4-pentylcyclohexanone

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

Ames test: The test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with or without addition of S9 mix (reference 7.6.1 -1).

Link to relevant study records

Reference

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May 16, to July 13, 1990

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:

1981

Deviations:

no

GLP compliance:

yes

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 TA 1538

Metabolic activation:

with and without

Metabolic activation system:

Due to migration, the value was transferred to one of the current document's attachments

Test concentrations with justification for top dose:

1st series of experiments:
5, 10, 50, 250, 500, and 750 µg/plate
2nd and additional series of experiments:
1, 5, 10, 50, 250, and 375 µg/plate (TA 98, TA 1535, TA 1537, and TA 1538)
1, 5, 10, 50, 250, and 500 µg/plate (TA 100)

The test material concentrations used were selected according to the OECD Guideline for this test system. They were chosen to cover a broad concentration range including, if possible, also a toxic concentration.

Vehicle / solvent:

- Vehicle/solvent used: The test material was dissolved in dimethylsulfoxide (DMSO).

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

methylmethanesulfonate

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

2-nitrofluorene

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 4-Nitro-1,2-phenylene diamine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

sodium azide

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

9-aminoacridine

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: Daunomycin

Remarks:

without S9 mix

Untreated negative controls:

no

Negative solvent / vehicle controls:

yes

True negative controls:

no

Positive controls:

yes

Positive control substance:

other: 2-Aminoanthracene

Remarks:

with S9 mix

Details on test system and experimental conditions:

NUMBER OF REPLICATIONS:
- Number of cultures per concentration: 4
- Number of independent experiments : 3

METHOD OF TREATMENT/ EXPOSURE:
- Test substance added in agar (plate incorporation)

TREATMENT AND HARVEST SCHEDULE:
- Exposure duration/duration of treatment: 48 h

METHODS FOR MEASUREMENT OF CYTOTOXICITY
- Method: reduction in the number of spontaneous revertants, clearing of the background lawn, reduced survival of treated cultures

Key result

Species / strain:

S. typhimurium TA 1535

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1537

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 100

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

from 500 µg/plate onwards with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 1538

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 500 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Key result

Species / strain:

S. typhimurium TA 98

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Remarks:

at 375 µg/plate with and without S9 mix

Vehicle controls validity:

valid

Untreated negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RANGE-FINDING/SCREENING STUDIES: In a range finding test (using S.typh. TA 100 and TA 1535 without metabolic activation) toxicity was investigated in a wide range of test material concentrations (50 - 10000 µg/plate). Toxicity normally becomes evident by a reduction in the number of spontaneous revertants, a clearing of the background lawn, or by reduced survival of treated cultures. These effects were observed with the test item in the range finding test at concentrations > 500 µg/plate.
Thus, in the first series of experiments the following concentrations were tested:
5, 10, 50, 250, 500, and 750 µg/plate.
As for the highest concentrations tested strong cytotoxic effects still were observed during the first series of experiments, the second and additional series of experiments were performed using the following concentrations:
TA 98, TA 1535, TA 1537, and TA 1538: 1, 5, 10, 50, 250, and 375 µg/plate
TA 100: 1, 5, 10, 50, 250, and 500 µg/plate

- Signs of toxicity
Because of cytotoxic effects only four concentrations were valid in the first series of experiments (except TA 100). As, however, five concentrations are recommended in the OECD- and EEC- Guidelines, an additional series of experiments was performed, using the same concentrations as in the second series.

- Individual plate counts
see table 1-4
- Mean number of revertant colonies per plate and standard deviation
see table 1-4

Table 1: Results of second series (TA100)

Without S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	124	127	140	144	4	134	9.7	-	-
	1.0	113	144	135	137	4	132	13.4	-	-
	5.0	123	123	120	125	4	123	2.1	-	-
	10.0	139	148	154	149	4	148	6.2	-	-
	50.0	153	162	152	163	4	158	5.8	-	-
	250.0	118	120	108	117	4	116	5.3	-	-
	500.0	55	74	70	59	4	65	9.0	-	+
	0.0	153	149	140	152	4	149	5.9	-	-
2-AA	1.0	150	140	138	163	4	148	11.4	-	-
MMS	500.0	1276	1295	1202	1128	4	1225	76.2	-	-
ENNG	4.0	906	866	721	855	4	837	80.4	-	-

 

With S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	192	164	192	213	4	190	20.1	-	-
	1.0	170	174	189	166	4	175	10.0	-	-
	5.0	189	226	200	208	4	206	15.6	-	-
	10.0	190	166	162	178	4	174	12.6	-	-
	50.0	165	194	217	162	4	185	26.0	-	-
	250.0	161	153	175	177	4	167	11.5	-	-
	500.0	0	0	0	0	4	0	0.0	-	+
	0.0	201	190	166	179	4	184	15.0	-	-
2-AA	1.0	541	624	528	637	4	583	55.9	-	-

Table 2: Results of second series (TA1535)

Without S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	7	16	13	19	4	14	5.1	-	-
	1.0	19	9	16	8	4	13	5.4	-	-
	5.0	26	21	18	13	4	20	5.4	-	-
	10.0	20	20	18	20	4	20	1.0	-	-
	50.0	16	28	8	7	4	15	9.7	-	-
	250.0	7	9	6	17	4	10	5.0	-	-
	375.0	2	9	8	1	4	5	4.1	-	+
	0.0	19	16	16	15	4	17	1.7	-	-
2-AA	1.0	13	17	21	27	4	20	6.0	-	-
ENNG	10.0	863	815	814	798	4	823	28.1	-	-
Ve-H2O		17	17	17	18	4	17	0.5	-	-
NaN3	1.0	448	479	463	378	4	442	44.5	-	-

 

 

With S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	15	14	13	16	4	15	1.3	-	-
	1.0	21	18	20	17	4	19	1.8	-	-
	5.0	14	18	27	22	4	20	5.6	-	-
	10.0	15	14	15	20	4	16	2.7	-	-
	50.0	20	9	14	13	4	14	4.5	-	-
	250.0	19	16	15	16	4	17	1.7	-	-
	375.0	9	7	5	7	4	7	1.6	-	+
	0.0	21	7	26	15	4	17	8.2	-	-
2-AA	1.0	141	122	118	137	4	130	11.2	-	-

 

 

Table 3: Results of second series (TA1537)

Without S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	5	3	4	6	4	5	1.3	-	-
	1.0	3	3	4	2	4	3	0.8	-	-
	5.0	5	6	5	2	4	5	1.7	-	-
	10.0	3	1	3	2	4	2	1.0	-	-
	50.0	6	7	7	10	4	8	1.7	-	-
	250.0	6	2	4	4	4	4	1.6	-	-
	375.0	1	0	1	2	4	1	0.8	-	+
	0.0	4	6	6	4	4	5	1.2	-	-
2-AA	1.0	5	7	8	5	4	6	1.5	-	-
EtOH		4	3	3	4	4	4	0.6	-	-
9-AA	20	30	35	43	42	4	38	6.1	-	-
9-AA	50	556	574	426	517	4	518	65.9	-	-

 

With S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	5	3	6	6	4	5	1.4	-	-
	1.0	6	4	5	14	4	7	4.6	-	-
	5.0	5	4	4	5	4	5	0.6	-	-
	10.0	7	13	3	2	4	6	5.0	-	-
	50.0	2	5	14	5	4	7	5.2	-	-
	250.0	4	6	3	6	4	5	1.5	-	-
	375.0	2	6	1	4	4	3	2.2	-	+
	0.0	9	4	5	4	4	6	2.4	-	-
2-AA	1.0	41	55	61	44	4	50	9.4	-	-

Table 4: Results of second series (TA1538)

Without S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	5	2	9	4	4	5	2.9	-	-
	1.0	8	6	3	7	4	6	2.2	-	-
	5.0	5	3	9	3	4	5	2.8	-	-
	10.0	2	4	4	8	4	5	2.5	-	-
	50.0	6	6	4	2	4	5	1.9	-	-
	250.0	3	7	6	3	4	5	2.1	-	-
	375.0	0	0	1	2	4	1	1.0	-	+
	0.0	6	1	5	5	4	4	2.2	-	-
2-AA	1.0	6	7	8	6	4	7	1.0	-	-
4-NP	10.0	1299	1320	1205	1177	4	1250	69.9	-	-
2-NF	5.0	783	741	543	388	4	614	183.3	-	-

 

 

With S-9 Mix

Conc.(µg/pl)		Individual Values				n	Mean	SD	Pc	Bg
	0.0	14	14	21	8	4	14	5.3	-	-
	1.0	7	14	7	4	4	8	4.2	-	-
	5.0	9	9	16	5	4	10	4.6	-	-
	10.0	c	c	c	c	0	-	-	-	-
	50.0	5	9	8	5	4	7	2.1	-	-
	250.0	6	5	7	8	4	7	1.3	-	-
	375.0	1	1	8	4	4	4	3.3	-	+
	0.0	15	18	14	15	4	16	1.7	-	-
2-AA	1.0	287	315	310	274	4	297	19.3	-	-

 

Conclusions:

Under the test conditions used, the test item was not mutagenic in Salmonella typhimurium TA 98, TA 100, TA 1535, TA 1537, and TA 1538 with or without addition of S9 mix.

Executive summary:

The investigations for mutagenic potential were performed according OECD TG 471 using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used.

The substance was tested in three series of experiments at the following concentrations:

First series of experiments: 5, 10, 50, 250, 500, and 750 µg/plate

Second and additional series of experiments: 

1, 5, 10, 50, 250, and 375 µg/plate (TA 98, TA 1535, TA 1537, and TA 1538)

1, 5, 10, 50, 250, and 500 µg/plate (TA 100)

9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, and sodium azide served as positive control compounds for testing the bacteria. 2-Aminoanthracene was used for testing the bacteria and the activity of the S-9 preparation.

The positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

With and without addition of S-9 as the metabolizing system, the substance did not show any mutagenic activity in the concentration range used.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Description of key information

In vivo micronucleus test: In a study according OECD TG 474, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. The test item is considered to be non-mutagenic in this micronucleus assay (reference 7.6.2 -1).

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

Study period:

May 14, 1990 - July 17, 1990

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

adopted May 26, 1983

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: EEC Directive 84/449, L 251, B 12, p. 137 - 139 Updating of Toxicological Methods Annex V of the Council Directive 79/831/EEC; Part B

Qualifier:

according to guideline

Guideline:

other: Environmental Protection Agency, Code of Federal Regulations, Title 40, Subpart F-Genetic Toxicity, Revision July 1, 1986 "In vivo mammalian bone marrow cytogenetics tests: Micronucleus assay."

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 Fullinsdorf, CH-4414 Fullinsdorf/Basel, Switzerland
- Age at study initiation: minimum 10 weeks
- Weight at study initiation: approximately 30 g
- Assigned to test groups randomly: yes
- Fasting period before study: approximately 18 hours before treatment with the test article the animals received no food but water ad libitum
- Housing: single, Makrolon Type I, with wire mesh top
- Diet: ad libitum, pelleted standard diet (ALTROMIN)
- 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(s)/solvent(s) used: polyethylene glycol PEG 400
- Justification for choice of solvent/vehicle: On the day of the experiment, the test article was suspended in PEG 400. The vehicle was chosen to its relative non-toxicity for the animals. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: Solution were prepared on the day of administration. All animals received a single standard dose volume of 10 mL/kg body weight orally.

Duration of treatment / exposure:

24 h or 48 h

Frequency of treatment:

once

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

24 h sampling time

Dose / conc.:

670 mg/kg bw/day (nominal)

Remarks:

24 h sampling time

Dose / conc.:

2 000 mg/kg bw/day (nominal)

Remarks:

24 and 48 h sampling time

No. of animals per sex per dose:

6

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: orally, once
- Doses / concentrations: 20 and 40 mg/kg b.w. (two groups; only 20 mg/kg b.w. dosing is reported)

Tissues and cell types examined:

bone marrow/erythrocytes

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: 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 48 hours.

TREATMENT AND SAMPLING TIMES: Sampling of the bone marrow was done 24 and 48 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 5 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, F.R.G.)/Giemsa (Gurr, BDH Limited Poole, Great Britain). Cover slips were mounted with EUKITT (KINDLER, D-7800 Freiburg F.R.G.). 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. 2000 polychromatic erythrocytes (PCE) were analysed per animal for micronuclei. To describe a cytotoxic effect the ratio between polychromatic and normochromatic erythrocytes was determined in same sample and expressed in normochromatic erythrocytes per 2000 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 either a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes or a reproducible statistically significant positive response for at least one of the test points.
A test article producing neither a statistically significant dose-related increase in the number of micronucleated polychromatic erythrocytes nor a statistically significant and reproducible positive response at anyone of the test points is considered non-mutagenic in this system.

Statistics:

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

Key result

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
In a first pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w.test substance suspended in PEG 400
All treated animals expressed toxic reactions: reduction of spontaneous activity
Two males and one female: abdominal position
Two males and one female: eyelid closure
Two males and one female: apathy
Two males died within 24 hours after treatment.

In a second pre-experiment 4 animals (2 males, 2 females) received orally a single dose of 2000 mg/kg b.w. test item suspended in PEG 400.
All treated animals expressed toxic reactions: reduction of spontaneous activity and apathy.
Two males and one female: eyelid closure
None of the treated animals died. Therefore, 2000 mg/kg b.w. test item was estimated to be a suitable dose.

Table 1: Summary of results

test group	dose mg/kg b.w.	sampling time (h)	PCE with micronuclei	range of micronuclei per 2000 PCE	PCE/NCE
suspending agent	0	24	0.06%	0-3	2000/1518
test article	200	24	0.07%	0-4	2000/1612
test article	670	24	0.06%	0 - 3	2000/1582
test article	2000	24	0.06%	0-4	2000/1907
cyclo- phosphamide	20	24	0.57%	6 -20	2000/1735
suspending agent	0	48	0.05%	0-3	2000/1601
test article	2000	48	0.06%	0-4	2000/1747

Conclusions:

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Executive summary:

A study according OECD TG 474 was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw . 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCE) per animal were scored.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw.

In a pre-experiment 2000 mg/kg was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the substance had weak cytotoxic effectiveness.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used.

20 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Additional information

Ames test

The investigations for mutagenic potential were performed using Salmonella typhimurium TA 100, TA 98, TA 1535, TA 1537, and TA 1538 as tester strains. The plate incorporation test with and without addition of rat liver S-9 fraction (Aroclor-induced) was used.

The substance was tested in three series of experiments at the following concentrations:

First series of experiments: 5, 10, 50, 250, 500, and 750 µg/plate

Second and additional series of experiments: 

1, 5, 10, 50, 250, and 375 µg/plate (TA 98, TA 1535, TA 1537, and TA 1538)

1, 5, 10, 50, 250, and 500 µg/plate (TA 100)

9-Aminoacridine, daunomycin, 1-ethyl-2-nitro-3-nitrosoguanidine, methyl methanesulfonate, 2-nitrofluorene, 4-nitro-1,2-phenylene diamine, and sodium azide served as positive control compounds for testing the bacteria. 2-Aminoanthracene was used for testing the bacteria and the activity of the S-9 preparation.

The positive controls showed normal reversion properties of all strains and good metabolic activity of the S-9 mix used.

With and without addition of S-9 as the metabolizing system, the substance did not show any mutagenic activity in the concentration range used (reference 7.6.1 -1).

In vivo micronucleus test

A study according OECD TG 474 was performed to investigate the potential of the substance to induce micronuclei in polychromatic erythrocytes (PCE) in the bone marrow of the mouse.

The test article was suspended in polyethylene glycol 400. This suspending agent was used as negative control. The volume administered orally was 10 mL/kg bw . 24 h and 48 h after a single application of the test article the bone marrow cells were collected for micronuclei analysis.

Ten animals (5 males, 5 females) per test group were evaluated for the occurrence of micronuclei. 2000 polychromatic erythrocytes (PCE) per animal were scored.

To describe a cytotoxic effect due to the treatment with the test article the ratio between polychromatic and normochromatic erythrocytes (NCE) was determined in the same sample and reported as the number of NCE per 2000 PCE.

The following dose levels of the test article were investigated:

24 h preparation interval: 200, 670 and 2000 mg/kg bw. 48 h preparation interval: 2000 mg/kg bw.

In a pre-experiment 2000 mg/kg was estimated to be suitable. The animals expressed toxic reactions. After treatment with the highest dose of the test article the number of NCEs was slightly increased as compared to the corresponding negative controls thus indicating that the substance had weak cytotoxic effectiveness.

In comparison to the corresponding negative controls there was no statistically significant enhancement in the frequency of the detected micronuclei at any preparation interval after application of the test article and with any dose level used.

20 mg/kg bw cyclophosphamide administered per os was used as positive control which showed a distinct increase of induced micronucleus frequency.

In conclusion, it can be stated that during the study described and under the experimental conditions reported, the test article did not induce micronuclei as determined by the micronucleus test with bone marrow cells of the mouse. Therefore, the test item is considered to be non-mutagenic in this micronucleus assay (reference 7.6.2 -1).

Justification for classification or non-classification

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

The available test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Thus, the test item is considered not to be classified for genotoxicity under Regulation (EC) No 1272/2008, as amended for the twelfth time in Regulation (EU) No 2019/521.