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 locus

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:

0.010, 0.033, 0.100, 0.333, 1.0, 3.333 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test substance well soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

TA98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation, agar plate

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days at 37 °C


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- relative total growth (background lawn)

Evaluation criteria:

Mutagenic (+) or weakly mutagenic (+w) if a reproducible, dose-related increase in revertants over the corresponding solvent controls in replicate trials was seen.
Questionable (?) if a reproducible increase in revertants did not meet the criteria of either "+" or "+w", or if single doses produced an increase in repeat trials.

Key result

Species / strain:

other: 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:

>= 3.333 mg/plate (see Report, Appendix 2, p. 79/80)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of this study 2-propanamine does not induce any mutations in bacterial strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium, when tested up to cytotoxic concentrations, both with and without metabolic activation.

Executive summary:

In a bacterial reverse gene mutation assay (equivalent to OECD guideline 471), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium were exposed to 2-propanamine (99% purity) in distilled water at concentrations ranging between 10 to 3333 µg/plate in the presence and absence of mammalian metabolic activation (S9 microsomal liver fraction, taken from Arochlor-induced livers of male SD rats and male Syrian hamsters). The positive controls induced the appropriate responses in the corresponding strains and the vehicle controls were considered valid.

The results showed a negative response,at any dose level tested. Therefore, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

Reference 7

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: read across to Klimisch 2 GLP , comparable to guideline study.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes (incl. QA statement)

Type of assay:

mammalian cell gene mutation assay

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

0, 0.6, 1, 1.8, 3, 6 µg/mL without S9
0, 6, 10, 18, 30, 60 µg/mL with S9

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

yes

Remarks:

H0 medium

Positive controls:

yes

Positive control substance:

other: ethyl methane sulfonate; 3-(20-)methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Preincubation period: 1 week
- Exposure duration: 4 hrs
- Expression time (cells in growth medium): 6 days at 37 degree C for cloning efficiency study, 9 days for mutation assay


STAIN (for cytogenetic assays): Giemsa


NUMBER OF REPLICATIONS: 2

Evaluation criteria:

A test substance was considered mutagenic if a statistically significant dose-related increase in mutant frequency was found in concentrations with greater than 20% survival rate. The mean mutant frequency must also be significantly above the maximum spontaneous mutant frequency.

Statistics:

Statistical significance was determined by the t-test.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

preliminary test showed cytotoxicity at >= 50 µg/mL without S9, and >= 100 µg/mL with S9.

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

COMPARISON WITH HISTORICAL CONTROL DATA:
In both the studies with and without S9, the mutant frequencies in the treated groups were statistically significantly higher than in the concurrent negative controls.However, the mutant frequencies in the treated groups were not significantly increased when compared to historical negative controls.There was also no dose-response relationship.The increased mutant frequency in treated groups was therefore not considered to be biologically significant.

Results of Test 1: Without S9 Mix            

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	82	3 ± 2
0.6	86	7 ± 1
1	85	3 ± 2
1.8	78	5 ± 2
3	86	1 ± 1
6	83	0 ± 1
EMS	83	277 ± 17

Results of Test 1: With S9 Mix     

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	88	1 ± 1
10	84	9 ± 4
18	78	5 ± 3
30	89	3 ± 2
60	89	7 ± 2
MCA	81	91 ± 9

Results of Test 2: Without S9 Mix

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	96	1 ± 1
0.6	92	2 ± 3
1	95	1 ± 1
1.8	93	5 ± 2
3	90	2 ± 1
6	91	6 ± 6
EMS	90	309 ± 20

Results of Test 2: With S9 Mix     

Concentration (ug/ml)	Absolute cloning efficiency (%)	Mutant frequency ( x 10^6)
0	90	2 ± 1
6	92	7 ± 3
10	88	9 ± 2
18	94	2 ± 1
30	93	2 ± 2
60	90	5 ± 1
MCA	95	89 ± 6

Conclusions:

The test substance is not mutagenic in either the presence or absence of metabolic activation.

Executive summary:

This study examined the potential of the test substance to cause mutations in mammalian cells. Chinese Hamster Ovary (CHO) cells were exposed to concentrations of 0, 0.6, 1, 1.8, 3, and 6 µg/mL without S9, and 0, 6, 10, 18, 30, and 60 µg/mL with S9. The cells were then examined for cytogenicity and mutation frequency. Ethyl methane sulfonate and 3-(20-)methylcholanthrene were used as positive control substances.

Preliminary tests show the test substance was cytotoxic at concentrations of 50 µg/mL or greater with metabolic activation, and 100 µg/mL or above without metabolic activation.

There was no biologically significant increase in mutation frequency in the treated groups. The test substance is considered not mutagenic to CHO cells both in the presence and absence of S9.

Reference 8

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

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, 80, 120, 210, 240, and 270 µg/mL (evaluated)
Experiment 2: 0, 100, 150, 175, 200, 250, 300, and 400 µg/mL (evaluated)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

benzo(a)pyrene

Remarks:

+S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

water as vehicle

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

Remarks:

-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 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 90 wells 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.

Key result

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

dose-related: The highest concentrations to provide >10 % RS were 148 µg/mL +S9 and 296 µg/mL -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 >= 240 µg/mL
- Effects of osmolality: no
- Evaporation from medium: no
- Precipitation: only at the highest concentration (which turned out to be too toxic)

RANGE-FINDING/SCREENING STUDIES: for cytotoxicity testing up to solublity limit of 500 µg/mL

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

Summary of mutation data [mean of two replicate cultures]:

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

[for individual replicates, see Appendix 2, Table 10 (-S9), and Appendix 3, Table 18 (+S9)]

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	1.28			0		100!	1.26
80		97	0.23		NS	40		85	1.79		NS
120		81	1.22		NS	200		66	2.11		NS
210		36	2.66		NS	240		58	0.51		NS
240		30	1.62		NS	280		45	1.94		NS
270		19	0.78		NS	300		33	2.20		NS
						350		17	0.39		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		44	19.72			2		54	10.78
0.15		34	40.90			3		20	48.09

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

[for individual replicates, see Appendix 4, Table 26 (-S9), and Appendix 5, Table 34 (+S9)]

Treatment (µg/mL)		-S-9				Treatment (µg/mL)		+S-9
		% RS	MF§					% RS	MF§
0		100	5.14			0		100	6.41
100		98	4.46		NS	200		90	7.21		NS
150		82	4.28		NS	250		93	3.43		NS
175		70	4.92		NS	300		60	4.95		NS
200		56	4.65		NS	325		53	5.67		NS
250		36	2.97		NS	350		32	4.64		NS
300		28	5.45		NS	375		20	5.70		NS
400		3	6.88		NS
Linear trend			NS			Linear trend			NS
NQO						B[a]P
0.1		59	54.25			2		66	80.64
0.15		58	66.29			3		39	104.52

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

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

NS  = not significant

!   = Based on one replicate only

         

Conclusions:

In a mammalian cell gene mutation assay conducted according to OECD 476, the test item 2-propanamine did not induce mutation at the hprt locus of L5178Y mouse lymphoma cells with and without metabolic activation under test conditions employed in this study at any tested concentrations. The test item can therefore be considered as non-mutagenic.

Executive summary:

In a mammalian cell gene mutation assay conducted according to OECD Guideline 476, mouse lymphoma L5178Y cells cultured in vitro were exposed 3 hours to 2-propanamine (99.91% purity) at concentrations of 0, 80, 120, 210, 240, and 270 µg/mL with and without S9 metabolic activation in the first experiment and at concentrations of 0, 100, 150, 175, 200, 250, 300, and 400 µg/mL with and without S9 metabolic activation in the second experiment.

Test item was tested up to solubility limit of 500 µg/mL. The positive controls induce the appropriate response.

Cytotoxicity was observed at the highest concentrations of 148 µg/mL and 296 µg/mL with and without S9 metabolic activation, respectively. There was no evidence of induced mutation at the hprt locus of L5178Y mouse lymphoma cells with and without metabolic activation under test conditions employed in this study at any tested concentrations. The test item can therefore be considered as non-mutagenic.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5300, OECD 476 for in vitro mutagenicity (mammalian forward gene mutation) data.

Reference 9

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

lymphocytes: human

Details on mammalian cell type (if applicable):

- Donators: two healthy humans with no recent X-ray exposure and believed to be free of viral infection
- Time: on day of of culture
- Culture: whole blood in sterile tubes containing heparin

Metabolic activation:

with and without

Metabolic activation system:

liver microsomal fraction (S9) of male SD rats induced with Arochlor 1254

Test concentrations with justification for top dose:

-S9 mix: 30, 100, and 300 µg/mL
+S9 mix: 250, 500, and 1000 µg/mL

Vehicle / solvent:

distilled water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

mitomycin C

Remarks:

without S9

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Exposure duration: 24 and 48 h (-S9); 2 h (+S9)
- Fixation time (start of exposure up to fixation or harvest of cells): 24 and 48 h(+/-S9)

SPINDLE INHIBITOR (cytogenetic assays): Colcemid, applied 2 h before harvest

NUMBER OF REPLICATIONS: 2 per dose and interval

NUMBER OF CELLS EVALUATED: 200 metaphases per dose and interval

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index, evaluated on 1000 cells each

OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes
- Other: gaps, chromatid / chromosomal breaks and exchanges, multiple aberrations and pulverisation

Evaluation criteria:

Validity:
- Aberrant cell frequencies (excluding gaps) in the controls fell within the range of historical data;
- The positive controls induced statistically significant increases in the aberrent cell frequencies that fall within the historical contrl ranges.

Evaluation criteria:
- a statistically significant increase in the aberrant cell frequency for at least one of the doses, for any harvest time.

Statistics:

X2 test (significance level of P = 0.05) to compare mean aberration frequencies with the controls.

Key result

Species / strain:

lymphocytes: human

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at >=300µg/mL (-S9), no cytotoxicity (+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: cytotoxicity

ADDITIONAL INFORMATION ON CYTOTOXICITY:
- At 300 and 1000 µg/ml, the pH values were 8 and 10, respectively.
- 5000 µg/ml were hemolytic to the cells.

MITOTIC INDEX:
- With metabolic activation (Report, Table 3 and 4):
1000 µg/ml (2 treatment / 24 h harvest): 87 % of the control
1000 µg/ml (2 treatment / 48 h harvest): 93 % of the control

- Without metabolic activation (Report, Table 1 and 2):
300 µg/ml (24 h treatment/24 h harvest): 66 % of the control
1000 µg/ml (24 h treatment/24 h harvest): 0 % of the control

300 µg/ml (48 treatment/48 h harvest): 76 % of the control
1000 µg/ml (48 h treatment/48 h harvest): 45 % of the control

The aberrant cell frequencies in the negative controls were within the range of the historical data (0.4 +/-0.5 %), gaps excluded.

The aberrant cell frequencies in the positive controls were significantly higher than that of the negative controls (P 0.001).

Table1: Overview of aberrant cell frequency (from Report, Table 1 and 2):

	Number of aberrations				% aberrant cells
Doses [µg/mL]	+ gaps		- gaps		+ gaps		- gaps
Without metabolic activation: 24 / 24 h or 48 / 48 h exposure / harvest time
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
0	3	3	1	2	1.5	1.5	1.5	1.0
30	4	6	2	2	2.0	3.0	1.0	1.0
100	4	1	0	1	2.0	0.5	0.0	0.5
300	1	3	1	2	0.5	1.5	0.5	1.0
MMC [0.2]	42	-	36	-	19	-	16	-

MMC = mitomycin

Table 2: Overview of aberrant cell frequency (from Report, Table 3 and 4):

	Number of aberrations				% aberrant cells
Doses [µg/mL]	+ gaps		- gaps		+ gaps		- gaps
With metabolic activation: 2 / 24 h or 2 / 48 h exposure / harvest time
	24 h	48 h	24 h	48 h	24 h	48 h	24 h	48 h
0	2	2	1	2	1.0	1.0	0.5	1.0
250	5	2	3	1	2.5	1.0	1.5	0.5
500	4	5	2	1	2.0	2.5	1.0	0.5
1000	1	2	0	1	0.5	1.0	0.0	0.5
CPA [50]	60	-	50	-	21.5	-	19.5	-

CPA = cyclophosphamide

Conclusions:

Under the conditions of this test 2-propanamine did not induce chromosomal aberrations in human lymphocytes when tested up to cytotoxic concentrations, both with and without metabolic activation.

Executive summary:

In a mammalian cell cytogenetics assay (chromosome aberration assay according to OECD Guideline 473), human lymphocyte cultures were exposed to 2-propanamine (99.7% purity) in water at concentrations of 0, 250,500 and 1000 µg/mL with metabolic activation and 0, 30, 100 and 300 µg/mL without metabolic activation. S9 microsomal liver fraction, taken from Arochlor 1254-induced livers of male SD rats was used as metabolic activation system in the assay.

2-propanamine was tested up to cytotoxic concentrations. Positive controls induced the appropriate responses. Negative control responses were within the range of the historical data.

There was no evidence of chromosome aberrations in human lymphocytes, both with and without metabolic activation. Based on the results, the test item is therefore considered to be non-clastogenic in the chromosome aberration assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline In vitro mammalian cytogenetics (chromosome aberration test) OPPTS 870.5375; OECD 473.

Reference 10

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

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

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

However, this study does not adequately address the results obtained at mildly cytotoxic concentrations.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Metabolic activation system:

S9 from Aroclor 1254 induced rat liver

Test concentrations with justification for top dose:

All concentrations in micrograms/mL

Test 1 with S9: 0.32, 0.63, 1.25, 2.5, 5, 10, 20, 39, 78 µg/mL
Test 1 without S9: 1.25, 2.5, 5, 10, 20, 39, 58,78, 156 µg/mL

Test 2 with S9: 2.5, 5, 10, 20, 26, 33, 39 µg/mL
Test 2 without S9: 20, 39, 58, 78, 130, 156 µg/mL

An additional test was done with S9 at the following dose levels:
2.5, 5, 7.5, 10, 15, 20, 25, and 30 µg/mL

Vehicle / solvent:

None

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Untreated negative controls:

yes

Negative solvent / vehicle controls:

no

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium


DURATION
- Exposure duration: 6 hrs with S9, 22 hrs without S9
- Expression time (cells in growth medium): 16-40 hrs with S9, 40 hrs without S9
- Selection time (if incubation with a selection agent): 2 hrs
- Fixation time (start of exposure up to fixation or harvest of cells): 24-48 hrs


SELECTION AGENT (mutation assays): Colcemid

NUMBER OF REPLICATIONS: 3


NUMBER OF CELLS EVALUATED: 100 metaphases


DETERMINATION OF CYTOTOXICITY
- Method: number of cells per culture


OTHER EXAMINATIONS:
- Determination of polyploidy: yes
- Determination of endoreplication: yes

Evaluation criteria:

A dosage was considered toxic if cell count was less then 60% of cell cultures. A test substance was considered clastogenic if a single dose caused the percentage of aberrant cells to be consistently greater than the 99% confidence limits of negative controls and there was also an increase at another dose level.

Statistics:

95% and 99% confidence limits

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>= 15 microgram/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

>=58 microgram/mL

Vehicle controls validity:

not examined

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

In the absence of S9, only one culture (Test 2, 24 hr harvest, 20 ug/ml) showed a suspicious result. This single result was considered sporadic, as other cultures at this concentration, or at higher concentrations did not show a positive response. In Test 1, in the absence of S9, cytoxicity was seen at 78 micrograms/mL and above. In Test 2, in the absence of S9, cytoxicity was seen at concentrations of 58 micrograms/mL and above.

In Test 1, in the presence of S9, no positive results were seen at concentrations of up to 20 micrograms/mL. Metaphases could not be analyzed due to severe cytotoxicity at the 39 and 78 microgram/mL concentrations. In Test 2, in the presence of S9, one of the cultures at the 5 microgram/mL concentration gave a suspicious result, and both cultures at the 10 microgram/mL concentrations gave positive responses. Mild cytotoxity was also seen at the 10 microgram/mL concentration. At concentrations at and above 20 micrograms/mL, metaphases could not be analyzed due to severe cytotoxicity. No positive results were seen in the Test 2, 48 hr harvest cultures grown in the presence of S9, though moderate cytotoxicity was seen in one of the 20 microgram/mL cultures, and severe cytotoxicity was seen in all cultures above this concentration.

A third test was done in the presence of S9, which showed positive results at the 15 micrograms/mL concentration. However, this concentration was also moderately cytotoxic with only 26% of cells survival. However, due to the low survival of cells, these results are not definitive for determining clastogenicity. Higher concentrations were completely cytotoxic. An additional assessment was then performed at 10 micrograms/ml in the presence of S9, with negative results.

Abbreviations used in tables:

T - Toxicity evident from morphological changes

TT- Toxicity evident from reduced cell count (<60% of vehicle)

TTT- Too toxic for metaphase assessment

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F10 medium	0.01	1	0	Nil
Hams F10 medium	0.02	1	1	Nil
0.32	-	-	-	Nil
0.32	-	-	-	Nil
0.63	-	-	-	Nil
0.63	-	-	-	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	0.01	1	0	Nil
2.5	0.00	0	0	Nil
5	0.00	0	0	Nil
5	0.05	5	0	Nil
10	0.01	1	0	Nil
10	0.01	1	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	-	-	-	TTT
39	-	-	-	TTT
78	-	-	-	TTT
78	-	-	-	TTT
Cyclophosphamide (20 micrograms/ml)	0.14	8	4	-
Cyclophosphamide (30 micrograms/ml)	0.06	4	4	-
Cyclophosphamide (40 micrograms/ml)	0.33	20	19	-

Test 1: Without S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F10 medium	0.00	0	0	Nil
Hams F10 medium	0.00	0	0	Nil
1.25	-	-	-	Nil
1.25	-	-	-	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
20	-	-	-	Nil
20	-	-	-	Nil
39	0.01	1	0	Nil
39	0.00	0	0	Nil
58	0.01	1	0	Nil
58	0.00	0	0	Nil
78	0.00	0	0	T
78	0.00	0	0	T
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.03	3	1	-
Cyclophosphamide (20 micrograms/ml)	0.16	14	10	-

Test 2 With S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.01	1	0	Nil
Hams F-10 medium	0.02	2	1	Nil
2.5	0.07	2	1	Nil
2.5	0.04	3	1	Nil
5	0.04	3	2	Nil
5	0.06	6	4	Nil
10	0.12	8	6	T
10	0.19	13	5	T
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.38	20	17	-
Cyclophosphamide (50 micrograms/ml)	0.31	18	11	-

Test 2: With S9 Mix, 48 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.00	0	0	Nil
Hams F-10 medium	0.00	0	0	Nil
2.5	0.01	1	0	Nil
2.5	0.01	1	1	Nil
5	0.00	0	0	Nil
5	0.02	2	2	Nil
10	0.03	2	1	Nil
10	0.02	2	1	TT
20	-	-	-	TTT
20	-	-	-	TTT
26	-	-	-	TTT
26	-	-	-	TTT
33	-	-	-	TTT
33	-	-	-	TTT
39	-	-	-	TTT
39	-	-	-	TTT
Cyclophosphamide (40 micrograms/ml)	0.03	3	2	-
Cyclophosphamide (50 micrograms/ml)	0.10	8	7	-

Test 2: Without S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.02	2	2	Nil
Hams F-10 medium	0.03	3	0	Nil
20	0.02	2	0	Nil
20	0.05	5	3	Nil
39	0.02	2	1	Nil
39	0.04	4	0	Nil
58	0.01	1	1	Nil
58	0.06	6	1	Nil
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (10 micrograms/ml)	0.30	21	14	-
Methyl methane-sulphonate (20 micrograms/ml)	0.71	33	28	-

Test 2: Without S9 Mix, 48 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytotoxicity
Hams F-10 medium	0.01	1	1	Nil
Hams F-10 medium	0.00	0	0	Nil
20	0.00	0	0	Nil
20	0.00	0	0	Nil
39	0.01	1	1	Nil
39	0.00	0	0	Nil
58	0.00	0	0	T
58	0.01	1	0	T
78	-	-	-	TTT
78	-	-	-	TTT
104	-	-	-	TTT
104	-	-	-	TTT
130	-	-	-	TTT
130	-	-	-	TTT
156	-	-	-	TTT
156	-	-	-	TTT
Methyl methane-sulphonate (20 micrograms/ml)	0.21	11	8	-
Methyl methane- sulphonate (40 micrograms/ml)	3.20	60	60	-

Test 3: With S9 Mix, 24 hr Harvest

Concentration (micrograms/mL)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.04	4	0	Nil
Ham¿s F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
10	-	-	-	Nil
10	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

 

Test 3: With S9 Mix, 24 hr Harvest

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.04	4	0	Nil
Ham¿s F-10 medium	0.04	4	0	Nil
2.5	-	-	-	Nil
2.5	-	-	-	Nil
5	-	-	-	Nil
5	-	-	-	Nil
7.5	-	-	-	Nil
7.5	-	-	-	Nil
15	0.20	12	8	TT
15	0.18	12	6	TT
20	-	-	-	TTT
20	-	-	-	TTT
25	-	-	-	TTT
25	-	-	-	TTT
30	-	-	-	TTT
30	-	-	-	TTT
Cyclophosphamide (30 micrograms/ml)	0.24	14	12	-
Cyclophosphamide (40 micrograms/ml)	0.32	17	11	-

Test 3: With S9 Mix, 24 hr Harvest - Additional Assessment

Concentration (micrograms/ml)	Aberration Frequency (lesions/cell)	Aberrant Cell Frequency (% Including Gaps)	Aberrant Cell Frequency (% Excluding Gaps)	Cytoxicity
Ham¿s F-10 medium	0.02	2	0	Nil
10	0.01	1	0	Nil
10	0.02	2	0	Nil
Cyclophosphamide (30 micrograms/ml)	0.22	14	10	-

Conclusions:

The test substance is not clastogenic in the absence of metabolic activation.The test substance is also not clastogenic in the presence of metabolic activation at non-cytotoxic concentrations. Chromosomal abberations were detected only at cytotoxic concentrations, with metabolic activation.

Executive summary:

This study examined the potential of the test substance Marlon A 350 to cause chromosomal aberrations in mammalian cells. Chinese hamster ovary cells were exposed to concentrations of 0.32 to 78 µg/mL with S9, and 1.25 to 156 µg/mL without S9. Methyl methanesuflphonate and cyclophosphamide were used as positive controls.

No biologically significant results were seen in treated cultures in the absence of metabolic activation. Positive responses were seen at cytotoxic concentrations in the presence of S9. Concentrations below the level of cytotoxicty with S9 did not show positive results. The test substance is not clastogenic in the absence of metabolic activation, or with metabolic activation below cytotoxic concentrations. These results indicate that LAS induces chromosomal aberrations at cytotoxic concentrations. However, this effect can be considered as secondary due to cytotoxicity.

Reference 11

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to K1 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Arochlor-induced S9 fraction

Test concentrations with justification for top dose:

8, 40, 200, 1000 and 5000 µg/plate

Vehicle / solvent:

Water solution at 50 g/L

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

water

True negative controls:

yes

Positive controls:

yes

Remarks:

aminoanthracene

Positive control substance:

other: nitrofluorene, sodium azide and aminoacridine

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 1537, TA 98, TA 100, TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

with and without activation

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 µg/plate. No precipitation of the product was observed at any concentration tested.

Conclusions:

The substance was not mutagenic in Salmonella typhimurium strains (TA98, TA100, TA1535, TA1537 and TA1538) in the presence and absence of metabolic activation in bacterial reverse mutation assay.

Executive summary:

The potential of the substance to induce reverse mutations in several strains of Salmonella typhimurium (TA98, TA100, TA1535, TA1537 and TA1538) was evaluated in the presence or absence of an exogenous mammalian metabolic activation system (S9). The concentrations of the substance employed were 8, 40, 200, 1000 and 5000 µg/plate.

 

During the pre-incubation test, signs of toxicity were noted at concentrations as low as 125 µg/plate. No precipitate was observed at the maximum concentration tested. No positive responses were induced in any of the strains. Based on the results of this study, the substance is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Reference 12

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

supporting study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Read-across to K2 study therefore K2 is the maximum Klimisch value.

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

not specified

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

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:

0.010, 0.033, 0.100, 0.333, 1.0, 3.333 mg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: water
- Justification for choice of solvent/vehicle: Test substance well soluble in water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

TA1535 and TA100 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

TA1537 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-nitro-o-phenylenediamine

Remarks:

TA98 without S9

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

distilled water

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene

Remarks:

All strains with S9

Details on test system and experimental conditions:

METHOD OF APPLICATION: preincubation, agar plate

DURATION
- Preincubation period: 20 min
- Exposure duration: 2 days at 37 °C


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- relative total growth (background lawn)

Evaluation criteria:

Mutagenic (+) or weakly mutagenic (+w) if a reproducible, dose-related increase in revertants over the corresponding solvent controls in replicate trials was seen.
Questionable (?) if a reproducible increase in revertants did not meet the criteria of either "+" or "+w", or if single doses produced an increase in repeat trials.

Key result

Species / strain:

other: 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:

>= 3.333 mg/plate (see Report, Appendix 2, p. 79/80)

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Conclusions:

Under the conditions of this study 2-propanamine does not induce any mutations in bacterial strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium, when tested up to cytotoxic concentrations, both with and without metabolic activation.

Executive summary:

In a bacterial reverse gene mutation assay (equivalent to OECD guideline 471), strains TA98, TA100, TA1535, and TA1537 of Salmonella typhimurium were exposed to 2-propanamine (99% purity) in distilled water at concentrations ranging between 10 to 3333 µg/plate in the presence and absence of mammalian metabolic activation (S9 microsomal liver fraction, taken from Arochlor-induced livers of male SD rats and male Syrian hamsters). The positive controls induced the appropriate responses in the corresponding strains and the vehicle controls were considered valid.

The results showed a negative response,at any dose level tested. Therefore, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

Reference 13

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Target gene:

Histidine locus

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and E. coli WP2

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 male rat liver microsomal fraction; animals induced with phenobarbitone/b-naphthoflavone

Test concentrations with justification for top dose:

Preliminary toxicity test: 0, 0.15, 0.5, 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Mutation test, Exp. I
all strains with S9: 1.5, 5, 15, 50, 150, 500, 1500 ug/plate
all strains without S9: 5, 15, 50, 150, 500, 1500 and 5000 ug/plate

Mutation test, Exp. II
Salmonella strains TA100 & TA1537 (without S9): 0.15, 0.5, 1.5, 5, 15, 50, 150, 500 ug/plate
Salmonella strains TA100 & TA1537 (with S9) and TA98 & TA1535 (without S9): 1.5, 5, 15, 50, 150, 500, 1500 and 5000 ug/plate
Salmonella strains TA1535 & TA98 (with S9) and E.coli strain WP2uvrA (without and with S9): 5, 15, 50, 150, 500, 1500, 5000 ug/plate

Vehicle / solvent:

- Vehicle used: tetrahydrofurane
- Justification for choice of vehicle: the test item was insoluble in water, DMSO, dimethyl formamide and acetonitrile at 50 mg/mL and acetone at 100 mg/mL, but fully soluble in tetrahydrofuran at 200 mg/mL

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

tetrahydrofuran

True negative controls:

no

Positive controls:

yes

Positive control substance:

4-nitroquinoline-N-oxide

9-aminoacridine

N-ethyl-N-nitro-N-nitrosoguanidine

benzo(a)pyrene

other: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: exp. I in agar (plate incorporation), exp.II pre-incubation method; according to the OECD Guideline

DURATION
- Preincubation period (exp. II): 20 min
- Expression time (cells in growth medium): 48 h

NUMBER OF REPLICATIONS: 3

DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn

Evaluation criteria:

Any, one, or all of the following can be used to determine the overall result of the study:
1. A dose-related increase in mutant frequency over the dose range tested
2. A reproducible increase at one or more concentrations.
3. Biological relevance against in-house historical control ranges.
4. Statistical analysis of data as determined by UKEMS (Mahon et al (1989)).
5. Fold increase greater than two times the concurrent solvent control for any tester strain (especially if accompanied by an out-of-historical range response).

A test item will be considered non-mutagenic in the test system if the above criteria are not met.
Although most experiments will give clear positive or negative results, in some instances the data generated will prohibit making a definite judgement about test item activity. Results of this type will be reported as equivocal.

Statistics:

UKEMS (Mahon GAT et al 1989; Analysis of data from microbial colony assays. In: Statistical Evaluation of Mutagenicity Test Data, UKEMS sub-committee on guidelines for mutagenicity testing)

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not applicable

Positive controls validity:

valid

Key result

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:

not applicable

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
The strains were checked for characteristics, viability and spontaneous reversion rate (all were found to be satisfactory). The amino acid supplemented top agar and S9-mix used in both experiments was shown to be sterile. The culture density for each bacterial strain was also checked and considered acceptable.
Precipitation: no precipitation seen at any doses
Preliminary toxicity test: the test material was toxic to TA100 from 500 ug/plate and to WP2urvA from 150 ug/plate onwards

Main tests:
Cytotoxiciy: The test material caused a reduction in the bacterial background lawn of all the tester strains, from 150 and 500 ug/plate with and without S9 mix.

Mutagenicity: no significant increases in the revertant colonies were seen at any dose level, both with and without metabolic activation

Positive and negative control results were considered valid.

Detailed results can be seen in the attached document.

Conclusions:

Under the conditions of this test the test material (LAS IPA) does not induce any mutations in bacterial strains, when tested up to cytotoxic concentrations, both with and without metabolic activation.

Executive summary:

In a bacterial reverse gene mutation assay conducted according to OECD guideline 471, strains TA98, TA100, TA1535, TA1537 of Salmonella typhimurium and E.coli WP2uvrA were exposed to LAS-IPA (97% purity) in tetrahydrofurane at concentrations ranging between 1.5 to 5000 µg/plate in the presence and absence of mammalian metabolic activation (S9 microsomal liver fraction, taken from male rats induced with phenobarbitone/b-naphthoflavone). The positive controls induced the appropriate responses in the corresponding strains and the vehicle controls were considered valid.

The results showed a negative response, i.e. no significant increase in the number of revertants was recorded at any dose level tested. Therefore, the test item is considered to be non-mutagenic in the bacterial reverse gene mutation assay.

This study is classified as acceptable. This study satisfies the requirement for Test Guideline OPPTS 870.5100; OECD 471 for in vitro mutagenicity (bacterial reverse gene mutation) data.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

There are no in vivo studies for genotoxicity available for LAS-IPA and therefore read-across substances were employed to fulfil this endpoint.

Benzenesulfonic acid, 4 -C10 -13 -sec-alkyl derivs.

The substance was negative in the in vivo mammalian erythrocyte micronucleus test performed in NMRI mice.

sodium 4-undecylbenzenesulfonate

The substance was negative in a mouse chromosome aberration assay employing JCL-ICR, mice at doses of 0 or 0.9% (equivalent to 1170 mg/kg bw) in the diet for 9 months.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unavailable - study report dated 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

comparable to guideline study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain NMRI. Animals were approximately 22-26 g (male) and 20-25 g (female) and acclimated for 1 week to the test conditions (20 =/- 3 degrees C, 30-70% relative humidity, 12 hour light/dark cycle). Food was given daily and water was ad libitum. All animals were healthy at the time of test initiation.

Route of administration:

oral: gavage

Vehicle:

NaCl

Duration of treatment / exposure:

72 hours

Frequency of treatment:

single dose

Dose / conc.:

1 122 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

40 males and 40 females per dose

Control animals:

yes

Positive control(s):

Endoxan (cyclophosphamid)

Tissues and cell types examined:

Cells were taken from the thigh.

Details of tissue and slide preparation:

Cells were mixed with cattle serum and suspended, then centrifuged. The sediment was then resuspended. The suspension was seperated in a cellulose chromatography column. This was centrifuged, and mixed with fetal calf serum and EDTA. This was air-dried for 24 hrs and stained with Giemsa.

Evaluation criteria:

number of polychromatid erythrocytes (PCE)
ratio of PCE to normochromatid erythrocytes (NCE)
number of cells with micronucleus

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.

Conclusions:

The substance was negative for genotoxicity in the in vivo mammalian erythrocyte micronucleus test.

Executive summary:

In a NMRI mouse erythrocyte micronucleus assay (equivalent to OECD guideline 474), 40 mice/sex/dose were treated via oral gavage with benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (97.3% purity.) at doses of 0 or 1122 mg/kg bw. The vehicle was NaCl.

 

There were no signs of toxicity during the study. The test item was tested at an adequate dose. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes. Based on the results, the test item was therefore negative for genotoxicity in the in vivo mammalian erythrocyte micronucleus test.

Reference 2

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

Unavailable - original publication dated 1976

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Qualifier:

no guideline followed

Principles of method if other than guideline:

A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Dose / conc.:

1 170 mg/kg bw/day (nominal)

Remarks:

corresponding to 0.9% test material in diet

No. of animals per sex per dose:

5 males

Control animals:

yes

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 degree C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.

Evaluation criteria:

Presence and absence of chromosomal aberrations. 50 metaphases per individual.

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was noted.

Table 1: Chromosome aberrations

	0.9% in Diet	Control
No. of cells with chromatid breaks	1	2
No. of cells with isochromatid breaks	1	0
No. of cells with chromatid gaps	4	5
No. of cells with isochromatid gaps	0	0
No. of cells with other aberrations	0	0

Conclusions:

Under the experimental conditions reported, sodium 4-undecylbenzenesulfonate did not cause an increase in chromosome aberrations in an in vivo chromosome aberration test in mice. Therefore, the test item is considered to be non-clastogenic in this in vivo chromosome aberration assay.

Executive summary:

In a JCL-ICR mouse chromosome aberration assay, 5 males/dose were fed sodium 4-undecylbenzenesulfonate at doses of 0 or 0.9% (equivalent to 1170 mg/kg bw) of the test item in the diet for 9 months. At the end of this period, the animals were sacrificed, and the femur bone marrow cells examined for chromosome aberrations

 

Signs of toxicity were not specified during the study. Sodium 4-undecylbenzenesulfonate was tested at an adequate dose. There was not a significant increase in chromosome aberrations in bone marrow compared to the control group after the treatment. Based on the results, the test item is therefore considered to be non-clastogenic in this in vivo chromosome aberration assay.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

GLP compliance:

yes (incl. QA statement)

Type of assay:

micronucleus assay

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

Strain NMRI. Animals were approximately 22-26 g (male) and 20-25 g (female) and acclimated for 1 week to the test conditions (20 =/- 3 degrees C, 30-70% relative humidity, 12 hour light/dark cycle). Food was given daily and water was ad libitum. All animals were healthy at the time of test initiation.

Route of administration:

oral: gavage

Vehicle:

NaCl

Duration of treatment / exposure:

72 hours

Frequency of treatment:

single dose

Dose / conc.:

1 122 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

40 males and 40 females per dose

Control animals:

yes

Positive control(s):

Endoxan (cyclophosphamid)

Tissues and cell types examined:

Cells were taken from the thigh.

Details of tissue and slide preparation:

Cells were mixed with cattle serum and suspended, then centrifuged. The sediment was then resuspended. The suspension was seperated in a cellulose chromatography column. This was centrifuged, and mixed with fetal calf serum and EDTA. This was air-dried for 24 hrs and stained with Giemsa.

Evaluation criteria:

number of polychromatid erythrocytes (PCE)
ratio of PCE to normochromatid erythrocytes (NCE)
number of cells with micronucleus

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

No significant increases in the number of polychromatic erythrocytes with micronuclei were observed.

Conclusions:

The substance was negative for genotoxicity in the in vivo mammalian erythrocyte micronucleus test.

Executive summary:

In a NMRI mouse erythrocyte micronucleus assay (equivalent to OECD guideline 474), 40 mice/sex/dose were treated via oral gavage with benzenesulfonic acid, 4-C10-13-sec-alkyl derivs. (97.3% purity.) at doses of 0 or 1122 mg/kg bw. The vehicle was NaCl.

 

There were no signs of toxicity during the study. The test item was tested at an adequate dose. The positive control induced the appropriate response. There was not a significant increase in the frequency of micronucleated polychromatic erythrocytes. Based on the results, the test item was therefore negative for genotoxicity in the in vivo mammalian erythrocyte micronucleus test.

Reference 4

Endpoint:

in vivo mammalian germ cell study: cytogenicity / chromosome aberration

Type of information:

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

Adequacy of study:

weight of evidence

Justification for type of information:

Read-across approach - see read-across justification in section 13.

Reason / purpose for cross-reference:

read-across source

Principles of method if other than guideline:

A group of 5 male mice was fed a diet containing 0.9% test substance for 9 months. At the end of this period, the animals were sacrificed, and the bone marrow cells examined for chromosome aberrations.

GLP compliance:

no

Type of assay:

mammalian germ cell cytogenetic assay

Species:

mouse

Strain:

other: JCL-ICR

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: CLEA Japan, Inc.
- Age at study initiation: 4 weeks
- Housing: individually, except during breeding
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 25 +/- 1
- Humidity (%): 55 +/- 5
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Details on exposure:

DIET PREPARATION
- Mixing appropriate amounts with (Type of food): feed powder CE-2

Duration of treatment / exposure:

9 months

Frequency of treatment:

daily

Dose / conc.:

1 170 mg/kg bw/day (nominal)

Remarks:

corresponding to 0.9% test material in diet

No. of animals per sex per dose:

5 males

Control animals:

yes

Tissues and cell types examined:

femur bone marrow cells

Details of tissue and slide preparation:

Animals were sacrificed by administration of 1 ml/kg of 1% colchine solution. Femurs were then removed, and bone marrow cells washed into centrifuge tubes. The cells were then treated with 0.075 M KCl solution at 37 degree C for 15 min, and then fixed with an acetic acid 1: ethanol 3 solution. Samples were then flame dried and treated with Giemsa.

Evaluation criteria:

Presence and absence of chromosomal aberrations. 50 metaphases per individual.

Statistics:

Rohrborn's method.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

not specified

Vehicle controls validity:

not applicable

Negative controls validity:

valid

Additional information on results:

No increase in chromosome aberrations was noted.

Table 1: Chromosome aberrations

	0.9% in Diet	Control
No. of cells with chromatid breaks	1	2
No. of cells with isochromatid breaks	1	0
No. of cells with chromatid gaps	4	5
No. of cells with isochromatid gaps	0	0
No. of cells with other aberrations	0	0

Conclusions:

Under the experimental conditions reported, sodium 4-undecylbenzenesulfonate did not cause an increase in chromosome aberrations in an in vivo chromosome aberration test in mice. Therefore, the test item is considered to be non-clastogenic in this in vivo chromosome aberration assay.

Executive summary:

In a JCL-ICR mouse chromosome aberration assay, 5 males/dose were fed sodium 4-undecylbenzenesulfonate at doses of 0 or 0.9% (equivalent to 1170 mg/kg bw) of the test item in the diet for 9 months. At the end of this period, the animals were sacrificed, and the femur bone marrow cells examined for chromosome aberrations

 

Signs of toxicity were not specified during the study. Sodium 4-undecylbenzenesulfonate was tested at an adequate dose. There was not a significant increase in chromosome aberrations in bone marrow compared to the control group after the treatment. Based on the results, the test item is therefore considered to be non-clastogenic in this in vivo chromosome aberration assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Taken together, all the above information, indicate that LAS IPA is likely to be not genotoxic.

The endpoint of genotoxicity is addressed with a weight of evidence approach. Therefore, one key study is not applicable. The database is considered sufficient to fulfil the specific requirement.

Justification for classification or non-classification

Based on the weight of evidence findings of in vitro and in vivo genetic toxicity studies employing both the substance LAS IPA and read-across substances, IPA and LAS Na, LAS IPA does not need to be classified for genotoxicity according to the Regulation (EC) No 1272/2008.