2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-03-23 to 1987-07-23

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with national standard methods

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

1988

Report date:

1988

Materials and methods

Principles of method if other than guideline:

Method: other: FDA-Guidelines for Safety Assessment of Direct Food Additives (App. II); FDA-Red Book

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

EC Number:

247-063-2

EC Name:

2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine

Cas Number:

25513-64-8

Molecular formula:

C9H22N2

IUPAC Name:

2,2,4(or 2,4,4)-Trimethyl-1,6-hexanediamine

Test material form:

other: liquid

Details on test material:

2,2,4(or 2,4,4)-trimethylhexamethylenediamine of Hüls AG, purity 99.7 % (mean of 9 potentiometric titration analyses).
Actually the test substance is identified in the reference with CAS No. 25620-58-0. However, the production process has been yielding the 2,2,4 (or 2,4,4) isomer mixture (CAS No. 25513-64-8) all the time since the beginning of the production of this substance at this site. In previous years the substance was not identified with a precision sufficient for REACH. The correct CAS No. would have been 25513-64-8 all the time.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Strain: Wistar derived SPF-Albinos of Han/WIST strain
- Source: Zentralinstitut für Versuchstierkunde Hannover
- Age: about 6 weeks
- Weight at study initiation:   males 99 to 158 g   females 96 to 141  g
- Number of animals: 20 per sex and dose group
- Housing: single in conventional Makrolon cages
- Diet:. ad libitum, standard fixed-furmula diet "Ssniff R"
- Water: ad libitum, tab water
- Acclimation period: 9 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 +/- 1.5 °C
- Humidity (%): 60 % +/- 10 %
- Air changes (per hr): 16 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h / 12 h

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

ADMINISTRATION / EXPOSURE 
- Vehicle: Deionized water
- Total volume applied: 10 ml/kg bw d
- Concentrations: 0.0, 0.1, 0.6, or 1.8 % (w/w), prepared before each  treatment.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

At study start and after 6 and 13 treatment weeks samples of test compound solutions were determined by potentiometric titration (0,1N HCl) for
analyses on identity, concentration and stability.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

once daily (midmorning), 7 days/week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw (total dose)

Remarks:

control

Doses / concentrations 2

Dose / conc.:

10 mg/kg bw (total dose)

Remarks:

low dose

Doses / concentrations 3

Dose / conc.:

60 mg/kg bw (total dose)

Remarks:

intermediate dose

Doses / concentrations 4

Dose / conc.:

180 mg/kg bw (total dose)

Remarks:

high dose

No. of animals per sex per dose:

20 test and control animals
Satellite groups: additional 5 animals for control group and high dose group III

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: preceding dose range finding study with decreasing body-weight-gain at a level of 200 mg/kg after 10 days
- Rationale for selecting satellite groups: 5 males and 5 females each  were attached to the high dose and control groups for recovery  observations
- Post-exposure period: core groups: none; satellite groups: 4 weeks

Positive control:

no

Examinations

Observations and examinations performed and frequency:

. CLINICAL OBSERVATIONS AND FREQUENCY: 
- Clinical signs: Daily: Sensory and motor behavior, hair coat, body  orifices, urine and fecal excretion, general health status and dose  responses.
- Mortality: Twice daily
- Body weight: Weekly and individually
- Food consumption: Weekly and individually
- Water consumption: ad libitum, not recorded
- Ophthalmoscopic examination: Cornea and episcleral vesels, anterior  chamber, pupil, lens, and retina (if possible) were examined using an  
ophthalmoscope fitted with switchable lens systems and a slit lamp before  the first dosing, after 6 weeks (10 animals per sex and dose group each)  and after the recovery period (remaining animals).
- Hematology: Before the first dosing, after 6 weeks (10 animals per sex  and dose group each) and after the recovery period (remaining animals):  
Erythrocytes, leukocytes, hemoglobin, hematocrit, platelet count, MCV,  MCH and MCHC, differential blood count, reticulocytes, inclusion bodies,  
prothrombin time.
- Biochemistry: Before the first dosing, after 6 weeks (10 animals per  sex and dose group each) and after the recovery period (remaining  animals): 
Albumin, alk. phosphatase, calcium, chloride, cholesterol,  creatinine, CK, glucose, AST (GOT), ALT (GPT), inorg. phosphorus, iron,  potassium, 
sodium, total bilirubin, total protein, triglyceride, urea  nitrogen (BUN), uric acid, GOT/GPT ratio, Na/K ratio. 
- Electrophoresis:  Albumin, alpha1+alpha2-globulin, beta-globulin, gamma-globulin.
- Urinalysis: Before the first dosing, after 6 weeks (10 animals per sex  and dose group each) and after the recovery period (remaining animals):  
20 ml/kg bw intragastric administration of water followed by 18 hours  sampling period in metabolic cages. Determination of color, protein,  
pH-value, glucose, bilirubin, urobilinogen, blood, nitrite, ketones,  sediment, spec. weight.
- Other: Hearing was tested by simple noise production, and reflex  examinations were proformed with special regard to awareness, emotion,  
coordination and autonomic functions (modified IRVING screen) before the  first dosing, after 6 weeks (10 animals per sex and dose group each) 
and  after the recovery period (remaining animals).

Sacrifice and pathology:

ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): 
- Macroscopic: Complete autopsy. Weights of cerebrum with cerebellum,  pituitary, heart, liver, kidney (l+r), adrenal (l+r), spleen, prostate  gland, 
testis (l+r) with epididymis, ovary (l+r), uterus.
- Microscopic: Skin, mammary gland, salivary gland, trachea, esophagus,  thyroid (2x), parathyroid (2x), thymus, heart, lungs, aorta, liver,  spleen, 
pancreas, kidney (2x), adrenal (2x), stomach, duodenum, jejunum,  ileum, cecum, colon, rectum, lymph node (mesenterialis and cervicalis),  
ovary (2x)/testis + epididymis (2x), prostate/uterus, urinary bladder,  sciatic nerve, skeletal muscle, bone with marrow (sternum + femur), eye  
with N. opticus (2x), cerebrum with brain stem (3x), cerebellum, spinal  cord, pituitary.

Other examinations:

no other examinations

Statistics:

STATISTICAL METHODS: Separately for males and females, Scheffé test for comparison of group mean values.
- Weight changes and food consumption: One- respectively two-factorial analysis of variance. 
- Organ weights: Analysis of co-variance
- Clinical chemistry and hematology: Analysis of variance for dose-effect  curves with factors group and time and interaction group/time.  
Epsilon-correction according to Greenhouse and Geisser.

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):

effects observed, treatment-related

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Details on results:

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL:
- Mortality and time to death:   
Control: 1 male sacrificed moribund (week 8).   
Low dose: No deaths.   
Mid dose: 2 males (weeks 7 and 14).   
High dose: 2 males (weeks 4 and 5) + 7 females (weeks 3, 3, 7, 8, 12,  13, 14).   Organ alterations noticed during necropsy and histopathological 
investigations of these animals were unspecific and did not point to  target organs. 
No animal died during the recovery period.
- Clinical signs: None in control, low dose and mid dose groups. High  dose group: Beginning after 4 weeks: Slightly reduced righting reflex,  
individuals with rough fur and salivation (males and females). The  reduction in righting reflex persisted throughout the recovery period.
- Body weight gain: The rate was significantly reduced only in high dose  males predominantly during the second half of the study 
(weeks 1-13:  -13.7 %, weeks 7-13: -33.9 %). It recovered immediately during the first  week of the recovery period.
- Food/water consumption: Significantly reduced food consumption was observed in high dose males during the second half of the study and also  
during the entire treatment period (weeks 1-13: -6.8 %, weeks 7-13: -5.2  %). The food conversion ratio was distinctly reduced in this group during   the last three weeks of treatment (-4.1 %; -2.3 %; -2.1 %). No distinct difference between treated and control was observed during the recovery  
period.
- Ophthalmoscopic examination: There were no findings which were considered to be treatment related.
- Clinical chemistry: Significant intergroup differences occurred in a  number of clinical chemistry parameters sporadically and mainly without a  
clear time- or dose-relation. These statistical results were therefore considered by the authors to be incidental. No abnormal values were  
observed at the end of the recovery period.
- Hematology: Slightly increased mean values of segmented neutrophilic granulocytes (statistically significant after 6 weeks: +33 %) were found  in 
the high dosed animals. Slight, statistically significant variations  in erythrocytes, reticulocytes, hematocrit, MCH, MCHC and prothrombin  time 
were existent within the normal range and mainly without any dose  specific tendency. These differences were therefore considered by the  authors 
to be without biological importance and not directly due to test substance administration. No abnormal values were observed at the end of  the 
recovery period.
- Urinalysis: No change was seen in the urine values in any group during  treatment and recovery.
- Organ weights: No distinct differences between treated and control were  found in males. Females of the mid and high dose groups exhibited  
significantly higher (than control) mean liver weights (absolute +6.0 and  +6.6 %, relative +9.4 and +13.9 %, respectively), and high dose females  
had higher mean spleen weights (absolute +18.7 %, relative +25.7 %). At  the end of the recovery period, there were no more significant  differences  between treated and control in the liver and spleen weights.  Reduced left adrenal weights (absolute -22.3 %, relative -23.7 %) in the  high dose 
female recovery rats are considered to be incidental because no  statistical significance had been found at the end of the treatment  period.
- Gross pathology: Gross pathological alterations were equally distributed among the groups. 
The predominant findings were:   
Hyperemia of the lungs. Male+female numbers: 4+5 (control), 3+4 (low),  3+4 (mid), 5+5 (high dose).   
Hydrometra in females: 5 (control), 5 (low), 8 (mid), 4 (high dose).   
No different types of findings were observed after the recovery period.
- Histopathology: No treatment related morphological changes were  revealed.

Effect levels

1

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effects

2

Dose descriptor:

LOAEL

Effect level:

60 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: females: significantly higher (than controls) mean liver weights

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

no further results

Applicant's summary and conclusion

Conclusions:

In this subchronic oral gavage study male and female Wistar rats were administered 0, 10, 60 and 180 mg 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine/kg bw /day in a 3 month investigation. In the 60 mg/kg bw/day group increased mean liver weights were seen in female rats. Therefore, the NOAEL derived from this study is considered to be 10 mg/kg bw/day for male and female rats.

Executive summary:

In this guideline-comparable study three groups of 20 male and 20 female albino wistar rats were dosed with 10, 60, 180 mg 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine/kg bw daily for 13 weeks by oral gavage. A concurrrent control group received deionized water only. The testmaterial was dissolved in deionized water and prepared fresh daily. Satellite groups of 5 animals per sex were added to the control and the high dose group and remained untreated after the 13 weeks application period for a 4 week recovery period. Mortality was developed as in control group 1 male sacrificed moribund at week 8, in low dose group were no deaths, in mid dose group died 2 males at weeks 7 and 14, in high dose died  2 males at weeks 4 and 5 and 7 females at weeks 3, 3, 7, 8, 12,  13, 14. Organ alterations noticed during necropsy and histopathological investigations of these animals were unspecific and did not  point to target organs.  There were no clinical signs in control, low dose and mid dose groups. In high dose group clinical signs started after 4 weeks: Slightly reduced righting reflex, individuals with rough fur and salivation (males and females). The reduction in righting reflex persisted throughout the recovery period. The body weight gain was significantly reduced only in high dose males predominantly during the second half of the study. It recovered immediately during the first week of the recovery period. Food consumption was significantly reduced in high dose males during the second half of the study and also during the entire treatment period. The food conversion ratio was distinctly reduced in this group during  the last three weeks of treatment. No distinct difference between treated and control was observed during the recovery  period. There were no findings concerning ophthalmoscopic examinations which were considered to be treatment related. The anaylsis of the determined clinical chemistry values showed significant intergroup differences occurred in a  number of clinical chemistry parameters sporadically and  mainly without a  clear time- or  dose-relation. These statistical results were therefore considered by the authors to be incidental.  No abnormal values were observed at  the end of the recovery period. Hematology analysis showed slightly increased mean values of segmented neutrophilic granulocytes in the high dosed animals. Slight,  statistically significant variations  in erythrocytes, reticulocytes, hematocrit, MCH, MCHC and prothrombin time were existent  within the normal range and mainly without any dose specific tendency. These differences were therefore considered by the authors  to be without biological importance and not directly due to test  substance administration. No abnormal values were observed at the  end of  the  recovery period. There were no changes in the urin values in any group during  treatment and recovery. Organ weights showed no distinct differences between treated and control were  found in males. Females of the mid and high dose groups exhibited significantly higher (than control) mean liver weights and high dose females had higher mean spleen weights. At the end of the recovery period, there were no more significant differences between treated and control in the liver and spleen weights. Reduced left adrenal weights in the high dose female recovery rats are considered to be incidental because no statistical  significance had been found at the end of the treatment  period. Gross pathological alterations were equally  distributed among the groups.The predominant findings were: Hyperemia of the lungs: Male and female numbers: 4+5 (control), 3+4 (low),  3+4 (mid), 5+5 (high dose). Hydrometra in females: 5 (control), 5 (low),  8 (mid), 4 (high dose). No different types of findings were observed after the recovery period. Histopathology: No treatment related morphological changes were   revealed. Under the experimental conditions of this study the "No-Observed Adverse Effect-Level" (NOAEL) of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine is considered to be 10 mg/kg bw/day and the "Lowest- Observed Adverse Effect-Level" (LOAEL) 60 mg/kg bw/day.