Fluorobenzene

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames-Test: negative; similar to OECD TG 471; 2021; no GLP; K2; Salmonella typhimurium strains TA 1535, TA 1538, TA 100, TA 1537 and TA 98; no Escherichia coli strain; with and without metabolic activation

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Remarks:

AMES

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

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

Species / strain / cell type:

S. typhimurium, other: TA 1538

Metabolic activation:

with and without

Metabolic activation system:

S9-mix from PCB-induced rat liver

Test concentrations with justification for top dose:

0.08, 0.16, 0.32, 0.64, 1.28, 2.56, 5.12 or 10.24 µI/plate

Vehicle / solvent:

DMSO

Untreated negative controls:

not specified

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

N-ethyl-N-nitro-N-nitrosoguanidine

other: 2-aminoantharcene

Details on test system and experimental conditions:

The test was performed in duplicate and repeated at least 3 times separately. Plates were inverted and incubated at 37°C in the dark for 3 days. Colonies of his+ revertants were counted after incubation.

Evaluation criteria:

Chemicals inducing more than twice the number of revertant colonies on the control plate were considered as mutagenic.

Species / strain:

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

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

≥ 5.12 µl/plate

Vehicle controls validity:

valid

Untreated negative controls validity:

not specified

True negative controls validity:

not specified

Positive controls validity:

valid

Number of revertant colonies in Ames test with fluorobenzene in S. typhimurium TA 98, TA 1538, TA 1537, TA 100 and TA 1535:

Compound	Dose/plate	His^+revertants/plate
		TA 98		TA 1538		TA 1537		TA 100		TA 1535
		- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9	- S9	+ S9
DMSO	0.05 ml	28±6	30±5	22±7	24±5	8±3	11±4	181±23	175±36	32±8	30±9
ENNG	2 µg	-	-	-	-	-	-	1994±377	-	-	-
	10 µg	-	-	-	-	-	-	-	-	2489±287	-
2-NF	2 µg	1798±258	-	-	-	-	-	-	-	-	-
	5 µg	-	-	1659±228	-	-	-	-	-	-	-
9-AA	100 µg	-	-	-	-	1288±198	-	-	-	-	-
2-AA	5 µg	-	1249±202	-	753±62	-	132±36	-	1549±269	-	174±21
Fluorobenzene	0.08 µl	28±3	29±4	28±4	32±6	10±3	9±2	184±19	196±21	33±3	31±3
	0.16 µl	31±4	33±8	24±3	22±4	10±2	10±3	165±12	188±16	30±3	51±7
	0.32 µl	33±5	28±4	21±3	27±5	12±4	8±1	157±22	195±19	40±5	41±8
	0.64 µl	30±3	26±3	23±4	27±4	10±3	8±2	180±16	204±28	38±4	31±4
	1.28 µl	28±3	33±5	27±5	20±3	11±3	9±2	160±19	172±15	31±3	32±3
	2.56 µl	26±3	21±3	18±2	24±4	9±2	7±1	204±18	185±17	33±4	30±3
	5.12 µl	15±4*	26±4	8±4*	18±2	2±4*	8±2	172±14	191±24	34±5	36±6
	10.24 µl	0*	0*	0*	0*	0*	0*	0*	0*	0*	0*

Data represent the results of 3 separate experiments and give the mean values of 3 plates. The number of revertant colonies indicates mean±SD. All these revertant colonies were counted after 68-72 h incubation. * indicates toxic effects.

Conclusions:

Fluorobenzene was not mutagenic in any of the strains tested either with or without metabolic activation.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

MNT: negative; according to OECD TG 474; 1991; GLP; K1; NMRI mice

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

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 474 (Mammalian Erythrocyte Micronucleus Test)

GLP compliance:

yes

Type of assay:

mammalian erythrocyte micronucleus test

Species:

mouse

Strain:

NMRI

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: BRL Tierfarm Füllinsdorf, CH-4414 Füllinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approx. 30 g
- Fasting period before study: 18 hours before treatment
- Housing: single housing in Makrolon Type I cages
- Diet: pelleted standard diet (ALTROMIN 1324, D-4937 Lage/Lippe) ad libitum except during fasting period
- Water: tap water ad libitum
- Acclimation period: minimum 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±3
- Humidity (%): 30-70
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

- Vehicle used: corn oil
- Justification for choice of solvent/vehicle: relative nontoxicity for the animals
- Concentration of test material in vehicle:
- Amount of vehicle: 10 ml/kg

Duration of treatment / exposure:

not specified

Frequency of treatment:

once

Post exposure period:

24, 48 and 72 hours

Dose / conc.:

5 000 mg/kg bw/day (nominal)

No. of animals per sex per dose:

6 animals/sex/dose were treated, 5 animals/sex/dose were evaluated

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide (CPA)
- Route of administration: orally, once
- Doses / concentrations: 30 mg/kg bw

Tissues and cell types examined:

1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION:
A preliminary study on acute toxicity .was performed with the same strain and under identical conditions as in the mutagenicity study. It is generally recommended to use the maximum tolerated dose or the highest dose that can be formulated and administered reproducibly. The volume to be administered should be compatible with physiological space available. The maximum tolerated dose level was determined to be the dose that caused toxic reactions, without having major effects on survival within 72 hours.

TREATMENT AND SAMPLING TIMES ( in addition to information in specific fields):
Approximately 18 hours before treatment with the test article the animals received no food but water ad libitum. At the beginning of the treatment the animals were weighed and the individual volume to be administered was adjusted to the animal's body weight. The animals received the test article once. Twelve animals, six males and six females, were treated per dose group. Sampling of the bone marrow was done 24, 48 and 72 hours after
treatment.

DETAILS OF SLIDE PREPARATION:
The animals were sacrificed by cervical dislocation. The femora were removed, the epiphyses were cut off and the marrow was flushed out with fetal calf serum, using a 5 ml syringe. The cell suspension was centrifuged at 1,500 rpm for 10 minutes and the supernatant was discarded. A small drop of the resuspended cell pellet was spread on a slide. The smear was air-dried and then stained with May-Grünwald (MERCK, D-6100 Darmstadt)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg). At least one slide was made from each bone marrow sample.

METHOD OF ANALYSIS:
Evaluation of the slides was performed using NIKON microscopes with 100x oil immersion objectives. 1000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in the same sample and expressed in normochromatic erythrocytes per 1000 the PCEs. The analysis was performed with coded slides. Five animals per sex and group were evaluated as described. The remaining animal of each test group was evaluated in case an animal had died in its test group spontaneously or due to gavage error.

Evaluation criteria:

A test article is classified as mutagenic if it induces a statistically significant increase in the number of micronucleated polychromatic erythrocytes at for at least one of the test points. A test article producing no statistically significant increase in the number of micronucleated polychromatic erythrocytes at any of the test points is considered non-mutagenic in this system. This can be confirmed by means of the nonparametric Mann-Whitney test. However, both biological and statistical significance should be considered together.

Statistics:

Statistical significance at the five per cent level (p < 0.05) was evaluated by means of the non-parametric Mann-Whitney test.

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

One out of 18 treated animals died.

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 5000 mg/kg bw Fluorbenzol suspended in corn oil. The volume administered was 10 ml/kg bw.
- Dose: 5000 mg/kg
- Clinical signs of toxicity in test animals: reduction of spontaneous activity (1 male and 1 female 24 hours post-treatment), eyelid closure (2 males and 1 female 6 hours post-treatment; 1 male and 1 female 24 hours post-treatment), apathy (1 female 24 hours post-treatment), death (1 female 48 hours post-treatment).

RESULTS OF DEFINITIVE STUDY
The mean number of normochromatic erythrocytes was increased after treatment with the test article at preparation interval 48 h as compared to the mean value of NCEs of the corresponding negative control, indicating that Fluorbenzol had cytotoxic properties. In comparison to the corresponding negative controls there was no significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article. The mean values of micronuclei observed after treatment with Fluorbenzol were in the same range as compared to the negative control groups.

Summary of results:

Test Group	Dose (mg/kg bw)	Sampling time (h)	PCEs with micronuclei (%)	Range ofmicronuclei in 1000 PCE per animal	PCE/NCE
Negative control	0	24	0.10%	0-4	1000/861
Fluorbenzol	5000	24	0.07%	0-2	1000/830
Positive control	30	24	1.36%	6-25	1000/766
Negative control	0	48	0.06%	0-3	1000/941
Fluorbenzol	5000	48	0.10%	0-3	1000/1316
Negative control	0	72	0.12%	0-4	1000/697
Fluorbenzol	5000	72	0.20%	0-3	1000/760

           

Conclusions:

The test article did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008.

 

The test substance was negative in the Ames assay when investigated in Salmonella typhimurium strains with and without metabolic activation. Also, the test substance did not induce micronuclei as determined by the micronucleus test in the bone marrow cells of the mouse (MNT). 

 

As a result, the substance is not considered to be classified for mutagenicity under Regulation (EC) No. 1272/2008, as amended for the seventeenth time in Regulation (EC) No. 2021/849.