2,6,10-trimethylundec-9-enal

Endpoint summary

Key value for chemical safety assessment

Genetic toxicity in vitro

Description of key information

In vitro gene mutation study in bacteria (Ames) - Perrothon 2003, negative

In vitro mammalian cell gene mutation study - Schulz 2008, negative

In vivo micronucleus test - National Toxicology Program 2003, negative

Link to relevant study records

Reference

Reference 1

Endpoint:

in vitro gene mutation study in bacteria

Type of information:

experimental study

Adequacy of study:

key study

Study period:

17 January 2003 - 10 February 2003

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

EU Method B.13/14 (Mutagenicity - Reverse Mutation Test Using Bacteria)

Deviations:

no

Qualifier:

according to guideline

Guideline:

OECD Guideline 471 (Bacterial Reverse Mutation Assay)

Deviations:

no

GLP compliance:

yes

Type of assay:

bacterial reverse mutation assay

Specific details on test material used for the study:

Batch 9000484970
Expiry date 18 March 2003

Species / strain / cell type:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Metabolic activation system:

S9-mix

Test concentrations with justification for top dose:

Dose range finding test (results used as part of the definitve test):
Strains TA100 and TA98; 3, 10, 33, 100, 333, 1000, 3330 and 5000 µg/plate (with and without metabolic activation).

Definitive test - preincubation method:
Strains TA1535, TA1537 and TA102; 10, 33, 100, 333, 1000 and 3330 µg/plate (with and without metabolic activation).

Definitive test - direct plate method:
Strains TA1535, TA1537 and TA102; 3, 10, 33, 100, 333 and 1000 µg/plate (with and without metabolic activation).

Vehicle / solvent:

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

Untreated negative controls:

yes

Remarks:

DMSO

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

not specified

Positive controls:

yes

Positive control substance:

9-aminoacridine

2-nitrofluorene

sodium azide

cumene hydroperoxide

other: daunomycin; methylmethanesulfonate; 2-aminoanthracene; 1,8 dihydroxy anthraquinone

Details on test system and experimental conditions:

METHOD OF APPLICATION: First experiment, preincubation method; Second experiment, direct plate method.

DURATION
- Preincubation period: 30 minutes by 70 rpm at 37 °C.
- Exposure duration: 48 hours, incubated in the dark at 37 °C.

NUMBER OF REPLICATIONS: Each concentration was tested in triplicate.

NUMBER OF CELLS EVALUATED: The revertant colonies were counted automatically with a Protos model 50000 colony counter or manually, if less than 40 colonies per plate were present. Plates with sufficient test material precipitate to interfere with automated colony counting were counted manually.

DETERMINATION OF CYTOTOXICITY
- Method: To determine the toxicity of the test material, the reduction of the bacterial background lawn, the increase in the size of the microcolonies and the reduction of the revertant colonies were observed.

DOSE RANGE FINDER
- Method: All doses were tested in triplicate in the preincubation and the direct plate assay.
- Results used for defining the definitive dosing range: The highest concentration of the test material used in the definitive mutation assay was the level at which the test material exhibited limited solubility. The dose range finding tests were reported as a part of the preincubation and direct plate assays for the definitive tests, respectively.

Key result

Species / strain:

S. typhimurium TA 1535, TA 1537, TA 98, TA 100 and TA 102

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Table 2: Toxicity of the Test Material in the Dose Range Finding Study (preincubation assay)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA98	333-3330 5000	Slight Slight	Slight Moderate	5000	-^1	-^2
TA100	10-5000	Slight	-^2	333-5000	Slight	-^2

-¹= No reduction of the bacterial background lawn

-²= No reduction in the number of revertant colonies.

Table 3: Toxicity to the Test Material in the Preincubation Assay (Definitive Test)

Strain	Without S9-mix			With S9-mix
	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies	Dose (µg/plate)	Bacterial Background Lawn	Revertant Colonies
TA1535	10	Slight	-^1	333	Slight	-^1
	33-100	Slight	-^2
	333-1000	Moderate	-^2	1000-3330	Moderate	-^1
	3330	Extreme	Microcolonies
TA1537	100	Slight	-^1	333-3330	Slight	-^1
	333	Moderate	Extreme
	1000	Moderate	Moderate
	3330	Extreme	Microcolonies
TA102	333-1000	-^3	-^2	3330	-^3	-^1
	3330	Slight	Moderate

 -¹= No reduction in the number of revertant colonies

-²= Reduction in the number of revertant colonies, but not less than the minimal value of the historical control data range

-³= No reduction of the bacterial background lawn

Table 4: Mutagenic Response of the Test Material in the Preincubation Assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± SD)
	TA1535	TA1537	TA98	TA100	TA102
	Without S9-mix
Positive control	777 ± 42	75 ± 6	377 ± 38	440 ± 24	594 ± 23
Solvent control	11 ± 3	6 ± 3	15 ± 2	139 ± 11	210 ± 4
3			12 ± 3	127 ± 14
10	9 ± 1^1	6 ± 3	14 ± 4	151 ± 15^1	219 ± 21
33	8 ± 2^1	6 ± 3	19 ± 1	186 ± 13^1	206 ± 5
100	5 ± 2^1	7 ± 2^1	10 ± 1	195 ± 14^1	202 ± 22
333	8 ± 3^2	2 ± 1^2	10 ± 1^1	190 ± 13^1	163 ± 7
1000	7 ± 1^2	3 ± 1^2	10 ± 2^1	247 ± 10^1	140 ± 15
3330^SP	MC^3	MC^3	9 ± 1^1	229 ± 27^1	109 ± 15^1
5000^SP			8 ± 3^1	222 ± 4^1
	With S9-mix
Positive control	99 ± 6	139 ± 24	344 ± 65	344 ± 21	693 ± 47
Solvent control	6 ± 2	6 ± 1	20 ± 3	137 ± 12	212 ± 14
3			20 ± 1	149 ± 25
10	9 ± 3	5 ± 2	17 ± 3	142 ± 12	268 ± 9
33	6 ± 4	5 ± 1	19 ± 2	138 ± 3	286 ± 44
100	9 ± 1	5 ± 2	18 ± 3	145 ± 18	230 ± 9
333	6 ± 2^1	6 ± 2^1	11 ± 2	159 ± 11^1	243 ± 12
1000	6 ± 2^2	6 ± 1^1	14 ± 2	159 ± 17^1	245 ± 19
3330^SP	7 ± 4^2	5 ± 1^1	14 ± 3	212 ± 27^1	221 ± 16
5000^SP			15 ± 2	180 ± 36^1

Solvent control: 0.1 mL DMSO

1 = Bacterial background lawn slightly reduced

2 = Bacterial background lawn moderately reduced

3 = Bacterial background lawn extremely reduced

SP = Slight Precipitate

MC = Microcolonies

 

Table 5: Mutagenic Response of the Test Material in the Direct Plate Assay

Dose (µg/plate)	Mean number of revertant colonies/ 3 replicate plates (± SD)
	TA1535	TA1537	TA98	TA100	TA102
	Without S9-mix
Positive control	1067 ± 54	605 ± 92	276 ± 20	998 ± 23	1403 ± 59
Solvent control	14 ± 3	6 ± 1	19 ± 2	140 ± 17	286 ± 22
3	12 ± 2	9 ± 3	20 ± 3	132 ± 6	184 ± 9
10	13 ± 2	7 ± 2	15 ± 3	131 ± 25	183 ± 16
33	14 ± 3	7 ± 2	17 ±3	123 ± 8	190 ± 7
100	12 ± 3	8 ± 3	18 ± 5	124 ± 16	239 ± 31
333	9 ± 1	8 ± 2	18 ± 4	114 ± 4	225 ± 6
1000^SP	7 ± 3	9 ± 1	17 ± 6	119 ± 8	211 ± 13
3330^SP			17 ± 2	124 ± 11
5000^SP			14 ± 1	128 ± 6
	With S9-mix
Positive control	281 ± 27	811 ± 57	1520 ± 39	1073 ± 61	1219 ± 48
Solvent control	13± 6	8 ± 3	22 ± 3	132 ± 10	269 ± 13
3	15 ± 5	9 ± 1	23 ± 6	149 ± 25	202 ± 12
10	15 ± 3	6 ± 2	24 ± 7	138 ± 25	212 ± 8
33	11 ± 2	9 ± 2	21 ± 3	147 ± 16	235 ± 15
100	12 ± 5	9 ± 6	22 ± 1	137 ± 5	205 ± 11
333	7 ± 4	7 ± 3	20 ± 3	123 ± 17	238 ± 19
1000^SP	9 ± 5	6 ± 3	24 ± 2	117 ± 8	214 ± 14
3330^SP			17 ± 4	99 ± 6
5000^SP			21 ± 3	120 ± 6

Solvent control: 0.1 mL DMSO

SP = Slight Precipitate

Conclusions:

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

Executive summary:

ADOXAL was tested in the Salmonella typhimurium reverse mutation assay with five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102). The test was performed in two separate experiments in the presence and absence of S9 -mix (Aroclor-1254 induced rat liver S9-mix). ADOXAL did not induce a dose-related increase in the number of revertant (His+) colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9 -metabolic activation. These results were confirmed in a separate experiment.

Reference 2

Endpoint:

in vitro gene mutation study in mammalian cells

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

27 September 2007 - 10 December 2007

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

guideline study without detailed documentation

Remarks:

The study was performed on a similar substance to the registered substance and as such is considered to be sufficient to address the endpoint by read-across.

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.17 (Mutagenicity - In Vitro Mammalian Cell Gene Mutation Test)

Deviations:

no

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5300 - In vitro Mammalian Cell Gene Mutation Test

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: Gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro.

Specific details on test material used for the study:

Name: Citral N
Batch No 01105997V0

Target gene:

HPRT locus

Species / strain / cell type:

Chinese hamster Ovary (CHO)

Details on mammalian cell type (if applicable):

The CHO (Chinese hamster ovary) cell line (substrain K2) (1, 2) is a permanent cell line derived from the Chinese hamster and has a:
- high proliferation rate (doubling time of about 12 - 16 hours)
- high plating efficiency (about 90 %)
- karyotype with a modal number of 20 chromosomes.
Stocks of the CHO cell line (1-mL portions) were maintained at -196 °C in liquid nitrogen using 7 % DMSO in culture medium as a cryoprotectant. Each batch used for mutagenicity testing was checked for mycoplasma contamination.

Metabolic activation:

with and without

Metabolic activation system:

Aroclor-induced rat liver S9 mix

Test concentrations with justification for top dose:

1st Experiment (4-hour exposure period)
without S9 mix: 0; 3.1; 6.3; 12.5; 25.0; 37.5; 50.0; 75.0; 100.0 μg/mL
with S9 mix: 0; 3.1; 6.3; 12.5; 25.0; 37.5; 50.0; 75.0; 100.0 μg/mL

2nd Experiment (4-hour exposure period)
without S9 mix: 0; 0.63; 1.25; 2.5; 5.0; 7.5; 10.0; 15.0; 20.0 μg/mL

3rd Experiment (4-hour exposure period)
without S9 mix: 0; 2.5; 5.0; 7.5; 10.0; 12.5; 15.0; 17.5; 20.0 μg/mL
with S9 mix: 0; 2.5; 5.0; 10.0; 20.0; 40.0; 60.0; 80.0 μg/mL

Vehicle / solvent:

- Vehicle(s)/solvent(s) used: DMSO
- Justification for choice of solvent/vehicle: Due to the insolubility of the test substance in water, dimethylsulfoxide (DMSO) was selected as the vehicle, which had been demonstrated to be suitable in the CHO/HPRT test and for which historical data are available.
The final concentration of the vehicle DMSO in the culture medium was 1 % (v/v).

Untreated negative controls:

yes

Negative solvent / vehicle controls:

yes

Remarks:

DMSO

True negative controls:

no

Positive controls:

yes

Positive control substance:

ethylmethanesulphonate

other: methylcholanthrene

Details on test system and experimental conditions:

METHOD OF APPLICATION: in medium

DURATION
- Pre-treatment: During the week prior to treatment, spontaneous HPRT-deficient mutants were eliminated by pretreatment with "HAT" medium. 3 – 5 x 10^5 cells were seeded per flask (75 cm²) and incubated with "HAT" medium for 3 - 4 days. After that, a passage in Ham's F12 medium incl. 10 % (v/v) FCS was followed with a subsequent incubation for a further 3 - 4 days.
- Attachment period: For each test group, about 1 x 10^6 logarithmically growing cells per flask (175 cm²; after the 2nd passage in the 1st and 2nd Experiments, respectively) were seeded into about 20 mL Ham's F12 medium supplemented with 10 % (v/v) FCS and incubated for about 20 - 24 hours. Two flasks (one flask referred to as A and one flask referred to as B) were used for each test group.
- Exposure duration: After the attachment period, the medium was removed from the flasks and the treatment medium was added (see table below). In all experiments the cells were exposed for 4 hours with and without S9 mix in an incubator.
- Expression time (cells in growth medium): After the exposure period, the cultures were rinsed several times with Hanks' balanced salt solution (HBSS). Then the flasks were topped up with at least 20 mL Ham's F12 medium with 10 % (v/v) FCS. After an incubation period of about 3 days the 1st passage was carried out. After an entire expression period of 7 – 9 days the cells were transferred into selection medium (2nd passage).
- Selection time (if incubation with a selection agent): For the selection of the mutants, six 75 cm² flasks with 3 x 10^5 cells each from every treatment group, if possible, were seeded in 10 mL selection medium ("TG" medium) at the end of the expression period. The flasks were returned to the incubator for about 6 - 7 days. At the end of the selection period, the medium was removed and the remaining colonies were fixed with methanol, stained with Giemsa and counted.

NUMBER OF REPLICATIONS: 6

NUMBER OF CELLS EVALUATED: six 75 cm² flasks with 3 x 10^5 cells each from every treatment group

DETERMINATION OF CYTOTOXICITY
Cloning efficiency (CE) (pre-experiment):
The procedure for the determination of the cloning efficiency in the pre-experiment was similar to that described for the determination of the cloning efficency 1 (CE1) in the main experiments, except every dose group contained only two cultures.

Cloning efficiency 1 (CE1; survival):
For the determination of the influence of the test substance directly after the exposure period, per dose group about 200 cells were seeded in 25 cm² flasks in duplicate using 5 mL Ham's F12 medium incl. 10 % (v/v) FCS. After an attachment period of 20 – 24 hours, the cells were treated with the vehicle, test substance or positive control for 4 hours. The exposure period was completed by rinsing several times with HBSS. Then the flasks were topped up with 5 mL Ham's F12 medium incl. 10 % (v/v) FCS.

Cloning efficiency 2 (CE2; viability):
The mutation rate after the expression period was determined in parallel to the selection of mutants. For each dose group about 200 cells were taken in duplicate, seeded in 25 cm² flasks using 5 mL Ham's F12 medium incl. 10 % (v/v) FCS.

In all cases, after seeding of the cells the flasks were incubated for 5 - 8 days to form colonies. These colonies were fixed, stained and counted.

Evaluation criteria:

Acceptance criteria:
The HPRT assay is considered valid if the following criteria are met:
• The absolute cloning efficiencies of the negative controls should not be less than 50 % (with and without S9 mix).
• The background mutant frequency in the negative controls should fall within historical negative control data range of 0 – 15 mutants per 10^6 clonable cells.
• The positive controls both with and without S9 mix must induce distinctly increased mutant frequencies (historical positive control data.
• At least 4 dose levels ranging up to a toxic concentration or up to or beyond the limit of solubility under culture conditions should be tested. Freely soluble and apparently non-toxic substances are not tested at concentrations higher than 5 mg/mL or 10 mM.

Key result

Species / strain:

Chinese hamster Ovary (CHO)

Metabolic activation:

with and without

Genotoxicity:

negative

Cytotoxicity / choice of top concentrations:

cytotoxicity

Vehicle controls validity:

valid

Untreated negative controls validity:

valid

Positive controls validity:

valid

Conclusions:

Under the experimental conditions, the conclusion is drawn that Citral N is not a mutagenic substance under in vitro conditions in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

Executive summary:

The substance Citral N was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of Aroclor-induced rat liver S9 mix (exogenous metabolic activation). In this study, in all experiments in the absence and the presence of metabolic activation at least the highest concentrations tested were clearly cytotoxic. On the basis from the results, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolizing system in three experiments performed independently of each other.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed (negative)

Genetic toxicity in vivo

Link to relevant study records

Reference

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

05 April 1993 - 08 April 1993

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

test procedure in accordance with national standard methods with acceptable restrictions

Remarks:

The study was performed on a similar substance to the registered substance and as such is considered sufficient to address the endpoint by read-across.

Qualifier:

according to guideline

Guideline:

other: http://ntp.niehs.nih.gov/testing/types/genetic/invivo/mn/index.html

Version / remarks:

study was conducted according to National Toxicology Program protocols

Deviations:

no

GLP compliance:

not specified

Type of assay:

other: in vivo bone marrow mouse micronucleus

Specific details on test material used for the study:

Citral was obtained from Aldrich Chemical Company, Inc. (Milwaukee, WI) in two lots. Lot 06930PG was used during the 14-week studies, and lot 04402AQ was used during the 2-year studies.

Species:

mouse

Strain:

B6C3F1

Sex:

male

Route of administration:

intraperitoneal

Vehicle:

corn oil

Details on exposure:

3 treatments of the chemical were administered at 24 hr. intervals over 72 hours

Duration of treatment / exposure:

72 hours

Frequency of treatment:

every 24 hours

Post exposure period:

none

Dose / conc.:

250 mg/kg bw/day

Dose / conc.:

500 mg/kg bw/day

Dose / conc.:

750 mg/kg bw/day

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Positive control(s):

5 animals treated with 25 mg/kg Cyclophosphamide

Tissues and cell types examined:

bone marrow derived polychromatic erythrocytes

Details of tissue and slide preparation:

bone marrow is flushed from the femurs and spread onto slides, which are air-dried, fixed, and stained with a fluorescent DNA-specific stain that easily illuminates any micronuclei that may be present

Evaluation criteria:

The acridine orange staining procedure that is used for micronucleus slides allows the scorer to differentiate between the recently formed, immature erythrocytes (polychromatic or PCE) that are less than 48 hrs old, and mature erythrocytes 2-35 days old (normochromatic or NCE) based on their staining characteristics. PCE contain residual RNA and thus they stain differently than the NCE that no longer have residual RNA. MN in PCEs arise from damage that occurred recently (within the past 48 hr).

Statistics:

A formal statistical analysis of the data is performed that includes a trend test, to determine if there is an overall increase across all doses in the frequency of cells containing micronuclei, and a pairwise comparison of each dose group to the corresponding control, to see if any one dose group is statistically different from the control group in frequency of micronucleated cells.

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

yes

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Conclusions:

Negative results were obtained in an in vivo bone marrow micronucleus test in male B6C3F1 mice treated by intraperitoneal injection with 250 to 750 mg/kg Citral daily for 3 days.

Executive summary:

Male B6C3F1 mice were exposed to 0, 250, 500 or 750 mg/kg citral in corn oil via intraperitoneal injection every 24 hours for 72 hours (i.e., total of three injections). Bone marrow was harvested 24 hours after the last dosing, flushed from femurs and spread onto slides. The slides were air-dried, fixed, and stained for scoring micronucleated cell frequency determination per animal. Citral was found not to increase the incidence of micronuclei in B6C3F1 male mice.

Additional information

In vitro gene mutation study in bacteria (Ames)

The in vitro genetic toxicity of the test material was determined in a bacterial reverse mutation assay, Ames test. The study was performed under GLP conditions and in accordance with the standardised guidelines OECD 471 and EU Test Method B.13/14. The study was well reported and assigned a reliability score of 1 in accordance with Klimisch at al (1997).

Five histidine requiring strains of Salmonella typhimurium (TA1535, TA1537, TA98, TA100 and TA102) were exposed to the test material at concentrations ranging from 3 to 5000 µg/plate in the presence and absence of the metabolic activation (S9-mix). Two separate experiments were performed the first using the preincubation method and the second using the direct plate method.

In the preincubation assay, at first the test material was tested in a dose range finding study up to concentrations of 5000 µg/plate in the strains TA98 and TA100. Precipitate was observed on the plates at dose levels of 3330 and 5000 µg/plate. In tester strain TA98, slight toxicity was observed at dose levels of 333 µg/plate and upwards in the absence of S9-mix only. In tester strain TA100, slight toxicity was observed at dose levels of 10 µg/plate and upwards in the absence of S9-mix and at 333 µg/plate and upwards in the presence of S9-mix.

Secondly, the test material was tested up to concentrations of 3330 µg/plate in the strains TA1535, TA1537 and TA102. The test material precipitated on the plates at this dose level. Toxicity was observed in all three tester strains in the absence and presence of S9-mix, except in strain TA102 (with S9-mix).

In the direct plate assay, at first the test material was tested in a dose range finding study up to concentrations of 5000 µg/plate in the strains TA98 and TA100. The test material precipitated on the plates at dose levels of 1000 µg/plate and upwards. The bacterial background lawn was not reduced at any of the concentrations tested and no biologically relevant decrease in the number of revertants was observed.

After that, the test material was tested up to concentrations of 1000 µg/plate in the strains TA1535, TA1537 and TA102. The test material precipitated on the plates at this dose level. The bacterial background lawn was not reduced at any of the concentrations tested and no decrease in the number of revertants was observed.

The test material did not induce a dose-related increase in the number of revertant colonies in each of the five tester strains (TA1535, TA1537, TA98, TA100 and TA102) both in the absence and presence of S9-metabolic activation. These results were confirmed in a separate experiment.

Under the conditions of the test it is concluded that the test material is not mutagenic in the Salmonella typhimurium reverse mutation assay.

In vitro mammalian cell gene mutation study

The test substance was tested for its ability to induce gene mutations at the hypoxanthine-guanine phosphoribosyl transferase (HPRT) locus in Chinese hamster ovary (CHO) cells in vitro. Three independent experiments were carried out, with and without the addition of Aroclor-induced rat liver S9 mix (exogenous metabolic activation. The study was conducted in accordance with OECD Guideline 476, EU Method B.17 and EPA OPPTS 870.5300 and to GLP standard. However, since the study was conducted with a structural analogue of the registered substance it was assigned a reliability score of 2 in accordance with Klimisch at al (1997).

The concentrations used ranged from 0 to 100.0 μg/mL. After an attachment period of 20 – 24 hours and a treatment period of 4 hours both with and without metabolic activation, an expression phase of about 7 - 9 days and a selection period of about 1 week followed. The colonies of each test group were fixed with methanol, stained with Giemsa and counted.

The vehicle controls gave mutant frequencies within the range expected for the CHO cell line. Both of the positive control substances, i.e. EMS and MCA, led to the expected increase in the frequencies of forward mutations.

In this study, in all experiments in the absence and the presence of metabolic activation at least the highest concentrations tested were clearly cytotoxic.

On the basis from the results of the present study, the test substance did not cause any relevant increase in the mutant frequencies either without S9 mix or after adding a metabolising system in three experiments performed independently of each other.

Under the experimental conditions of this study, the test substance is considered not to be a mutagenic substance under in vitro conditions in the HPRT locus assay using CHO cells in the absence and the presence of metabolic activation.

In vivo micronucleus test

Testing was performed according to the methods reported in Shelby et al. (1993). The study was conducted to sound scientific principles with a sufficient level of detail to assess the quality of the relevant results; however, since the study was conducted with a structural analogue of the registered substance it was assigned a reliability score of 2 in accordance with Klimisch at al (1997). This study is regarded as key over the cytogenicity study since it was an in vivo chromosome aberration study rather than an in vitro chromosome aberration study.

Male B6C3F1 mice were injected intraperitoneally three times at 24-hour intervals with 250 to 1,000 mg test substance/kg body weight dissolved in corn oil. Solvent control animals were injected with corn oil only. The positive control animals received injections of cyclophosphamide. The animals were killed 24 hours after the third injection, and blood smears were prepared from bone marrow cells obtained from the femurs. Air-dried smears were fixed and stained; 2,000 polychromatic erythrocytes (PCEs) were scored for the frequency of micronucleated cells in each of four or five animals per dose group.

Negative results were obtained in an in vivo bone marrow micronucleus test in male B6C3F1 mice treated by intraperitoneal injection with 250 to 750 mg/kg daily for 3 days.

Justification for selection of genetic toxicity endpoint
No one study can be selected as key, as all are required to assess the genetic toxicity potential of the test substance, as each of the studies examines a different area of genetic toxicity.

Short description of key information:
Key Study: In vitro gene mutation study in bacteria (Ames): Perrothon 2003 - Negative in the presence and absence of metabolic activation
Key Study: In vitro mammalian cell gene mutation study: Schulz 2008 - Negative in the presence and absence of metabolic activation
Key Study: In vivo micronucleus test: NTP 2003 - Negative

Endpoint Conclusion: No adverse effect observed (negative)

Justification for classification or non-classification

In view of the overall lack of genetic toxicity displayed in the studies available for the test material and its structural analogue, no classification in accordance with Regulation (EC) No. 1272/2008 is required for this endpoint.