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

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1998-01-13 to 1998-02-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1998

Report date:

1998

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

1995

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

(1996)

Deviations:

no

GLP compliance:

yes

Limit test:

no

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

Details on test material:

2,2,4(or 2,4,4)-Trimethylhexanedinitrile of Creanova Spezialchemie GmbH. Purity 96.5 % (GC-FID area), ID 0637/81824, produced January 1997.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Source: Harlan Winkelmann GmbH, 33176 Borchen (Germany)
- Age: 6-8 weeks
- Weight at study initiation: males mean 143 g, females mean 124 g
- Number of animals: 5 per dose group and sex, total 60 including  satellite groups
- Diet: ad libitum, R10 special diet for rats, SSniff R
- Water: ad libitum, tap water
- Acclimation period: at least 5 days
ENVIRONMENTAL CONDITIONS
- Temperature: 22 +/- 3 °C
- Humidity: 30 - 705 %
- Photoperiod: 12 hours artificial light, 12 hours dark
- Air changes: 15 per hour

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

ADMINISTRATION / EXPOSURE
- Vehicle: Corn oil
- Concentration in vehicle: 0, 7.5, 25, or 75 mg/ml
- Total volume applied: 2 ml/kg bw/treatment
- dosing formulations were prepared weekly
- test substance and vehicle was homogenized by means of a magnetic stirrer for 15 minutes

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentrations of test substance and stability were determined by gas-liquid chromatography. Results showed a satisfactory concordance between
measured and nominal concentration and a stability over one week.
Homogenity of the samples was calculated by density-determination. The results showed that the samples were homogeneous.

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Doses / concentrations

Remarks:

Doses / Concentrations:
15; 50; 150 mg/(kg bw * d)
Basis:
other: nominal in corn oil

No. of animals per sex per dose:

5 control and dosed groups
5 control satellite and high dose satellite

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 2 days
SATELLITE GROUPS AND REASONS THEY WERE ADDED: Control (group 5) and high dose (group 6) satellite groups were treated similary to replace
control or high dose animals in case of mortalities in these groups.

Positive control:

no

Examinations

Observations and examinations performed and frequency:

CLINICAL OBSERVATIONS AND FREQUENCY:
- Clinical signs: General observation once daily, considering the peak of  anticipated effects after dosing; at weekends one hour after application.
- Mortality: Twice daily, preferably early morning and late afternoon
- Body weight: Days 0, 7, 14, 21, 28, 30.
- Food consumption: Once each week. Meaningful evaluation was not  possible due to mortality in high dose males and females and medium 
dose  females.
- Water consumption: Daily
- Hematology: End of treatment: Red blood cell count (RBC); total white  blood cell count (WBC); platelet count (PLT); hemoglobin (HGB); 
 hematocrit (HCT); erythrocyte mean corpuscular volume (MCV), mean  corpuscular hemoglobin (MCH), mean corpuscular hemoglobin 
concentration  (MCHC); differential white blood cellcount (granulocyte, lymphocyte,  monocyte, plasmacyte); coagulation.
- Biochemistry: End of treatment: Sodium; potassium; calcium; aspartate  aminotransferase (AST); alanine aminotransferase (ALT); alkaline  
phosphatase (AP); glucose (GLUC); triglycerides (TRIG); cholesterol  (CHOL); total bilirubin (TBIL); blood urea nitrogen (BUN); creatinine  (CREA); 
total protein (TPROT); albumin (ALB).
- Urinalysis: End of treatment: Volume (VOL); specific gravity (SPGR);  pH; color.   Semiquantitative: Protein (PROT); glucose (GLUC); keton; 
urobilinogen  (UBG); blood ingredients.  Microscopical urine sediment analysis (all samples): Leucocytes  (LEUCO); erythrocytes (ERY); bacteria
(BAC); epithelial cells, squamous  (ECSQ) and renal (ECRE); oxalate crystals (OXA); triple phosphate  crystals (TRIP); urate crystals (URA); 
carbonate (CARB); granular  cylinders (GRACYL); phosphate (PHOS).

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: Complete autopsy of all animals that
(a) died during the study or
(b) survived in groups 1-4 and 6.
Weights of adrenals, brain, epididymides, heart, kidneys, liver,  spleen, testes, thymus from animals of groups 1-4 (incl. substitutes of  group 6).
- Microscopic: Kidney, liver, lung, gastrointestinal tract of all high  dose animals which died during the study; selected additional samples.

Other examinations:

OTHER EXAMINATIONS: Functional observational batteries (FOB) in home cage (HCO) and in open field (OFO) once each week, extended version in 
the 4th week.
HCO parameters: Posture; coordination; tremor/convulsion; abnormal  behavior.
OFO parameters: Behaviour; autonomic functions; fur; skin / appearance;  muscle tone; posture; coordination; respiration; activity / attention;  tremor / convulsion; abnormal behavior; eyes / palpepral closure; feces;  urine.
Extended OFO parameters: Vision test; pupillary reflex; winking reflex;  pinna-reflex; hearing test; sense of smell; examination of catalepsy;  
coordination of movement; sensitivity to pain; rearing; grip strength;  landing foot-splay test.   
Motoractivity assessment in 4th week.

Statistics:

STATISTICAL METHODS:
- Kruskal Wallis non parametric analysis of variance and in case of  significance Wilcoxon, Mann, and Whitney U-test: Rearing; landing  foot-splay; 
grip strength; motoractivity; absolute body weights; body  weight changes; absolute and relative organ weights; differential blood  count; urine 
analysis data.
- One way analysis of variance (ANOVA) incorporating a Bartlett's test  for homogeneity of variance and in case of heterogeneous variances a  
Kruskal Wallis test / in case of significant ANOVA a Scheffe test:  Hematological data (except differential blood count) and serum clinical  chemistry 
data.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

NOAEL (NOEL), LOAEL (LOEL):
Oral adminstration of trimethyladiponitrile  for a period of 28 days at dose levels of 150 and 50 mg/(kg bw * d)  resulted in effects of toxicological importance. Administration of  trimethyladiponitrile at a nominal dose level of 15 mg/(kg bw * d) caused  no effects of toxicological importance. Therefore, under the experimental  conditions, the NOAEL is 15 mg(kg bw * d).
ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX
  Throughout the study the analytical results showed a satisfactory  concordance between measured and nominal concentrations (94.5 - 100 %  recovery).
TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death (first treatment = day 0):
medium dose: 1/5 females (day 18), 0/5 males
high dose: 4/10 females (days 5, 12, 13, 18), 5/10 males (days 1, 1, 6,  7, 9)
Time of death: Between day 1 and day 18, approximately 1/2 - 2 hours  after applications.
- Clinical signs:
Control groups: No clinical signs of toxicological importance were  observed.
Low dose group: No clinical signs of toxicological importance were  observed.
Medium dose group: Squatting position, convulsions, increased activity,  prone position and closed eyes were observed on some days of the  
treatment period.
High dose groups: The same symptoms as in the medium dose group were  observed more often and more severe.
- Body weight gain: No differences of toxicological importance were found  in females. The male high dose group had a statistically significantly 
 (p<0.05) lower body weight gain (-23.8 %) and body weight during / after  the first week. Both body weight gain and body weight remained below  
those of the control throughout the study, but the differences were no  longer statistically significant.
- Food/water consumption: No overt intergroup differences in food or  water consumption were found. Determination of food consumption was 
not  possible in groups with mortalities. 
- Clinical chemistry: No significant differences to controls were found  in treated females. The male high dose group showed an increase of sodium  
when compared with control, which was statistically significant but minor  (+2.0 %) and within the historical controls.
- Hematology: Statistically significant findings were found in females  and considered to be of toxicological importance:    
Medium dose: Decrease of red blood cell count (-7.8 %), decrease of  lymphocytes (-8.6 %)   
High dose: Decrease of red blood cell count (-5.8 %), decrease of  hemoglobin (-7.8 %)
- Urinalysis: The only statistically significant finding was an increase  of pH in high (+1.9) and medium (+2.4) dose females, which was, however,  
in the normal range and thus considered to be of minor toxicological  importance. 
- Organ weights: Changes in female liver weights and in male gonad and  epididymis weights were considered to be of toxicological importance:   
Liver weights were increased in medium (absolute +15 %; relative +11.8  %) and high dose (absolute +15 %; relative +11.5 %) females. Statistical  
significance was found only for the medium group relative liver weights  (p<0.05).    
Absolute weights of gonads (-18 %) and epididymis (-28 %) were  statistically significantly decreased in high dose males. The poor  general 
health state of the high-dose males might be an explanation for  the reduced development of the male gonads.    
Due to absence of a dose-relationship and a clear pattern, a  statistically significant decrease in relative heart weight of medium  dose 
males (-7.3 %) was considered to be of no toxicological importance.
- Gross pathology: The macroscopical findings in treated and control  animals consisted of lesions such as pelvic dilatation in the kidneys of  one 
male animal, sperm granuloma in five males and hydrometra in one  female. The lung of individual animals of the different dose and control  groups  showed small foci (0.1 mm diameter). These findings were not  dose-related. One male of the high dose group showed unilateral testis  atrophy. 
The lung of the animals which died during the study showed  congestion and emphysema. The liver of three high dose animals (one male,  two 
females) showed an abnormal appearance and parts of the  gastrointestinal tract of individual high dose male and female animals  showed 
hyperemic / hemorrhagic mucosa. 
- Histopathology: Histopathological examination of animals of the high dose group revealed changes of  toxicological importance predominantly in li ver (centrilobular  vacuolation, one female: centrilobular hypertrophy) and kidney (tubular necrosis). Inflammatory foci in the  lungs were not dose- related and most likely caused as part of a virus  infection. They were not observed in animals that died during the study.  
Histopathological examination of testes and epididymis in high-dose males  showed no significant findings apart from the already macroscopically   detected unilateral atrophy of testes and epididymis in one animal. The  unilateral testes atrophy of this animal does not explain the significant  
reduction of the group mean gonad weight because testes and epididymis of  all animals in this group showed a decreased weight when compared 
with  control, low- or medium-dose group. 
- Other: Functional observational batteries (FOB)   
Home cage (HCO) and open field (OFO): Only mild clinical effects in  individual animals of the high and medium dose groups were observed:  
Minimal hyper-and hypoactivity, piloerection, eructation and  hunchback/squatting position.   
Extended OFO: Animals of all groups showed a normal response. Female  and male animals of all dose groups showed no significant findings  
concerning number or rearings, landing foot-splay test and grip strength  when compared with controls.    
Motoractivity assessment: Differences were minor and not dose-related  in treated animals of both sexes.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

15 mg/kg bw/day (nominal)

Basis for effect level:

gross pathology

haematology

organ weights and organ / body weight ratios

2

Dose descriptor:

LOAEL

Effect level:

50 mg/kg bw/day (nominal)

Basis for effect level:

haematology

organ weights and organ / body weight ratios

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:

Oral administration of 2,2,4(or 2,4,4)-trimethylhexanedinitrile to rats for a period of 28 days at dose levels of 150 and 50 mg/kg bodyweight resulted in effects of toxicological importance. Administration of 2,2,4(or 2,4,4)-trimethylhexanedinitrile at a nominal dose level of 15 mg/kg/day caused no effeccts of toxicological importance. Therefore, in our experimantal conditions, the no effect level (NOEL) is 15 mg/kg/day.

Executive summary:

In this guideline study (OECD 407 (1995); 96/54/EC B.7) three groups of 5 male and 5 female Wistar rats, additional a control and a high dose satellite group each consisting of five males and females, were dosed with 15, 50, and 150 mg/kg/day 2,2,4(or 2,4,4)-trimethylhexanedinitrile once daily by oral gavage for 4 weeks. 2,2,4(or 2,4,4)-Trimethylhexanedinitrile treatment of 15 mg/kg/day caused no effects of toxicological importance. Administration of the test substance of 50 and 150 mg/kg/day caused clinical symptoms associated with a high mortality rate especially in high dose animals (only one medium dose female died). The high dose groups and the high dose satellite groups showed during the application period the clinical symptoms more often and more severe when compared with the medium dose group. Beside these clinical symptoms other effects of toxicological importance were noted in medium and especially in high dose animals. The high dose male group showed a slightly reduced bodyweight gain. Medium and high dose females showed a decrease of Red Blood Cell Count and high dose females showed also a decrease of Haemoglobin. The relative and absolute liver weights were increased in high and medium dose females. The male high dose group showed statistically significantly decreased weight of gonads and epididymides when compared with control. Macroscopical and especially histopathological examination revealed changes of toxicological importance predominantly in liver and kidney. Histopathological examination of testis and epididymis in high dose males showed no significant findings, apart from the already macroscopically detected unilateral atrophy of testis and epididymis in one animal. The unilateral testis atrophy of this animal does not explain the significant reduction of the group mean gonad weight because testis and epididymis of all animals in this group showed a decreased weight when compared with control, low or medium dose groups. The poor general health state of the high dose males might be an explanation for the reduced development of the male gonads.

Oral administration of 2,2,4(or 2,4,4)-trimethylhexanedinitrile to rats for a period of 28 days at dose levels of 150 and 50 mg/kg bodyweight resulted in effects of toxicological importance. Administration of 2,2,4(or 2,4,4)-trimethylhexanedinitrile at a nominal dose level of 15 mg/kg/day caused no effeccts of toxicological importance. Therefore, in our experimantal conditions, the no effect level (NOEL) is 15 mg/kg/day.