Amines, C12-14-tert-alkyl, compds. with 1-[2-[5-(1,1-dimethylpropyl)-2-hydroxy-3-nitrophenyl]diazenyl]-2-naphthalenol 1-[2-[2-hydroxy-4(or 5)-nitrophenyl]diazenyl]-2-naphthalenol chromium complexes

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

The test substance was found to be positive in the standard Ames test (OECD 471, GLP). No genotoxic potential was found in the chromosomal aberration Test in vitro (OECD 473, GLP), in the Mammalian Cell Gene Mutation Test in vitro (OECD 476, GLP) and in the Erythrocyte Micronucleus Test in vivo (OECD 474, GLP).

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2015

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Version / remarks:

(21 Jul 1997)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

(BASF SE, Experimental Toxicology and Ecology, Ludwigshafen, Germany)

Type of assay:

bacterial reverse mutation assay

Target gene:

his+ / trp+

Species / strain / cell type:

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

Additional strain / cell type characteristics:

not specified

Metabolic activation:

with and without

Metabolic activation system:

phenobarbital and beta-naphthoflavone induced rat liver S9 microsomal fraction

Test concentrations with justification for top dose:

0; 33; 100; 333; 1000; 2500 and 5000 μg/plate

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the limited solubility of the test substance in ultrapure water, DMSO was used as vehicle, which had been demonstrated to be suitable in bacterial reverse mutation tests and for which historical control data are available.

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-aminoanthracene (2-AA), N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 4-nitro-o-phenylenediamine (NOPD), 9-aminoacridine (AAC), 4-nitroquinoline-N-oxide (4-NQO)

Details on test system and experimental conditions:

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

Standard plate test:
- Exposure duration: 48 – 72 hours

Preincubation test:
- Preincubation period: 20 min
- Exposure duration: 48 – 72 hours


NUMBER OF REPLICATIONS: 3


DETERMINATION OF CYTOTOXICITY
- Method: decrease in the number of revertants, clearing or diminution of the background lawn, reduction in the titer


OTHER:
Titer determination: The titer was determined only in the experimental parts with S9 mix both for the negative controls (vehicle only) and for the two highest doses in all experiments.

Positive controls:

With S9 mix: 2-aminoanthracene (2-AA), 2.5 μg/plate, dissolved in DMSO / TA 1535, TA 100, TA 1537, TA 98; 60 μg/plate, dissolved in DMSO / Escherichia coli WP2 uvrA
Without S9 mix: N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), 5 μg/plate, dissolved in DMSO / TA 1535, TA 100; 4-nitro-o-phenylenediamine (NOPD), 10 μg/plate, dissolved in DMSO / TA 98; 9-aminoacridine (AAC), 100 μg/plate, dissolved in DMSO / TA 1537; 4-nitroquinoline-N-oxide (4-NQO) (SIGMA, N-8141), 5 μg/plate, dissolved in DMSO / E. coli WP2 uvrA

Evaluation criteria:

Acceptance criteria:
Generally, the experiment is considered valid if the following criteria are met:
• The number of revertant colonies in the negative controls was within the range of the historical negative control data for each tester strain.
• The sterility controls revealed no indication of bacterial contamination.
• The positive control substances both with and without S9 mix induced a distinct increase in the number of revertant colonies within the range of the historical positive control data or above.
• Fresh bacterial culture containing approximately 10^9 cells per mL were used. For approval the titer of viable bacteria was ≥ 10^8 colonies per mL.

Assessment criteria:
The test substance is considered positive in this assay if the following criteria are met:
• A dose-related and reproducible increase in the number of revertant colonies, i.e. about doubling of the spontaneous mutation rate in at least one tester strain either without S9 mix or after adding a metabolizing system.

A test substance is generally considered non-mutagenic in this test if:
• The number of revertants for all tester strains were within the historical negative control range under all experimental conditions in at least two experiments carried out independently of each other.

Species / strain:

E. coli WP2 uvr A

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Increase of revertants at concentrations of 33, 333, 1000, 2500 and 5000 μg/plate (without S9) or at all concentrations (with S9)

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

Increase of revertants at concentrations of 333, 1000, 2500 and 5000 μg/plate

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

positive

Remarks:

at all concentrations

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix.

COMPARISON WITH HISTORICAL CONTROL DATA: yes

ADDITIONAL INFORMATION ON CYTOTOXICITY: No bacteriotoxic effect was observed under all test conditions.

Tests without S9 -mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of revertants at 333, 1000, 2500 and 5000 μg/plate (factors 2.2, 3.1, 4.9 and 5.8, respectively).

TA 1537: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 3.5, 2.9, 4.8, 7.3, 13.4 and

20.8, respectively).

TA 98: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 12.1, 14.6, 19.5, 26.2, 30.6

and 38.3, respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Tests with S9 -mix

TA 1535: No relevant increase in the number of his+ revertants.

TA 100: Increase in the number of revertants at 333, 1000, 2500 and 5000 μg/plate (factors 2.4, 4.0, 5.5 and 5.6, respectively).

TA 1537: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 5.0, 6.0, 8.9, 11.8, 14.7 and

19.1, respectively).

TA 98: Increase in the number of revertants at 33, 100, 333, 1000, 2500 and 5000 μg/plate (factors 11.8, 13.9. 20.7, 28.8, 33.4

and 35.9, respectively).

E. coli WP2 uvrA: No relevant increase in the number of trp+ revertants.

Conclusions:

Interpretation of results (migrated information):
positive

The substance is mutagenic in the Ames test.

Executive summary:

The Ames test (OECD 471, GLP) was performed with Salmonella typhimuriam strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with doses between 33 μg - 5000 μg/plate in the standard plate assay with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test either with or without S9 mix using tester strains TA 1535 and E.coli WP2uvrA.

A distinct and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation. Using tester strain TA 1537, a distinct and dose depending increase of revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed in the standard plate test with and without metabolic activation.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Remarks:

Type of genotoxicity: gene mutation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. October - 11. November 1988

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:

no

GLP compliance:

yes

Type of assay:

mammalian cell gene mutation assay

Target gene:

Hypoxanthine-guanine phosphoribosyl transferase (HPRT) gene

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver prepared 5 days after single i.p. treatment with 500 mg/kg bw Aroclor in olive oil.

Test concentrations with justification for top dose:

The cell cultures were evaluated at the following concentrations:
Experiment I and II
without S9 mix: 6; 10; 15; and 20 µg/mL
with S9 mix: 6; 10; 30; 60; and 100 µg/mL

Rationale for dose selction:
Dose selection for the cytogenetic experiments was performed considering the toxicity data and the occurrence of precipitation.

Vehicle / solvent:

- Vehicle used: ethanol
- Justification for choice of solvent/vehicle: The solvent was chosen according to its solubility properties and its relative nontoxicity for the cells. The final concentration of the solvent in the culture medium did not exceed 1% (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

Without metabolic activation Migrated to IUCLID6: 1 mg/ml

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

7,12-dimethylbenzanthracene

Remarks:

With metabolic activation Migrated to IUCLID6: 15.4 µg/ml

Details on test system and experimental conditions:

Two independent assays were performed. In each experimental group two parallel cultures were set up. Per culture at least
100 metaphase plates were scored for structural chromosome aberrations.

METHOD OF APPLICATION: in medium

SELECTION AGENT: For the selection of mutant cells the medium is supplemented with 11 µg/mL 6-thioguanine.

DETERMINATION OF CYTOTOXICITY
A pre-test was performed in order to determine the concentration range for the mutagenicity experiments. In this pre-test the colony forming ability of approximately 500 single cells (duplicate cultures per concentration level) after treatment with the test item (with or without S9-mix) was observed and compared to the controls. Toxicity of the test item is indicated by a reduction of the relative plating efficiency [%].
Plating efficiency (about 500 cells/flask): Subculturing in two 25 cm^2 flasks.

Evaluation criteria:

A test item is classified as positive if it induces either a concentration-related increase of the mutant frequency or a reproducible and positive response at one of the test points.
A test item producing neither a concentration-related increase of the mutant frequency nor a reproducible positive response at any of the test points is considered non-mutagenic in this system.
A positive response is described as follows:
A test item is classified as mutagenic if it reproducibly induces a mutation frequency that is three times above the spontaneous mutation frequencyat least at one of the concentrations in the experiment.
The test item is classified as mutagenic if there is a reproducible concentration-related increase of the mutation frequency. Such evaluation may be considered also in the case that a threefold increase of the mutant frequency is not observed.

However, in a case by case evaluation this decision depends on the level of the corresponding solvent control data. If there is by chance a low spontaneous mutation rate in the range normally found (5 – 45 mutants per 1xE6 cells) a seemingly concentration-related increase of the mutations within this range is regarded as to be irrelevant.

Statistics:

A statistical evaluation of the results was not necessary to perform as the mutant colony numbers per 10^6 cells of the groups treated with the test article were in the range of the negative controls.

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at the maximum concentration of 20 µg/ml without S9-mix

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Remarks on result:

other: strain/cell type: As described above

Remarks:

Migrated from field 'Test system'.

In both experiments, treatment with the highest concentration (20.0 µg/mL) of the test article reduced clearly the plating efficiency of the cells only in the absence of S9 mix. In the presence of S9 mix the test article did not reduce the plating efficiency even after treatment with 100 µg/mL. Higher concentrations precipitated in the culture medium. The mutation rates found in the groups treated with the test article were considered not to be enhanced significantly as compared with the negative controls. The test article did not induce a reproducible concentration-related increase in mutant colony numbers. The mutant values of the groups treated with the test article were in the range of the controls. In experiment I and II in the samples treated with 60.0 µg/mL (exp. I, with S9 mix) and 20 µg/mL (exp. II, without S9 mix) the mutation rates (18.1 and 8.4 mutants/10E6 cells, respectively) were certainly increased by a factor 3. However, these increasments could not be reproduced in the independent experiments. In addition, these mutation frequencies are in the range of the control values of this study and in the range of our historical controls: 5 - 45 mutants per 10E6 cells. In this study the range of the controls was from 2.7 up to 29.2 mutants per 10E6 cells; the range of the groups treated with the test article was from 1.2 up to 21.4 mutants per 10E6 cells. EMS (1.0 mg/mL) and DMBA (15.4 µg/mL) were used as positive controls and showed a distinct increase in induced mutant colonies.

Conclusions:

Interpretation of results (migrated information):
negative

Reference 3

Endpoint:

in vitro cytogenicity / chromosome aberration study in mammalian cells

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17. January - 05. April 2007

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 473 (In Vitro Mammalian Chromosome Aberration Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

in vitro mammalian chromosome aberration test

Target gene:

Not applicable

Species / strain / cell type:

Chinese hamster lung fibroblasts (V79)

Details on mammalian cell type (if applicable):

- Type and identity of media: Cells were grown in Minimal Essential Medium (MEM), supplemented with 10% foetal calf serum (FCS), at 37'C with 1.5% C02 in air. The lymphocytes of fresh heparinised whole blood were stimulated to divide by the addition of phytohaemagglutinin-M (PHA) at 90 µg/ml final concentration.
- Properly maintained: yes
- Periodically checked for Mycoplasma contamination: yes
- Periodically checked for karyotype stability: yes

Metabolic activation:

with and without

Metabolic activation system:

S-9 mix from rat liver prepared 24 hours after treatment with 80 mg/kg bw Phenobarbital i.p. and b-Naphtoflavone p.o., respectively.

Test concentrations with justification for top dose:

Concentrations were in the range of 0.7- 350 µg/mL

In the first experiment:
4 hours exposure, 18 hours harvest time, without S-9 mix: 0.7; 1.4; 2.7; 5.5; 10.9; 21.9; 43.8 (this experimental part was repeated due to missing precipitation of the test item.
4 hours exposure, 18 hours harvest time, without S-9 mix: 1.4; 2.7; 5.5; 10.9; 21.9; 43.8; 87.5; 175.0
4 hours exposure, 18 hours harvest time, with S-9 mix: 0.7; 1.4; 2.7; 5.5; 10.9; 21.9; 43.8; 87.5; 175; 350

In the second experiment:
18 hours exposure, 18 hours harvest time, without S-9 mix: 5.5; 10.9; 21.9; 43.8; 87.5; 175 µg/mL
18 hours exposure, 28 hours harvest time, without S-9 mix: 5.5; 10.9; 21.9; 43.8; 87.5; 175 µg/mL
4 hours exposure, 28 hours harvest time, with S-9 mix: 5.5; 10.9; 21.9; 43.8; 87.5; 175 µg/mL

Vehicle / solvent:

- solvent used: Acetone 0.5% (v/v) in culture medium
- Justification for choice of solvent: The solvent was chosen to its solubility properties and its relative non-toxicity to the cell cultures.

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

Remarks:

without S9-mix

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

cyclophosphamide

Remarks:

with S9-mix

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

SPINDLE INHIBITOR (cytogenetic assays): Colcemid (0.2 µg/mL culture medium)
STAIN (for cytogenetic assays): GIEMSA

NUMBER OF REPLICATIONS: Duplicate cultures

NUMBER OF CELLS EVALUATED:
Per culture at least 100 metaphase plates were scored for structural chromosome abberations.
The number of polyploid cells was determined of each test group in a sample of 500 cells per culture
The mitotic index was determined in a sample of 1000 cells per culture of each test group in %

DETERMINATION OF CYTOTOXICITY
- Method: mitotic index; cell count

OTHER EXAMINATIONS:
The number of polyploid cells in 500 metaphase plates per culture was determined (% polyploid metaphases; in the case of this aneuploid cell line polyploid means a near tetraploid karyotype).

Evaluation criteria:

The test substance is to be considered positive in this assay if the following criteria are met:
1. A dose-related and reproducible significant increase in the number of structural chromosomal aberrations.
2. The proportion of aberrations exceeded both the concurrent negative control range and the negative historical control range.

A test substance is generally considered nonclastogenic in this test system if:
1. There was no significant increase in the number of chromosomally damaged cells at any dose above concurrent control frequencies.
2. The aberration frequencies were within the historical control range.

Statistics:

Statistical significance was confirmed by means of the Fisher's exact test (p < 0.05).

Species / strain:

Chinese hamster lung fibroblasts (V79)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Remarks on result:

other: all strains/cell types tested

Remarks:

Migrated from field 'Test system'.

Results on dose selection:

Test substance concentrations between 21.9 and 2800 µg/mL (with and without S9 mix) were chosen for the evaluation of cytotoxicity. Precipitation of the test item after 4 hrs treatment was observed at 21.9 µg/mL and above. In the pre-test, toxic effects indicated by a reduced cell number to 51.1 % of control were observed after treatment with 350 µg/mL in the presence of S9 mix. Therefore, 350 µg/mL (with S9 mix) was chosen as top concentration in Experiment I. In contrast, no relevant toxicity was observed in the absence of metabolic activation up to the highest applied test item concentration. Therefore, the test substance was tested up to 43.8 µg/mL in Experiment I, a concentration exhibiting clear test item precipitation as recommended in the OECD Guideline 473. This experimental part was repeated with a top concentration of 175 µg/mL due to missing precipitation of the test item. Dose selection of Experiment II was also influenced by test substance toxicity. In the range finding experiment clearly reduced cell numbers were observed after 24 hrs exposure with 175 µg/mL and above. Therefore, 175 µg/mL was chosen as top treatment concentration for continuous exposure in the absence of S9 mix. In the presence of S9 mix 175 µg/mL was chosen as top treatment concentration with respect to the results obtained in Experiment I.

Table 1: Summary of results of the chromosomal abberation study with the test substance.

Experiment	Preparation interval	Test item concentration in µg/ml	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	Aberrant cells in %
						incl. Gaps*	excl. Gaps*	with exchanges
Exposure period 4hrs without S9 mix
I	18 hrs	Solvent control (1)	3	100**	100	2	1.5	0
		Positive control (2)	4	n.t.	108.6	8.5	8 (s)	2
		10.9	2.4	103.7**	83.3	1	0.5	0
		21.9	4.2	98.6**	102.7	3.5	2	0
		43.8 (p)	3.1	85.2**	76.9	4	3.5	0
Exposure period 18 hrs without S9 mix
I	18 hrs	Solvent control	3.4	100**	100	1	1	0
		Positive control (1)	3.5	n.t.	98.7	16	13.5 (s)	7
		43.8	4.7	82.5	117.9	1	0.5	0
		87.5 (p)	2	118.1	120.1	1.5	1.5	0
		175 (p)	2.4	5.8	94.8	2.5	2.5	0.5
Exposure period 28 hrs without S9 mix
I	28 hrs	Solvent control (1)	3.2	100**	100	3.5	3	0
		Positive control (2)	2.2	n.t.	104.4	30	26 (s)	12.5
		21.9	2.7	104.7**	114.8	1	0.5	0.5
		43.8 (#)	3.4	77.8**	83.6	4.8	4	0
		87.5 (p)	2.4	59**	85.5	4.5	4	0

* Inclusive cells carrying exchanges

# Evaluation of 200 metaphase plates per culture

n.t.: not tested

P: Precipitation occured

S: Abberation frequency statistically significant higher than corresponding control values

1: Acetone 0.5 % (v/v)

2: EMS 500 µg/ml

Table 2: Summary of results of the chromosomal abberation study with the test substance.

Experiment	Preparation interval	Test item concentration in µg/ml	Polyploid cells in %	Cell numbers in % of control	Mitotic indices in % of control	Aberrant cells in %
						incl. Gaps*	excl. Gaps*	with exchanges
Exposure period 4hrs with S9 mix
I	18 hrs	Solvent control (1)	1.9	100	100	4.5	3	0
		Positive control (2)	3.8	n.t.	90.1	10	10 (s)	0
		5.5	2.3	75.3	100.9	4	3.5	1.5
		10.9	1.6	102.4	115.5	5	4	1
		21.9 (p)	2.7	90.5	120.2	4	3.5	1.5
II	28 hrs	Solvent control (1)	3.8	100	100	3	3	0.5
		Positive control (3)	1.9	n.t.	100	11.5	11 (s)	1
		5.5	3.8	191.4	107.5	3	3	0.5
		10.9	3.1	144	99.4	1	1	0
		21.9 (p#)	1.6	219.4	113.1	3	2.8	0.8

* Inclusive cells carrying exchanges

# Evaluation of 200 metaphase plates per culture

n.t.: not tested

P: Precipitation occured

S: Abberation frequency statistically significant higher than corresponding control values

1: Acetone 0.5 % (v/v)

2: CPA 1.4 µg/ml

3: CPA 2 µg/ml

Conclusions:

Interpretation of results (migrated information):
negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

The test substance was found to be positive in the standard Ames test (OECD 471, GLP). No genotoxic potential was found in the chromosomal aberration Test in vitro (OECD 473, GLP), in the Mammalian Cell Gene Mutation Test in vitro (OECD 476, GLP) and in the Erythrocyte Micronucleus Test in vivo (OECD 474, GLP).

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

16. August - 30. September 1988

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Species:

mouse

Strain:

Swiss

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Wiga, Sulzfeld, FRG
- Age at study initiation: approx. 8 weeks of age
- Weight at study initiation: males: 25.1 - 33.8 g and females: 18.9 - 28 g
- Fasting period before study: Feed was withheld overnight before dsing until about 4 hours after administration of the test substance.
- Housing: groups of 5 in polycarbonate cages
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2 °C
- Humidity (%): 60-80 %
- Photoperiod: 12 hrs dark / 12 hrs light

IN-LIFE DATES: 09. August - 30. September 1988

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: Corn oil
- Concentration of test material in vehicle: 5000 mg/kg bw
- Amount of vehicle (if gavage or dermal): 10 mL/kg bw

Duration of treatment / exposure:

single stomach intubation

Frequency of treatment:

once

Post exposure period:

Test item group and vehicle group animals dosed at 5000 mg/kg bw were sacrificed 24, 48 and 72 hrs following dosing.
Positive control animals were sacrificed 48 hrs following dosing.

Remarks:

Doses / Concentrations:
5000 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide dissolved in 0.9% NaCl solution was used as a positive control and dosed once orally at a level of 50 mg/kg bw.

Tissues and cell types examined:

Bone marrow smears from femur.

Details of tissue and slide preparation:

Preparation of the bone marrow smears
The cell suspension was centrifuged at 1000 rpm (approximately 100 g) for 5 min. The supernatant was removed with a Pasteur pipette. A drop of serum was left on the pellet. The cells in the sediment were carefully resuspended in the serum. A drop of the cell suspension was placed on the end of a slide which was previously cleaned (24 hours immersed in a 1:1 mixture of 96% ethanol/ether and cleaned with a tissue) and marked (with the animal number). The drop was spread by moving a clean slide with round-whetted sides at an angle of 45" over.the slide with the drop of bone marrow suspension."The preparations were then air-dried and thereafter 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 stained for 3 min in undiluted May-Grünwald solution followed by 2 min in May-GrOnwald solution diluted 1:1 with Sörensen buffer pH 6.8. Thereafter slides were rinsed in this buffer and stained for 25 min in 5% (v/v) Giemsa solution in Sörensen buffer pH 6.8. The preparations were rinsed for 1 min in
running tap-water and blotted dry between filter paper. The dry slides were cleared by dipping them in xylol before they were embedded in DePeX and mounted with a coverslip.

Analysis of the bone marrow smears for micronuclei
All slides were randomly coded before examination. At first the slides were screened at a magnification of 100 x for regions of suitable technical quality, i.e. where the cells are well spread, undamaged and well stained. Slides were scored at a magnification of 1000x. The number of micronuclei was counted in 1000
polychromatic erythrocytes. The ratio polychromatic to normochromatic erythrocytes was determined by counting and differentiating the first 1000 erythrocytes at the same time. Micronuclei were counted in polychromatic erythrocytes only.

Evaluation criteria:

Acceptability of assay and criteria for response
A micronucleus test is considered acceptable if it meets the following criteria:
a) The positive control substance induced a statistically significant (Wilcoxon rank-sum test at P<0.05) increase in the frequencies of micronuclei.
b) The incidence of micronuclei in the control animals should reasonably fall within the laboratory historical control data range.
A test substance is considered positive in the micronucleus test if:
a) It induces a statistically (P<0.05) as well as biologically significant increase in the frequencies of micronuclei (at any dose or at any sampling time) in the combined data for both sexes, or in the data for male or female groups separately.
A test substance is considered negative in the micronucleus test
if:
a) None of the tested concentrations or sampling times showed a statistically significant (at P<0.05) increase in the incidence of micronuclei either in the combined data for both sexes or in the data for male or female groups alone. The preceding criteria are not absolute and other modifying factors may enter into the final evaluation decision.

Statistics:

The Wilcoxon rank-sum test was used to assess significant differences between the numbers of micronuclei in the treatment and control groups, in which P<0.05 was used as the lowest level of significance.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a preliminary study 12 animals (3 males and 3 females per group) were dosed orally with 5000 and 2000 mg/kg bw. No signs of reaction to treatment were observed. Based on the results of this pilot study 5000 mg/kg bw was selected as an appropriate dose for the Micronucleus test.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei (for Micronucleus assay):
No increase in the frequency of micronuclei was observed. The incidenceof micronuclei in the test substance and control animals were found to be in the range of the historical data (0.75 +/- 0.97; mean +/- deviation, N = 700). The positive control substance induced in both sexes a statistically sgnificant (P < 0.05) increase in the number of micronuclei.

- Ratio of PCE/NCE (for Micronucleus assay):
The ratio of polychromatic to normochromatic erythrocytes was always in the same range as that of the control values in all test substance dose groups.
The groups that were treated with cyclophosphamide showed a decrease in the ratio of polychromatic to normochromatic erythrocytes, which reflects a toxic effect of this compound on the erythropoiesis.

- Statistical evaluation:
No statistically significant differences in the frequency of erythrocytes containing micronuclei either between the solvent control and the dose group (5000 mg/kg bw) or between the various sacrifice intervals (24, 48 and 72 hours) was observed.

Conclusions:

Interpretation of results (migrated information): negative

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

In vitro

Ames test:

Two Ames tests are available. The first Ames test was performed in 1988 when the substance underwent new substance registration in the EU. The second Ames test was performed in 2015 and is representative of the test material manufactured at a new production site.

The first mutagenicity of the test substance was evaluated in the standard Ames test according to OECD TG 471 and GLP in the bacteria strains TA 1535, TA 1537, TA 98 and TA 100 of S. thyphimurium (RCC Notox 1988). The test substance was tested both in the presence and absence of metabolic activation (S9 mix from induced rat liver) at concentrations of 100, 333, 1000, 3330 and 5000 µg/plate. A preliminary toxicity test was carried out in the tester strain TA100 with concentrations ranging from 1 to 5000 µg/plate, from which it was concluded that 5000mg/plate could be used as the highest concentration. 0.1 ml DMSO/plate was used as solvent control. Three Petri dishes were prepared per strain per concentration and in order to confirm the results, the experiments were repeated. In the mutagenicity assay the test substance induced a 5 to 45 fold increase in the number of revertant (His+) colonies in the tester strains TA1537, TA98 and TA100 both in the absence and presence of S9-mix in two independent experiments. Tester strain TA1535 was considered negative in both experiments. The positive controls gave the expected results. Based on the results of this study it was concluded that the test substance can be considered as mutagenic in the Ames test.

The more recent Ames test was performed according to OECD TG 471 and GLP in 2015 (BASF 2015) and it confirmed the pattern of mutagenic effects in bacteria. The Ames test was performed with Salmonella typhimuriam strains TA 1535, TA 100, TA 1537, TA 98 and E. coli WP2 uvrA with doses between 33 μg - 5000 μg/plate in the standard plate assay with and without metabolic activation (liver S9 mix from induced rats). Precipitation of the test substance was found from 333 μg/plate onward with and without S9 mix. No bacteriotoxic effect was observed under all test conditions. A biologically relevant increase in the number of his+ or trp+ revertants (factor ≥ 2: TA 100, TA 98 and E.coli WP2 uvrA or factor ≥ 3: TA 1535 and TA 1537) was not observed in the standard plate test either with or without S9 mix using tester strains TA 1535 and E.coli WP2uvrA.

A distinct and dose dependent increase in the number of his+ revertants exceeding a factor of 2 compared to the concurrent vehicle control was observed with TA 100 and TA 98 with and without metabolic activation. Using tester strain TA 1537, a distinct and dose depending increase of revertants exceeding a factor of 3 compared to the concurrent vehicle control was observed in the standard plate test with and without metabolic activation. The highest increase in revertants observed was 38fold and is therefore in a comparable range to the test performed in 1988.

Chromosome aberration test:

In a mammalian cell cytogenetics assay according to OECD TG 473 and GLP (RCC 2007), the test substance formulated in acetone was assessed for its potential to induce structural chromosome aberrations in V79 cells up to cytotoxic and/or precipitating concentrations in the absence and the presence of metabolic activation by S9 mix. Two independent experiments were performed, each set up with two parallel cultures, in which the first experiment included an exposure time of 18 hrs and the second a exposure and/or harvest time of 28 hrs. Per culture at least 100 metaphase plates were scored for structural chromosome aberrations. The concentrations of the test substance in both experiments ranged from 0 to 350 µg/mL depending on the exposure time and the use of metabolic activation. The concentrations used were chosen according to an initial range–finding cytotoxicity test. The negative controls (vehicle controls) gave frequencies of aberrations within the range expected for the V79 cell line. Both of the positive controls, i.e. cyclophosphamide and ethylmethanesulfonate, led to the expected increase in the number of cells containing structural chromosomal aberrations. The test substance did not cause any increase in the number of structurally aberrant metaphases incl. and excl. gaps in both experiments, with and without a metabolizing system. In addition no increase in the frequency of cells containing numerical aberrations was noted.  In conclusion, it can be stated that under the experimental conditions reported, the test substance did not induce structural chromosome aberrations in V79 cells when tested up to cytotoxic and/or precipitating concentrations.

Mammalian Cell Gene Mutation test:

In a cell gene mutation assay in accordance with OECD TG 476 and under GLP, the potential of the test substance to induce mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus using the cell line V79 was investigated (CCR 1989). The test was performed with the same batch that gave the positive result in the Ames test. The treatment period was 4h in two independent experiments (each with duplicate cultures) at concentrations of 6; 10; 15; and 20 µg/ml with metabolic activation and concentrations of 6; 10; 30; 60 and 100 µg/ml without metabolic activation. Appropriate reference mutagens were used as positive controls and showed a distinct increase in induced mutant colonies, indicating that the tests were sensitive andvalid. No substantial and reproducible dose dependent increase in mutant colony numbers was observed in both main experiments. Cytotoxic effects were observed in the absence of metabolic activation (relative plating efficiency of 84.8%, 80.5% and 49.1% at 10, 15 and 20 µg/ml, respectively), whereas with metabolic activation no cytotoxicity was seen up to the highest test concentration of 100 µg/ml. Higher concentrations precipitated in the culture medium. In conclusion it can be stated that under the experimental conditions the test substance did not induce mutations in the HPRT-locus with or without metabolic activation.

In vivo

Micronucleus test:

In a micronucleus test in accordance with OECD TG 474 and GLP in Swiss mice (5 animals/sex/ dose) the test substance was dosed once by gavage at 5000 mg/kg bw (RCC NOTOX 1988). The dose was selected based on the results in a preliminary toxicty study. The tested batch was the same that gave the positive result in the Ames test. The dosing volume was 10 ml/kg bw and corn oil was used as vehicle. Bone marrow cells were harvested 24, 48 and 72 hours (negative control and all dose levels) and 48 hours post-treatment (for positive control). There were no signs of toxicity during the study. The test substance did not cause any significant decreases in the ratio of polychromatic to normochromatic erythrocytes at the 24, 48 and 72 hour sampling time. No significant increase in the frequency of micronucleated erythrocytes in bone marrow at a dose of 5000 mg/kg bw after treatment with the test substance was observed. The positive control cyclophosphamide induced the appropriate response. In conclusion the test substance was found to be not clastogenic or aneugenic in this test.

Justification for classification or non-classification

The test substance was found to be positive in the standard Ames test. However, no genotoxic potential was found in the Chromosomal Aberration Test in vitro, in the Mammalian Cell Gene Mutation Test in vitro and in the Erythrocyte Micronucleus Test in vivo. Based on these results, the test substance was not classified and labelled for mutagenicity according to Directive 67/548/EEC when it was included in Annex I (Index no. 611-044-00-0) which was then transferred to Annex VI of Regulation 1272/2008/EC.