Registration Dossier

Diss Factsheets

Administrative data

Endpoint:
two-generation reproductive toxicity
Remarks:
based on test type (migrated information)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1987-03-23 to 1988-01-04
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1988
Report date:
1988

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Principles of method if other than guideline:
and U.S. EPA Guidelines, Fed. Reg. 47, No. 100 (1982)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
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-trimethylhexane-1,6-diamine; 2,4,4-trimethylhexane-1,6-diamine
Test material form:
other: liquid
Details on test material:
2,2,4(or 2,4,4)-trimethylhexamethylenediamine of Hüls AG, purity 97.9 % (mean of 12 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: Han/WIST (SPF) Wistar rats
- Origin: Zentralinstitut für Versuchstierkunde (Hannover, Germany)
- Number: 28 per sex and dose = 112 males + 112 females in P generation  (F0)
- Age at first dosing: males 5-6 weeks, females 9-10 weeks
- Weight at study initiation: males 86-153 g, females 150-210 g
- Housing: single in conventional Makrolon cages, escept during mating and breeding periods
- Diet:. ad libitum, standard fixed-furmula diet "Ssniff R"
- Water: ad libitum, tab water
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21.5 +/- 1.5 °C
- Humidity (%): 65 % +/- 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:
other: aqua deionized
Details on exposure:
ADMINISTRATION / EXPOSURE
- Duration of test/exposure: Generally, exposure was continuous from  pre-mating treatment until death / sacrifice. 
Treatment of offspring was installed at the age of 4 weeks.
- Vehicle: Deionized water
- Concentration in vehicle: 0.1, 0.6, or 1.2 %, solutions prepared each  day before treatment
- Total volume applied: 10 ml/kg bw per dose, adapted to weekly  determined body weights
Details on mating procedure:
MATING PROCEDURES:
- Monogamous mating was allowed for a maximum of 21 days by caging  partners together only at night. Each morning every female was checked  
for copulation plugs or subjected to a vaginal smear preparation. When a  plug or sperms was found, partners were separated. 
- A second (similar) mate of the P generation was started 10 days post  weaning for the teratology studies. All mated P females were  hysterectomized  after 20 gestation days for a teratological assessment.
- Mating of F1 animals was begun at 16 weeks of age avoiding  brother-sister mating.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of compound solutions were analyzed upon identity (by gas-chromatography), concentration and stability at study start, after 16 and 26
weeks. In a stability study prior to the study start was found out that the test substance was stable in water for at least 18 days, analysis method:
potentiometric titration. Concentration analyses resulted in recovery values within acceptable limits and were performed by potentiometric titration
Duration of treatment / exposure:
Exposure period: see Test Conditions
Premating exposure period (males): 10 weeks during growth into adulthood
Premating exposure period (females): 2 weeks including two complete estrous cycles
Duration of test: Until day 21 post partum of F2 generation = 287 days
Frequency of treatment:
once daily (midmorning), 7 days/week
Details on study schedule:
Number of generation studies: 2
Doses / concentrations
Remarks:
Doses / Concentrations:
10, 60, 120 mg/kg bw d
Basis:
nominal conc.
No. of animals per sex per dose:
F0: 28 males and females in parent generation
F1: 24 males and females selected on day 21 postpartum
Control animals:
yes, concurrent vehicle
Details on study design:
OTHER EXAMINATIONS: Teratology study, see separate entry in chapter 7.8.2 of IUCLID

Positive control:
no positive control

Examinations

Parental animals: Observations and examinations:
PARAMETERS ASSESSED DURING STUDY P AND F1: 
- Clinical signs: Daily recording, weekly reports: Sensory and motor  behavior, hair coat, body orifices, urine and fecal excretion, general  health 
status and dose responses. Reflex examinations were performed  weekly in 10 rats per dose group and sex with special regard to  awareness, 
emotion, coordination and autonomic functions (modified IRVING  screen). Body weight and food consumption were determined weekly and  
individually (body weights daily during mating period).
- Mortality: Twice daily
Oestrous cyclicity (parental animals):
Mean mating period data did not differ significantly between control and dose groups
Litter observations:
STANDARDIZATION OF LITTERS: Determination of number, potential loss  (mortalities / cannibalism), body weight (days 1+4 post partum per  litter, 
day 21 individually), sex (days 1+21). Selection of 24 males + 24  females from F1 generation on day 21 post partum for the next generation,  
weights as close as possible to weight mean of each sex. Sacrifice of  excessive F1 pups / all F2 pups on day 21 post (respective) partum for  gross 
pathology examination.
PARAMETERS ASSESSED DURING STUDY F1 AND F2: 
- Clinical observations and frequency:    F1, day 21 post partum: Special behavior examinations (Irvin screen,  righting reflex, visual-placing test, 
startle-response test and  pupil-reflex test) on 2 male and 2 female pups of each litter.
Postmortem examinations (parental animals):
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC):    
Sacrifice and autopsy of all parents when no longer needed for mating  or rearing purposes. Females showing no signs of implantations in the  
teratogenicity study were disregarded from evaluation.  
P and F1:  Complete macroscopic examination.
- Organ weights P and F1:    F1 and F2: Heart, liver, kidneys, spleen (2 pups per sex and litter).
- Histopathology P and F1: Preservation of organs from animals which died  or were were killed in extremis during the study.
- Histopathology F1 not selected for mating, F2: Preservation of heart,  liver, kidneys, spleen (2 pups per sex and litter) plus macroscopically  
changed organs.
Postmortem examinations (offspring):
F1, day 21 post partum: Physical anomalies, behavior, development;  including hairgrowth, pinna unfolding, eye opening and teething.
Statistics:
STATISTICAL METHODS:
- Body weight changes, food consumption, fetal weights, placenta weights,  number of corpora lutea, implantations, fetuses and offspring: One  
factorial analysis of variance
- Comparison of group mean values: Method of Tukey
- Organ weights: Covariance analysis, correction for body weight,  comparison of mean values with Scheffé test.
- Comparison of indices: Mann-Whitney U-test
- Group means of gestation length and copulation attempts: Multiple  t-test.
Reproductive indices:
Fertility index: Significantly reduced in high-dose P females (56.0% vs  78.6% in control), not notably affected in F1 females (85.0% in high-dose  vs 
83.3% in control). During delivery, one low-dose female and one  high-dose female of the P generation plus two low-dose females of the F1  
generation destroyed their litters by cannibalism.   The second mating of the P animals, which was done for teratology  studies, led to a reduced 
number of pregnant females in all dosed groups,  which was in the normal range only at the low-dose level: 81.5% / 72% /  64% / 60% in control / 
low / mid / high dose groups. It was associated  with a significantly increased pre-implantation loss of ova for the  high-dose females 
(61.0% vs 36.% in control).
Offspring viability indices:
F1: Mean number of pubs per dam and groups and indices did not differ remarkably between testgroups, and from day 4 to day 21 the only change (dead pups) occured in the control group
F2: Mean number of pups was nearly equal in all testgroups and during the entire 3-week breeding period. No great drop of viability was found in
any testgroup

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Other effects:
not examined

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"

Details on results (P0)

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: Parental data and F1
- Body weight:   During the growth period (weeks 1-10), gain rates of P males were  unaffected in the low-dose group and significantly reduced in the   mid-dose (-8.3%) and high-dose (-12.1%) groups. In the P females, body  weight changes were comparable between controls and dose groups 
during  the growth period. During gestation, the weight development of high-dose  P females was at the borderline of statistical significance 
(-15.6%).  During lactation, no great differences were observed in P females. During  the subsequent teratology study, the bodyweight gain in the 
high-dose  females was significantly reduced (-29.0%).   In the F1 generation, the body weight gain of high-dose males (-14.1%)  and females 
(-11.3%) was significantly reduced during the first 6 weeks  of the treatment period. During gestation, the bodyweight gain rates of  high-dose 
females were insignificantly lower than those of the controls.  During lactation, the initially determined weight deficiencies of mid-  and high-dose 
F1 females were compensated. This led to a statistically  significant increase in the mid-dose group which is considered to be  coincidental.
- Food/water consumption:   During the growth period of the P generation (weeks 1-10), food intake  (-8.0%) and food conversion ratio were 
significantly reduced only the in  high-dosed males. No great intergroup differences were observed in P  females during their growth period. 
During gestation, the reduction in  the high-dose females did not attain statistical significance, and during  lactation no great intergroup 
differences were observed. During the  second gestation, food consumption of high-dose females was significantly  reduced (-9.3%).   In the F1 
generation, food consumption was significantly reduced in the  high-dose males during growth (all 12 weeks: -7.4%; weeks 1-6: -9.2%). No  
significant differences in food consumption and food conversion rate  between treated and control were observed in the F1 females from growth  
through lactation.
- Description, severity, time of onset and duration of clinical signs:   
In the P generation, male mid-dose and high-dose rats showed a slightly  reduced righting-reflex from treatment week 5 on. There were no 
abnormal  findings in low-dose males and in all females, also in the teratology  phase.   
In the F1 generation, all mid- and high-dose animals had salivation shortly after the daily treatment during the entire growth period. This  effect 
continued in the females during gestation and breeding.
- Fertility index: Significantly reduced in high-dose P females (56.0% vs  78.6% in control), not notably affected in F1 females (85.0% in high-dose  vs 
83.3% in control). During delivery, one low-dose female and one  high-dose female of the P generation plus two low-dose females of the F1  
generation destroyed their litters by cannibalism. The second mating of the P animals, which was done for teratology  studies, led to a reduced 
number of pregnant females in all dosed groups,  which was in the normal range only at the low-dose level: 81.5% / 72% /  64% / 60% in control / 
low / mid / high dose groups. It was associated  with a significantly increased pre-implantation loss of ova for the  high-dose females 
(61.0% vs 36.% in control).
- Precoital interval: Mean mating period data did not differ significantly between control and dose groups both in the P and the F1  generation.
- Duration of gestation: Almost equal in all groups (P: 21.3 - 21.6 days;  F1: 21.5 - 21.8 days)
- Mortality, P generation:   
Mid-dose: Two females died, one during the first mating period, the  other one day after weaning.   
High-dose: Three males died, one after two weeks of the 10-week  treatment phase, the second four weeks later, the third after the first  mating 
period. Four females died, one during the first treatment week,  two during the mating period, one during the second gestation period.   
F1 generation: Two high-dose males died, one during treatment week 1,  the second after treatment week 12. Three high-dose females died  
during/after treatment weeks 1, 2, and 6.     
Mortalities were considered to be treatment related.
- Gross pathology incidence and severity: Hyperemic lungs were the most common finding in the P animals that died  during the study (8/9). All 
other findings were identified in only one  animal each. At terminal necropsy, no macroscopic organ changes in P  animals were found which could 
be attributed to test compound  administration.   
Findings occurring in more than one of the F1 animals that died during  the study were hyperemic lungs (3/5), tympanitis (3/5), and redness in  
gastric or intestinal mucous membranes (2/5). At terminal necropsy, no  macroscopic treatment related findings were observed in F1 animals.
- Organ weight changes: No significant intergroup differences were found  in the terminal weights of the testes of P and F1 males. Terminal  
placenta weights of P females were also comparable, and uterus weights  decreased insignificantly with increasing dose level.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain
Dose descriptor:
LOEL
Effect level:
60 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical signs
body weight and weight gain

Results: F1 generation

General toxicity (F1)

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results (offspring)"
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (offspring)"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see "Details on result (parental animals)"
Histopathological findings:
not examined

Details on results (F1)

TOXIC RESPONSE/EFFECTS BY DOSE LEVEL: Offspring toxicity F1 and F2: 
- Litter size: In the fertility study, there were no remarkable  differences between test groups (F1: 8.1 - 9.6; F2: 8.9 - 9.4). A  distinct, though not 
statistically significant reduction was observed in  the second gestation of high-dose P females (teratology study: 4.1 vs 7.6  in control).
- Sex and sex ratios: Statistically insignificant differences between  test groups (F1: m/f = 0.96, 0.99, 0.91, 1.53 in control, low-, mid-, and  high-dose  groups, respectively. F2: 0.92, 1.17, 1.02, 0.86. Second mate  of P: 1.2, 1.4, 1.4, 1.5).
- Viability index: No remarkable differences between test groups (F1:  96.7 - 99.0%; F2: 95.8 - 98.9%)
- Post natal survival until weaning: All litters remaining (after initial  cannibalism) developed normally.
- Effects on offspring: No adverse effect due to P maternal treatment was  observed in any F1 litter during the breeding period.
- Postnatal growth, growth rate: Mean bodyweights of F1 pups were similar  in all groups on days 1, 4, and 21 of breeding. A slight weight  deficiency  of high-dose F2 pups (-4.5% on day 1) became statistically  significant on breeding day 21 (-11.0%). Mean weights of fetuses from the  second P 
mate were comparable in all groups.
- Organ weights: F1 high-dose males selected for organ weight  determinations had significantly increased terminal liver (+8.9%) and  kidney 
(left +10.6%, right +8.1%) weights. The liver weights of the F2  rats showed a dose-dependent increase at the mid-dose and high-dose  levels:    
Males: Mid-dose +9.7%, high-dose +10.2%   
Females: Mid-dose +9.6%, high dose +11.5%
- Terminal necropsy: Findings in F2 animals were considered to be unrelated to treatment with an incidence within the normal spontaneous  
variation range in rats.   
No external abnormalities were found in the fetuses from the dosed  groups of the teratology study (1 small hematoma in a control fetus).  
Skeletal examination revealed a comparable development stage for all test  groups and variations (wavy ribs) at incidences often seen in this strain   of rats. One cystic dilated renal pelvis each in low- and mid-dose  fetuses was considered to be within the normal rate of spontaneously  
occurring anomalies.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Results: F2 generation

Effect levels (F2)

Key result
Dose descriptor:
NOAEL
Generation:
F2
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

no further remarks

Applicant's summary and conclusion

Conclusions:
The results of the two generation study indicate that a daily oral treatment with 10 mg/kg of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine to rats over two generations did not adversely affect maternal or fetal litter development in any stage of the study. Thus a 10 mg/kg dose-level revealed a clear no-effect level (NOAEL) for 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine.
Executive summary:

In a two generation study with integrated teratological investigation, dose levels of 10, 60, 120 mg/kg 2,2,4(or 2,4,4)- trimethylhexane-1,6-diamine were administered once daily by oral gavage to male and female rats. Following a 10-weeks pre-mating treatment period for male animals and a 2 -weeks treatment period for females, this generation of animals (F0) was mated to produce the F1 generation. After gestation, delivery of F1, and a 3-week lactation period, F0 females were mated for a second time. F0 -generation males were sacrificed after this second mating period and F0 -generation females were sacrificed for hysterectomy after 20 gestation days.

24 males and 24 femals of F1 generation were selected per dose-group to form the basis of next generation. Treatment was installed at the age of 4 weeks.

The results of the present study indicate that a daily oral treatment with 10 mg/kg of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine to rats over two generations did not adversely affect maternal or fetal litter development in any stage of the study. Thus a 10 mg/kg dose-level revealed a clear no-effect level (NOAEL) for 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine.

Dose levels of 60 mg/kg (group II) and 120 mg/kg (group III) led to dose-related maternal and paternal reduced increase of weight gains and reduced food consumption. Some mortalities (3 males and 4 females in parental F0- generation and 2 males and 3 females in the F1 -generation of the highest dose group) most likely are due to the test compound administration. At this high (slight toxic) dose level pregnancy rate was distinctly reduced among F0 -females (probably due to pre-implantation loss), which was found to be significantly increased among high dosed females in the teratology segment (120 mg/kg). Above all no adverse effect on litter development, e.g. malformations, behaviour dysfunctions or increased death rates were noticed in any phase of the study. Only liver-(and kidney) weights increased in both, F1- and F2- pups and F2 -pups showed a slight tendency towards decreased weight gains in the highest dose group.