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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The results of the two generation study (IBR, 1988) indicate that a daily oral treatment with 10 mg/kg of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamin 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.

Link to relevant study records

Reference

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:

guideline study

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

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

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Description (incidence and severity):

see "Details on result (parental animals)"

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

see "Details on result (parental animals)"

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.

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

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

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.

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

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

Reproductive effects observed:

not specified

no further remarks

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.

Effect on fertility: via oral route

Dose descriptor:

NOAEL

10 mg/kg bw/day

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a two generation study (OECD TG 416, IBR, 1988) 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.

Short description of key information:
The results of the two generation study (IBR, 1988) indicate that a daily oral treatment with 10 mg/kg of 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamin 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.

Effects on developmental toxicity

Description of key information

Under the conditions of a 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. In a second oral teragonicity study, a daily exposition to a 0.05 %  2,2,4(or  2,4,4)-trimethylhexane-1,6-diamine concentration in drinking water did not exert signs of maternal toxicity and a 0.25 % concentration did not adversely affect fetal development of New Zealand White-rabbits.

Link to relevant study records

Reference

Reference 1

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:

guideline study

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

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

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

Dose / conc.:

0 mg/kg bw/day

Remarks:

control

Dose / conc.:

10 mg/kg bw/day

Remarks:

low dose

Dose / conc.:

60 mg/kg bw/day

Remarks:

intermediate dose

Dose / conc.:

120 mg/kg bw/day

Remarks:

high dose

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

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

Details on maternal toxic effects:

Maternal toxic effects:yes

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

Dose descriptor:

NOAEL

Effect level:

10 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

Dose descriptor:

NOAEL

Effect level:

120 mg/kg bw/day

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

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.

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.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1987-02-16 to 1987-05-12

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Test procedure in accordance with generally accepted scientific standards and described in sufficient detail

Principles of method if other than guideline:

Teratogenicity study designed according to the FDA-Guideline for Safety Assessment of Direct Food Additives (App. II), FDA-Red Book.

GLP compliance:

yes

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ORGANISMS
- Source: H. Schriever, Rabbitfarm, Bremervörde (Germany)
- Age: Approx. 4 months at study initiation
- Weight at study initiation: 2.6 - 4.6 kg
- Number of animals: 12 per dosed group, 10 in control group
- Housing: single
- Diet:. ad libitum, standard fixed-furmula diet "Ssniff Mümmel Z"
- Water: ad libitum, tab water
- Acclimation period: 7 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 3 °C
- Humidity (%): 50 % - 85 %
- Air changes (per hr): 16 air changes per hour
- Photoperiod (hrs dark / hrs light): 12h / 12 h

Route of administration:

oral: drinking water

Vehicle:

other: Tap water (aqua fontana)

Details on exposure:

ADMINISTRATION / EXPOSURE
- Vehicle: Tap water (aqua fontana)
- Concentration in vehicle: 0.01; 0.05; 0.25 % (w/w)   (significantly reduced water intake at 0.3 % in dose finding study)
- Doses: Proportional to water consumption (ad libitum), which was  monitored daily during dosing period

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of compound solutions were analyzed upon identity (gas-chromatographically), concentration and stability at study start.
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: Females were mated with untreated, fertile and proven  bucks (day 0 of gestation). Non-gravid females were substituted by  
additional, gravid animals.

Duration of treatment / exposure:

13 days (days 6 - 18 of gestation)

Frequency of treatment:

permanent

Duration of test:

Duration of test: 29 days

No. of animals per sex per dose:

12 female per dose group
10 female control group

Control animals:

yes, concurrent vehicle

Details on study design:

Sex: female
Duration of test: 29 days

Maternal examinations:

PARAMETERS ASSESSED DURING STUDY: 
- Mortality: Daily
- Clinical signs: Daily over the entire study period with special regard  to sensory and motor behavior, hair coat, urine and fecal excretion, and  
conditions of orifices.
- Body weight gain: Days 0, 5, 10, 15, 20
- Food consumption: Days 0-5, 5-15, 15-20 - 
ORGANS EXAMINED AT NECROPSY (MACROSCOPIC AND MICROSCOPIC): "Complete"  autopsy and macroscopic examination of organs in all 
pregnant females.  Weights of placentae and uteri.

Ovaries and uterine content:

Examination of uterine content (sacrifice on day 29 of gestation):   For each uterine horn: Fetuses (alive / dead); birth position (anterior  /posterior); 
early- and late resorptions; placentae; implantation sites   
For each ovary: Number of corpora lutea

Fetal examinations:

Examination of fetuses: Individual weights and sex (ano-genital  distance), gross external examination (all), evaluation for visceral  imperfections 
(approx. 1/3 of fetuses in each litter), evaluation for  skeletal defects (approx. 2/3 of fetuses in each litter)

Statistics:

STATISTICAL METHODS: One factorial analysis of variance for growth  parameters and reproduction parameters (number of fetuses, implantations,  
corpora lutea, weights of fetuses, placentae and uteri). Comparison of  group mean values according to the method of Tukey. Comparison of indices  with Mann-Whitney U-test.

Indices:

Abortion Rate, Runts Index, Life Birth Index, Resorption Index, Post-Implnatation Loss Index, Still birth Index, Variation Index, Malformation Index

Historical control data:

own laboratory experiences

Details on maternal toxic effects:

Maternal toxic effects:no effects

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

Dose descriptor:

NOAEL

Effect level:

0.05 other: %

Based on:

dissolved

Basis for effect level:

other: maternal toxicity

Dose descriptor:

NOAEL

Effect level:

44 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

0.25 other: %

Based on:

dissolved

Basis for effect level:

other: maternal toxicity

Dose descriptor:

LOAEL

Effect level:

77.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

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

Dose descriptor:

NOAEL

Effect level:

0.25 other: %

Based on:

dissolved

Basis for effect level:

other: teratogenicity

Dose descriptor:

NOAEL

Effect level:

77.9 mg/kg bw/day (actual dose received)

Based on:

test mat.

Basis for effect level:

other: teratogenicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Result: not teratogenic

ACTUAL DOSE RECEIVED BY DOSE LEVEL BY SEX: 9.1 / 44.0 / 77.9 mg/kg bw day
MATERNAL TOXIC EFFECTS BY DOSE LEVEL: No treatment related effects on the  reproductive system were observed. 
- Mortality and day of death: No mortality occurred during the study.
- Description, severity, time of onset and duration of clinical signs: No  differences between control and dosed animals (healthy habit 

and  appearance, normal defecations of all animals)
- Food/water consumption: Water consumption was significantly reduced  (-55.3 %) in the high dose group during the exposure 

period.
- Body weight: Reduced in high dose group during exposure period (mean  -15.0 g vs +292.0 g in control group). No significant 

intergroup  differences during days 0-6, 18-29, or 0-29.
- Gross pathology incidence and severity: No signs of treatment related  organ alterations were found.
- Organ weight changes: No significant intergroup differences (placenta  and uteri)
- Number pregnant per dose level: 100 % (Non-gravid females were  substituted leaving only 10 pregnant females for the control 

group).
- Number aborting: None in any group; 1 low dose animal with only one  implantation site
- Number of resorptions: No significant intergroup differences. Index  slightly (not statistically significant) increased in high dose 

females  due to two dams showing 6 or 5 early resorptions
- Number of implantations: No significant intergroup differences
- Pre and post implantation loss: No significant intergroup differences.  Post implantation loss index slightly (not statistically significant)  increased in high dose females due to two dams showing 6 or 5 early  resorptions.
- Number of corpora lutea: No significant intergroup differences
FETAL DATA: The data obtained did not indicate treatment related  teratogenic effects.
- Litter size and weights: No significant intergroup differences
- Number viable: No significant intergroup differences (no dead fetuses  in any group)
- Sex ratio: No significant intergroup differences
- Grossly visible abnormalities: One malformed fetus in low dose group  only
- External abnormalities: None except same malformed fetus in low dose  group
- Soft tissue abnormalities: No adverse effects of treatment was  indicated from the fetal examinations by free-hand serial sectioning.
- Skeletal abnormalities: None except same malformed fetus in low dose  group, which showed:
  Head: Left side rudimentary, right side missing
  Thorax, abdomen: Sternochisis, umbilical hernia with a prolaps of the  liver.
STATISTICAL RESULTS: Type and incidence of the observed malformations is  considered to be within the spontaneous 

malformation rate for this strain  of rabbits.

Conclusions:

Under the experimental conditions of this study, a daily exposition to a 0.05 % 2,2,4(or 2,4,4)-trimethylhexamethylenediamine concentration in drinking water did not exert signs of maternal toxicity and a 0.25 % concentration did not adversely affect fetal development of New Zealand White-rabbits.

Executive summary:

2,2,4(or 2,4,4)-trimethylhexamethylenediamine was administered daily via drinking water to pregnant rabbits during the organogenetic phase of gestation (Day 6 - 18), concentrations of 0.01 %, 0.05 % and 0.25 % were used, a concurrent control group received tap water only.

The only sign of maternal response to treatment were reduced bodyweights and reduced water consumption in the high dose group. Maternal uterine and intrauterine parameters (e.g. number and weight of fetuses, number of resorptions, implantations, corpora lutea and weights of placentae or uteri) did not reveal significant differences between groups. The index of resorptions and thus the postimplantion-loss index was slightly increased for dose group III - females. This was, however, only due to two dams showing 6 or 5 early resorptions. Since the difference to the controls did not attain statistical significance, this increase is considered to be coincidental and not treatment related.

Examination of fetuses (external, skeletal and visceral) demonstrated one malformed fetus in dose group I only. Therefore no treatment related effect was indicated from data obtained during this study.

Under the experimental conditions of this study, a daily exposition to a 0.05 % 2,2,4(or 2,4,4)-trimethylhexamethylenediamine concentration in drinking water did not exert signs of maternal toxicity and a 0.25 % concentration did not adversely affect fetal development of New Zealand White-rabbits.

Effect on developmental toxicity: via oral route

Dose descriptor:

NOAEL

120 mg/kg bw/day

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

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.

In a second oral teratogenicity study 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine was administered daily via drinking water to pregnant rabbits during the organogenetic phase of gestation (Day 6 - 18), concentrations of 0.01 %, 0.05 % and 0.25 % were used, a concurrent control group received tap water only.

The only sign of maternal response to treatment were reduced bodyweights and reduced water consumption in the high dose group. Maternal uterine and intrauterine parameters (e.g. number and weight of fetuses, number of resorptions, implantations, corpora lutea and weights of placentae or uteri) did not reveal significant differences between groups. The index of resorptions and thus the postimplantion-loss index was slightly increased for dose group III - females. This was, however, only due to two dams showing 6 or 5 early resorptions. Since the difference to the controls did not attain statistical significance, this increase is considered to be coincidental and not treatment related.

Examination of fetuses (external, skeletal and visceral) demonstrated one malformed fetus in dose group I only. Therefore no treatment related effect was indicated from data obtained during this study.

Under the experimental conditions of this study, a daily exposition to a 0.05 % 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine concentration in drinking water did not exert signs of maternal toxicity and a 0.25 % concentration did not adversely affect fetal development of New Zealand White-rabbits.

Justification for classification or non-classification

Regarding toxicity to reproduction (embryotoxicity and fertility) the substance 2,2,4(or 2,4,4)-trimethylhexane-1,6-diamine is not classified according to the criteria of EC Directive 67/548/EEC and EC Regulation 1272/2008.