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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
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

Principles of method if other than guideline:
OECD Guideline 416 (1983) 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
Details on test animals or test system and environmental conditions:
TEST ORGANISMS
- Strain: Han/WIST (SPF) = Crl:Wi/Br Wistar rats
- Source: Zentralinstitut für Versuchstierkunde (Hannover, Germany)
- Number: 28 per sex and dose = 112 males + 112 females in P generation  (F0). 
For the teratology study, 27 mated control females and 25 mated  females per dosed group were available.
- 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: deionized water
Details on exposure:
ADMINISTRATION / EXPOSURE
- Duration of test/exposure: Exposure was continuous from pre-mating  treatment until death / sacrifice. Pre-mating treatment was 10 weeks  
during growth into adulthood for males and 2 weeks including two complete  estrous cycles for females.
- 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
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
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. The first mating 
was  for the fertility study.
- A second (similar) mate of the P generation with identical partners was  started 10 days post weaning for the teratology studies. All mated P  females  were sacrificed on day 20 of pregnancy for hysterectomy and  judgment of the uterine content.
- For the fertility study, 24 males + 24 females from the F1 generation  (resulting from the first P mating) were selected on day 21 post partum  for 
the next generation, with their weights as close as possible to the  weight mean of each sex. Further data are presented in a separate entry  in 
chapter 7.8.1.
Duration of treatment / exposure:
see Test Conditions
Frequency of treatment:
once daily (midmorning), 7 days/week
Duration of test:
Duration of test: Until gestation day 20 of a second F1 generation
No. of animals per sex per dose:
28
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: male/female
OTHER EXAMINATIONS: Fertility study, see separate entry in chapter 7.8.1

Examinations

Maternal examinations:
PARAMETERS ASSESSED DURING STUDY: 
- Mortality: Twice daily
- 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 gain: weekly and individually, daily during mating period,  every 5th day during second gestation period
- Food consumption: weekly and individually, for 5 day intervals during  second gestation period
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 of organs.
Ovaries and uterine content:
- Examination of uterine content: For each uterus horn: Fetuses (alive /  dead), birth position (anterior / posterior), early and late resorptions,  
placentae, implantation sites.
- Examination of ovaries: Number of corpora lutea
Fetal examinations:
P (and F1):  Complete macroscopic examination of organs.
- Examination of fetuses: Individual weight and sex (ano-genital  distance), externally visible deviations from normal, visceral  imperfections 
(approx. 1/3 of the fetuses, slightly thawn frozen  samples), skeletal defects (approx. 2/3 of the  fetuses).
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.
Indices:
Fertility Index, , Viability Index, Resorption Index, Pre-Implantation Loss Index, Post-Implantation Loss Index, Runts Index, Variation Index,
Number of litters having abnormalities, Number of abnormalities per litter

Historical control data:
no data

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
see below: "Any other information on results..."

Effect levels (maternal animals)

Dose descriptor:
NOAEL
Effect level:
10 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
see below: "Any other information on results..."

Effect levels (fetuses)

Dose descriptor:
NOAEL
Effect level:
120 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: teratogenicity

Fetal abnormalities

Abnormalities:
not specified

Overall developmental toxicity

Developmental effects observed:
not specified

Any other information on results incl. tables

Result: not teratogenic

MATERNAL TOXIC EFFECTS BY DOSE LEVEL: 
- Mortality and day of death, 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 (not pregnant) during 

the second  gestation period.
- Mortalities were considered to be treatment related.
- 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. In the teratology phase,  all mated females showed a healthy 

general habit without clinical signs  throughout gestation.
- 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%).
- 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 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%).

- 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.
- 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.

- Number pregnant per dose level: 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. 
- Number aborting: No abortions or premature delivery in any group.
- Number of resorptions: No relevant effect (1.1 / 0.9 / 0.4 / 0.8 in  control / low / mid / high dose groups, 0.8 / 0.8 / 0.4 / 0.5 

thereof  early resorptions).
- Number of implantations: Lower values at mid- and high-dose levels were  not significant 

(8.7 / 7.9 / 6.8 / 4.9 in control / low / mid / high dose  groups).
- Pre and post implantation loss: Pre-implantation loss of ova was  significantly increased for the teratology study high-dose females 

(61.0%  vs 36.% in control). Post implantation loss varied insignificantly  between groups (11.9% / 13.8% / 8.9% / 34.8% in 

control / low / mid /  high dose groups)
- Number of corpora lutea: Similar in all groups (12.3 - 13.7 per dam).
- Duration of pregnancy: Terminated after 20 days

FETAL DATA: F1 and F2 data are presented in chapter 7.8.1. Only data  resulting from the second mating of the P animals are 

presented here:
- Litter size: A distinct, though not statistically significant reduction  was observed at the high dose level: 4.1 vs 7.6 in control. Left 

and  righ  uterine horns were similarly affected. Anterior or posterior birth  position was equally distributed.
- Litter weights: Mean weights of fetuses from the second P mate were comparable in all groups (3.49 - 3.81).
- Number viable: Only in the high-dose group there was one dead fetus.
- Sex ratio: Not notably affected by treatment (1.2, 1.4, 1.4, 1.5 in  control, low-, mid-, and high-dose groups, respectively).
- External abnormalities: No external abnormalities were found in the  fetuses from the dosed groups of the teratology study 

(1 small hematoma  in a control fetus). 
- Soft tissue abnormalities: One cystic dilated renal pelvis each in one  low- and one mid-dose fetus was considered to be within the 

normal rate  of spontaneously occurring anomalies.
- Skeletal abnormalities: Skeletal examination revealed a comparable  development stage for all test groups and variations 

(wavy ribs) at  incidences often seen in this strain of rats.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this oral two generation study with integrated teratological investigation with rats, 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine was not teratogenic up to the highest test concentration of 120 mg/kg bw.
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 in any stage of the study. Therefore, under the conditions of this oral teratogenicity study with rats, the NOAEL for maternal toxicity was determined to be 10 mg/kg bw.

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). No external abnormalities were found in the fetuses from the dosed groups of the teratology study (1 small hematoma  in a control fetus). One cystic dilated renal pelvis each in one  low- (10 mg/kg bw) and one mid-dose (60 mg/kg bw) fetus was considered to be within the normal rate of spontaneously occurring anomalies. Skeletal examination revealed a comparable development stage for all test groups and variations 

(wavy ribs) at  incidences often seen in this strain of rats.

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.

Therefore, under the conditions of this oral two generation study with integrated teratological investigation with rats,

2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine was not teratogenic up to the highest test concentration of 120 mg/kg bw.