N,N'-propane-1,3-diylbis[3-(3,5-di-tert-butyl-4-hydroxyphenyl)propionamide]

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Mutagenic effects - bacterial: OECD 471; Ames study with read-across substance. Negative. Reliability = 2.

Mutagenic effects - mammalian: OECD 476; HPRT study in Chinese Hamster V79 cells. Negative. Reliability = 1.

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Remarks:

The test substance was concluded to be negative in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537. No indications of mutagenicity were observed at any dose level in any tester strain when tested up to 10000 μg/plate in the absence or presence of S9. The assay was conducted prior to the 1997 adaptation of the current OECD 471, and did not include an E.coli WP2 or S. typhimurium TA102 tester strain. These strains are known to specifically detect certain oxidising mutagens, cross-linking agents and hydrazines that other Salmonella tester strains may not be sensitive to. However, based on the physico-chemical characteristics of the test substance, the inclusion of a fifth tester strain would not have changed the clearly negative outcome of the Ames assay. In addition, there was no indication of in vitro or in vivo mutagenicity for any other genetic toxicology endpoint.

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

not specified

GLP compliance:

no

Type of assay:

bacterial reverse mutation assay

Species / strain / cell type:

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

Metabolic activation:

with and without

Metabolic activation system:

S9 fraction from liver of rats induced with Aroclor 1254 and solution of Co-factors

Test concentrations with justification for top dose:

Experiment I: 20, 78, 313, 1250 and 5000 µg/0.1 mL
Repeat Experiment: 500, 1000, 2000, 4000 and 8000 µg/0.1 mL
Concentrations were based on results from a preliminary toxicity test carried out with strain TA100 without activation at concentrations ranging from 0.08 to 5000 µg/0.1 mL.

Vehicle / solvent:

- Vehicle used: Ethanol

Untreated negative controls:

yes

Remarks:

ethanol

Positive control substance:

4-nitroquinoline-N-oxide

sodium azide

cyclophosphamide

other: 2-aminoanthracene (TA98, TA100, and TA1537 with S9); daunorubicin-HCl (TA100 without S9); 9(5)-aminoacridine hydrochloride monohydrate (TA1537 without S9)

Details on test system and experimental conditions:

In the experiments with and without the addition of microsomal activation mixture three petri dishes were prepared per strain per group (i.e., per concentration or per control group). The plates were incubated for about 48 hours at 37 ± 1.5°C in darkness.

Evaluation criteria:

The test substance is considered to be positive in this test system if one or both of the following conditions are met:
- at least a reproducible doubling of the mean number of revertants per plate above that of the negative control at any concentration level for one or more of the following strains:TA 98, TA 1535 and TA 1537,
- a reproducible increase of the mean number of revertants per plate for any concentration above that of the negative control by at least a factor of 1.5 for strain TA 100.
Generally a concentration-related effect should be demonstrable.

Statistics:

Mean values and standard deviation for each bacterial strain and each concentration were calculated.

Key result

Species / strain:

other: S. typhimurium TA98, TA100, TA1535, TA1537

Metabolic activation:

with and without

Genotoxicity:

negative

Remarks:

At the concentrations of 313 μg/0.1 mL and above the substance precipitated in soft agar

Cytotoxicity / choice of top concentrations:

not specified

Vehicle controls validity:

valid

Positive controls validity:

valid

Table-1: Number of back-mutant colonies per plate (arithmetic mean) (without metabolic activation)

	TA98	TA100	TA1535	TA1537
Control	27	135	18	9
500 µg/0.1 ml	29	109	14	7
1000 µg/0.1 ml	26	129	13	5
2000 µg/0.1 ml	26	132	12	6
4000 µg/0.1 ml	24	118	22	7
8000 µg/0.1 ml	22	105	16	9
Positive controls
Daunorubicin-HCl
Control	26
5 µg/0.1 ml	160
10 µg/0.1 ml	----
4-nitroquinoline-N-oxide
control		139
0.125 µg/0.1 ml		683
0.25 µg/0.1 ml		1184
Sodium azide
Control			12
2.5 µg/0.1 ml			461
5.0 µg/0.1 ml			699
9(5)aminoacridine-hydrochloride
Control				7
50 µg/0.1 ml				1553
100 µg/0.1 ml				2608

Table-2: Number of back-mutant colonies per plate (arithmetic mean) (with metabolic activation)

	TA98	TA100	TA1535	TA1537
Control	45	145	20	19
500 µg/0.1 ml	42	127	16	17
1000 µg/0.1 ml	40	113	20	20
2000 µg/0.1 ml	34	123	18	19
4000 µg/0.1 ml	41	117	19	20
8000 µg/0.1 ml	54	96	25	20
Positive control of microsomal activation
cyclophosphamide
control			16
250 µg/0.1 ml			983
2-aminoanthracene
control	30	147		18
5 µg/0.1 ml	1171	586		165

Conclusions:

Negative with and without metabolic activation upto 8000 µg/0.1 mL.

Executive summary:

The study was conducted according to OECD Guideline 471 to determine mutagenic effects of the test substance on histidine-auxotrophic mutants of Salmonella typhimurium.

The investigations were performed on strains TA98, TA100, TA1535 and TA1537 without and with microsomal activation with concentrations of 20, 78, 313, 1250 and 5000 µg/0.1 mL in the first and 500, 1000, 2000, 4000 and 8000 µg/0.1 mL in the repeat experiment. These tests permit the detection of point mutations in bacteria induced by chemical substances. Any mutagenic effects of the substances are demonstrable on comparison of the number of bacteria in the treated and control cultures that have undergone back-mutation to histidine-prototrophism. To ensure that mutagenic effects of metabolites of the test substances formed in mammals would also be detected, experiments were performed in which the cultures were additionally treated with an activation mixture (rat liver microsomes and co-factors). In the experiments performed without and with microsomal activation, none of the tested concentrations of test substance led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

yes

Remarks:

To clarify equivocal results an independent repetition experiment (without metabolic activation) with closer spaced concentrations and double cell cultures was performed on sponsor’s request.

GLP compliance:

yes

Type of assay:

other: HPRT assay in Chinese Hamster V79 cells

Target gene:

HPRT locus using V79 cells of the Chinese Hamster

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

CELLS USED
- Source of cells: Eurofins BioPharma Product Testing Munich GmbH stock cultures
- Cell cycle length, doubling time or proliferation index:12 - 14 h doubling time; high cloning efficiency of untreated cells, usually more than 50%
- Methods for maintenance in cell culture if applicable: The V79 cells (ATCC, CCL-93) were stored over liquid nitrogen (vapour phase) in the cell bank at the testing facility.

MEDIA USED
- Type and identity of media including CO2 concentration if applicable: stored over liquid nitrogen (vapour phase); minimal essential medium (MEM)
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes (via PCR)
- Periodically checked for karyotype stability: yes
- Periodically 'cleansed' against high spontaneous background: yes

Metabolic activation:

with and without

Metabolic activation system:

rat liver (S9)

Test concentrations with justification for top dose:

Toxicity test: 1, 2.5, 5, 10, 25, 50, 100, 250 µg/mL
Experiment I (without activation): 0.05, 0.1, 0.25, 0.5, 1, 2.5, 5, 10, 25, 50, 100 and 200 µg/mL
Experiment I (with activation): 0.5, 1, 2.5, 5, 10, 25, 50, and 100 µg/mL
Experiment I repeat assay (without activation): 10, 25, 50, 60, 70, 80, 90 and 100 µg/mL
The selection of the concentrations was based on data from the pre-experiments.

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the nature of the test item, it was not possible to prepare a solution of the test item with an appropriate solvent and with the recommended concentration of 2 mg/mL. By lowering the highest concentration to 0.25 mg/mL and based on the results of the solubility test, the best suited vehicle was DMSO (1% v/v).

Untreated negative controls:

yes

Remarks:

treatment medium

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

ethylmethanesulphonate

Details on test system and experimental conditions:

Seeding of the Cultures: Prior to use, cultures were cleansed of pre-existing cells. Two or three day-old exponentially growing stock cultures (more than 50% confluent) were trypsinised at 37°C for 5 minutes. Then the enzymatic digestion was stopped by adding complete culture medium and a single cell suspension was prepared. The trypsine concentration for all subculturing steps was 0.05%. Approximately 10-20e6 cells per concentration, solvent/negative and positive control, were seeded in complete culture medium (MEM supplemented with 10% FBS) in a culture flask, respectively.

Treatment: Approximately 24 hours after seeding the cells were exposed to designated concentrations of the test item either in the presence or absence of metabolic activation in the mutation experiment. After 4 hours exposure the cultures (single cell culture for experiment I and double cell culture for the repetition of experiment I without metabolic activation) were checked for precipitation and the treatment medium containing the test item was removed. The cells were washed twice with PBS, trypsinised and counted with a cell counter. During the following expression period most of the cells were subcultured in complete culture medium (MEM supplemented with 10% FBS) in a sufficient number of cells (at least 2e6 cells per treatment group). In addition, for determination of the relative survival (RS) two 25 cm2 flasks were seeded with approx. 200 cells in complete culture medium for each treatment group. After incubation for an appropriate time (6 - 7 days) colonies were fixed with methanol, stained with Giemsa and counted. Cytotoxicity (relative survival) was calculated based on the cloning efficiency of cells plated immediately after treatment adjusted by any loss of cells during treatment.

Selection: At the end of the expression period (i.e. after 7 to 9 days) for selection the mutants, about 4e5 cells for each treatment group were seeded in cell culture petri dishes (diameter 90 mm) with selective medium containing 11 µg/mL 6-thioguanine (TG) for further incubation (additional 9 - 11 days). The cloning efficiencies (CE) were determined in parallel to the selection of mutants. For each treatment group two 25 cm2 flasks were seeded with approximately 200 cells in complete culture medium to determine the cloning efficiencies after additional 6 - 8 days. After incubation for an appropriate time (9 - 11 days for mutant frequency and 6 - 8 days for CE) colonies were fixed with methanol, stained with Giemsa and counted. The mutant frequency was calculated based on the number of mutant colonies corrected by the cloning efficiency at the time of mutant selection.

Assessment of Cytotoxicity and Mutant Frequency: Cytotoxicity was evaluated by relative survival (RS). The cloning efficiency (CE) of cells plated immediately after treatment was adjusted by any loss of cells during treatment as compared with adjusted cloning efficiency in negative / solvent controls (assigned a survival of 100%). The mutant frequency (MF) is the cloning efficiency of mutant colonies in selective medium divided by the cloning efficiency in non-selective medium measured for the same culture at the time of selection

Evaluation criteria:

A test chemical is considered to be clearly negative if, in all experimental conditions examined
- none of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control,
- there is no concentration-related increase when evaluated with an appropriate trend-test
- all results are inside the distribution of the historical negative control data

A test chemical is considered to be clearly positive if, in any of the experimental conditions examined
- at least one of the test concentrations exhibits a statistically significant increase compared with the concurrent negative control, and
- the increase is concentration-related when evaluated with an appropriate trend test, and
- any of the results are outside the distribution of the historical negative control data.
- if there is by chance a low spontaneous mutation rate in the corresponding negative and solvent controls a concentration related increase of the mutations within their range has to be discussed.

According to the OECD guideline, the biological relevance is considered first for the interpretation of results.

Statistics:

The non-parametric Mann-Whitney test was applied to the mutation data to prove the dose groups for any significant difference in mutant frequency compared to the negative/solvent controls. Mutant frequencies of the negative/solvent controls were used as reference.

Key result

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

Exp. I (with and without activation): no growth inhibition observed. Exp. I repeat (without activation): relative survival <70%; relative survival for all concentrations except highest was within range 50-63% (slightly below non-toxic section)

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Additional information on results:

Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL.
For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed.
DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency.

Experiment I – Toxicity, without metabolic activation
Dose Group	Concentration [µg/mL]	Number of cells at		Number of colonies per flask			CE [%]	Adjusted CE [%]	Relative Survival (RS) [%]
		beginning of treatment	endo of treatment	I	II	mean
NC1	0	10000000	12665000	140	165	153	76	97	120
NC2		10000000	12818000	159	172	166	83	106	132
S1	0	10000000	10642000	141	120	131	65	69	100
S2		10000000	10846000	166	170	168	84	91
1	0.05	10000000	10625000	147	150	149	74	79	98
2	0.1	10000000	9537000	157	176	167	83	79	99
3	0.25	10000000	10013000	174	177	176	88	88	109
4	0.5	10000000	9622000	144	158	151	76	73	90
5	1	10000000	10217000	141	147	144	72	74	92
6	2.5	10000000	10591000	146	152	149	75	79	98
7	5	10000000	9418000	177	195	186	93	88	109
8	10	10000000	9265000	178	218	198	99	92	114
9	25	10000000	8993000	166	170	168	84	76	94
10	50	10000000	8041000	159	164	162	81	65	81
11 P	100	10000000	8041000	191	180	186	93	75	93
12 P	200	10000000	7854000	202	188	195	98	77	95
EMS	300	10000000	13107000	181	168	175	87	114	142
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I – Mutagenicity, without metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE              [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	185	183	184	92	18	11	17	13	14	14.6	2.6	0.0037	39.7
NC2		184	186	185	93	7	14	15	9	13	11.6	3.1	0.0029	31.4
S1	0	148	187	168	84	14	12	7	17	7	11.4	3.9	0.0029	34.0
S2		143	186	165	82	19	9	11	13	13	13.0	3.3	0.0033	39.5
7	5	182	164	173	87	16	13	14	15	11	13.8	1.7	0.0035	39.9
8	10	187	181	184	92	10	14	16	17	10	13.4	2.9	0.0034	36.4
9	25	185	149	167	84	11	15	7	12	16	12.2	3.2	0.0031	36.5
10	50	189	186	188	94	15	15	19	18	21	17.6	2.3	0.0044	46.9
11 P	100	183	160	172	86	12	8	14	26	11	14.2	6.2	0.0036	41.4
EMS	300	134	115	125	62	78	89	92	90	94	88.6	5.6	0.0222	355.8
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I – Toxicity, with metabolic activation
Dose Group	Concen-tration	Number of cells at the		Number of colonies per flask			CE     [%]	Adjusted CE [%]	Relative Survival (RS)
	[µg/mL]	beginning of treatment	end of treatment	I	II	mean			[%]
NC1	0	10000000	11288000	175	148	162	81	91	97
NC2		10000000	10863000	157	158	158	79	86	91
S1	0	10000000	11458000	143	179	161	81	92	100
S2		10000000	12257000	147	168	158	79	97
1	0.5	10000000	11237000	173	175	174	87	98	104
2	1	10000000	11424000	154	156	155	78	89	94
3	2.5	10000000	11900000	153	167	160	80	95	101
4	5	10000000	11730000	125	138	132	66	77	82
5	10	10000000	11305000	200	203	202	101	114	121
6	25	10000000	10744000	189	195	192	96	103	109
7	50	10000000	10591000	204	207	206	103	109	115
8:00 PM	100	10000000	9979000	177	160	169	84	84	89
DMBA	1	10000000	11441000	151	132	142	71	81	86
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency DMBA: 7,12-dimethylbenz(a)anthracene

Experiment I – Mutagenicity, with metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE  [%]	Number of colonies per flask							CE  [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	199	215	207	104	21	17	21	19	11	17.8	3.7	0.0045	43.0
NC2		166	179	173	86	12	16	14	14	10	13.2	2.0	0.0033	38.3
S1	0	174	197	186	93	5	16	8	5	7	8.2	4.1	0.0021	22.1
S2		189	194	192	96	16	15	15	12	17	15.0	1.7	0.0038	39.2
4	5	157	173	165	83	15	15	12	14	12	13.6	1.4	0.0034	41.2
5	10	174	167	171	85	9	9	10	10	15	10.6	2.2	0.0027	31.1
6	25	157	154	156	78	10	13	15	10	12	12.0	1.9	0.0030	38.6
7	50	168	162	165	83	8	15	12	11	7	10.6	2.9	0.0027	32.1
8 P	100	157	173	165	83	8	14	8	8	10	9.6	2.3	0.0024	29.1
DMBA	1.0	134	142	138	69	93	101	104	78	81	91.4	10.4	0.0229	331.2
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency DMBA: 7,12-dimethylbenz(a)anthracene

 

Experiment I (repetition) – Toxicity, without metabolic activation
Dose Group	Concen-tration [µg/mL]	Number of cells at the		Number of colonies per flask			CE             [%]	Adjusted CE          [%]	Relative Survival (RS) [%]
		beginning of treatment	end of treatment	I	II	mean
NC1	0	10000000	9010000	73	73	73	37	33	78
NC2		10000000	10081000	92	93	93	46	47	111
S1	0	10000000	7633000	125	128	127	63	48	100
S2		10000000	8058000	86	91	89	44	36
1	10	20000000	13090000	67	65	66	33	22	51
2	25	20000000	11424000	81	68	75	37	21	51
3	50	20000000	10914000	100	84	92	46	25	60
4	60	20000000	11322000	69	80	75	37	21	50
5	70	20000000	11356000	90	97	94	47	27	63
6 P	80	20000000	12512000	104	126	115	58	36	86
7 P	90	20000000	13430000	142	131	137	68	46	109
8 P	100	20000000	12580000	120	141	131	65	41	98
EMS	300	10000000	11475000	139	134	137	68	78	187
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

 

Experiment I (repetition) – Mutagenicity, without metabolic activation
		CE in non-selective medium				CE in selective medium
Dose Group	Concen-tration [µg/mL]	Number of colonies per flask			CE              [%]	Number of colonies per flask							CE              [%]	Mutant Frequency per 10^6cells
		I	II	mean		I	II	III	IV	V	mean	SD
NC1	0	155	160	158	79	8	6	6	11	3	6.8	2.6	0.0017	21.6
NC2		147	144	146	73	13	15	13	12	10	12.6	1.6	0.0032	43.3
S1	0	133	130	132	66	7	5	8	9	8	7.4	1.4	0.0019	28.1
S2		152	151	152	76	7	11	11	14	9	10.4	2.3	0.0026	34.3
2	25	162	158	160	80	14	8	11	8	6	9.4	2.8	0.0024	29.4
3	50	179	165	172	86	6	12	12	5	12	9.4	3.2	0.0024	27.3
4	60	141	152	147	73	7	8	11	15	7	9.6	3.1	0.0024	32.8
5	70	150	140	145	73	12	12	15	5	6	10.0	3.8	0.0025	34.5
6 P	80	169	151	160	80	6	7	7	3	6	5.8	1.5	0.0015	18.1
EMS	300	138	152	145	73	91	80	96	80	86	86.6	6.2	0.0217	298.6
NC: negative control S: solvent control (1% DMSO) P: precipitation at the end of treatment CE: cloning efficiency EMS: Ethylmethanesulfonate

Conclusions:

Under the experimental conditions reported, the test item is non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Executive summary:

The test substance was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster in accordance with OECD Guideline 476. The selection of the concentrations was based on data from the pre-experiments. Experiment I (with and without metabolic activation) and repetition experiment I (without metabolic activation) were performed as a 4 h short-term exposure assay. In consultation with the Sponsor and due to the equivocal results in experiment I, an independent repetition of experiment I without metabolic activation with different experimental conditions (closer spaced concentrations and double cultures) was conducted. The test item was investigated at the following concentrations: 5.0, 10, 25, 50 and 100 µg/mL (Experiment I, without and with metabolic activation) and 25, 50, 60, 70 and 80 µg/mL (Experiment I (repetition), without metabolic activation).

Precipitation of the test item was noted in the pre- and main experiment at concentration 100 µg/mL. In the repetition of experiment I precipitation occurred at concentration 80 µg/mL. No growth inhibition was observed in the experiment I without and with metabolic activation. A growth inhibition (relative survival < 70%) was observed in the repetition experiment without metabolic activation: the relative survival for all concentrations evaluated, with the exception of the highest concentration, was found slightly below the non-toxic section (within the range of 50-63 %). For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed. DMBA and EMS were used as positive controls and showed distinct and biologically relevant effects in mutation frequency. In conclusion, in the described mutagenicity test under the experimental conditions reported, the test substance is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

Clastogenic effects (mammalian): Hamster micronucleus study with read-across substance. Equivalent to OECD 474; Negative. Reliability = 2.

Clastogenic effects (mammalian): Rat dominant lethal with read-across substance. Equivalent to OECD 478; Negative; Reliability = 2.

DNA damage and/or repair (mammalian): Sister chromatid exchange with read-across substance. Scientifically valid study; Negative; Reliability = 2.

Link to relevant study records

Reference

Reference 1

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:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The test was conducted to evaluate the cytotoxic or mutagenic effects on male germinal cells produced by test substance.

GLP compliance:

not specified

Type of assay:

rodent dominant lethal assay

Species:

mouse

Strain:

other: Tif: MAG f (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Animals were obtained from closed breeding colony
- Age at study initiation: Female: 2 months; Males: 2 1/2 to 6 months
- Diet: Standard diet (NAFAG No .890)
- Water: Tap water ad litium

ENVIRONMENTAL CONDITIONS
- Temperature : 21 ± 1°C
- Humidity: 60 ± 5%
- Photoperiod (hrs dark / hrs light): 14 hours dark and 10 hours light

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: CMC (carboxymethyl cellulose)
- Amount of vehicle: 0.2 mL/10 g of body weight

Duration of treatment / exposure:

Single exposure

Post exposure period:

Each group consisted of 20 males, each of which was placed in a cage with 2 untreated females immediately after treatment. At the end of 1 week, the females were removed and replaced by another group of 2 females. The procedure was continued for six consecutive weeks. The females were daily examined for successful mating indicated by the occurrence of a vaginal plug. The day that the vaginal plug was observed was designated as "day 0" of gestation. The whole time of six "mating periods" comprises all the stages of the maturation of the germ cell from the A-spermatogonia to the mature spermatozoon.
- Males were observed first week after administration of test substance for general condition and symptomatology.
- Autopsy of females was done and progeny were examined.

Dose / conc.:

1 000 other: mg/kg

Dose / conc.:

3 000 other: mg/kg

No. of animals per sex per dose:

20 males/group

Control animals:

yes, concurrent vehicle

Statistics:

- Student's t test or Mann-Whitney u-test is used to compare the total no of implanatations indicating possible pre-implantation losses
- X²-test or fischer's exact test is used to compare total number of mated and pregnant dams or embryonic deaths.
- Experimental data, particularly on the number of implantations and early embryonic deaths were compared with spontaneous data of a cummulative of treated controls observed over a longer period of time (autopsy on day 18 of pregnancy).

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks on result:

other: No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.

Additional information on results:

The females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions).

Conclusions:

No evidence of dominant lethal effects were observed in progeny of male mice tretaed with test substance.

Executive summary:

The test was conducted to evaluate the cytotoxic or mutagenic effects on male germinal cells produced by test substance The test substance was administered orally by intubation in single doses to male albino mice which were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The test was done to evaluate any cytotoxic or mutagenic effects on the male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development.

The results showed that the females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.

Reference 2

Endpoint:

genetic toxicity in vivo, other

Remarks:

Nucleus anomaly test

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo.

GLP compliance:

no

Type of assay:

other: Nuclues anomaly in bone marrow cells

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Female: 20-26 g, Male: 22-30 g
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22-24
- Humidity (%): 51-59
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.7% aqueous solution of Sodium-Carboxymethylcellulose (CMC)
- Amount of vehicle: 20 mL/kg

Duration of treatment / exposure:

2 consecutive days

Frequency of treatment:

Once daily

Dose / conc.:

1 500 mg/kg bw/day

Dose / conc.:

3 000 mg/kg bw/day

Dose / conc.:

6 000 mg/kg bw/day

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Route of administration: Oral
- Doses: 128 mg/kg

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Bone marrow was harvested from the shafts of both femurs. In a siliconized pipette filled with approximately 0.5 µL rat serum the bone marrow was drawn up. In order to receive a homogeneous, suspension the content of pipette was aspirated gently about three times. Small drops of the mixture were transferred on the end of a slide, spread out by pulling it behind a polished cover glass and the preparations were air-dried. Three hours later, the slides were stained in undiluted May-Grunwald solution for 2 min then in May-Grunwald solution/water 1/1 for 2 min and then in Giemsa's, 40% for 20 min. After being rinsed in methanol 55% for 5-8 sec and washed off twice in water, they were left immersed in water for approximately 2 min. After rinsing with distilled water and air-drying, the slides were cleared in xylol and mounted in Eukitt.

SCORING: 1000 bone marrow cells each were scored per animal and the following anomalies were registered: Single jolly bodies, fragments of nuclei in erythrocytes, micronuclei in erythroblasts, micronuclei in leucopoietic cells, polyploid cells.

Statistics:

The significance of difference was assessed by Chi²-test

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The positive control (cyclophosphamide, 128 mg/kg) yielded a marked increase of the percentage of cells with anomalies. The mean percentage of anomalies was 8.52, whereas the negative control yielded a percentage of 0.12.

Table-1: Animals sacrificed 24 h after the second application. Percent of cells with anomalies of nuclei

	No. of animals	Sex of animals	Single jolly bodies	Fragments of nuclei in erythrocytes	Micronuclei in erythroblasts	Micronuclei in leucopoietic cells	Polyploid cells	Total
Control (CMC 0.7%)	1	Female	-	-	-	-	-	0.0
	2	Female	-	-	-	-	-	0.0
	3	Female	0.2	-	-	-	-	0.2
	4	Male	-	-	-	0.1		0.1
	5	Male	0.3	-	-	-	-	0.3
	6	Male	0.1	-	-	-	-	0.1
Cyclophosphamide (128 mg/kg)	1	Female	8.9	0.9	0.8	0.2	0.2	11.0
	2	Female	8.4	1.7	0.5	0.5		11.1
	3	Female	10.0	0.7	2.1	0.9	0.1	13.8
	4	Male	3.2	0.9	0.3	0.4	0.1	4.9
	5	Male	3.1	1.5	0.7	0.2	-	5.5
	6	Male	3.6	0.2	0.6	0.4	-	4.8
1500 mg/kg	1	Female	-	-	-	-	-	0.0
	2	Female	0.3	-	-	-	-	0.3
	3	Female	0.1	-	-	-	-	0.1
	4	Male		-	-	-	-	0.0
	5	Male	0.1	-	-	-	-	0.1
	6	Male	-	-	-	-	-	0.0
3000 mg/kg	1	Female	-	-	-	0.1	-	0.1
	2	Female	-	-	-	-	-	0.0
	3	Female	-	-	-	-	-	0.0
	4	Male	-	-	-	-	-	0.0
	5	Male	-	-	-	-	-	0.0
	6	Male	0.1	-	-	-	-	0.1
6000 mg/kg	1	Female	0.1	-	-	-	-	0.1
	2	Female	0.1	-	-	-	0.1	0.2
	3	Female	-	-	-	-	-	0.0
	4	Male	-	-	-	-	-	0.0
	5	Male	-	-	-	-	-	0.0
	6	Male	0.1	-	-	-	-	0.1

Conclusions:

Negative for nuclear anomalies in Chinese Hamester bone marrow cells

Executive summary:

The mutagenic effects ot the test substance to the bone marrow of Chinese hamsters was evaluated. The test substance was administered by gavage at a daily dose of 1500, 3000 or 6000 mg/kg for two consecutive days. The animals were sacrificed 24 h after the second administration. From the bone marrow, smears were made. The experiment was performed to evaluate any mutagenic effect on somatic interphase cells in vivo. Mutagenic effects present themselves in interphase cells in form of nucleus anomalies of bone marrow cells.

The bone marrow smears from animals treated with various doses of the test substance showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponds to the frequency observed in the control group. By contrast, a positive control experiment with cyclophosphamide (128 mg/kg) yielded 8.52% cells with anomalies of nuclei. This is significantly different from the controls treated with the vehicle (0.7 CMC) alone. It was concluded that under the conditions of this experiment, no evidence of mutagenic effects was obtained in Chinese hamsters treated with the test substance.

Reference 3

Endpoint:

in vivo mammalian cell study: DNA damage and/or repair

Type of information:

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

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

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

Justification for type of information:

Additional documentation, provided within the IUCLID Assessment Reports (Section 13), supports the read-across approach.

Reason / purpose for cross-reference:

read-across: supporting information

Qualifier:

no guideline followed

Principles of method if other than guideline:

The experiments were performed in order to detect a possible mutagenic property of the substance, manifested in somatic cells in vivo in the form of sister chromatid exchanges (SCE).

GLP compliance:

no

Type of assay:

sister chromatid exchange assay

Species:

hamster, Chinese

Strain:

other: Cricetulus griseus

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Weight at study initiation: Female: 20-28 g, Male: 25-30 g
- Diet: ad libitum
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 22-24°C
- Humidity: 54-62%
- Photoperiod (hrs dark / hrs light): 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: 0.7% aqueous solution of Sodium-Carboxymethylcellulose (CMC)
- Amount of vehicle: 20 mL/kg

Duration of treatment / exposure:

Single dose

Post exposure period:

Animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg).

Dose / conc.:

1 500 mg/kg bw/day

Dose / conc.:

3 000 mg/kg bw/day

Dose / conc.:

6 000 mg/kg bw/day

No. of animals per sex per dose:

2

Control animals:

yes, concurrent vehicle

Positive control(s):

7,12 Dimethylbenz(a)anthracene (DMBA)
- Route of administration: Oral
- Doses / concentrations: 100 mg/kg in 0.7% aqueous solution of sodium-carboxymethylcellulose (CMC)

Tissues and cell types examined:

Bone marrow cells

Details of tissue and slide preparation:

DETAILS OF SLIDE PREPARATION: Animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg). Bone marrow from the shafts of both femurs was suspended in balanced salt solution and diluted to hypotonicity with distilled water, kept in a water bath at 4 to 6°C for 23 min and then centrifuged for 10 min at 200 x g. The pellets were then fixed in methanolacetic acid 3:1 for a period of 30 min, resuspended, centrifuged for 5 min at 150 x g, and stored in renewed fixative overnight at 4°C. Finally, the pellets again were centrifuged for 5 min at 150 x g, and resuspended in some 0.5 ml fixative in order to obtain a more concentrated cell suspension.
These specimens were pipetted onto wet slides and air-dried. The air-dried slides then were treated with a solution of bisbenzimide for 15 min, rinsed in Mcilvaine-buffer pH 8.0 and irradiated in this buffer at 50°C with UV-light of 350 nm. Following the development of the fluorochrome-UV light reaction in 60°C 2 x SSC (standard sodium citrate) for 90 min, the slides were stained in 40% Giemsa for 20-40 min, well rinsed, cleared in Xylol and mounted in Eukitt.

Scoring of the slides: The slides of two females and two male animals each of the treatment groups and of the control groups were examined. Twenty-five differently stained metaphases of the second cell cycle with BUdR-substitution were analysed per animal for the number of SCE's.

Statistics:

The significance of differences of the treatment groups compared to the negative control group was assessed by t-test on the level of one percent.

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Positive controls validity:

valid

Additional information on results:

The positive control group showed a highly significant increase of SCE's per cell (12.8) in comparison with the negative control (5.28 SCE's/cell).

Table 1: Effects of the test substance on Bone marrow cells of Chinese Hamster

Group	No-of animals	Sex	Group SCE’s per cell (mean and SD)	Observed t value
Negative Control	4	2M + 2F	5.28 (2.95)	-
DMBA (positive control)	4	2M + 2F	12.79 (6.25)	10.88*
1500 mg/Kg	4	2M + 2F	6.02 (3.16)	1.72
3000 mg/Kg	4	2M + 2F	4.81 (2.37)	1.24
6000 mg/Kg	4	2M + 2F	4.88 (3.91)	0.82

*statistically significant at 1% (critical t-value = 2.34, d.f. = 198)

Conclusions:

The test substance does not provoke any effect interpretable as being suggestive of a mutagenic property.

Executive summary:

The experiments were performed in order to detect a possible mutagenic property of the substance, manifested in somatic cells in vivo in the form of sister chromatid exchanges (SCE). The test substance was administered by gavage. Treatment consisted of a single dose of 1500, 3000 or 6000 mg/kg. The animals were sacrificed 24 h after the application and 2 h after an intraperitoneal injection of colcemide (10 mg/kg). From the bone marrow, drop-preparations were made and stained according to a modified fluorochrome plus Giemsa technique.

The number of SCE's in animals from the control group and in those treated with the various doses of the test substance was not significantly different. A "positive control" experiment with DMBA (100 mg/kg) yielded a mean value of 12.8 SCE's per cell. This is significantly different from the controls treated with the vehicle (0.7% CMC) alone.

The results obtained indicate that under the given experimental conditions, the test substance does not provoke any effect interpretable as being suggestive of a mutagenic property.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In an Ames test according to OECD Guideline 471, the test substance was tested for gene mutations in Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537 with or without metabolic activation at concentrations up to 5000 or 8000 µg/mL in the first and repeat assays, respectively. None of the tested concentrations led to an increase in the incidence of histidine-prototrophic mutants by comparison with the negative control. No evidence of the induction of point mutations by test substance or by the metabolites of the substance formed as a result of microsomal activation was detectable in the strains of S. typhimurium used in these experiments. Therefore, the test substance is considered not mutagenic in the Ames test.

 

The test substance was assessed for its potential to induce mutations at the HPRT locus using V79 cells of the Chinese Hamster in accordance with OECD Guideline 476. The test item was investigated at the following concentrations: 5.0, 10, 25, 50 and 100 µg/mL (Experiment I, without and with metabolic activation) and 25, 50, 60, 70 and 80 µg/mL (Experiment I (repetition), without metabolic activation). For experiment I without metabolic activation the mutant frequencies for the two highest concentrations (50 and 100 µg/mL) were found above the historical data of the test facility. As there was no statistically significant increase, these results needed to be clarified in an independent repetition experiment. In the repetition of experiment I without metabolic activation none of the observed mutant frequencies was significantly increased over those of the negative controls. No dose-response relationship was observed. The test substance is considered to be non-mutagenic at the HPRT locus using V79 cells of the Chinese Hamster.

 

The test substance was administered by gavage daily at doses of 1500, 3000 or 6000 mg/kg on two consecutive days to evaluate the mutagenic effect on somatic interphase cells in vivo. The animals were sacrificed 24 h after the second application and bone marrow smears were made. The bone marrow smears showed no significant difference from the control. The incidence of bone marrow cells with anomalies of nuclei corresponded to the frequency observed in the control group. Therefore, the test substance is considered negative in this test.

 

The potential of the test substance was examined to evaluate the cytotoxic or mutagenic effects on male germinal cells as expressed by the loss of pre-implantation zygotes as well as by the rate of deaths of post-implantation stages of embryonic development. The test substance was administered orally by intubation in single doses to male albino mice which were then mated to untreated females from the same strain over a period of six weeks. At the end of each week the females were replaced by new ones. Doses of 1000 and 3000 mg/kg were given. The results showed that the females mated to males which had been treated with the test substance did not differ significantly from the females mated to control, neither in mating ratio nor in the number of implantations and embryonic deaths (resorptions). No evidence of dominant lethal effect was observed in the progeny of male mice treated with test substance.

 

The ability of the test substance to manifest changes in somatic cells was studied in vivo in the form of sister chromatid exchanges (SCE). The test substance was administered by gavage at a single dose of 1500, 3000, or 6000 mg/kg. The results obtained indicate that under the given experimental conditions, the test substance does not provoke any effect interpretable as being suggestive of a mutagenic property and was negative in this test.

Justification for classification or non-classification

The test substance did not produce mutagenicity when evaluated in cell culture or laboratory animals. The substance does not need to be classified for mutagenicity according the EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008.