2,2,4(or 2,4,4)-trimethylhexanedinitrile

Genetic toxicity: in vivo

Administrative data

Endpoint:

in vivo mammalian somatic cell study: cytogenicity / erythrocyte micronucleus

Remarks:

Type of genotoxicity: chromosome aberration

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1999-07-16 to 1999-09-09

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1999

Report date:

1999

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:

(1992)

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

EPA OTS 798.5395 (In Vivo Mammalian Cytogenics Tests: Erythrocyte Micronucleus Assay)

Version / remarks:

(1987)

Deviations:

no

GLP compliance:

yes

Type of assay:

micronucleus assay

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4(or 2,4,4)-trimethylhexanedinitrile

EC Number:

283-810-9

EC Name:

2,2,4(or 2,4,4)-trimethylhexanedinitrile

Cas Number:

84713-17-7

Molecular formula:

C9H14N2

IUPAC Name:

2,2,4-trimethylhexanedinitrile; 2,4,4-trimethylhexanedinitrile

Constituent 2

Reference substance name:

Reference substance 001

Details on test material:

2,2,4(or 2,4,4)-Trimethylhexanedinitrile of Degussa-Hüls AG, batch no. TMND7 240699-10:00. Purity not reported.

Test animals

Species:

mouse

Strain:

CD-1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS:
- Source: Harlan Italy S.r.l., San Pietro al Natisone (UD, Italy)
- Weight at study initiation: males 20.6 - 29.9 g; females 20.0 - 24.9 g
- No. of animals per dose: 5 males + 5 females per group
positive control, low and mid dose 1 group each;
negative control 2 groups; high dose 2 + 1 reserve groups
- Diet: ad libitum, Altromin MT
- Water: ad libitum, tap water
- Acclimation period: 4-8 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 2 °C
- Humidity: 55 +/- 10 %
- Photoperiod: 12 hours artificial light, 12 hours dark

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

ADMINISTRATION:
- Vehicle: corn oil
- Control groups and treatment:
negative: vehicle
positive: 3 mg mitomycin-C (MMC)/kg bw in sterile distilled water (injectable grade)
- Total volume applied: 10 ml/kg bw
- Duration of test: 24 hours; 48 hours
- Sampling times and number of samples: 24 hours; 48 hours
EXAMINATIONS:
- Clinical observations: daily

Duration of treatment / exposure:

single dose

Frequency of treatment:

1 time

Post exposure period:

24 hours, additional animals 48 hours

Doses / concentrations

Remarks:

Doses / Concentrations:
30; 60; 120 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

10 control
5 test concentration low and medium
15 test concentration high
5 positive control

Control animals:

yes, concurrent vehicle

Positive control(s):

3 mg mitomycin-C (MMC)/kg bw in sterile distilled water  (injectable grade)

Examinations

Tissues and cell types examined:

polychromatic erythrocytes of the bone marrow from femur

Details of tissue and slide preparation:

Animals were sacrified at appropriate sampling times. Femurs were removed and bone marrow cells obtained by flushing with foetal calf serum.
The cells were centrifuged and a concentrated suspension prepared to make smears on slides. Slides were air-dried and then stained with May-Gruenwald and Giemsa. Three slides were made from each animal,   >= 2000 PCE (polychromatic erythrocytes) per animal were analysed for  micronuclei.

Evaluation criteria:

Statistically significant (P<0.05) increase in micronucleus incidence in polychromatic erythrocytes at any dose level for one sex or for both  
sexes pooled as compared to the corresponding negative control    AND   biological significance as compared to the historical control data.
- Criteria for selection of M.T.D.: Maximum dose with expected low  mortality within 24 hours.

Statistics:

BIOMETRY:
- Normally 2000 polychromatic erythrocytes were counted per animal.
-Using original observations a modified chi-squared calculation was employed to compare treated and control groups
- Degree of heterogeneity within each group was first calculated and where this was significantt it was taken into account in the comparision between groups
- Variance ratios or Chi-squared values are taken to show the significance of any difference between each treated group and the controls.

Results and discussion

Test results

Sex:

male/female

Genotoxicity:

negative

Toxicity:

yes

Remarks:

see under: "additional information on results"

Vehicle controls validity:

valid

Negative controls validity:

not applicable

Positive controls validity:

valid

Additional information on results:

MORTALITY: 
- Phase 1 of dose finding, 300 and 600 mg/kg bw: 2/2 males and 2/2 females per dose level died  within 24 hours.
- Phase 2 of dose finding, 150 mg/kg bw: 1/2 males and 1/2 females survived and were sacrificed 24  hours post dosing.
75 mg/kg bw: 2/2 males and 2/2 females survived and were sacrificed 24  hours post dosing.
- Main test, 120 mg/kg bw: 4/15 males and 6/15 females died within 24 hours.
CLINICAL SIGNS:
- Dose finding
600 mg/kg bw: Moribund
300 mg/kg bw: Moribund, lethargy, convulsions, breathing difficulty,  ataxia, salivation, hunched posture, decreased activity, piloerection.
150 mg/kg bw: Piloerection, hunched posture, salivation, soiling of  urogenital region, swollen abdomen, liquid feces, increased breathing,  
pronation, cold to touch.
75 mg/kg bw: Piloerection.
- Main study
120 mg/kg bw: Piloerection, lethargy, decreased activity, ataxia,  increased breathing, tremors, convulsions, hunched posture, pronation,  
swollen abdomen, liquid feces. Surviving animals recovered completely  within 24 hours after treatment.
60 mg/kg bw: Piloerection, liquid feces.
30 mg/kg bw: Piloerection BODY WEIGHT was not affected in any treatment group.
BONE MARROW CELL TOXICITY:
The ratio of mature to immature erythrocytes and the proportion of immature erythrocytes to total were analysed to evaluate the bone marrow cell toxicity. No inhibitory effect on erythropoietic cell division was observed.
GENOTOXIC EFFECTS:    
No statistically significant increases in the numbers of micronucleated  PCE's were observed in any group treated with the test substance at any  
sampling time. Statistically significant increases in the frequency of  micronucleated PCE's were observed in the positive control groups.

Any other information on results incl. tables

EFFECT ON MITOTIC INDEX OR PCE/NCE RATIO: 
  No inhibitory effect on erythropoietic cell division was observed.
GENOTOXIC EFFECTS: 
  No statistically significant increases in the numbers of micronucleated  PCE's were observed in any group treated with the test substance at any  sampling time. Statistically significant increases in the frequency of  micronucleated PCE's were observed in the positive control groups.
---------------------------------------------------------
Treatment    Sex   Time   Micronucl. PCE/1000     NCE/PCE
---------------------------------------------------------
Vehicle       m    24 h      0.9 +- 0.2             1.28
 30 mg/kg bw  m    24 h      1.3 +- 0.5             1.33
 60 mg/kg bw  m    24 h      0.8 +- 0.2             1.02
120 mg/kg bw  m    24 h      1.4 +- 0.4             1.01
Pos. control  m    24 h      6.9 +- 1.9 **          1.31
Vehicle       f    24 h      1.3 +- 0.5             1.27
 30 mg/kg bw  f    24 h      0.3 +- 0.2             0.90
 60 mg/kg bw  f    24 h      1.2 +- 0.3             1.15
120 mg/kg bw  f    24 h      1.2 +- 0.3             0.98
Pos. control  f    24 h      5.9 +- 1.7 *           1.14
Vehicle       m    48 h      0.9 +- 0.2             0.92
120 mg/kg bw  m    48 h      0.7 +- 0.1             0.87
Vehicle       f    48 h      0.9 +- 0.2             1.01
120 mg/kg bw  f    48 h      1.0 +- 0.2             0.94
Pos. control m+f   24 h      6.4 +- 1.2 ***         1.23
---------------------------------------------------------
* p < 0.05; ** p < 0.01; *** p < 0.001
---------------------------------------------------------
Clinical signs and mortality observed at the highest dose level provide evidence of bioavailability of the test substance and adequate exposure.

Applicant's summary and conclusion

Conclusions:

Interpretation of results (migrated information): negative
The test item Trimethyladiponitrile was found to be non-mutagenic in vivo in a micronucleus test on male and female Swiss CD-1 mice.

Executive summary:

A micronucleus test was conducted according to OECD TG 474 (1997) and EU method 92/69/EEC B.12 to investigate the test substance Trimethyladipoinitrile in male and female Swiss CD-1 mice for a possible clastogenic effect on the chromosomes of bone-marrow erythroblasts. 30.0, 60.0 and 120 mg/kg test substance was administered once oral to 5 male and 5 female CD-1 mice per group, with the exception of 5 additional mice in the control group and 10 additional mice in the high-dose group. 5 males and 5 females per dose group were sacrificed 24 hours after treatment, the additional animals 48 hours after treatment. Bone marrow polychromatic erythrocytes were examined microscopically for micronucleated polychromatic erythrocytes (PCE). No significant increase in the frequency of PCE over the control was found with any group treated  with the test substance. For the positive control Mitomycin-C a significant increase in the frequency of PCE was observed. Therefore it is concluded that, Trimethyladiponitrile is not a mutagenic substance under the in vivo conditions in this micronucleus assay using male and female CD-1 mice. Clincal signs and mortality observed at the highest dose-level provide evidence of bioavailability of the test substance and adequate exposure.