4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Genetic toxicity: in vitro

Administrative data

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11 Jan - 29 Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study with acceptable restrictions

Remarks:

Comparable to guideline study with acceptable restrictions (no TA102 or E.coli. WP2 tester strain, only 2-Aminoanthracene was used as positive control with S9 mix). Also in accordance with GLP principles.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

Endpoint:

in vitro DNA damage and/or repair study

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

11 Jan - 29 Mar 1988

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Remarks:

Also in accordance with GLP principles

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 480 (Genetic Toxicology: Saccharomyces cerevisiae, Gene Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

mitotic recombination assay with Saccharomyces cerevisiae

Target gene:

enzyme of adenine metabolizing pathway

Species / strain / cell type:

yeast, other: Saccharomyces Cerevisiae D3

Additional strain / cell type characteristics:

not applicable

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

0.05, 0.1, 0.5, 1.0, 2.5% (w/v)

Vehicle / solvent:

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

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: SMC (Sterigmatocystin, 0.005% (w/v), +S9); 1,2,3,4-Diepoxybutane (DEB, 0.025% (w/v), -S9)

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium then plating on agar

DURATION
- Preincubation period: 4 hours
- Exposure duration: 3 days at 30°C, 1 day at 4°C

NUMBER OF REPLICATIONS: triplicates (1E-05 dilution) and quintuplicates (1E-03 dilution)

DETERMINATION OF CYTOTOXICITY
- Method: survival rate

Evaluation criteria:

Positive: A positive response in this assay is indicated by a dose-related increase of more than threefold in the absolute number of mitotic recombinants per milliliter and in the relative number of mitotic recombinants per 1.0E+05.
Negative: When no reproducible recombinogenic activity is obtained in any of the assays perfomred, the test results are considered to be negative.
Inconcluisve: When a test article cannot be identified clearly as causing a positive or negative response, the results are classified as inconclusive.

Species / strain:

Saccharomyces cerevisiae

Remarks:

D3

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: A preliminary assay with dose levels of 0.01, 0.1, and 1% in the presence and absence of metabolic activation was done, no toxicity was seen.

Table 1: Results of Experiment I

compound	S9 (%)	Conc. (%) (w/v)	Surviving cells/mL (x 10^-7)	Survivors (%)	Mitotic Recombinants/mL (x 10^-3)	Mitotic Recombinants per 10^5 survivors
DMSO	0		8.4	100	14	17
	10		7.6	100	8.0	11
DEB	0	0.025	7.6	90	1100	1400
SMC	0	0.005	8.2	98	8.0	9.8
	10	0.005	7.7	100	240	310
Test substance	0	0.05	7.8	93	7.0	9.0
	0	0.1	8.1	96	10	12
	0	0.5	7.7	92	12	16
	0	1.0	8.6	100	9.0	10
	0	2.5	7.7	92	10	13
	10	0.05	10.3	100	12	12
	10	0.1	9.7	100	9.0	9.3
	10	0.5	9.2	100	9.0	9.7
	10	1.0	8.5	100	8.0	9.4
	10	2.5	8.7	100	4.0	4.6

Table 2: Results of Experiment II

compound	S9 (%)	Conc. (%) (w/v)	Surviving cells/mL (x 10^-7)	Survivors (%)	Mitotic Recombinants/mL (x 10^-3)	Mitotic Recombinants per 10^5survivors
DMSO	0		5.8	100	8.0	14
	10		5.3	100	9.0	17
DEB	0	0.025	6.0	100	720	1200
SMC	0	0.005	4.8	83	14	29
	10	0.005	6.9	100	130	190
Test substance	0	0.05	6.4	100	10	16
	0	0.1	5.3	91	13	24
	0	0.5	4.6	79	6.0	13
	0	1.0	5.5	95	12	22
	0	2.5	5.5	95	8.0	14
	10	0.05	7.3	100	20	27
	10	0.1	5.0	94	10	20
	10	0.5	6.4	100	5.0	7.8
	10	1.0	5.7	100	12	21
	10	2.5	6.6	100	12	18

Conclusions:

Interpretation of results: negative
The test material was not detected as being recombinogenic in the yeast Saccharomyces cerevisiae D3 assay with and without metabolic activation.

Executive summary:

The test material was tested for recombinogenic activity in the yeast Saccharomyces cerevisiae D3 assay for mitotic recombination. A preliminary assay was conducted at dose levles of 0.01, 0.1 and 1.0% in the presence and absence of metabolic activation. No toxicity was seen. Based on these results and on the solubility limitations of the compound, the two assays were performed at dose levels from 0.05 through 2.5% both in the presence and absence of 10% metabolic activation.

In the first assay, a slight increase, not dose-related, occurred in the number of mitotic recombinants, both with and without metabolic activation. Because these increases were slight, not dose-related, and not reproducible, they were not attributed to recombinogenic activity.

Therefore, the test material was not detected as being recombinogenic in the yeast Saccharomyces cerevisiae D3 assay with and without metabolic activation.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

yes

Remarks:

no TA102 or E.coli. WP2 tester strain, only 2-Aminoanthracene was used as positive control with S9 mix

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Test material

Test material information

Constituent 1

Reference substance name:

4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

EC Number:

407-560-9

EC Name:

4,4'-(9H-fluoren-9-ylidene)bis(2-chloroaniline)

Cas Number:

107934-68-9

Molecular formula:

C25H18Cl2N2

IUPAC Name:

4-[9-(4-amino-3-chlorophenyl)-9H-fluoren-9-yl]-2-chloroaniline

Test material form:

solid: particulate/powder

Method

Target gene:

his operon

Species / strain

Species / strain 1

Species / strain / cell type:

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

Species / strain 2

Species / strain / cell type:

S. typhimurium TA 1538

Metabolic activation:

with and without

Metabolic activation system:

cofactor supplemented post-mitochondrial fraction (S9 mix), prepared from the livers of rats treated with Aroclor 1254

Test concentrations with justification for top dose:

preliminary assay: 10, 50, 100, 500, 1000, 5000 µg/plate (with and without metabolic activation)
experiment I: 10, 50, 100, 500, 1000, 5000 µg/plate (with and without metabolic activation)
experiment II: 5, 10, 50, 100, 500, 1000 µg/plate (with and without metabolic activation)

Vehicle / solvent:

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

Controls

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

other: 2-Aminoanthracene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in agar (plate incorporation)

DURATION
- Exposure duration: 48 hours

NUMBER OF REPLICATIONS: triplicates each in 2 independent experiments

DETERMINATION OF CYTOTOXICITY
- Method: reduced background lawn, reduced number of colonies; formation of pinpoint colonies

Evaluation criteria:

Positive: A test article is considered a mutagen when it produces a reproducible, dose-related increase in the number of revertants in one or more strains. This increase should occur for at least three dose levels.
Negative: A test article is considered a nonmutagen when no dose-related increase in the number of revertants is observed in at least two independent experiments. The highest dose level tested for nontoxic solid substances and pure liquids is usually 5000µg/plate. For toxic compounds only the highest dose level tested should show evidence of toxicity.
Inconclusive: When a test article cannot be identified clearly as a mutagen or a nonmutagen in the assay, the results are classified as inconclusive.

Statistics:

No statistical analysis was performed.

Results and discussion

Test results

Test results 1

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 2

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 3

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 4

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Remarks:

Cytotoxicity only for TA1537 without metabolic activation in the first experiment at 5000 µg/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Test results 5

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

no cytotoxicity, but tested up to precipitating concentrations

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

TEST-SPECIFIC CONFOUNDING FACTORS
- Precipitation: precipitation was seen from 500µg/plate and upwards, for 1000 and 5000 µg/plate it interfered with counting of colonies wherefore plates were handcounted at those concentrations

RANGE-FINDING/SCREENING STUDIES: TA100 was used to screen for toxicity induced by the test article, with (4% v/v) and without metabolic activation, since no toxicity was seen the main experiments were carried out with 10% v/v S9 mix for metabolic activation.

Any other information on results incl. tables

Table 1: Results of Experiment I

with or without S9 mix	Test substance concentration	Mean number of revertant colonies per plate (average of 3 plates  ± SD)
	[µg/plate]	Base-pair substitution type		Frameshift type
		TA100	TA1535	TA98	TA1537	TA1538
-	0	119 ± 15	17 ± 9	17 ± 5	6 ± 1	18 ± 3
-	10	114 ± 1	16 ± 3	20 ± 5	7 ± 2	12 ± 3
-	50	114 ± 14	10 ± 1	24 ± 4	4 ± 1	14 ± 3
-	100	104 ± 6	15 ± 1	19 ± 4	6 ± 1	6 ± 1
-	500 P	92 ± 17	14 ± 8	15 ± 2	5 ± 3	9 ± 5
-	1000 P	103 ± 15	13 ± 4	17 ± 6	8 ± 3	15 ± 5
-	5000 P	125 ± 13	11 ± 3	15 ± 7 N	5 ± 1 T	17 ± 6
positive control, -S9	Name	9-AA	SA	NF	9-AA	NF
	Conc. [µg/plate]	1	1	5	50	5
	Mean number of colonies/plate	457 ± 30	448 ± 9	1107 ± 80	571 ± 215	1503 ± 105
		TA100	TA1535	TA98	TA1537	TA1538
	0	153 ± 3	13 ± 1	34 ± 5	8 ± 2	32 ± 4
	10	137 ± 11	11 ± 11	42 ± 6	8 ± 4	39 ± 4
	50	134 ± 8	16 ± 7	42 ± 2	8 ± 1	32 ± 4
	100	175 ± 16	14 ± 1	42 ± 8	7 ± 3	28 ± 3
	500 P	162 ± 16	16 ± 3	41 ± 10	7 ± 3	25 ± 2
	1000 P	159 ± 10	11 ± 3	34 ± 4 N	9 ± 6	27 ± 7
	5000 P	132 ± 14 N	9 ± 3 N	39 ± 11 N	10 ± 4 N	30 ± 9 N
positive control, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Conc. [µg/plate]	1	2.5	1	2.5	1
	Mean number of colonies/plate	489 ± 64	158 ± 45	200 ± 16	68 ± 12	178 ± 5

P = Precipitated

N = Normal background lawn

T = Thinning of background bacterial lawn, indicating toxicity

9 -AA = 9 -Aminoacridine

SA = Sodium Azide

NF = 2 -Nitrofluorene

2 -AA = 2 -Aminoanthracene

Table 2: Results of Experiment II

with or without S9 mix	Test substance concentration	Mean number of revertant colonies per plate (average of 3 plates ±SD)
	[µg/plate]	Base-pair substitution type		Frameshift type
		TA100	TA1535	TA98	TA1537	TA1538
-	0	114 ± 21	10 ± 4	14 ± 1	6 ± 2	14 ± 6
-	5	125 ± 30	20 ± 5	21 ± 11	10 ± 1	15 ± 1
-	10	130 ± 10	17 ± 2	19 ± 6	7 ± 2	13 ± 3
-	50	133 ± 7	14 ± 1	19 ± 3	7 ± 3	14 ± 1
-	100	128 ± 22	15 ± 6	19 ± 4	7 ± 3	13 ± 5
-	500 P	121 ± 6	12 ± 2	18 ± 7	8 ± 2	11 ± 2
-	1000 P	110 ± 11	10 ± 3	14 ± 3	5 ± 3	11 ± 3
positive control, -S9	Name	9-AA	SA	NF	9-AA	NF
	Conc. [µg/plate]	1	1	5	50	5
	Mean number of colonies/plate	344 ± 22	207 ± 9	944 ± 49	414 ± 81	1060 ± 53
		TA100	TA1535	TA98	TA1537	TA1538
	0	142 ± 6	9 ± 3	37 ± 6	8 ± 1	29 ± 8
	5	164 ± 15	12 ± 3	37 ± 2	7 ± 3	35 ± 4
	10	156 ± 22	8 ± 2	35 ± 5	8 ± 1	27 ± 1
	50	137 ± 6	7 ± 5	35 ± 7	11 ± 2	25 ± 5
	100	146 ± 6	9 ± 2	39 ± 1	6 ± 1	22 ± 5
	500 P	144 ± 7	10 ± 3	32 ± 4	9 ± 2	27 ± 7
	1000 P	115 ± 1	8 ± 6	28 ± 16	5 ± 1	27 ± 9
positive control, +S9	Name	2 -AA	2 -AA	2 -AA	2 -AA	2 -AA
	Conc. [µg/plate]	1	2.5	1	2.5	1
	Mean number of colonies/plate	745 ± 56	123 ± 8	660 ± 14	173 ± 41	623 ± 13

P = Precipitated

N = Normal background lawn

T = Thinning of background bacterial lawn, indicating toxicity

9 -AA = 9 -Aminoacridine

SA = Sodium Azide

NF = 2 -Nitrofluorene

2 -AA = 2 -Aminoanthracene

Applicant's summary and conclusion

Conclusions:

Interpretation of results: negative
The test material was not detected as being mutagenic with or without metabolic activation in the standard Ames Salmonella/microsome assay with strains TA1535, TA1537, TA1538, TA98 and TA100 of the bacterium Salmonella typhimurium.

Executive summary:

The mutagenic activity of the test material in the Ames Salmonella/microsome assay was evaluated with a method similar to OECD TG 471 with strains TA1535, TA1537, TA1538, TA98 and TA100 of the bacterium Salmonella typhimurium and under GLP conditions.

The test material did not induce a significant dose-related increase in the number of revertant (His+) colonies in each of the five S. typhimurium tester strains (TA1538, TA1535, TA1537, TA98 and TA100) both in the absence and presence of S9-metabolic activation.  

In this study, the positive and solvent control values were within the laboratory historical control data ranges indicating that the test conditions were adequate and that the metabolic activation system functioned properly.

Based on the results of this study it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay.