(2Z)-2-phenylhex-2-enenitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Type of information:

experimental study

Adequacy of study:

key study

Study period:

11-07-2006 to 06-11-2006

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

Guideline study performed under GLP. All relevant validity criteria were met. The test was consistent with the guideline methodology applicable at the time.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2006

Report date:

2006

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

(1997), noting that the requirement to evaluate 4000 erythrocytes for micronuclei, was not implemented until a later version (2014) of the guideline. This would not be considered a deviation as the test was conducted to the methodology applicable at the time.

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.12 (Mutagenicity - In Vivo Mammalian Erythrocyte Micronucleus Test)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OPPTS 870.5395 (In Vivo Mammalian Cytogenetics Tests: Erythrocyte Micronucleus Assay)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

inspected: August 2005 ; signature: November 2005

Type of assay:

mammalian erythrocyte micronucleus test

Test material

Test material information

Constituent 1

Reference substance name:

-

EC Number:

482-160-5

EC Name:

-

Cas Number:

130786-09-3

Molecular formula:

C12H13N

IUPAC Name:

(2Z)-2-phenylhex-2-enenitrile

Test material form:

liquid

Details on test material:

- Physical state: liquid
- Storage condition of test material: approximately 4°C in the dark, under nitrogen
- Other: colourless

Test animals

Species:

mouse

Strain:

other: Crl:CD-l (ICR)BR

Details on species / strain selection:

The species and strain was selected in accordance with the OECD TG 474 guideline.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Recognised supplier (reported in the full study report)
- Age at study initiation: approximately 5 to 8 weeks
- Weight at study initiation: definitive test: males 22 - 30 g (males only were used in the definitive test following a preliminary toxicity test using males/females which indicated no marked intersex differences in toxicity)
- Assigned to test groups randomly: Yes.
- Fasting period before study: No.
- Housing: Group housed (up to 7 per group)
- Diet (e.g. ad libitum): Certified Rat and Mouse diet 5LF2, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: > 7 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 15 per hour
- Photoperiod (hrs dark / hrs light): 12 h light / 12 h dark

IN-LIFE DATES: From: 2006-07-11 To: 2006-08-30

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

- Vehicle(s)/solvent(s) used: arachis oil
- Justification for choice of solvent/vehicle: Applicant assessment indicates: Aqueous vehicle was not applicable due to limited solubility. Arachis oil BP was considered as appropriate based on test item solubility. Formulations were freshly prepared prior to dosing.
- Concentration of test material in vehicle: Range-finding test: 2000 mg/kg and 1000 mg/kg (or 200 mg/mL and 100 mg/mL respectively); Definitive test: 1000, 500, 250 mg/kg (or 100, 50, 25 mg/mL respectively)
- Amount of vehicle (if gavage or dermal): Treatment volume was 10 mL/kg for control (negative, untreated groups) and all treatment groups with applicable test item concentrations per group; positive control was 10 mL/kg. For further information see 'Doses / concentrations'.
- Type and concentration of dispersant aid (if powder): Not applicable.

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
Arachis oil BP was considered as appropriate based on test item solubility. Formulations were freshly prepared prior to dosing.

DIET PREPARATION
- Rate of preparation of diet (frequency): Not applicable.
- Mixing appropriate amounts with (Type of food): Not applicable.
- Storage temperature of food: Not applicable.

Duration of treatment / exposure:

Dosed once, then termination at 24 hours (vehicle, positive control and 1000 mg/kg bw groups), and/or 48 hours (vehicle and 1000 mg/kg bw duplicate groups, plus 500 mg/kg bw, 250 mg/kg bw dose levels)

Frequency of treatment:

Once by oral gavage

Post exposure period:

Termination at 24 hours (vehicle, positive control and 1000 mg/kg bw groups), and/or 48 hours (vehicle and 1000 mg/kg bw duplicate groups, plus 500 mg/kg bw, 250 mg/kg bw dose levels)

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Vehicle control - Group

Doses / concentrations 2

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

Low - Group

Doses / concentrations 3

Dose / conc.:

500 mg/kg bw/day (nominal)

Remarks:

Intermediate - Group

Doses / concentrations 4

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

High - Group

No. of animals per sex per dose:

7 male mice per group (14 males for vehicle and 1000 mg/kg dose levels, in two groups for 24 and 48 hour termination and analysis).

Control animals:

yes, concurrent vehicle

Positive control(s):

Cyclophosphamide
- Justification for choice of positive control(s): The choice of positive control was made in accordance with the OECD TG 474 guideline.
- Route of administration: Oral gavage
- Doses / concentrations: 1 dose; 50 mg/kg bw
- Other: five (5) males - single dose were used for micronucleus test, treated with cyclophosphamide at 50 mg/kg bw.

Examinations

Tissues and cell types examined:

Bone marrow was extracted and smear preparations were made and stained Polychromatic (PCE) and Normochromatic (NCE) erythrocytes were scored for the presence of micronuclei and PCE/NCE ratio was calculated as an indicator for (genetic) toxicity.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Following a range-finding test at: 2000 mg/kg and 1000 mg/kg, the test item dose levels used in the Definitive test were 1000, 500, 250 mg/kg. This was based on significant clinical signs being seen in the range finding test at and above 1000 mg/kg: Hunched posture, lethargy, ataxia, ptosis, decreased respiratory rate, laboured respiration, splayed gait, prostration and hypothermia. With the clinical signs of hunched posture, ptosis, lethargy, ataxia and decreased respiratory rate: 1000 mg/kg was selected as the maximum tolerated dose (MTD) of the test item for use in the main test, with 500 and 250 mg/kg as the lower dose
levels.
Applicant assessment indicates: the maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study based on the observed toxicity that limited the maximum dose level to the MTD dose level of 1000 mg/kg bw.

TREATMENT AND SAMPLING TIMES (in addition to information in specific fields):
Treatment and Control groups were treated once. 24-hours and/or 48-hours following the treatment the respective groups were terminated and then bone marrow was extracted from the femurs as part of sampling. Smear preparations were subsequently made and stained.

DETAILS OF SLIDE PREPARATION:
24-hours and/or 48-hours following treatment (as applicable), femurs were extracted, aspirated with foetal bovine serum and bone marrow smears were prepared following centrifugation and re-suspension. The smears were air-dried, fixed in absolute methanol, stained in May-Grunwald / Giemsa. Then allowed to air-dry and a cover slip applied using mounting medium.

METHOD OF ANALYSIS:
Stained smears were coded and examined blind using microscopy (x1000) magnification. The incidence of micronucleated cells per 4000 polychromatic erythrocytes (PCE-blue stained immature cells) per animal was scored. In addition, normochromatic erythrocytes (NCE-pink stained mature cells) associated with 1000 erythrocytes was counted; these cells were scored for incidence of micronuclei. The ratio of polychromatic to normochromatic erythrocytes was calculated together with the appropriate group mean values and standard deviations.

OTHER: Not applicable.

Evaluation criteria:

1. Comparison was made between the number of micronucleated polychromatic erythrocytes occurring in each of the test item groups and the number occurring in the vehicle control group:
- A positive response would be demonstrated when: there is a statistically significant dose response, toxicologically relevant increases in the number of micronucleated polychromatic erythrocytes observed at the 24-hour or 48-hour termination time compared with the corresponding vehicle control group
- A negative response would be fulfilled if the positive response criteria were not fulfil (no statistically significant dose responses, no toxicologically relevant increases in micronucleated polychromatic erythrocytes.

2. A positive response for bone marrow toxicity is demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically lower than the vehicle control group.

Data is subject to statistical analysis, where appropriate. References: UKEMS sub-committee on Guidelines of Mutagenicity Testing Reports, Part III (1989).

Statistics:

Wherea appropriate, data is subject to statistical analysis using appropriate methods as recommended in UKEMS Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a transformation using Student's t-test (two tailed). Any significant results would be examined by one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Remarks:

No statistically significant PCE/NCE decreases at up to 1000 mg/kg bw observed

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Remarks on result:

other: In the range finder: There was no marked difference in toxicity of the test material between the sexes; therefore the definitive test was performed using only male mice.

Additional information on results:

RESULTS OF DEFINITIVE STUDY
- Types of structural aberrations for significant dose levels (for Cytogenetic or SCE assay): Not applicable.
- Induction of micronuclei (for Micronucleus assay):
Vehicle Control (24-h), 0 mg/kg bw: Group mean (SD) = 1.4 (2.9) PCE with micronuclei per 2000 PCE
Vehicle Control (48-h), 0 mg/kg bw: Group mean (SD) = 1.0 (1.0) PCE with micronuclei per 2000 PCE
Positive Control (24-h), 50 mg/kg bw/day: Group mean (SD) = 55.2 *** (12.9) PCE with micronuclei per 2000 PCE
Low dose (24-h), 250 mg/kg bw: Group mean (SD) = 3.1 (3.0) PCE with micronuclei per 2000 PCE
Intermediate dose (24-h), 500 mg/kg bw: Group mean (SD) = 1.6 (1.1) PCE with micronuclei per 2000 PCE
High dose (24-h), 1000 mg/kg bw: Group mean (SD) = 2.4 (1.3) PCE with micronuclei per 2000 PCE
High dose (48-h), 1000 mg/kg bw: Group mean (SD) = 1.4 (1.9) PCE with micronuclei per 2000 PCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes ; *** P < 0.001
- Ratio of PCE/NCE (for Micronucleus assay):
Vehicle Control (24-h), 0 mg/kg bw: Group mean (SD) = 0.99 (0.53) PCE/NCE
Vehicle Control (48-h), 0 mg/kg bw: Group mean (SD) = 2.00 (1.65) PCE/NCE
Positive Control (24-h), 50 mg/kg bw/day: Group mean (SD) = 1.33 (0.49) PCE/NCE
Low dose (24-h), 250 mg/kg bw: Group mean (SD) = 1.56 (0.82) PCE/NCE
Intermediate dose (24-h), 500 mg/kg bw: Group mean (SD) = 1.13 (0.18) PCE/NCE
High dose (24-h), 1000 mg/kg bw: Group mean (SD) = 1.65 (0.56) PCE/NCE
High dose (48-h), 1000 mg/kg bw: Group mean (SD) = 1.50 (0.50) PCE/NCE
Where: PCE = polychromatic erythrocytes ; NCE = normochromatic erythrocytes
- Appropriateness of dose levels and route: The maximum dose level was considered to be more than adequate to investigate the genotoxic endpoint in this study based on the observed toxicity that limited the maximum dose level to the MTD dose level of 1000 mg/kg bw. The appropriateness of the dose and route was confirmed during the study (recorded in the full study report). With adequate toxicity being observed in the range-finding test via the oral route it was considered to be unnecessary to investigate the intraperitoneal route of administration. Furthermore, in the definitive test: clinical signs were observed when dosed with the test item at and above 250 mg/kg in both the 24 and 48-hour groups where applicable, these were as follows: Hunched posture, ptosis, ataxia, lethargy and splayed gait. The observation of clinical signs was taken to indicate that systemic absorption had occurred.
- Statistical evaluation: No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw relative to the concurrent vehicle control.
- Other: A statistically significant (P < 0.001) response was observed in PCE with micronuclei per 2000 PCE therefore confirming the sensitivity of the test system under the conditions of the test.

Applicant's summary and conclusion

Conclusions:

Interpretation of results:
Negative
Under the conditions of this study the test item was considered to be non-genotoxic.

Executive summary:

The study was performed according the requirements of OECD TG 474, EU Method B.12 and US EPA OPPTS 870.5395 and Japan MHLW/METI guidelines under GLP conditions. Within the study a preliminary range-finding test was performed to find suitable dose levels of the test item, route of administration and to investigate to see if there was a marked difference in toxic response between the sexes at 2000 mg/kg and 1000 mg/kg dose levels. There was no marked difference in toxicity of the test item between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the oral route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1000 mg/kg and with 500 and 250 mg/kg. Exposure was via oral gavage and then terminations were completed at 24 or 48 hours, as applicable. The femoral bone marrow was extracted, and smear preparations made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Within the vehicle controls further groups of seven males were dosed with the vehicle (arachis oil) and then terminations were completed at 24 or 48 hours, as applicable. A positive control group utilising cyclophosphamide at 50 mg/kg was also employed and terminated at 24 hours. There were no premature deaths seen in any of the dose groups. Clinical signs were observed in animals dosed with the test item at and above 250 mg/kg in both the 24 and 48 hour groups where applicable, these were as follows: Hunched posture, ptosis, ataxia, lethargy and splayed gait. No statistically significant decreases in the PCE/NCE ratio were observed in the 24 or 48 hour test item dose groups when compared to their concurrent control groups. However, the observation of clinical signs was considered to indicate that systemic absorption had occurred. No statistically significant responses were observed in the test item treatment groups up to 1000 mg/kg bw relative to the concurrent vehicle control. The positive control group showed .a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system. Under the conditions of this study, the test item would not be considered as genotoxic.