Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-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:

2004-01-08 to 2004-02-13

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2004

Report date:

2004

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 474 (Mammalian Erythrocyte Micronucleus Test)

Version / remarks:

1997

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Type of assay:

other: mammalian cell micronucleus assay

Test material

Test material information

1

Reference substance name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

EC Number:

919-489-5

Molecular formula:

C13H23N

IUPAC Name:

Reaction mass of (2E)-Tridec-2-enenitrile and (2Z)-Tridec-2-enenitrile and (3E)-Tridec-3-enenitrile and (3Z)-Tridec-3-enenitrile

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Age at study initiation: 5-8 weeks
- Weight at study initiation: 21-30 g
- Assigned to test groups randomly: yes
- Housing: in groups
- Diet: ad libitum certified rat and mouse diet 5LF2, BCM, IPS, UK
- Water: ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-25
- Humidity (%): 30-70
- Air changes (per hr): appr. 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Route of administration:

intraperitoneal

Vehicle:

- Vehicle/solvent used: arachis oil

Details on exposure:

TEST MATERIAL
- Amount applied: 10 mL/kg bw
- Concentration: Dose level 1000 mg/kg bw: 100 mg/mL, dose level 500 mg/kg bw: 50 mg/mL, dose level 250 mg/kg bw: 25 mg/mL

Duration of treatment / exposure:

The animals were treated once with the test material, one group of each dose level was killed after 24 hours, the other animals were killed after 48 hours.

Frequency of treatment:

single treatment

Post exposure period:

The animals were observed for signs of toxicity and death one hour after dosing and once daily and immediately before termination.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

500 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

7 male mice per dose group were used.

Control animals:

yes, concurrent vehicle

Positive control(s):

cyclophosphamide
- Route of administration: oral
- Doses / concentrations: 50 mg/kg bw

Examinations

Tissues and cell types examined:

bone marrow smears from the femurs were examined.

Details of tissue and slide preparation:

CRITERIA FOR DOSE SELECTION: Doses were selected based on the results of a range-finding toxicity test.

DETAILS OF SLIDE PREPARATION: The smears were air-dried, fixed in absolute methanol, stained in May-Grünwald/Giemsa, allowed to air-dry and coverslipped using mounting medium.

METHOD OF ANALYSIS: light microscopy at 1000 x magnification

Evaluation criteria:

A positive mutagenic response was considered when a statistically significant, dose-responsive, toxicologically relevant increase in the number of microucleated polychromatic erythrocytes was observed for either the 24 or 48-hour kill times when compared to their corresponding control group.
A positive response for bone marrow toxicity was demonstrated when the dose group mean polychromatic to normochromatic ratio was shown to be statistically significantly lower than the vehicle control group.

Statistics:

The data were statistically analysed using appropriate statistical methods as recommended by the UKEMS Sub-committee on Guidelines for Mutagenicity Testing Report, Part III (1989). The data was analysed following a (x+1)^(1/2) transformation using student's t-test (two tailed) and any significant results were confirmed using the one way analysis of variance.

Results and discussion

Test results

Key result

Sex:

male

Genotoxicity:

negative

Toxicity:

no effects

Vehicle controls validity:

valid

Negative controls validity:

not examined

Positive controls validity:

valid

Additional information on results:

RESULTS OF RANGE-FINDING STUDY
- Dose range: 2000 (i.p. and oral), 1500 (i.p.), 1000 (i.p.) mg/kg bw
- Clinical signs of toxicity in test animals: In animals dosed with the test material via the intraperitoneal route premature deaths occurred at and above 1500 mg/kg bw, though in most instances the animals were killed in extremis due to the severity of the clinical signs they exhibited. Clinical signs were observed at and above 1500 mg/kg bw as follows: hunched posture, prostration, ptosis, decreased respiratory rate, laboured respiration, elevated tail, pallor of the extremities, splayed gait, hypothermia, lethargy and ataxia.

RESULTS OF DEFINITIVE STUDY
- Induction of micronuclei: There were no statistically significant increases in the frequency of micronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups.
- Ratio of PCE/NCE: There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups.
- Other observations: There were no premature deaths or clinical signs in any of the dose groups.

Any other information on results incl. tables

Treatment Group	Number of PCE with Micronuclei per 2000 PCE		PCE/NCE Ratio
	Group Mean	SD	Group Mean	SD
Vehicle Control 48 hour sampling time	1.3	1.4	1.21	0.45
Vehicle Control 24 hour sampling time	1.4	1.9	1.19	0.42
Positive Control 24 hour sampling time	53.8*	26	1.07	0.44
Test substance 1000 mg/kg bw 48 hour sampling time	1.1	1.5	1.22	0.54
Test substance 1000 mg/kg bw 24 hour sampling time	2.9	2.7	1.12	0.50
Test substance 500 mg/kg bw 24 hour sampling time	1.1	1.3	1.01	0.23
Test substance 250 mg/kg bw 24 hour sampling time	0.9	1.1	1.01	0.25

*P<0.001

Applicant's summary and conclusion

Conclusions:

The test material was considered to be non-genotoxic in this in vivo micronucleus test.

Executive summary:

The study, according to OECD Guideline 474, was performed to assess the potential of the test material to produce, damage to chromosomes or aneuploidy when administered to mice. A range-finding toxicity test was performed to find suitable dose levels of the test material, route of administration and to investigate if there was a marked difference in toxic response between the sexes. There was no marked difference in toxicity of the test material between the sexes; therefore the main test was performed using only male mice. The micronucleus test was conducted using the intraperitoneal route in groups of seven mice (males) at the maximum tolerated dose (MTD) 1000 mg/kg bw and with 250 and 500 mg/kg bw as the two lower dose levels. Animals were killed 24 or 48 hours later, the bone marrow was extracted, and smear preparations were made and stained. Polychromatic (PCE) and normochromatic (NCE) erythrocytes were scored for the presence of micronuclei. Further groups of mice were given a single intraperitoneal dose of arachis oil (7 mice) or dosed orally with cyclophosphamide (5 mice), to serve as vehicle and positive controls respectively. Vehicle control animals were killed 24 or 48 hours later, and positive control animals were killed after 24 hours. There were no statistically significant decreases in the PCE/NCE ratio in the 24 or 48-hour test material groups when compared to their concurrent vehicle control groups. There were no statistically significant increases in the frequency of nucronucleated PCEs in any of the test material dose groups when compared to their concurrent vehicle control groups. The elevated value seen in the 24-hour 1000 mg/kg bw test material dose group was the result of two animals having individual scores that were at the high end of the acceptable range for vehicle control animals. It is possible that they were most likely due to the chance scoring of clusters of micronuclei, and with the other animals in the group having low individual scores, with no evidence of a response in the 48-hour group and with the value being within the historical range for vehicle controls it was, therefore, considered to have no toxicological significance. The positive control group showed a marked increase in the incidence of micronucleated polychromatic erythrocytes hence confirming the sensitivity of the system to the known mutagenic activity of cyclophosphamide under the conditions of the test. Based on these results, the test material was considered to be non-genotoxic in the in vivo micronucleus assay.