1-(3,5-dihydroxyphenyl)-2-[[2-(4-hydroxyphenyl)-1-methylethyl]amino]ethan-1-one hydrobromide

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance gave positive results for genotoxicity in the three Ames assays (with and without metabolic activation). It was positive for mutagenicity in the in vitro mammalian cell gene mutation test with L5178Y mouse lymphoma cells. However, it was negative in the vitro chromosome aberration test with human lymphocytes.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

31 July 2003 to 18 September 2003

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test substance was not reported. Batch No: 0003A/RCC 0122A.
- Expiration date of the lot/batch: October 2004
- Purity test date: 10 October 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark (ambient humidity).
- Stability under test conditions: Not reported
- Solubility and stability of the test substance in the solvent/vehicle: Solutions were freshly prepared for each experiment. The stability of the test substance can be assumed.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium:Not applicable.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Solutions were freshly prepared for each experiment.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Target gene:

Strain TA1537 (his C3076)(no R factor) detects frameshift mutations
Strain TA98 (his D3052) R factor pKM101 detects frameshift mutations
Strain TA100 (hisG46) R factor pK,101 detects basepair substitutions
Strain TA 1535 (his G46) (no R factor) detects basepair subsitutions
Strain TA 102 (hist G428) R factor pKM101, pAQ1 detects basepair substitutions.

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

100, 300,1000, 3000, 5000 µg/plate with and without metabolic activation (experiment 1)
500, 1000, 1500 µg/plate with and without metabolic activation (experiment 2, only with strain TA 1537)
All concentrations are given as free base equivalent.

Vehicle / solvent:

- Vehicle(s) used: DMSO and dist. water

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

in experiment 2, two independent experiments were conducted, one with DMSO as solvent and as solvent control and one with distilled water as solvent and as solvent control.

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

mitomycin C

other: 2-aminoanthracene

Remarks:

without S9: 2-nitrofluorene, sodium azide, mitomycin C, 9-aminoacridine; with S9: 2-aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

EXPOSURE DURATION: 48 hrs

NUMBER OF REPLICATIONS: 3 each per concentration level and positive control. 6 per vehicle control


DETERMINATION OF CYTOTOXICITY
- Method: bacterial background lawn, microcolonies

Evaluation criteria:

A reproducible, concentration-dependent increase in the number of revertants of at least one tester strain over the vehicle control value and/or outside the historical control range is indicative of genotoxic activity. Since the results were unequivocal, no detailed statistical analysis was performed.

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Key result

Species / strain:

other: S. typhimurium TA 1535, TA 98 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

TA 98 without metabolic activation, TA 102 with metabolic activation

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

No precipitation was observed up to the top concentration tested. There was, however, a concentration-dependent and strain-specific bacteriotoxicity, as seen by a reduced background lawn and/or a decrease of absolute revertants.

 

Mutagenicity

The test item did not increase consistently the number of revertant colonies in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the concentration range from 100 to 5000 µg/plate (free base) compared to the negative control in the standard plate test. There was no substantial difference in the mutation frequency in the presence and absence of metabolic activation.

There was a slight mutagenic response (up to 1.6-fold) observed in S. typhimurium TA 100 (nonactivation) at 1000 µg/plate. A marked increase of revertants (up to 22.7-fold) was seen in TA 1537 (with metabolic activation) at 500 µg/plate and above, using the standard plate test, although no dose response was observed. The effect was more pronounced in the absence of metabolic activation. This positive result in TA 1537 was confirmed in a repeat assay.

All revertant counts were within the historical background data. The positive controls caused the expected mutagenic response showing the sensitivity and validity of the assay.

Table 1: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). Without metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO	10	11	32	77	301
100	10	90	26	84	289
300	11	153	38	91	315
1000	16	250	17 T	127	308
3000	18	1 T	0 T	51 T	343
5000 T	0	0	0	0	231
NaN3 NaN3 5	538	-	-	703	-
9-AA 50	-	238	-	-	-
2-Nf 10	-	-	488	-	-
MMC 0.5	-	-	-	-	737

Table 2: Mutagenic activity of the test item. Mean values Experiment 1 (Plate incorporation). With metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium
	TA 1535	TA 1537	TA 98	TA 100	TA 102
DMSO	15	13	34	82	285
100	15	15	35	89	289
300	12	23	40	90	334
1000	15	49	39	76	232
3000	16	31T	50	89	219 T
5000	14	18 T	31	68 T	50 T
9-AA 4	257	215	797	985	-
9-AA 10					1131
P: Precipitation; T: Toxic; -: Not used; Underlined values are regarded as increased
Historical Range	8 - 15	5 -21	32 - 54	49 - 119	285 - 418

Table 3: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). Without metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium TA 1537
	DMSO	Dist. Water
Control	9	8
500	144	126
1000	164	151
1500	147	137
9-AA 50	187	186

Table 4: Mutagenic activity of the test item. Mean values Experiment 2 (Plate incorporation). With metabolic activation

Compound (µg/plate)	Mean Revertants/Plate
	S. typhimurium TA 1537
	DMSO	Dist. Water
Control	11	9
500	19	30
1000	27	34
1500	27	44
9-AA 4	172	169
Historical Range	5 - 21

Conclusions:

Based on the results of this study it is concluded that the test substance, when tested up to bacteriotoxic concentrations, caused no mutations in the tester strains S. typhimurium TA 1535, TA 98 and TA 102 in the presence and absence of metabolic activation. However, there was a weak increase of revertants in TA 100 and a reproducible mutagenic response in S. typhimurium TA 1537.
Based on the results of this study, it was concluded that the test substance was mutagenic.

Reference 2

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

28 July 2003 to 20 January 2004

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Version / remarks:

1997

GLP compliance:

yes

Type of assay:

in vitro mammalian chromosome aberration test

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test item was not provided. Batch No: 0003A/RCC 0122A.
- Expiration date of the lot/batch: Retest date: October 2004
- Purity test date: 10 October 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark (ambient humidity).
- Stability under test conditions: The test item was dissolved in the vehicle immediatley before use. The stability of the test substance solutions during the exposure can be assumed.
- Solubility and stability of the test substance in the solvent/vehicle: No details reported.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test item was dissolved in the vehicle immediatley before use
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Target gene:

Not applicable

Species / strain / cell type:

other: human lymphocytes

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10, 30, 100, 300, 1000, 1500, 2000 µg/mL (free base equivalent)

Vehicle / solvent:

- Vehicle used: DMSO
- Justification for choice of solvent/vehicle: DMSO was selected because the test item showed limited solubility in water

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

other: adriamycin/ADR

Details on test system and experimental conditions:

DURATION
- Exposure duration: Experiment I, 4 hours with and without metabolic activation; Experiment II, 24 hours without
- Expression time (cells in growth medium): mitogen-stimulation time, 48 hours
- Fixation time (start of exposure up to fixation or harvest of cells): Experiment I and II, harvesting time 72 hours; Experiment II, delayed harvesting time 96 hours.

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (added two hours prior to harvest)
STAIN (for cytogenetic assays): Giemsa stain

NUMBER OF REPLICATIONS: 2

NUMBER OF CELLS EVALUATED: 200 cells/concentration

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index (MI)

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

Evaluation criteria:

The clastogenic potential of the test compound was evaluated by an increase in the percentage of cells showing structural chromosomal aberrations excluding gaps. A positive response is defined, if there is a reproducible and concentration dependent increase in the aberration frequency in the exposed cultures. Comparisons are made with the negative control values (vehicle) in the respective test. Additionally, historical control frequencies obtained in similar lymphocyte experiments are also taken into consideration. Historical data may prove useful in deciding whether effects not observed or observed at a low incidence in the particular culture resulted by chance or were treatment-related.
Generally, an assay will be considered acceptable for evaluation if the vehicle control data were within historical ranges and if positive controls showed significant increases in cells with chromosomal aberrations.

Statistics:

The statistical calculation were performed by the Group Biostatistics, Dept. Medical Data Services, Biberach. The percentages of aberrant cells of the treated cultures were compared with the concurrent control means using the 2-sided Fisher's Exact Test without an adjustment for multiple comparisons. A probability of P<= 5% was considered statistically significant.

Species / strain:

other: cultured human lymphocytes

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: no
- Definition of acceptable cells for analysis: At least 3 concentrations were selected for chromosomal analysis on the basis of solubility, mitotic inhibition and/or metaphase quality.

HISTORICAL CONTROL DATA
- Positive historical control data: yes
- Negative (solvent/vehicle) historical control data: yes

The negative and positive controls used in this study fulfilled the requirements for a valid test. The test item was tested up to clearly cytotoxic concentration levels as shown by mitotic inhibition and/or morphological cellular changes. The results showed that non-toxic concentrations of the test item did not induce a reproducible increase of structural chromosomal aberrations and polyploid cells in human peripheral lymphocytes in vitro when tested in the presence and absence of metabolic activation. However, at the toxic concentration of 1000 µg/mL, the test item induced a significant increase of aberrant cells excl. gaps incl. chromosomal rearrangements compared with the concurrent negative control. There is convincing evidence that compounds which are clastogenic only at highly cytotoxic concentrations (high toxicity clastogens) do not have the same biological importance as those inducing chromosomal aberrations at low levels of cytotoxicity, e.g. low toxicity clastogens. This conclusion is also supported by negative oral bone marrow micronucleus tests (IUCLID section 7.6.2).

Summary Table - Structural Chomosome Aberrations (% Aberrant Cells excl. Gaps) without Metabolic Activation

Compound	Cult	Treatment 4 hrs	Treatment 24 hrs
(µg/mL)	No.	Harvest: 72 hr	72 hr	96 hr
Controls:
Negative: DMSO	A	0	0	2.0
	B	1.0	3.0	1.0
	Mean	0.5	1.5	1.5
Positive: ADR 0.05	A	14.0	17.0	35.0
	B	18.0	10.8	34.0
	Mean	16.0^* P<0.01	14.2^* P<0.01%	34.5^* P<0.01%
MEN 1616 free base	A		1.0
30	B		2.0
	Mean	na	1.5 P=100%	nd
100	A	2.0	2.0
	B	1.0	1.0
	Mean	1.5 P=62.3%	1.5 P=100%	na
300	A	1.0	2.0
	B	0	1.0
	Mean	0.5 P=100%	1.5 P=100%	na
1000t	A	3.0		3.0
	B	7.0		8.0
	Mean	5.0^* P=1.1%	ne	5.5 P=5.3%
1500	A
	B
	Mean	nd	ne	ne
2000t	A
	B
	Mean	ne	nd	nd

t: toxicity (mitotic inhibition and/or cellular toxicity)

nd: not done; na: not analyzed; ne: not evaluable

*: Significantly different from the vehicle control (P <=5%)

Historical negative control values (% aberrant cells excl. gaps): 0.8 (range 0 -4)

Summary Table - Structural Chromosome Aberrations (% Aberrant Cells excl. Gaps) with Metabolic Activation

Compound	Cult.	Treatment 4 hrs
(µg/mL)	No.	Harvest: 72 hr
Controls:
Negative: DMSO	A	1.0
	B	1.0
	Mean	1.0
Positive: CP 7	A	26.0
	B	34.0
	Mean	30.0^* P<0.01
MEN 1616 free base	A	1.0
100	B	1.0
	Mean	1.0 P=100%
300T	A	2.0
	B	0
	Mean	1.0 P=100%
1000T	A	9.0
	B	4.0
	Mean	6.5^* P=0.6%
1500T	A
	B
	Mean	ne

T: toxicity (mitotic inhibition and/or cellular toxicity)

ne: not evaluable

*: Significantly different from the vehicle control (P<=5%)

Historical negative control values (% aberrant cells excl. gaps): 0.6 (range 0 -3)

Conclusions:

Based on the results of this study, the test substance is not considered to be clastogenic with and without metabolic activation.

Reference 3

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Study period:

20 September 2000 to 09 October 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test item was not reported. Batch No: 228.
- Expiration date of the lot/batch: 01 June 2001
- Purity test date: No details reported.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Stable. No further details reported.
- Solubility and stability of the test substance in the solvent/vehicle: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

FORM AS APPLIED IN THE TEST (if different from that of starting material)

TYPE OF BIOCIDE/PESTICIDE FORMULATION (if applicable)

OTHER SPECIFICS:

Target gene:

TK locus (forward mutation)

Species / strain / cell type:

mouse lymphoma L5178Y cells

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

1, 3, 10, 33, 100, 180, 333, 420 and 560 µg/mL (with and without metabolic activation)

Vehicle / solvent:

- Vehicle(s): DMSO

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

N-dimethylnitrosamine

ethylmethanesulphonate

Details on test system and experimental conditions:

METHOD OF APPLICATION: Culture medium

DURATION
- Exposure duration: 3 hours
- Expression time (cells in growth medium): 3 days
- Selection time (if incubation with a selection agent): 10 days

SELECTION AGENT (mutation assays): trifluorothymidine (TFT)

NUMBER OF REPLICATIONS: 1 dose range finding test and 1 mutagenicity test. No details on the number of replicates were reported.

DETERMINATION OF CYTOTOXICITY
- Method: cell survival

Evaluation criteria:

A mutation assay was considered acceptable if it met the following criteria: a) The absolute cloning efficiency of the solvent controls was >= 50%; b) In at least seven of the eight doses of the test substance, an acceptable number of surviving cells (10exp6) could be analysed for expression of the TK mutation; c) the spontaneous mutant frequency in the untreated or solvent control was < 10 per 10exp5 clonable cells; d) the positive controls (ethylmethanesulfonate and dimethylnitrosamine) induced significant (at least three fold) increases in the mutant frequencies.

Statistics:

No formal hypothesis testing was done. A test substance was considered positive (mutagenic) in the mutation assay if: It induced at least a 3-fold increase in the mutant frequency compared to the solvent control in a dose-dependent manner.
A test substance was considered negative (not mutagenic) in the mutation assay if: None of the tested concentrations showed a mutant frequency of at least three-fold compared to the solvent control.
The preceding criteria were not absolute and other modifying factors might enter into the final evaluation decision.

Species / strain:

mouse lymphoma L5178Y cells

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Effects of pH: no effects
- Effects of osmolality: no effects
- Precipitation: yes (at 3330 µg/mL in the range finding test)


RANGE-FINDING/SCREENING STUDIES:
Test concentration: 33, 100, 333, 1000, 3330 µg/mL
Without metabolic activation, the toxicity in the suspension growth was 32% at 333 µg/mL compared to the suspension growth of the solvent control. No cell survival was observed at the test substance concentrations of 1000 and 3330 µg/mL after 24 hours of subculture.
With metabolic activation, the toxicity in the suspension growth was 23% at 333 µg/mL compared to the suspension growth of the solvent control. Hardly any cell survival was observed at 1000 µg/mL after the 3 hours treatment and no cells survived the 3 hours treatment period at 3330 µg/ml.

Evaluation of the toxicity

In the absence of S9 metabolic activation, the dose level of 1 µg/ml was not regarded relevant for mutant frequency measurement. The dose level of 750 µg/ml was not used for mutant frequency measurement, since this dose level was too toxic for further testing. In the presence of S9 metabolic activation, the dose levels of 1 and 3 µg/ml were not regarded relevant for mutant frequency measurement.

Dose levels selected to measure mutant frequencies at the TK-locus:

3, 10, 33, 100, 180, 333, 420 and 560 µg/ml (without metabolic activation)

10, 33, 100, 180, 333, 420, 560 and 750 µg/m (with metabolic activation)

In the absence of S9 -mix, after 3 hours treatment the actual cell survival of the highest test substance concentration was reduced by more than 99% compared to the actual cell survival of the solvent controls.

In the presence of S9 -mix, after 3 hours treatment the actual cell survival of the highest test substance concentration was reduced by 96% compared to the actual cell survival of the solvent controls.

Evaluation of the mutagenicity

TH 1165 Keton II induced 5.3 -and 8.6 -fold, dose related increases in the mutant frequency at the TK-locus in the absence and presence of S9 -mix respectively.

CYTOTOXIC AND MUTAGENIC RESPONSE OF IN THE MOUSE LYMPHOMA L5178Y TEST SYSTEM

Dose (µg/ml)	Cell count after treatment % of controls	CE after treatment % of controls	Actual survival of the cells % of controls (1)	CE at day 3 absolute %	Total no. of wells with mutants   Total ( small large)	Mutation frequency x 10^5   Total ( small large)
Without metabolic activation
SC1	100	100	100	121	30 (12s 18l)	2.7 (1.1s 1.6l)
SC2				131	30 (16s 14l)	2.5 (1.3s 1.2l)
3	69	96	66	145	29 (17s 12l)	2.2 (1.3s 0.9l)
10	86	90	77	127	36 (8s 28l)	3.1 (0.7s 2.4l)
33	93	75	70	165	48 (17s 31l)	3.2 (1.1s 2.1l)
100	88	63	55	157	50 (15s 35l)	3.6 (1.1s 2.5l)
180	82	65	53	131	34 (16s 18l)	2.8 (1.3s 1.5l)
333	67	50	34	127	57 (19s 38l)	5.1 (1.7s 3.4l)
420	66	29	19	98	97 (37s 60l)	11.9 (4.5s 7.4l)
560	11	2	<1	66	78 (25s 53l)	13.8 (4.4s 9.4l)
EMS	86	62	53	123	206 (31s 175l)	25.0 (3.8s 21.2l)
With 8% (v/v) metabolic activation
SC1	100	100	100	125	16 ( 4s121)	1.4 (0.4s 1.1l)
SC2				137	23 ( 4s 19l)	1.8 (0.3s 1.5l)
10	98	90	88	143	21 ( 7s 14l)	1.6 (0.5s 1.1l)
33	94	92	86	123	57 (16s 41l)	5.2 (1.5s 3.7l)
100	109	87	95	120	43 (10s 33l)	4.0 (0.9s 3.1l)
180	87	108	94	133	69 (22s 47l)	6.0 (1.9s 4.1l)
333	98	65	64	118	70 (22s 48l)	6.8 (2.1s 4.7l)
420	84	83	70	152	72 (25s 47l)	5.5 (1.9s 3.6l)
560	86	29	25	152	114 (31s 83l)	9.3 (2.5s 6.8l)
750	57	7	4	98	110 (30s 80l)	13.8 (3.8s 10.0l)
DMN	91	70	64	80	144 (43s 101l)	23.5 (7.0s 16.5l)

 

CE = Cloning Efficiency; EMS=Ethylmethanesulphonate; DMN = Dimethylnitrosamine; s = small colonies; I = large colonies

SC = Solvent control = dimethylsulfoxide

(1) The actual survival of cells (% of controls) = the cell count after treatment (% of controls) x the CE after treatment (% of controls)

Conclusions:

The test substance induced 5.3 and 8.6 -fold, dose related increases in the mutant frequencey at the TK-locus in the absence and presence of S9 -mix respectively. Based on the results of this study, the test substance is mutagenic in the TK mutation test system.

Reference 4

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

08 December 1993 to 03 June 1994

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

Only four test strains (TA1535, TA1537, TA100 and TA98) were tested

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1992

Deviations:

yes

Remarks:

Only four test strains (TA1535, TA1537, TA100 and TA98) were tested

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Two batches of test material were used in this study (sources not provided). The batches are referred to as 'Batch B' and 'Batch GD 240194'.
- Expiration date of the lot/batch: Batch B - January 1994. Batch GD 240194- August 1995
- Purity test date: Batch B - 4 November 1992. Batch GD 240194 - 3 November 1994.


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Ambient temperature in the dark
- Stability under test conditions: The solution was freshly prepared for each experiment.
- Solubility and stability of the test substance in the solvent/vehicle: No details reported.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The solution was freshly prepared for each experiment.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Target gene:

Strain TA1535 (his G46)
Strain TA1537 (his C3076)
Strain TA100 (hisG46, Rfactor)
Strain TA98 (his D3052 Rfactor).

Details on the types of mutation that each gene was investigating were not provided in the report.

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10, 100, 500, 1500, 5000 µg/plate (Experiment)
100, 500, 1500, 3000, 5000 µg/plate (Experiment)
1500, 3000, 5000 µg/plate (Extension study)
100, 500 µg/plate (Additional test )

Vehicle / solvent:

Solvent used: DMSO (positive controls); dist. water (9-Aminoacridine, test item)

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

other: 2-aminoanthracene; 1-methyl-3-nitro-1-nitrosoguanidine;

Details on test system and experimental conditions:

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

DURATION
- Preincubation period: 0, 1, 2 and 3 days, respectively if preincubated
- Exposure duration: 48 hours (37°C in the dark)

NUMBER OF REPLICATIONS: 5

Evaluation criteria:

The absolute numbers of revertant colonies on the plates, with and without substance, were compared to evaluate mutagenicity.

Statistics:

Not necessary to perform as there is a clear and dose-dependent enhancement of the mutation frequencies after the treatment with 100 µg/plate and more of the test substance in both the absence and presence of S9 mix (observed in at least one tester strain).

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

other: bacteriotoxic in strain 1537 in the abscence of metabolic activation at the highest concentration

Vehicle controls validity:

valid

Positive controls validity:

valid

The test substance poured onto minimal medium plates in amounts >= 500 (without S9 mix) or >= 1500 (with S9 mix) µg resulted in the formation of a brown-tinted agar medium at the end of the 2- or 3-day incubation at 37°C (whereas the yellowish stock solution at 50 mg/ml the test item didn't show such an alteration after a 3-day stay at 37°C). The test item was bacteriotoxic in strain TA 1537 in the absence of S9 mix at the highest concentration tested.

The test substance was bacteriotoxic in strain TA 1537 without metabolic activation at the highest concentration tested. No mutagenicity of the test item was observed in strain TA1535. But increases in mutation frequencies were seen at concentrations >= 100 µg/plate, both without and with S9 mix, in the strains TA1537, TA100 and TA98. Of these three tester strains, the highest mutagenic response was found in strain TA1537, and the results show a pronounced dose-dependent increase in mutation frequency between 100 and 3000 µg/plate and the confirmation of the clear mutagenic activity with batch the second batch.

Differences in the stability of the bacterio-mutagenic principle could be detected in aqueous test item solutions preincubated at 37°C in the dark: nearly no decrease of the mutagenic activity in water up to 3 days and loss of the clear mutagenic effect after 1 day in “Vobel-Bonner Medium E (10x)” salt solution.

Conclusions:

It was concluded that the test substance was mutagenic in the standard plate incorporation assay (strains TA 1537, TA100 and TA98); both with and without metabolic activation.Negative results for mutagenicity were reported in strain TA1535. Based on the results of this study, it was concluded that the test susbtance was mutagenic in the standard plate incorporation assay; both with and without metabolic activation.

Reference 5

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

16 November 1999 to 18 April 2000

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

1997

Deviations:

yes

Remarks:

The study was performed only with strain T98 and strain TA100

Qualifier:

according to guideline

Guideline:

other: EU Method B 14

Version / remarks:

Directive 92/69. 1992

Deviations:

yes

Remarks:

The study was performed only with strain T98 and strain TA100

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5265 (The Salmonella typhimurium Bacterial Reverse Mutation Test)

Version / remarks:

1996

Deviations:

yes

Remarks:

The study was performed only with strain T98 and strain TA100

GLP compliance:

yes (incl. QA statement)

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material:No details on the source of the test material were provided. Batch number: 206.
- Expiration date of the lot/batch: Not reported
- Purity test date: 15 June 1999

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature, protected from light.
- Stability under test conditions: Stable
- Solubility and stability of the test substance in the solvent/vehicle: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing:The formulations were prepared within 4 hours before dosing. Homogeneity was accimplished to a visually acceptable level. Adjustment was made for specific gravity of the vehicle.
- Preliminary purification step (if any) No details reported.
- Final dilution of a dissolved solid, stock liquid or gel. No details reported.
- Final preparation of a solid:No details reported.

Target gene:

Strain TA98 characterises the histidine mutation 'hisD3053/R-factor8' for frameshift mutations
Strain TA100 characterises the histidine mutation 'hisG46/R-factor' for base pair substitutions.

Species / strain / cell type:

S. typhimurium TA 98

Species / strain / cell type:

S. typhimurium TA 100

Metabolic activation:

with and without

Metabolic activation system:

S9 mix

Test concentrations with justification for top dose:

10.0, 31.6, 100.0, 316.2, 1000.0, 2500.0 and 5000.0 µg/plate (TA 100 und TA 98, experiment I and II with and without S9 mix)
100.0, 200.0, 400.0, 600.0, 800.0 and 1000.0 µg/plate (TA 100, experiment III; without S9 mix)
500.0, 1000.0, 2000.0, 3000.0, 4000.0 and 5000.0 µg/plate (TA 100, experiment III; with S9 mix)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

other: 2-aminoanthracene; 4-nitro-o-phenylene-diamine

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation, experiment I and III; preincubation, experiment II)

EXPOSURE DURATION: 48 hours (37°C in the dark)

NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: backgound lawn

Evaluation criteria:

A test item is considered mutagenic if:
- a dose-related increase in the number of revertants occurs and/or
- a reproducible biologically relevant positive response for at least one of the test points occurs
in at least one strain with or without metabolic activation.
A test item producing neither a dose related increase in the number of revertants nor a reproducible biologically relevant positive response at any of the test points is considered non-mutagenic in this system.
A biologically relevant increase is described as follows:
- if in test strain TA 100 the number of reversions is at least twice as high
- if in test strain TA 98 the number of reversions is at least three times higher
as compared to the spontaneous reversion rate.

Statistics:

The biological relevance of the results will be the criterion for the interpretation of results, a statistical evaluation of the results was not regarded necessary.

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

without

Genotoxicity:

positive

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Positive controls validity:

valid

In experiment I and II without metabolic activation, the test item was toxic in both tester strains at the higher concentrations. With metabolic activation no toxicity was observed in either tester strain in any experiments. In experiment III in which the dose selection was carried out according to the results of experiment I, no toxic effects were observed up to the highest investigated concentration (1000 µg/plate without metabolic activation, 5000 µg/plate with metabolic activation).

In strain TA 100 a reproducible and biologically relevant increase in revertant colony numbers was obtained in the presence of metabolic activation in the plate incorporation test (experiment I and III). Without metabolic activation a slight increase in revertant colony numbers (factor 1.7) was obtained in experiment I at 316.2 µg/plate in TA 100. After adjusting the concentrations in order to select a more narrow concentration range, a clear dose-dependent and distinct increase was obtained in experiment III (reaching a factor 2.3 at the highest concentration).

As positive controls reference mutagens were tested in parallel to the test item. They showed a distinct increase of induced revertant colonies.

Conclusions:

Based on the results of this study, it is concluded that the test substance is mutagenic in the Salmonella typhimurium reverse mutation assay (Strain TA100 only). Negative results for mutatgenicity were reported in strain TA98.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed (positive)

Genetic toxicity in vivo

Description of key information

The test substance was tested negative in all of the three in vivo micronucleus tests (one key, two supporting studies). The key study was conducted in combination with an in vivo comet assay using liver cells, which also showed negative results.

Link to relevant study records

Reference

Reference 1

Endpoint:

genetic toxicity in vivo, other

Remarks:

in vivo combined mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and comet assay

Type of information:

experimental study

Adequacy of study:

key study

Study period:

03 December 2013 to 05 March 2014

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

other: In vivo Mammalian Alkaline Comet Assay - Draft OECD Guideline for the Testing of Chemicals

Qualifier:

according to guideline

Guideline:

other: ICH S2 (R1)

Version / remarks:

2011

GLP compliance:

yes

Type of assay:

other: in vivo combined mammalian somatic cell study: cytogenicity / erythrocyte micronucleus and comet assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test substance was not reported. Batch No: 236/2013
- Expiration date of the lot/batch: 28 Feb 2015
- Purity test date: No details reported.

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Determined by the Sponsor. No further details reported.
- Stability under test conditions: Determined by the Sponsor. No further details reported.
- Solubility and stability of the test substance in the solvent/vehicle: Suspensions of the test item were freshly prepared before testing.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No details reported.



TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: Suspensions of the test item were freshly prepared before testing.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD), Italy
- Age at study initiation: 8-10 weeks
- Weight at study initiation: approximately 300 grams
- Assigned to test groups randomly: not specified
- Fasting period before study: not specified
- Housing: up to 5 animals/cage, in polisulphone H-temp solid bottomed cages with nesting material provided into suitable bedding bags
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C +- 2°C
- Humidity (%): 55% +- 15%
- Photoperiod (hrs dark / hrs light): 12 hour light/ 12 hour dark

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: 0.5% methylcellulose
- Justification for choice of solvent/vehicle: based on results obtained from solubility trials and the preliminary tests

Details on exposure:

Test item:
Treatment: three times
Dose volume: 20 mL/kg body weight.

Positive control (comet assay):
Treatment: twice
Dose volume: 10 mL/kg body weight.

Positive control (micronucleus assay):
Treatment: once
Dose volume: 10 mL/kg body weight.

A reserve group of 3 males was dosed at the high dose level to substitute animals dying during the treatment period. Moreover, a satellite group of 3 males was included for blood sampling.

Duration of treatment / exposure:

- Animals were sacrifced 3-4 hours after last dosing (test item group and positive control group of comet assay)
- Animals were sacrifced 24 hours after dosing (positive control group micronucleus assay)

Frequency of treatment:

test item: at 0 (Day 1), 24 (Day 2) and 45 (Day 3) hours
positive control (comet assay): 21 hour interval
positive control (micronucleus assay):once

Dose / conc.:

500 mg/kg bw/day (nominal)

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Dose / conc.:

2 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

5 males

Control animals:

yes, concurrent vehicle

Positive control(s):

Comet assay: EMS
Micronucleus assay: Mitomycin C:
- Route of administration: oral gavage (test item, positive control comet assay), intraperitoneal injection (positive control micronucleus assay)

Tissues and cell types examined:

Comet assay: liver tissue
Micronucleus assay: bone marrow

Details of tissue and slide preparation:

Collection of blood samples for exposure determination:
Before the first dosing, blood samples were collected pre-dose. Additional blood samples were collected 1 and 3 hours after dosing from 3 animals treated at the high dose level (satellite group). Approximately 1 mL of blood was collected from the tail of each animal and placed into a tube containing lithium heparin as anticoagulant. Blood samples were centrifuged at room temperature. The plasma was separated immediately after centrifugation and transferred into two separate pre-labelled tubes. Approximately 250 mL of plasma was transferred in the first set tube (aliquot A) and the remaining plasma in the second set tube (aliquot B). The plasma aliquots were stored at -80°C until analysis. Plasma analysis was performed by the Analytical Chemistry Department at RTC according to a validated method.

Comet assay:

Preparation of single cells from liver:
The animals were sacrifced at the appropriate time after last treatment by asphyxiation with carbon dioxide. From each animal, a section of liver was removed, cut into 3 or more pieces and gently agitated in a Petri dish full of ice-cold phosphate buffered saline (DPBS) containing 20 mM EDTA and 10% DMSO (mincing solution) to remove as much blood as possible. The sample was placed into ice-cold mincing solution and minced into finer pieces. The cell suspension was filtered and centrifuged at 500 rpm for 10 minutes at approximately +4°C. The pellet was resuspended in ice-cold mincing solution and the cell concentration was adjusted to a final concentration of approximately 1×106 cells/mL using mincing solution. The cell suspension was used for slide preparation. Prior to preparation of single cell suspensions, a sample of liver from each vehicle control and test item treated animal was taken. These samples were fixed and preserved in 10% neutral buffered formalin. No samples were retained from rats treated with the positive control solution.

Slide preparation:
For each cell suspension, 60 µL of cell suspension was added to 390 µL of 0.7% (w/v) low melting point agarose (LMA), pre-warmed at 37°C. An aliquot of 75 µL of each suspension was dispensed onto glass microscope slides pre-coated with a layer of 1% (w/v) normal melting point agarose (NMA). The slides were covered with a clean coverslip and the gels were allowed to solidify. The coverslips were removed and further 75 µL of LMA added over the cell layer and covered with a clean coverslip. The gels were allowed to solidify and when applied the coverslips were carefully removed. At least four slides were prepared. Theslideswereimmersedinfreshlypreparedpre-cooledlysissolutionovernight at approximately 4°C in the dark. After the lysis step, one slide from each animal was rinsed three times for 5 minutes in 0.4 M Tris, pH 7.5 and then dried and stored at room temperature. These ‘diffusion’ slides were used to estimate the degree of highly damaged cells in the cell suspensions. All slides, with the exception of the “diffusion” slides, were incubated for 25 minutes in alkaline (pH>13) electrophoresis buffer to produce single-stranded DNA and to express ALS and SSB. After alkali unwinding, electrophoresis was carried out at 1-10°C for 25 minutes, at 30 V (1 V/cm) and 300 mA, using a Bio-Rad power supply. Since not all slides can be processed at the same time, a block design was employed for the unwinding and electrophoretic steps in order to avoid excessive variation across the groups for each electrophoretic run. After electrophoresis, the slides were immersed in 0.3 M sodium acetate in ethanol for 30 minutes. Microgels were dehydrated in absolute ethanol for 2 hours and immersed for 5 min in 70% ethanol. Slides were air-dried and stored at room temperature. Immediately before scoring, slides were stained with 12 µg/mL ethidium bromide.

Micronucleus assay:

Slide preparation:
One femur of each animal was rapidly dissected out and cleaned of surrounding tissue. In order to extract the bone marrow, the bone was cut at the proximal end and irrigated with 2 mL foetal calf serum using a syringe. The suspension of cells was aspirated and this procedure was repeated several times. The cells were centrifuged and a concentrated suspension prepared. The suspension thus obtained was centrifuged at 1000 rpm for 5 minutes and the supernatant completely removed. The cells of the sediment were then resuspended and transferred onto clean microscope slides as smear preparations. They were air-dried overnight and then fixed with methanol for 10 minutes. Subsequently slides were stained with haematoxylin and eosin solutions. Finally slides were rinsed in distilled water and allowed to dry.

Evaluation criteria:

Comet Assay:

Acceptance criteria:
The assay is considered to be valid if the following criteria are met:
– The positive control treatment shows a statistically signifcant increase in comet parameters over the concurrent vehicle control.
– At least 4 animals per group are available for slide analysis.

Criterion for outcome of the assays:
For the test item to be considered positive, a significant increase (indicative of strand breaks) or decrease (indicative of cross-links) in DNA migration must be observed in any tissue evaluated, in at least one dose group. In addition there must be evidence of a dose-related increase or decrease in DNA migration measurements. It is important to consider any cytotoxic effect in conjunction with a positive response in DNA migration.

Micronucleus Assay:

Acceptance criteria:
The assay is considered to be valid if the following criteria are met:
- The incidence of micronucleated PCEs of the vehicle control group falls within the historical negative control range.
- The positive control item induces a significant increase in the frequency of micronucleated PCEs.
- At least 5 animals per group are available for slide analysis.

Criterion for outcome of the assays:
The test item is considered to induce micronuclei if a statistically significant and biologically meaningful increase in micronucleus incidence (at p<0.05) is observed in any treatment group. Where increases in the incidence of micronucleated PCEs are observed which are statistically signifcant, but fall within the range of negative control values of this laboratory, then these data are used to demonstrate that these increases do not have biological significance. Historical controls are available.

Statistics:

Comet assay:
DNA damage was evaluated as extent of DNA migration and the metric end points used were tail moment and tail intensity. The statistical significance of the differences amongst groups was assessed by analysis of variance followed by Dunnett’s or Cochran’s test.

Micronucleus assay:
Two thousand polychromatic erythrocytes (PCEs) per animal were examined for the presence of micronuclei. The results obtained were subjected to statistical analysis using a modifed Chi-squared test.

Key result

Sex:

male

Genotoxicity:

negative

Remarks:

Micronucleus Assay

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Key result

Sex:

male

Genotoxicity:

other: Comet assay: All treatments demonstrated low levels of cytotoxicity. Micronucleus assay: No inhibitory effect on erythropoietic cell division was induced by the test item in any treatment group.

Toxicity:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Comet assay:

Comet parameters of the vehicle control treatment are within historical control values with the exception of one animal which presented high values of tail intensity and tail moment. These values were markedly higher than those observed in animals of the same group or in animals from historical negative controls and were probably attributable to an e¿ect induced by mechanical reasons (e.g cell suspension preparation). No increases in tail migration were observed in any treatment group. Good responses were observed after treatment with the positive control item ethyl methanesulfonate, indicating the correct response of the comet test system.

At least four animals per group were available for slide analysis. The positive control ethyl methanesulphonate induced clear and statistically significant increases in DNA migration parameters over the concurrent controls. The study was accepted as valid. No evidence of a dose-related increase or decrease in DNA migration measurements was observed, nor statistically significant difference between vehicle and test item treated groups. Based on the stated criteria, the test was considered valid and the test substance was not considered positive in the comet assay.

Micronucleus assay:

An increase in the incidence of micronucleated PCEs over the control value was observed at the low dose level. The incidence was slightly above the range of historical values observed in our laboratory . No increases in the numbers of micronucleated PCEs were observed in other treatment groups. For one animal, possibly due to misdosing during Mitomycin-C administration, no increase of micronucleated PCEs was observed. The animal was excluded from the evaluation of data. Marked increases in the incidence of micronucleated PCEs were observed in other positive control group animals, indicating the correct functioning of the test system.

The incidence and distribution of micronucleated PCEs in vehicle control groups were consistent with the laboratory’s historical control data. A statistically significant increase in the incidence of micronucleated PCEs over the control values was seen in the positive control group, indicating the correct functioning of the test system. Five animals were available for slide analysis in all treatment groups. The study was accepted as valid. Following treatment with the test item, a statistically significant increase (p<0.05) in the incidence of micronucleated cells over the control was observed. The incidence slightly exceeded the range of historical values for negative controls.

Since the increase was only observed in the low dose group and was attributable to the incidence noted in one animal, it was considered to be due to a chance event and therefore not biologically relevant.

Conclusions:

It is concluded that the test substance did not induce DNA damage in the liver of male rats following oral gavage administration of doses of 500, 1000 and 2000 mg/kg/day. At these same dose levels, the test item did not induce significant increases in micronucleated polychromatic erythrocytes in the bone marrow.

Reference 2

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

01 August 1994 to 03 February 1995

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1983

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1992

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test substance was not reported. Batch No: GD 240194
- Expiration date of the lot/batch: August 1995
- Purity test date: 3 November 1994

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At ambient temperature in the dark
- Stability under test conditions: The test substance solutions were freshly prepared just prior to use.
- Solubility and stability of the test substance in the solvent/vehicle: No details reported.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance solutions were freshly prepared just prior to use.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Species:

rat

Strain:

other: Chbb:THOM

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Experimental Animal Breeding Station of Dr. Karl Thomae GmbH, Biberach/Riss, Germany
- Age at study initiation: 7 weeks
- Weight at study initiation: 210 - 242 g
- Housing: individually in Makrolon cages, type III
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 4 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22°C
- Humidity: from 50 % to 65 %
- Photoperiod (hrs dark / hrs light): 12h day/12h night light phase from 6.00 to 18.00 h

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: distilled water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
A dose volume of 25 ml/kg (test substance and vehicle) or 10 ml/kg (positive control substance) was administered orally by gavage. Two treatment (test substance) groups were used together with a vehicle and a positive (cyclophosphamide) control group. Sampling was performed at 24 and 48 h.

Duration of treatment / exposure:

24 and 48 hours, respectively

Frequency of treatment:

once

Post exposure period:

24 h (vehicle/positive control and test group)
48 h ( test group)

Dose / conc.:

1 800 mg/kg bw/day (nominal)

No. of animals per sex per dose:

4 males for test substance and 5 males per control (pos. and neg.)

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide (20 mg/kg bw)

Tissues and cell types examined:

Both femora from each animal were dissected out and freed of adherent tissue. The femur was cut medially with scissors and the bone marrow removed with a paint-brush and smeared on a slide: a red marten brush, size 2, was dipped into a suitable medium (phosphate-buffered saline salt solution), excess fluid wiped off on a tissue and the tip dipped into the open femur.

Details of tissue and slide preparation:

The bone marrow thus obtained was smeared in four parallel strokes across a clean slide.
The preparation was air-dried for 24 h, subsequently fixed for 10 min in absolute methanol and dried gently (up to 2 h) in a chamber which was moist to start with. The preparation was then air-dried for another 24 h. The subsequent staining was carried out in a darkened room in the following stages: phosphate buffer pH 6.8; acridine orange solution; phosphate buffer pH 6.8, renewed phospate buffer pH 6.8.
Immediately afterwards the damp slides were mounted using the phosphate buffer. Two slides were made per animal (1 slide/femur).

Evaluation criteria:

2000 polychromatic erythrocytes were counted per animal and the number of micronucleated cells determined. Simultaneously, the number of normochromatic erythrocytes with micronuclei was checked in the field analyzed. The ratio of polychromatic to normochromatic cells was determined for each animal by counting a total of 2000 erythrocytes.

Statistics:

The number of micronucleated polychromatic erythrocytes in each treatment group was compared with the concurrent vehicle control by the one-sided Fisher-Pitman test. The exact p-values were calculated with the algorithm of Streitberg and Röhmel (1986). Statistical analysis was performed on an HP-Vectra using SAS, version 6.08.

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Positive controls validity:

valid

After dosing, the animals of the test substance groups showed exophthalmos.

The test item did not cause an increase in the frequency of micronucleated polychromatic erythrocytes at any treatment time. The incidence of micronucleated polychromatic erythrocytes of the animals treated with the test item ranges from 0 -3.0% (control 0 -2.0%). These results are within the normal range of the vehicle control obtained in this laboratory. In contrast to this, cyclophosphamide significantly increased the frequency of micronucleated polychromatic erythrocytes.

The frequencies of micronucleated normochromatic erythrocytes were not increased above the usual low level.

The slight change in the ratio of polychromatic to normochromatic erythrocytes observed in the test item/24 h-treatment group (38.2% polychromatic erythrocytes vs. 50.2% polychromatic erythrocytes in the vehicle control group) was an indication of moderate cytotoxicity of the test substance.

Conclusions:

The test substance was found to be negative in the rat micronucleus assay.

Reference 3

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

15 January 2001 to 21 February 2001

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

2000

GLP compliance:

yes

Type of assay:

micronucleus assay

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: Source of the test substance was not reported. Batch No: 228.
- Expiration date of the lot/batch: 01 May 2001
- Purity test date: No details reported


STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature in the dark.
- Stability under test conditions: Stable. No further details provided.
- Solubility and stability of the test substance in the solvent/vehicle: Stability of the vehicle was not indicated by the Sponsor.
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: No details reported.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: No details reported.
- Preliminary purification step (if any): No details reported.
- Final dilution of a dissolved solid, stock liquid or gel: No details reported.
- Final preparation of a solid: No details reported.

Species:

mouse

Strain:

NMRI

Details on species / strain selection:

NMRI BR mice (SPF)

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River, Sulzfeld Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: from 30.3 to 31.4 g
- Assigned to test groups randomly: [no/yes, under following basis: ]
- Housing: group of 5 animals per cage (polycarbonate cage) containing purified sawdust
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature: 21 +- 3°C
- Humidity: 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 hours artifical fluorescent light/dark per day

Route of administration:

intraperitoneal

Vehicle:

- Vehicle(s) used: corn oil

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
TH test item was suspended in corn oil (OPG, Utrecht, The Netherlands). The test item concentrations were blended and treated with ultra-sonic waves to obtain a homogenous suspension and were dosed within 4 hours after preparation.
Dosing volume: 10 mL/kg bw

Duration of treatment / exposure:

24 and 48 hours, respectively

Frequency of treatment:

single intraperitoneal injection

Post exposure period:

24 h (vehicle control and test group)
48 h ( positive control and test group)

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

based on range finding test

No. of animals per sex per dose:

dose range finding: 2 males and 2 females per dose group
main test: 5 male mice per sampling time in each dose group

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide dissolved in physiological saline
- Route of administration: intraperitoneal injection
- Doses / concentrations: 50 mg salt/kg bw

Tissues and cell types examined:

- animals were sacrificed by cervical dislocation
- both femurs were removed and freed of blood and muscles
- both ends of the bone were shortened until a small opening to the marrow canal become visible
- the bone was flushed withh approx. 2 mL of foetal claf serum
- the cell suspension was collected and centrifuged at 1000 rpm (approx. 100 g) for 5 min

Details of tissue and slide preparation:

After centrifugation, the supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully mixed with the serum by aspiration with the remaining serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 h immersed in a 1:1 mixture of 96% (v/v) ethanol/ether and cleaned with a tissue) and marked (with the testing factlity's study identification number and the animal identification number). The drop was spread by moving a clean slide with round-whetted sides at an angle of approx. 45° over the slide with the drop of bone marrow suspension. The preparations were air-dried, fixed for 5 min in 100% methanol and air-dried overnight. Two slides were prepared per animal.

Staining of the bone marrow smears:
The slides were automatically stained using the 'Wright-stain-procedure' in an 'Ames' HEMA-tek slide stainer (Miles, Bayer Nederland B. V.). The dry slides were dipped in xylene before they were embedded in MicroMount and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei:
All slides were randomly coded before examination. An adhesive label with a study identification number and code was stuck over the marked slide. At first the slides were screened at a magnification of 100x for regions of suitable technical quality, i. e. where the cells were well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronucleated polychromatic erythrocytes was counted in 2000 polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were only counted in polychromatic erythrocytes. Averages and standard deviations were calculated.

Evaluation criteria:

Equivocal results should be clarified by further testing using modification of experimental conditions.
A test substance is considered positive in the micronucleus test if:
- It induced a biologically as well a statistically significant (Wilcoxon Rank Sum Test; two-sided test at P < 0.005) increase in the frequency of micronucleated polychromatic erythrocytes.
A test substance is considered negative in the micronucleus test if:
- None of the tested concentrations or sampling times showed a statistically significant (P < 0.05) increase in the incidence of micronucleated polychromatic erythrocytes.
The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

A Wilcoxon Rank Sum Test was used as part of the evaluation criteria. No further details reported.

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Micronucleus test:
- The animals of the groups which were treated with the test item showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis.

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000, 500, 250 mg/k (2 males and two females per test group)
- Clinical signs of toxicity in test animals: in the highest test group of 2000 mg/kg, all animals died on day 2. In test group 500 mg/kg one animal died on day 2 and two animals on day 4. Ataxia and lethargy was found in all test groups at different times. Ventro-lateral recumbency, rough coat, ptosis, was observed in test group 250 and 500 mg/kg. Hunched posture and hypothermia was observed only in the test group of 500 mg/kg.
- Based on the results of the range finding study, the dose level of 250 mg/kg body weight was selected as appropriate dose for the micronucleus test.

Mortality and systemic toxic signs:

The following clinical observations were made in the groups treated with 250 mg/kg bw: During the first hour after dosing all animals were lethargic. Within 20 hours after dosing all animals were lethargic, had a rough coat and showed a hunched posture. Within 44 hours after dosing, one animal died and the other animals were still lethargic, had a rough coat and showed a hunched posture.

 

Micronucleated polychromatic erythrocytes:

No increase in the frequency of micronucleated polychromatic erythrocytes was observed in the polychromatic erythrocytes of the bone marrow of the treated animals compared to the vehicle treated animals.

The incidence of micronucleated polychromatic erythrocytes in the bone marrow of all negative control animals were within the historical solvent control data range.

Cyclophosphamide, the positive control substance, induced a statistically significant increase in the number of micronucleated polychromatic erythrocytes. Hence, the acceptability criteria of the test were met.

 

Ratio polychromatic to normochromatic erythrocytes:

The animal treated with the test substance showed no decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a lack of toxic effects of this compound on the erythropoiesis. The animals of the positive control groups showed a decreases in the ration of polychromatic to normochromatic erythrocytes.

Conclusions:

Based on the results of the study, the authors concluded that the test was valid and the test substance is not mutagenic in the micronucelus test.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

From the available in vitro genotoxicity data, the combined in vivo bone marrow micronucleus comet assay (MN-CMT) is deemed an appropriate follow up model to understand the biological relevance of the adverse in vitro genotoxicity data.

In the available MN-CMT study, the test substance was dosed at the highest practical volume that could be administered. Based on the results it can be concluded that no significant increase in the incidence of micronucleated polychromatic erythrocytes in bone marrow was observed. However, the available data obtained from the comet assay are inadequate to address the in vitro gene mutation data for the following reasons:

- Liver vehicle control group mean median %tail intensity values exceeds the OECD test guideline range (6%), with a cross over between individual animal vehicle and positive control values. The sensitivity of this endpoint is therefore questionable.

- In the absence of the glandular stomach being assessed for the CMT endpoint, the positive in vitro bacterial reverse gene mutation data in the absence of S9 is not adequately addressed.

Finally, the DNA reactivity is unknown. Therefore, the suitability of the comet assay is questionable.

For the reasons stated above no firm conclusion can be drawn on the liver comet assay endpoint, with concerns raised over the sensitivity and specificity of the data.

Justification for classification or non-classification

Data available for the endpoint cytogentoxicity do not justify a classification. The data for the endpoint gene mutation are not sufficient for classification, due the inadequacies of the comet assay data. Further investigations to assess the substance for genotoxicity are not required because the substance is registered according to REACH Article 18 (no exposure to humans). In conclusion, the data available do not allow a clear assessment as positive / negative. According to CLP regulation classification based only on in vitro data is considered not sufficient. Thus, the substance is not classified. Reason for no classification: data inconclusive.