Registration Dossier
Registration Dossier
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EC number: 222-037-3 | CAS number: 3323-53-3
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
No data are available for the AH-salt.
Data for fertility are only available for 1,6-hexanediamine and for
di(2-ethylhexyl) adipate, which is metabolized to adipic acid in vivo.
The REACh dossier for adipic acid refers to a study proposal for an
extended one-generation toxicity study, which will be included in this
dossier, as soon as a final study report is available.
1,6-hexanediamine (or its dihydrochloride salt) had no effect on
fertility of rats in a two-generation study after administration with
the diet in doses up to 150 mg/kg bw/day (NOAEL for risk assessment) and
after inhalation of up to 160 mg/m³ of HDDC (corresponding to 100 mg/m3
of HMD) for 13 weeks in rats and mice.
In the 2-generation study the top dose (500 mg/kg bw/day) was associated
with a small reduction in litter size in the F1 and F2 generation,
however, without histological changes in the reproductive organs of
males and females, and in the presence of paternal as well as maternal
toxicity.
With the exception of a slight reduction of the litter size,
reproductive parameters were not adversely influenced in rats fed with
di(2-ethylhexyl) adipate up to exposure levels of 12000 ppm in the diet
(corresponding to ca. 240-480 mg adipic acid/kg bw/day). 1800 ppm (36
-72 mg adipic acid/kg bw/day or corresponds to 65-129 mg/kg AH salt) was
a clear NOAEL for fertility effects.
Conclusion: The overall conclusion is that AH salt may present a hazard
to fertility only at doses which are parentally toxic.
Link to relevant study records
- Endpoint:
- two-generation reproductive toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- study well documented, meets generally accepted scientific principles, acceptable for assessment
- Principles of method if other than guideline:
- Sprague-Dawley rats were treated with the test compound by oral feeding over two generations for about 40 weeks. Litters were examined for size, stillbirths, live births, and gross anomalies. Pubs were selected for F1 parents of the F2 offspring. After 98 days of treatment the F1 parents were mated. F2 pubs were killed on day 21. Gross necropsies were performed on F0 and F1 parents as well as F2 pubs. Histological evaluation of tissue of different organs was conducted.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Breeding Laboratories, Portage, MI)
- Age at study initiation: (P) ca. 8 weeks; (F1) 3 weeks
- Housing: individually in wire mesh cages or plastic cages with wood chip bedding; except for the 20-day cohabitation (mating)
- Diet (ad libitum): control or experimented diet prepared with ground Purina Certified Rodent Chow No. 5002 (Ralston Purina Co., St. Louis, MO)
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
All rats were housed in environmentally controlled rooms; no further details
- Photoperiod (hrs dark / hrs light): 12/12
No further data - Route of administration:
- oral: feed
- Vehicle:
- water
- Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): Test diets for the control, low, and mid dose groups were prepared weekly, while the diet for the high dose group was prepared every 4 days in order to maintain adequate levels of test material.
- Mixing appropriate amounts with (Type of food): ground Purina Certified Rodent Chow No. 5002
A weighed amount of test material was diluted with ethanol and mixed with ground Purina Certified Rodent Chow to form a premix. This premix was further diluted with additional feed to obtain diets formulated to provide 0, 50, 150, and 500 mg/kg/day of hexamethylenediamine. Dietary concentrations were based on the most recent weekly body weights except during the 20-day mating period when concentrations were based on the most recent female values obtained prior to mating.
Diets were analyzed for homogeneity, stability, and concentration of test material. - Details on mating procedure:
- - M/F ratio per cage: 1/1
- Length of cohabitation: 20 days
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- Diets were analyzed for homogeneity, stability, and to the concentration of test material. A dansylated derivative of hexamethylenediamine was prepared and quantified at 254 nm using a UV detector.
- Duration of treatment / exposure:
- Exposure period: over two generations
Duration of test: 40 weeks - Frequency of treatment:
- continuously in the diet
- Details on study schedule:
- After a minimum of 56 days of treatment, the F0 rats were mated to produce the F1 offspring.
After a minimum of 98 days of treatment the F1 parents were mated to produce the F2 offspring. - Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 150 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 500 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 26 males and 26 females per group
- Control animals:
- yes, plain diet
- Positive control:
- no
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: No data
DETAILED CLINICAL OBSERVATIONS: No data
BODY WEIGHT: Yes
- Time schedule for examinations: weekly
FOOD CONSUMPTION: Yes
- Time schedule for examinations: weekly
WATER CONSUMPTION: No data - Oestrous cyclicity (parental animals):
- no
- Sperm parameters (parental animals):
- no
- Litter observations:
- Pregnant F0 females were allowed to give birth to F1 pups and the day all pups were delivered was designated Day 0 of lactation.
Litters were examined for size, stillbirths, live births, and gross anomalies. Litter size was reduced to a total of 8 pups of equal sex, when possible, on Day 4 of lactation. Pups were housed with their mothers and weighed at intervals for 3 weeks after birth. Afterward, 26 pups of each sex from group were selected to become F1 parents of the F2 offspring. - Postmortem examinations (parental animals):
- Gross necropsies were performed on F0 and F1 parents as well as F2 pups. The following tissues were taken from F0 and F1 rats for histopathological evaluation: kidneys, liver, lung, ovaries, prostate, seminal vesicles, spleen, testes with epididymes, uterus, and vagina. Tissues were preserved in neutral buffered formalin and stained with hematoxylin and eosin. No further data.
- Postmortem examinations (offspring):
- The F2 pups were sacrificed on day 21 of lactation and subjected to gross necropsy. No further data.
- Statistics:
- Several different statistical methods were used to compare measurements made on test animals to the corresponding values determined for controls. The methods and the measurements to which they applied were analysis of variance and Dunnett's test for body weight and litter size, Chi square test with Yates' correction or Fisher's exact probability test for fertility indices; and Mann-Whitney U test for pup survival. The level of significance was selected as p < 0.05.
- Dose descriptor:
- NOAEL
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- 150 mg/kg bw/day (actual dose received)
- Sex:
- male/female
- Basis for effect level:
- body weight and weight gain
- other: reduced litter size
- Reproductive effects observed:
- not specified
- Executive summary:
In a two generation study 26 males and 26 females SD- rats/group received Hexamethylenediamine (HMD) orally in the diet at dosage levels of 0, 50, 150 or 500 mg/kg/day for 56 days prior to mating, then throughout the study in accordance with Series 83 -4 of the Environmental protection Agency Pesticide Assessment Guidelines, Subdivision F., Hazard Evaluation: Human and Domestic Animals, issued November, 1982 (similar to OECD 416) and in accordance with GLP.
The parental rats and pups were observed twice each day for signs of overt toxicity, changes in general appearance and behavior, and mortality. Individual body weights were recorded weekly for the adult rats; In addition, females were weighed on gestation days 0, 6, 15 and 20 and lacation days 0, 4, 7, 14 and 21. Parental food consumption was measured weekly for individual parental rats except during mating. Specific reproductive observations included tabulation of male and female fertility indices, and the length of cohabitation and gestation were recorded.Gross necropsies were performed on F0 and F1 parents as well as F2 pups. The following tissues were taken from F0 and F1 rats for histopathological evaluation: kidneys, liver, lung, ovaries, prostate, seminal vesicles, spleen, testes with epididymis, uterus, and vagina.
The results of this study were:
- Dietary analysis indicated that greater than 90% of the target concentration of hexamethylenediamine were fed to rats in all groups. The actual doses consumed, however, averaged between 123 and 132% of the target doses in these groups.
- No treatment-related mortality was observed in any of the groups. Some deaths occurred, however, they were singular events in specific groups and there was no pattern indicative of a dose-response relationship. Mortality ratios for the F0 males were 0/26, 1/26, 1/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F0 females were 0/26, 0/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F1 males were 0/26, 0/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively. Mortality ratios for the F1 females were 1/26, 1/26, 0/26, and 0/26 for the 0, 50, 150, and 500 mg/kg groups, respectively.
- Body weights of male F0 and F1 rats in the 500 mg/kg group were reduced by about 10%, relative to control values, at the end of study weeks 15 and38. The body weights of females, in contrast, were comparable to control values at these intervals. During gestation, the female weight gain was reduced by about 10% in the high-dose group. Decreased body weight is correlated with a decreased food consumption. Therefore, this effect was likely due to the unpalatability of HMD.-Fertility was not adversely affected by the dietary administration of hexamethylenediamine over 2 generations (See Table ). The F0 and the F1 litter size in the 500 mg/kg group was significantly reduced without an increase in the number of dead pups. There was no biological meaningful or statistically significant differences in the number of viable and dead pups on lactation day 1, as compared to control for either generation in the mid and low-dose treatment groups. Pup survival was not significantly reduced in any of the treated groups. At birth, pup body weights were not adversely affected by treatment, but during lactation, reduced weights were apparent in pups of each sex from the high dose group
-No meaningful differences were noted between the control and treated rats of either generation with regard to antemortem observations, copulatory interval, gestation length, nesting and nursing behavior, and appearance of the pups. No treatment related effects were noted on testes weights and no effects were noted by macroscopic or microscopic examination of tissues evaluated.
- Endpoint:
- fertility, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- no
- Limit test:
- no
- Species:
- mouse
- Strain:
- B6C3F1
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
male and female B6C3F1 mice
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 20.6 - 24.8 g (males); 17.9 - 19.9 g (females)
- Fasting period before study: no
- Housing: in individual compartments of multi-compartment stainless steel wire mesh cages; during exposure: in Hazelton H-2000 stainless steel and glass exposure chambers (Hazelton Systems, Inc., Aberdeen, MD) of 2 m³ volume
- Diet (ad libitum): pelleted NIH-07 feed
- Water (ad libitum): tap water
- Acclimation period: 11 - 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 - 24 °C (original value: 72 +/- 3°F); during exposure: ca. 22 - 26 °C (72 - 78°F)
- Humidity (%): 35 - 65 % ; during exposure: 70 - 80 %
- Air changes (per hr): 12 - 15; during exposure: 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
For the inhalation studies, 1,6-hexanediamine was converted to 1,6-hexanediamine dihydrochloride (HDDC) by acidification with concentrated hydrochloric acid under a stream of nitrogen. The final pH was adjusted within the range of 4 .5 to 5.5 before storage and again before use in the inhalation chambers.
The aqueous HDDC solution was placed in a 9-liter glass reservoir and pressurized with N2 gas. HDDC was delivered to 5 Sonimist Ultrasonic Spray Nozzles by a positive displacement metering pump. Up to this point, stainless steel lines carried the test substance. The nebulizer reservoir was kept in a separate exposure chamber. This chamber served as a mixing plenum where large droplets and nonnebulized liquid were impacted or sedimented out of the test atmosphere before the aerosol was delivered to the inhalation chambers. The HDDC aerosol was mixed with compressed breathing air that had been filtered and supplied at 50 psi to generate an aerosol at a concentration equal to the highest exposure concentration. The resulting
aerosol was transported to the inhalation chambers through a manifold constructed of 3-inch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount of HEPA/charcoal-filtered room air to obtain the desired test concentration, then delivered to the inhalation chamber. After exiting the chambers, the test atmospheres were delivered to a common duct and
cleansed of the test substance.
TEST ATMOSPHERE
Concentrations of HDDC in the exposure chamber, exposure room, and exhaust were monitored by measuring the forward light scatter with RAM-S real-time aerosol monitors and by gravimetric analyses of filter samples collected from each exposure chamber. Six RAM-S readings and 3 gravimetric samples were taken from each exposure chamber on each day of exposure. Gravimetric sampling was conducted with 25 mm glass fiber filter paper. Gravimetric analysis was performed by weighing filters to the nearest 0.01 mg before and after sampling and again after storing the filters in a desiccator overnight. Measured concentrations of HDDC in the exposure chambers were within 6% of the target concentrations in all samples.
Spatial homogeneity of the aerosol within the exposure chambers was determined using the calibrated RAM-S monitors. Chamber concentrations were measured at 12 points within each chamber and then were compared to a fixed reference point. Time spans required to reach stable concentrations after start up and to reach background concentrations at the end of exposure were determined by taking measurements of aerosol concentrations every 60 seconds. The time span required after start up to reach 90% of the target concentration was identified as the T90; the time span required after the end of the exposure period to reach 10% of the target concentration was identified as the T10.
Triplicate particle size measurements were obtained for each exposure chamber. The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 microns, with a geometric standard deviation of 1.52 to 1.53 . All control chamber respirable mass concentration values were less than 0.005 mg/m³. - Details on mating procedure:
- - M/F ratio per cage: 1/2
- Length of cohabitation: 10 nights (days 68 to 80 of the study)
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy
Mating trials were performed on mice from the control group and from the 3 highest exposure groups (0, 16, 50, and 160 mg/m³) in the 13-week inhalation studies. These exposure groups were selected based on the lack of significant clinical findings (body weight changes or clinical signs of toxicity) in all exposure groups. Mating trial animals were bred for 10 nights (approximately study days 68 to 80, weekdays only) prior to the end of the 13-week exposure period. Females were removed from the inhalation chambers and housed overnight in polycarbonate cages with males from the same treatment group (2 females per male). Trios selected for breeding were not altered during the mating trial. These animals were returned to the inhalation chambers each day and exposed in the same manner as the base-study animals. Each morning during the mating period, females were examined for evidence of copulation by vaginal lavage. Females not showing evidence of copulation were mated again each night until they were sperm positive or for a maximum of 10 nights. Day 0 of gestation was considered to be the clay sperm were observed in the lavage samples. Females not showing signs of copulation by the end of the breeding period were monitored for signs of pregnancy for an additional 23 days. If no clinical signs of pregnancy were seen, the animals were killed, and the uteri were examined for signs of pregnancy. If implantation was not evident, the uterus was stained with ammonium sulfide and was examined for signs of early implantation. Following the last day of exposure, females were housed individually in polycarbonate cages until parturition. - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hours plus T90 (30 minutes) per day; 5 days per week
- Dose / conc.:
- 1.6 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 1 mg HMD/m³
- Dose / conc.:
- 5 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 3.1 mg HMD/m³
- Dose / conc.:
- 16 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 10 mg HMD/m³
- Dose / conc.:
- 50 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 31 mg HMD/m³
- Dose / conc.:
- 160 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 100 mg HMD/m³
- No. of animals per sex per dose:
- 10 males and 10 females per base group;
20 male and 40 females per satellite group (mating trial) - Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: The test concentrations were chosen based on the reported inhalation LCLo of 750 mg/m³ in mice and on the results of a 2-week inhalation study (weight gain depression and the inflammation and ulceration of the nasal cavity and larynx seen in both sexes; see other entry in this section).
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: mating trials - Positive control:
- no
- Parental animals: Observations and examinations:
- BODY WEIGHT: Yes
- Time schedule for examinations: Adult females (dams) were weighed on gestation Days 0 and 20 and on lactation Days 0, 5, 14, and 21. Adult males were weighed at the end of the mating period.
For further examinations carried out during the course of the study, see section 7.5.3 - Oestrous cyclicity (parental animals):
- Vaginal cytology was evaluated in base-study mice from the control, 16, 50, and 160 mg HDDC/m³ exposure groups.
- Sperm parameters (parental animals):
- Sperm morphology was evaluated in base-study mice from the control, 16, 50, and 160 mg HDDC/m³ exposure groups.
- Litter observations:
- Pups were individually weighed on lactation Days 0, 5, 14, and 21. Pups were examined at birth for morphological abnormalities, viability, and gender. The number of live/dead offspring, percent neonatal survival, mean live pup weight, and sex ratio were recorded on lactation Days 0, 5, 14, and 21.
- Postmortem examinations (parental animals):
- Necropsies were performed only on mating-trial females selected for breeding and examined for pregnancy 23 days after the conclusion of breeding as described above (--> details on mating procedure). Tissues from mating-trial animals were not fixed or retained.
- Dose descriptor:
- NOAEC
- Effect level:
- > 160 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose administered
- Dose descriptor:
- NOAEC
- Effect level:
- > 100 mg/m³ air
- Based on:
- other: corresponding HMD concentration
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- > 160 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose administered
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- > 100 mg/m³ air
- Based on:
- other: corresponding HMD concentration
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose
- Reproductive effects observed:
- not specified
- Executive summary:
The only statistically significant changes of the test substance in reproductive parameters of mice were a slight increase in gestation length in the 50 mg/m³ and 160 mg/m³ exposure groups and a decrease in mean pup weight on Day 21 in the highest exposure group. These changes were not considered to be biologically significant. The NOAEC was >160 mg/m³ for both parental and offspring animals.
- Endpoint:
- fertility, other
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- comparable to guideline study with acceptable restrictions
- Qualifier:
- equivalent or similar to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Fischer 344
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
male and female Fischer 344/N rats
- Source: Taconic Laboratory Animals and Services (Germantown, NY)
- Age at study initiation: 6-7 weeks
- Weight at study initiation: 142 - 150 g (males); 112 - 114 g (females)
- Fasting period before study: no
- Housing: in individual compartments of multi-compartment stainless steel wire mesh cages; during exposure: in Hazelton H-2000 stainless steel and glass exposure chambers (Hazelton Systems, Inc., Aberdeen, MD) of 2 m³ volume
- Diet (ad libitum): pelleted NIH-07 feed
- Water (ad libitum): tap water
- Acclimation period: 11 - 14 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): ca. 21 - 24 °C (original value: 72 +/- 3°F); during exposure: ca. 22 - 26 °C (72 - 78°F)
- Humidity (%): 35 - 65 % ; during exposure: 70 - 80 %
- Air changes (per hr): 12 - 15; during exposure: 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- inhalation: aerosol
- Type of inhalation exposure (if applicable):
- whole body
- Vehicle:
- unchanged (no vehicle)
- Details on exposure:
- GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
For the inhalation studies, 1,6-hexanediamine was converted to 1,6-hexanediamine dihydrochloride (HDDC) by acidification with concentrated hydrochloric acid under a stream of nitrogen. The final pH was adjusted within the range of 4 .5 to 5.5 before storage and again before use in the inhalation chambers.
The aqueous HDDC solution was placed in a 9-liter glass reservoir and pressurized with N2 gas. HDDC was delivered to 5 Sonimist Ultrasonic Spray Nozzles by a positive displacement metering pump. Up to this point, stainless steel lines carried the test substance. The nebulizer reservoir was kept in a separate exposure chamber. This chamber served as a mixing plenum where large droplets and nonnebulized liquid were impacted or sedimented out of the test atmosphere before the aerosol was delivered to the inhalation chambers. The HDDC aerosol was mixed with compressed breathing air that had been filtered and supplied at 50 psi to generate an aerosol at a concentration equal to the highest exposure concentration. The resulting
aerosol was transported to the inhalation chambers through a manifold constructed of 3-inch diameter PVC tubing. At each chamber, a metered amount of aerosol was removed from the manifold and mixed with the appropriate amount of HEPA/charcoal-filtered room air to obtain the desired test concentration, then delivered to the inhalation chamber. After exiting the chambers, the test atmospheres were delivered to a common duct and
cleansed of the test substance.
TEST ATMOSPHERE
Concentrations of HDDC in the exposure chamber, exposure room, and exhaust were monitored by measuring the forward light scatter with RAM-S real-time aerosol monitors and by gravimetric analyses of filter samples collected from each exposure chamber. Six RAM-S readings and 3 gravimetric samples were taken from each exposure chamber on each day of exposure. Gravimetric sampling was conducted with 25 mm glass fiber filter paper. Gravimetric analysis was performed by weighing filters to the nearest 0.01 mg before and after sampling and again after storing the filters in a desiccator overnight. Measured concentrations of HDDC in the exposure chambers were within 6% of the target concentrations in all samples.
Spatial homogeneity of the aerosol within the exposure chambers was determined using the calibrated RAM-S monitors. Chamber concentrations were measured at 12 points within each chamber and then were compared to a fixed reference point. Time spans required to reach stable concentrations after start up and to reach background concentrations at the end of exposure were determined by taking measurements of aerosol concentrations every 60 seconds. The time span required after start up to reach 90% of the target concentration was identified as the T90; the time span required after the end of the exposure period to reach 10% of the target concentration was identified as the T10.
Triplicate particle size measurements were obtained for each exposure chamber. The mass median aerodynamic diameter values for each chamber ranged from 1.62 to 1.72 microns, with a geometric standard deviation of 1.52 to 1.53 . All control chamber respirable mass concentration values were less than 0.005 mg/m³. - Details on mating procedure:
- - M/F ratio per cage: 1/2
- Length of cohabitation: 10 nights (days 68 to 80 of the study)
- Proof of pregnancy: sperm in vaginal lavage referred to as day 0 of pregnancy
Mating trials were performed on rats from the control group and from the 3 highest exposure groups (0, 16, 50, and 160 mg/m³) in the 13-week inhalation studies. These exposure groups were selected based on the lack of significant clinical findings (body weight changes or clinical signs of toxicity) in all exposure groups. Mating trial animals were bred for 10 nights (approximately study days 68 to 80, weekdays only) prior to the end of the 13-week exposure period. Females were removed from the inhalation chambers and housed overnight in polycarbonate cages with males from the same treatment group (2 females per male). Trios selected for breeding were not altered during the mating trial. These animals were returned to the inhalation chambers each day and exposed in the same manner as the base-study animals. Each morning during the mating period, females were examined for evidence of copulation by vaginal lavage. Females not showing evidence of copulation were mated again each night until they were sperm positive or for a maximum of 10 nights. Day 0 of gestation was considered to be the clay sperm were observed in the lavage samples. Females not showing signs of copulation by the end of the breeding period were monitored for signs of pregnancy for an additional 23 days. If no clinical signs of pregnancy were seen, the animals were killed, and the uteri were examined for signs of pregnancy. If implantation was not evident, the uterus was stained with ammonium sulfide and was examined for signs of early implantation. Following the last day of exposure, females were housed individually in polycarbonate cages until parturition. - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- 13 weeks
- Frequency of treatment:
- 6 hours plus T90 (30 minutes) per day; 5 days per week
- Dose / conc.:
- 1.6 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 1 mg HMD/m³
- Dose / conc.:
- 5 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 3.1 mg HMD/m³
- Dose / conc.:
- 16 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 10 mg HMD/m³
- Dose / conc.:
- 50 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 31 mg HMD/m³
- Dose / conc.:
- 160 other: mg HDDC/m³ (analytical)
- Remarks:
- corresponding to 100 mg HMD/m³
- No. of animals per sex per dose:
- 10 males and 10 females per base group;
20 male and 40 females per satellite group (mating trial) - Control animals:
- yes, sham-exposed
- Details on study design:
- - Dose selection rationale: The test concentrations were chosen based on the reported inhalation LCLo of 750 mg/m³ in mice and because of the lack of information on inhalation toxicity of HDDC in rats and on the results of a 2-week inhalation study (weight gain depression and the inflammation and ulceration of the nasal cavity and larynx seen in both sexes; see other entry in this section).
- Rationale for animal assignment: random
- Rationale for selecting satellite groups: mating trials - Positive control:
- no
- Parental animals: Observations and examinations:
- BODY WEIGHT: Yes
- Time schedule for examinations: Adult females (dams) were weighed on gestation Days 0 and 20 and on lactation Days 0, 5, 14, and 21. Adult males were weighed at the end of the mating period.
For further examinations carried out during the course of the study, see section 7.5.3 - Oestrous cyclicity (parental animals):
- Vaginal cytology was evaluated in base-study rats from the control, 16, 50, and 160 mg HDDC/m³ exposure groups.
- Sperm parameters (parental animals):
- Sperm morphology was evaluated in base-study rats from the control, 16, 50, and 160 mg HDDC/m³ exposure groups.
- Litter observations:
- Pups were individually weighed on lactation Days 0, 5, 14, and 21. Pups were examined at birth for morphological abnormalities, viability, and gender. The number of live/dead offspring, percent neonatal survival, mean live pup weight, and sex ratio were recorded on lactation Days 0, 5, 14, and 21.
- Postmortem examinations (parental animals):
- Necropsies were performed only on mating-trial females selected for breeding and examined for pregnancy 23 days after the conclusion of breeding as described above (--> details on mating procedure). Tissues from mating-trial animals were not fixed or retained.
- Dose descriptor:
- NOAEC
- Effect level:
- > 160 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose administered
- Dose descriptor:
- NOAEC
- Effect level:
- > 100 mg/m³ air
- Based on:
- other: corresponding HMD concentration
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- > 160 mg/m³ air (analytical)
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose administered
- Dose descriptor:
- NOAEC
- Generation:
- F1
- Effect level:
- > 100 mg/m³ air
- Based on:
- other: corresponding HMD concentration
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects even at the highest dose
- Reproductive effects observed:
- not specified
- Executive summary:
In a mating trial as a part of a 13-week subchronic toxicity study, the test substance demonstrated no adverse effects on reproduction of rats.
Referenceopen allclose all
No treatment-related mortality was observed in any of the groups.
The
ability of rats to successfully mate and produce litters was not
adversely affected by daily doses of up to 500 mg/kg.
The weight of male F0 and F1 parent animals was significantly reduced by
about
10% at 500 mg/kg at the end of the treatment period. The body
weight
of the females was not altered at that time but the weight gain
was
reduced by about 10% during gestation (no further data).
The
litter size
at birth was significantly reduced in the F1 generation (13.8 vs 11.7)
and not significantly reduced in the F2 generation (13.0 vs. 11.0)
at
500 mg/kg. Pup weight was normal at birth but was significantly lower at
day 21 in male F1 pups and female F2 pups at 500 mg/kg. There was no
effect on their survival and they appeared normal during lactation.
The
treatment with up to 150 mg/kg did not adversely affect reproduction or
fertility. No differences between control and treated rats in either
generation with regard to clinical observations, copulation, gestation
length, nesting behaviour, appearance of pups. No treatment related
effects on testes weights, no microscopic and macroscopic effects on
tissues.
No
data are given on malformations.
Table 7.8.1/2: F0 Males: Group Mean Body Weight, Standard Deviation and Survival.
|
Hexamethylene Diamine (mg/kg/day) |
|||||||||||
Week of Study |
0 (Control) |
50 |
150 |
500 |
||||||||
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
|
0 |
269 |
11.4 |
26/26 |
274 |
10.1 |
26/26 |
272 |
11.6 |
26/26 |
267 |
10.0 |
26/26 |
1 |
317 |
15.4 |
26/26 |
320 |
13.8 |
26/26 |
320 |
15.7 |
26/26 |
308 |
14.4 |
26/26 |
2 |
347 |
17.3 |
26/26 |
351 |
17.3 |
26/26 |
352 |
19.3 |
26/26 |
332 |
15.8 |
26/26 |
3 |
371 |
19.2 |
26/26 |
376 |
19.8 |
26/26 |
381 |
22.3 |
26/26 |
353 |
16.9 |
26/26 |
4 |
392 |
21.5 |
26/26 |
398 |
22.1 |
26/26 |
401 |
25.8 |
26/26 |
367 |
18.7 |
26/26 |
5 |
415 |
25.1 |
26/26 |
422 |
23.2 |
26/26 |
424 |
27.9 |
26/26 |
389 |
20.4 |
26/26 |
6 |
432 |
26.6 |
26/26 |
438 |
26.8 |
26/26 |
438 |
33.9 |
26/26 |
404 |
20.9 |
26/26 |
7 |
446 |
30.1 |
26/26 |
451 |
31.6 |
25/26 |
454 |
33.0 |
26/26 |
413 |
21.9 |
26/26 |
8 |
458 |
34.8 |
26/26 |
464 |
28.7 |
25/26 |
467 |
38.2 |
26/26 |
421** |
23.6 |
26/26 |
9 |
452 |
34.1 |
26/26 |
465 |
30.8 |
25/26 |
468 |
37.5 |
26/26 |
425 |
22.7 |
26/26 |
10 |
470 |
34.1 |
26/26 |
476 |
30.8 |
25/26 |
474 |
39.3 |
26/26 |
429 |
23.1 |
26/26 |
11 |
482 |
35.0 |
26/26 |
486 |
30.5 |
25/26 |
486 |
42.0 |
26/26 |
438 |
25.5 |
26/26 |
12 |
488 |
35.0 |
26/26 |
487 |
47.6 |
25/26 |
492 |
44.8 |
26/26 |
439 |
26.8 |
26/26 |
13 |
501 |
36.7 |
26/26 |
510 |
35.2 |
25/26 |
505 |
45.8 |
25/26 |
446 |
27.5 |
26/26 |
14 |
509 |
38.8 |
26/26 |
519 |
36.8 |
25/26 |
515 |
44.7 |
25/26 |
450 |
28.1 |
26/26 |
15 |
513 |
39.3 |
26/26 |
523 |
36.9 |
25/26 |
521 |
46.4 |
25/26 |
450** |
26.4 |
26/26 |
** Significantly different from the control group, p < 0.01
S.D. : Standard Deviation
Table 7.8.1/3: F0 Females: Group Mean Body Weight, Standard Deviations and Survival
|
Hexamethylene Diamine (mg/kg/day) |
|||||||||||
Week of Study |
0 (Control) |
50 |
150 |
500 |
||||||||
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
Mean Body Weight (g) |
± S.D. |
Survival |
|
0 |
193 |
10.1 |
26/26 |
194 |
11.2 |
26/26 |
199 |
8.9 |
26/26 |
1199 |
10.7 |
26/26 |
1 |
216 |
12.1 |
26/26 |
216 |
13.5 |
26/26 |
222 |
10.0 |
26/26 |
220 |
12.3 |
26/26 |
2 |
226 |
18.1 |
26/26 |
229 |
15.2 |
26/26 |
236 |
13.7 |
26/26 |
229 |
12.9 |
26/26 |
3 |
238 |
11.9 |
26/26 |
239 |
18.1 |
26/26 |
247 |
13.0 |
26/26 |
239 |
12.9 |
26/26 |
4 |
247 |
14.1 |
26/26 |
248 |
17.5 |
26/26 |
258 |
21.7 |
26/26 |
249 |
15.8 |
26/26 |
5 |
256 |
14.2 |
26/26 |
260 |
20.5 |
26/26 |
265 |
14.7 |
26/26 |
259 |
13.9 |
26/26 |
6 |
265 |
16.2 |
26/26 |
265 |
19.9 |
26/26 |
274 |
13.0 |
26/26 |
263 |
15.0 |
26/26 |
7 |
272 |
15.9 |
26/26 |
274 |
21.4 |
26/26 |
263 |
15.7 |
26/26 |
268 |
14.6 |
26/26 |
8 |
274 |
15.2 |
26/26 |
276 |
22.4 |
26/26 |
286* |
16.6 |
26/26 |
270 |
16.5 |
26/26 |
9 |
289 |
15.5 |
26/26 |
291 |
20.0 |
26/26 |
298 |
17.5 |
26/26 |
282 |
14.1 |
26/26 |
10 |
310 |
17.9 |
26/26 |
315 |
23.7 |
26/26 |
320 |
21.8 |
26/26 |
299 |
14.9 |
26/26 |
11 |
351 |
31.5 |
26/26 |
358 |
36.3 |
26/26 |
356 |
36.7 |
26/26 |
343 |
26.6 |
26/26 |
12 |
325 |
23.7 |
26/26 |
327 |
33.1 |
26/26 |
327 |
21.9 |
26/26 |
309 |
19.1 |
26/26 |
13 |
34 |
29.3 |
26/26 |
352 |
28.5 |
26/26 |
347 |
30.3 |
26/26 |
325 |
22.7 |
26/26 |
14 |
334 |
22.5 |
26/26 |
342 |
25.1 |
26/26 |
343 |
22.6 |
26/26 |
343 |
26.9 |
26/26 |
15 |
311 |
22.5 |
26/26 |
315 |
25.2 |
26/26 |
317 |
16.9 |
26/26 |
314 |
17.0 |
26/26 |
16 |
310 |
22.2 |
26/26 |
314 |
19.4 |
26/26 |
317 |
17.7 |
26/26 |
312 |
18.6 |
26/26 |
17 |
314 |
17.4 |
26/26 |
321 |
21.5 |
26/26 |
321 |
18.7 |
26/26 |
312 |
18.7 |
26/26 |
18 |
315 |
18.1 |
26/26 |
321 |
19.9 |
26/26 |
325 |
19.5 |
26/26 |
310 |
20.1 |
26/26 |
* Significantly different from the control group, p<0.05
S.D. : Standard Deviation
Reproductive effects of the test substance (HDDC) on mice were minimal. There was no effect on male or female body weights or body weight gains, and no effect on male or female fertility. Three female mice exposed to 16 mg/m³ and 1 female and 1 male mouse exposed to 50 mg/m³ died before scheduled termination; however, these deaths were not considered compound related. A statistically significant increase in the mean gestation length of mice in the 50 and 160 mg/m³ exposure groups was noted; however, in the absence of other reproductive toxicity, this effect was not considered biologically significant. HDDC had no effect on litter size, neonatal survival, sex ratio of pups, or pup morphology in mice. Pups in the 160 mg/m³ exposure group had mean weights similar to that of controls at birth and on lactation Day 5; however, mean weights for pups in this exposure group were lower than that of controls on lactation Days 14 and 21.
Administration of the test substance to mice by inhalation caused no changes in the sperm morphology parameters evaluated, with the exception of an increase in sperm motility in the 16 and 160 mg/m³ exposure groups. However, this change was not dose related, and the values for sperm motility were all well within the range for historical controls for NTP studies. Consequently, the increase in sperm motility was not interpreted as an adverse effect.
The test substance demonstrated no reproductive toxicity. There was no effect on male or female fertility, body weights or body weight gains, gestation length, litter size, neonatal survival, pup weights, sex ratios of pups, or pup morphology in rats exposed to HDDC. Administration of HDDC to rats by inhalation caused no changes in any of the sperm morphology or vaginal cytology parameters evaluated.
Effect on fertility: via oral route
- Dose descriptor:
- NOAEL
- 150 mg/kg bw/day
Effect on fertility: via inhalation route
- Dose descriptor:
- NOAEC
- 100 mg/m³
Effect on fertility: via dermal route
- Endpoint conclusion:
- no study available
Additional information
AH salt was not tested for its toxicity to fertility and development and there is only limited information available from a 28-day study on the effects on reproductive organs. Studies with 1,6-hexanediamine (or its dihydrochloride), one of the two compounds that constitute AH salt are therefore used to cover this endpoint. Since a fertility study with the second component of AH-salt, adipic acid, is lacking, a one-generation study with di-2-(ethylhexyl)adipate which is rapidly metabolised to adipic acid is used to cover this endpoint.
In addition to that, the REACh dossier for adipic acid refers to a study proposal for an extended one-generation toxicity study, which will be included in this dossier, as soon as a final study report is available.
Repeated dose studies were performed with 1,6-hexanediamine (or its dihydrochloride salt), one component of AH salt.
Dietary treatment with up to 150 mg/kg bw/day 1,6-hexanediamine (purity: 70% in water) for 56 days (F0 rats, 8 weeks old) and 98 days (F1 rats, 3 weeks old) prior to mating had no effect on fertility or reproduction in a two-generation study in Sprague-Dawley rats (groups of 36 rats/sex in each generation). The highest dose of 500 mg/kg bw/day which was tested, showed no influence on the animal mating performance and the number of litters. However, litter size in the F1 generation showed a significant reduction to 11.7 vs. 13.8 in the control, in the F2 generation there was a numerical reduction (11.0 vs. 13.0). The weight of male F0 and F1 parents was significantly reduced by about 10% in this dose at the end of the treatment period. The body weight of the females was not altered at that time but the weight gain was reduced by about 10% during gestation. The pup weights were normal at birth, but significantly reduced at day 21 in male F1 and female F2 pups. There was no effect on their survival and they appeared normal during lactation. No macroscopic and microscopic effects on the sex organs were observed. NOAEL for fertility and all other parameter is 150 mg/kg bw/day (Short et al, 1991) and the starting point for risk assessment.
The effect of 1,6 -hexanediamine on reproductive parameters was also studied in the course of a 13 -week inhalation study with rats and mice (Herbert CD, 1993).
Inhalation of 1,6-hexanediamine dihydrochloride (as aerosol) 6 h/day, 5 d/week for 13 weeks at exposure levels of up to 160 mg/m³ (corresponding to 100 mg/m3 of HMD) did not influence sperm motility and vaginal cytology in B6C3F1 mice or F344 rats (10 animals/sex/group of each species). A subsequent mating trial (20 males and 40 females per group of each species) did not impair the fertility and reproductive outcome of rats and mice (Herbert CD 1993). 160 mg/m³ corresponds to 38 mg/kg bw assuming an inhalation volume of 12 l/h, a body weight of 300 g and a retention of 100%. The dose is therefore much lower than those used by Short et al (1991).
A one-generation study with di(2-ethylhexyl)adipate (DEHA) is also taken to cover this endpoint. DEHA was administered to ca. 21 day old rats, each dose and control group consisted of 30 female and 10 male rats. DEHA was given in the feed at 300, 1800 and 12000 ppm. The authors do not specify the effective dose levels; however, as a general rule, the dose ranges within the experiment varied between 15 -30, 90 -180 and 600 -1200 mg/kg bw/day, (according to a conversion factor of 10 and 20; WHO, 1987), depending on the age and body weight of the animals for a period of 10 weeks prior to mating, during mating and during the gestation and lactation periods. These doses correspond to 6 -12, 36 -72, 240 -480 mg adipic acid/kg bw/day. Necropsy was performed on male animals immediately after successful mating, on females after the pups had been weaned, and on the progeny after day 36 of life. The following organs were histologically examined: cervix, epididymis, liver, mammary gland, ovaries, seminal vesicle, prostate, testes, uterus and all other organs if showing macroscopic changes. No clinical symptoms of intoxication occurred in the parent animals. Only the females in the high dose group suffered slight, but nonsignificant, inhibition of body weight gain during the pretreatment period (ca. 3%) and a significant reduction during pregnancy. Data on body weight of females in the lactation period are lacking. The males of the high dose group showed a slight but significant increase in feed consumption from weeks 6 to 9 with simultaneous reduction in feed efficiency. Male and female fertility, length of gestation and the pre-coital interval were not affected. The parental animals did not show any signs of substance-related histopathological organ change. Both males and females in the high dose group, however, had significantly higher absolute and relative liver weights. There were four whole litter losses, none in control, one in the 300 ppm group, two in the 1800 ppm group and one in the 12000 ppm dose group. Only in the high dose group, there was there a slight but nonsignificant reduction in litter sizes (day 1: 9.7 vs. 10.9; day 36: 8.5 vs. 10.0). None of the pups showed any clinical signs, substance-related macroscopic or histopathologic changes or gross malformations. Pup weight at birth was not different from controls. In the highest dose group, a significant inhibition (10 -23 %) of the mean body weight gain of pups in the postnatal follow-up period (day 1 -36) was observed, as well as a reduction in the total litter weight of both males and females. The author derived a NOAEL for fertility parameters in both generations of 12000 ppm; pup body weight reduction in the postnatal phase, however, was recorded at 12000 ppm. There are no data on maternal body weight gain during that phase and the pup body weight at term was not different from controls. Therefore, maternal toxicity cannot explain this effect. Thus, 1800 ppm, (36 - 72 mg adipic acid/kg bw/day or corresponds to 65 -129 mg AHsalt/kg bw/day ) was shown as a clear-cut NOAEL for all effects (Tinston, 1988).
Effects on developmental toxicity
Description of key information
Data for developmental toxicity are only available for the components of
AH salt.
In various species (rat, mouse, rabbit), studies with adipic acid, one
of the two constituents of AH salt, did not indicate an adverse effect
on development up to the highest doses tested (gavage ; 250 - 288 mg/kg
bw/day). In none of these studies, signs of maternal or fetal toxicity
have been observed (NOAEL rat, mouse, rabbit (maternal/developmental
toxicity) 250 - 288 mg/kg bw/day).
The other constituent, 1,6-hexanediamine, caused some retardation
in fetal development of rats in the presence of maternal toxicity. No
teratogenic effects were found up to the highest tested dose level of
300 mg/kg bw/day, which was already associated with pronounced maternal
toxicity (NOAEL maternal/developmental toxicity: 112 mg/kg bw/day). An
impairment of body weight gain of rat pups in the postnatal period was
shown for 1,6-hexanediamine at a dose of 500 mg/kg bw/day in the absence
of maternal toxicity . The NOAEL for this effect is 150 mg/kg bw/day.
These doses correspond to 1130 mg and 338 mg AH-salt/ kg bw/day,
respectively.
However, as no adverse effects on pup body weights occurred on the day of birth (day 0) and on the day 4 after birth, but only on day 21 after birth (less than 10 %; no data presented on days 7/14 after birth) it cannot be excluded that the effect on pup body weight data is a consequence of the food intake rather than lactation of the pups, particularly between days 14-21 after birth.
Therefore, there is insufficient evidence that AH salt may act as developmental toxicant by impairment of body weight gain of progeny during lactation found at non maternally toxic but high doses of 1,6-hexanediamine .
In a prenatal developmental toxicity study in rabbits according to OECD guideline 414, Hexamethylenediamine was administered by gavage, once daily, from Days 6 to 28 p.c. to mated female New Zealand White rabbits at dosages of 12.5, 25 or 50 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 25 mg/kg/day (based on mortality at 50 mg/kg/day), the NOAEL for embryo-fetal development was considered to be 50 mg/kg/day based on absence of adverse effects at this dose-level. Based on the results of this study, Hexamethylenediamine did not elicit a teratogenic potential.
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study did not include a high dose that caused maternal toxicity, low number of animals per group, no statistical evaluation. Data on purity of adipic acid are lacking, but as no effects were observed up to the highest dose tested this is thought not to impair the validity of the results.
- Principles of method if other than guideline:
- On gestation day 6 through 18, pregnant rabbits were dosed daily with the test substance by oral intubation. Animals were observed daily for clinical signs, food consumption and body weight were determined. On Day 29 all does were subjected Caesarean section, the uterine contents were examined, and the foetuses were examined for fetotoxicity and teratogenicity.
- GLP compliance:
- no
- Remarks:
- pre-GLP data
- Limit test:
- no
- Species:
- rabbit
- Strain:
- Dutch
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
Virgin adult, Dutch-belted female rabbits
- Housing: individually in mesh bottom cages
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
temperature and humidity controlled quarter
No further data. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: artificial insemination
On Day 0, each doe was given an injection of 0.4 ml of human chorionic gonadotropin (400 IU) via the marginal ear vein. Three hours later, each doe was inseminated artificially with 0.3 ml of diluted semen from a proven donor buck using approximately 20 x 10E6 motile sperm.
No further details. - Duration of treatment / exposure:
- days 6 through 18 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- until day 29 of gestation
- Dose / conc.:
- 2.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 12 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 54 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 250 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 13 females (2.5 mg/kg bw/d);
15 females (54 mg/kg bw/d);
16 females per group (12 mg/kg bw/d, positive control);
19 females (sham-treated control);
20 females (250 mg/kg bw/d) - Control animals:
- yes, sham-exposed
- other: Positive control: 6-Aminonicotinamide, 2.5 mg/kg bw (actual ingested), dosed on day 9 of gestation
- Maternal examinations:
- CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All animals were observed daily for appearance and behavior.
BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 12, 18, and 29 of gestation.
FOOD CONSUMPTION: Yes
- Time schedule: daily
All animals were observed daily with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
WATER CONSUMPTION: No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 29
All does were subjected to Caesarean section under surgical anesthesia.
- Organs examined: gravid uterus, urogenital tract - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- The live fetuses of each litter were placed in an incubator for 24 hours for the evaluation of neonatal survival.
- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: No
Other: The body weights of the live pups were recorded. - Statistics:
- The results were not evaluated statistically.
- Mortality:
- no mortality observed
- Description (incidence):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on nidation or on maternal survival.
- Early or late resorptions:
- no effects observed
- Description (incidence and severity):
- No differences between treatment and control groups were found for total number of resorptions.
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No differences between treatment and control groups were found for fetal weight. - Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- No differences between treatment and control groups were found for total number of live litters.
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on fetal survival.
- Skeletal malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Visceral malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Details on embryotoxic / teratogenic effects:
- No differences between treatment and control groups were found for corpora lutea, implantations, and total number of fetuses.
- Dose descriptor:
- NOAEL
- Effect level:
- 250 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Executive summary:
The administration of the test substance, adipic acid, at doses of up to 250 mg/kg bw/d to pregnant rabbits for 13 consecutive days (gd 6 - 18) had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: No statistical evaluation. Data on purity of adipic acid are lacking, but as no effects were observed up to the highest dose tested this is thought not to impair the validity of the results.
- Principles of method if other than guideline:
- On gestation day 6 through day 15 pregnant mice were dosed daily with the test substance by oral intubation. Animals were observed for clinical signs. Food consumption and body weight were determined. On day 17 all dams were subjected Caesarean section, the uterine contents were examined, and the foetuses were examined for fetotoxicity and teratogenicity.
- GLP compliance:
- no
- Remarks:
- pre-GLP data
- Limit test:
- no
- Species:
- mouse
- Strain:
- CD-1
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
Virgin adult female albino CD-1 outbred mice
- Housing: individually in disposable plastic cages
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
temperature and humidity controlled quarter
No further data. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- The females were mated with young adult males.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
No further details - Duration of treatment / exposure:
- days 6 through 15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- until day 17 of gestation
- Dose / conc.:
- 2.6 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 12 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 56 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 263 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 25 females per group (2.6, 12, 56 mg/kg bw/d; sham-treated control);
31 females (263 mg/kg bw/d);
30 females (positive control)
) - Control animals:
- yes, sham-exposed
- other: Positive control: Aspirin, 150 mg/kg bw/d (actual ingested)
- Maternal examinations:
- CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All animals were observed daily for appearance and behavior.
BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 11, 15, and 17 of gestation.
FOOD CONSUMPTION: Yes
- Time schedule: daily
All animals were observed daily with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
WATER CONSUMPTION: No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 17
- Organs examined: gravid uterus, urogenital tract - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: No
Other: The body weights of the live pups were recorded. - Statistics:
- The results were not evaluated statistically.
- Mortality:
- no mortality observed
- Description (incidence):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on nidation or on maternal survival.
- Dose descriptor:
- NOAEL
- Effect level:
- 263 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No differences between treatment and control groups were found for fetal weight. - Reduction in number of live offspring:
- no effects observed
- Description (incidence and severity):
- No differences between treatment and control groups were found for total number of fetuses.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- No differences between treatment and control groups were found for total number of live litters.
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on fetal survival.
- Skeletal malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Visceral malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Details on embryotoxic / teratogenic effects:
- No differences between treatment and control groups were found for corpora lutea, implantations and total number of resorptions.
- Dose descriptor:
- NOAEL
- Effect level:
- 263 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Executive summary:
The administration of the test substance, adipic acid, at doses of up to 263 mg/kg bw/d to pregnant mice for 10 consecutive days (gd 6 - 15) had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: see 'Remark'
- Remarks:
- Study did not include a high dose that caused maternal toxicity, no statistical evaluation. Data on purity of adipic acid are lacking, but as no effects were observed up to the highest dose tested this is thought not to impair the validity of the results.
- Principles of method if other than guideline:
- On gestation day 6 through day 15, pregnant rats were dosed daily with the test substance by oral intubation. Animals were observed for clinical signs. Food consumption and body weight were determined. On day 17 all dams were subjected Caesarean section, the uterine contents were examined, and the foetuses were examined for foetotoxicity and teratogenicity.
- GLP compliance:
- no
- Remarks:
- pre-GLP data
- Limit test:
- no
- Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
Virgin adult female albino rats (Wistar derived stock)
- Housing: individually in mesh bottom cages
- Diet: ad libitum
- Water: ad libitum
ENVIRONMENTAL CONDITIONS
temperature and humidity controlled quarter
No further data. - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- The females were mated with young adult males.
- Proof of pregnancy: vaginal plug referred to as day 0 of pregnancy
No further details - Duration of treatment / exposure:
- days 6 through 15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- until day 20 of gestation
- Dose / conc.:
- 2.9 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 13 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 62 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 288 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 25 females per group (2.9, 13.0, 62.0 mg/kg bw/d; sham-treated control);
24 females (288 mg/kg bw/d);
28 females (positive control)
) - Control animals:
- yes, sham-exposed
- other: Positive control: Aspirin, 250 mg/kg bw/d (actual ingested)
- Maternal examinations:
- CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
All animals were observed daily for appearance and behavior.
BODY WEIGHT: Yes
- Time schedule for examinations: days 0, 6, 11, 15, and 20 of gestation.
FOOD CONSUMPTION: Yes
- Time schedule: daily
All animals were observed daily with particular attention to food consumption and weight, in order to rule out any abnormalities which may have occurred as a result of anorexic effects in the pregnant female animal.
WATER CONSUMPTION: No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 20
- Organs examined: gravid uterus, urogenital tract - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: one third per litter
- Skeletal examinations: Yes: two thirds per litter
- Head examinations: No
Other: The body weights of the live pups were recorded. - Statistics:
- The results were not evaluated statistically.
- Mortality:
- no mortality observed
- Description (incidence):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on nidation or on maternal survival.
- Dose descriptor:
- NOAEL
- Effect level:
- 288 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed
Field "Description (incidence and severity)" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.DescriptionIncidenceAndSeverityFetalPupBodyWeightChanges): No differences between treatment and control groups were found for fetal weight. - Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- No differences between treatment and control groups were found for total number of live litters.
- Changes in postnatal survival:
- no effects observed
- Description (incidence and severity):
- The administration of the test substance up to the highest dose level had no clearly discernible effect on fetal survival.
- Skeletal malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Visceral malformations:
- no effects observed
- Description (incidence and severity):
- The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Details on embryotoxic / teratogenic effects:
- No differences between treatment and control groups were found for corpora lutea, implantations and total number of resorptions.
- Dose descriptor:
- NOAEL
- Effect level:
- 288 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Executive summary:
The administration of the test substance, adipic acid, at doses of up to 288 mg/kg bw/d to pregnant rats for 10 consecutive days (gd 6 - 15) had no clearly discernible effect on nidation or on maternal or fetal survival. The number of abnormalities seen in either soft or skeletal tissues of the test groups did not differ from the number occurring spontaneously in the sham-treated controls.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- no data
- Reliability:
- 2 (reliable with restrictions)
- Rationale for reliability incl. deficiencies:
- other: Limited documentation of variations, data on skeletal redardation were not shown.
- Principles of method if other than guideline:
- On day 6 through 15 pregnant rats were dosed daily with the test compound. On day 21 dams were killed and were examined for embryotoxic/fetotoxic and teratogenic effects.
- GLP compliance:
- not specified
- Limit test:
- no
- Species:
- rat
- Strain:
- Sprague-Dawley
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
male (untreated) and female Sprague-Dawley derived albino rats
- Source: Charles River
- Housing: individually in wire-bottom steel rat cages, except for cohabitation (mating)
- Diet (ad libitum): Wayne Lab meal
- Water (ad libitum): tap water (automatic watering device)
ENVIRONMENTAL CONDITIONS: no data
No further data - Route of administration:
- oral: gavage
- Vehicle:
- water
- Analytical verification of doses or concentrations:
- not specified
- Details on mating procedure:
- - Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/1 or 1/2
- Proof of pregnancy: sperm in vaginal smear referred to as day 1 of pregnancy
No further data - Duration of treatment / exposure:
- days 6 through 15 of gestation
- Frequency of treatment:
- daily
- Duration of test:
- until day 21 of gestation
- Dose / conc.:
- 112 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 184 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- groups of 22 pregnant females
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: Doses were selected on the basis of a pilot teratology study.
In this pilot study, aqueous solutions of HMD were administered at a constant volume of 10 ml/kg to groups of four to six pregnant rats by oral intubation on days 6-15 of gestation. Dosages administered were equivalent to 0, 112.5, 225, 450 and 900 mg HMD/kg/d. Body weight and the health of each dam were recorded daily; food consumption was monitored at 3-day intervals throughout gestation. Dams surviving to Day 21 of gestation were killed and the number and replacement of early resorptions, late resorptions and live and dead fetuses were determined. Live fetuses were weighed, examined externally and their length and sex recorded.
RESULTS:
All dams administered 450 or 900 mg/HMD kg died within 6 days of initiation of treatment; many exhibited severe internal hemorrhaging. No deaths occurred at 225 or 112.5 mg HMD/kg. Reduced body weight gains were observed in dams given 225 mg HMD/kg while no observable effect was seen in dams from the 112.5 mg/kg group. Neither embryotoxicity nor fetotoxicity nor external fetal malformations were apparent up to 225 mg HMD/kg/d.
- Rationale for animal assignment: random - Maternal examinations:
- CAGE SIDE OBSERVATIONS and DETAILED CLINICAL OBSERVATIONS:
Dams were checked for survival twice daily. No further data.
BODY WEIGHT: Yes
- Time schedule for examinations: Body weights, actual and adjusted [minus fetal, uterine and placental weights], were recorded on Days 6-15 and 21.
FOOD CONSUMPTION: Yes
Daily food intake was recorded at 3-day intervals
WATER CONSUMPTION: No data
POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day # 21
- Organs examined: Uterine horns were examined for number and placement of early resorptions, late resorptions and fetal survival. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Uterine horns were examined for number and placement of early resorptions, late resorptions and fetal survival. - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Live fetuses were sexed, weighed, measured and examined externally. Half of the fetuses in each litter were fixed in Bouin's solution and examined for visceral abnormalities. The remaining fetuses were fixed in 95% ethanol, cleared, stained with alizarin-red-S and examined for skeletal abnormalities. - Statistics:
- Fischer Exact Probability Test (incidences of specific maternal and fetal observations), analysis of variance and, where necessary, tests for multiple comparison (body weight, food consumption, organ weights). Significance level: p < 0.05.
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- In the 300 mg/kg dosage group, a single death and one animal killed in a moribund condition were considered to have resulted from treatment. Single deaths at other test levels were considered the result of dosing accidents.
- Body weight and weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- Pregnant rats given 300 mg/kg/d gained less weight than control dams throughout the test period. Statistically significant changes were observed through gestation Day 15 and again on Day 21 after adjustment.
- Food consumption and compound intake (if feeding study):
- effects observed, treatment-related
- Description (incidence and severity):
- Transient reductions in food consumption were also noted at this test level (300 mg/kg bw/d). No effects on maternal toxicity were observed at or below 184 mg/kg/d.
- Dose descriptor:
- NOAEL
- Effect level:
- 112 mg/kg bw/day (actual dose received)
- Basis for effect level:
- body weight and weight gain
- Fetal body weight changes:
- effects observed, treatment-related
- Description (incidence and severity):
- A statistically significant decrease in fetal body weights of both male and female pups was observed at 300 mg/kg/d.
- Changes in sex ratio:
- no effects observed
- Description (incidence and severity):
- The test substance had no effect on sex ratio.
- Changes in litter size and weights:
- no effects observed
- Description (incidence and severity):
- The test substance had no effect on mean litter size.
- External malformations:
- no effects observed
- Description (incidence and severity):
- The overall incidence of minor and major malformations observed in this study was low none of which were judged related to treatment. The incidence of external observation of pups from each of the treated groups was not increased above background levels.
- Skeletal malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Three types of anatomical variations and ossification delays differed significantly between control and treated groups. These were: poor development of (1) hyoids and (2) cervical vertebral centra and (3) the lack of fusion in the posterior sacral and anterior caudal vertebra. These retardations were limited in that no other significant correlative alterations in ossification were observed. There was no dose-related pattern for hyoid development; thus, this is not considered related to treatment. The occurrence of fetuses with poorly or unossified cervical centra or sacral/caudal vertebra indicate slight retardation in skeletal development observed at both the 184 and 300 mg/kg/d dosage levels.
- Visceral malformations:
- effects observed, non-treatment-related
- Description (incidence and severity):
- Visceral examinations revealed a significant increase in the number of pups with spotty livers in the high dosage level. There also was a significant increase in bladder distension in the mid dosage group only. Since there was no dose-related pattern, it was concluded that this latter observation is not related to treatment.
- Details on embryotoxic / teratogenic effects:
- The test substance had no effect on the number of implantation sites per dam, incidence of resorptions or fetal length.
- Dose descriptor:
- NOAEL
- Remarks:
- fetotoxicity
- Effect level:
- 112 mg/kg bw/day (actual dose received)
- Basis for effect level:
- fetal/pup body weight changes
- other: increase in skeletal retardations
- Dose descriptor:
- NOAEL
- Remarks:
- teratogenicity
- Effect level:
- 300 mg/kg bw/day (actual dose received)
- Basis for effect level:
- other: no adverse effects observed up to and including the highest tested dose
- Abnormalities:
- not specified
- Developmental effects observed:
- not specified
- Conclusions:
- According to the conditions of the test, the test substance Hexamethylene diamine did not induce teratogenic effects. However, after treatment with the the substance maternal toxicity and fetotoxicity was observed.
- Executive summary:
The administration of hexamethylene diamine (HMD) by gavage to pregnant rats at 0, 112, 184 and 300 mg/kg/d on days 6-15 of pregnancy (day 1 = day sperm detected) did not induce any teratogenic effects. Signs of maternal toxicity were observed only at 300 mg/kg/d. Fetotoxicity was observed at both the 300 and 184 mg/kg/d dosage levels. No treatment-related effects were observed at 112 mg/kg/d.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- August 2015 - June 2017
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 414 (Prenatal Developmental Toxicity Study)
- Version / remarks:
- 22 January 2001
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Species:
- rabbit
- Strain:
- New Zealand White
- Remarks:
- New Zealand White, INRA, A 1077, Specific Pathogen Free (S.P.F.)
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Centre LAGO (Vonnas, France)
- Age at study initiation: 18-20 weeks
- Weight at study initiation: mean 3761 g (range: 3070 g to 4460 g)
- Fasting period before study: no
- Housing: individually (noryl cages (Tecniplast, 65.3 cm x 65.3 cm x 45 cm))
- Diet: ad libitum; breeding pelleted diet "type 110C", batch Nos. 15114 and 15159 (SAFE, Augy, France)
- Water: ad libitum, tap water (filtered with a 0.22 µm filter)
- Acclimation period: at least 4 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 ± 3
- Humidity (%): 50 ± 20
- Air changes (per hr): 5 to 15
- Photoperiod (hrs dark / hrs light): 8h dark/16h light
IN-LIFE DATES: From: 22 September 2015 To: 6 November 2015 - Route of administration:
- oral: gavage
- Vehicle:
- other: Phosphate Buffered Saline (PBS 1X pH 7.4)
- Remarks:
- pH of vehicle adjusted to pH 7.2 +/- 0.5 with hydrochloric acid after addition of test item
- Details on exposure:
- Route = oral
VEHICLE
- Justification for use and choice of vehicle (if other than water): to neutralise the alkaline test item
- Concentration in vehicle: 0, 2.5, 5, 10 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day
- Lot/batch no.: 1691215 and 1685701 - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- - analytical method: Gas Chromatography with FID detection (GC-FID)
- time schedule: once in Weeks 1 and 5 a sample was taken from control and test item dose formulations and analyzed using the validated method
- acceptance criterion: measured concentration = nominal concentration +/- 10% - Details on mating procedure:
- - Impregnation procedure: purchased timed pregnant
- Proof of pregnancy: confirmed mating (visual assessment) was designated as Day 0 p.c. - Duration of treatment / exposure:
- Day 6 to Day 28 p.c.
- Frequency of treatment:
- daily
- Duration of test:
- till Day 29 p.c. (scheduled sacrifice)
- Dose / conc.:
- 12.5 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 25 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 50 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 24 females per dose group in main study, 3 females per dose group in satellite study
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: based on results of pre-tests
Maximum Tolerated Dose study: non-pregnant animals, 7 repeated exposures towards
- 200 mg/kg bw/d (all 3 animals died or sacrificed after single exposure),
- 50 mg/kg bw/d (no animal died, no obvious signs of in vivo toxicity); these animals were afterwards treated with 125 mg/kg bw/day (see below)
- 125 mg/kg bw/day (one animal sacrificed after 4 days due to blood loss, two other animals survived till end of exposure period)
Preliminary study: exposure of pregnant animals, Day 6-28 p.c.
- 90 mg/kg bw/day: all animals prematurely sacrificed due to blood loss, loud breathing and/or dyspnea, reduced food consumption and body weight loss
- 60 mg/kg bw/day: one animal prematurely sacrificed due to blood loss, loud breathing and dyspnea, food consumption not clearly affected
- 30 mg/kg bw/day: well tolerated - Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes (principal and satellite animals)
- Time schedule: once a day checked for clinical observations; twice a day check for mortality and morbidity during treatment period
DETAILED CLINICAL OBSERVATIONS: No
BODY WEIGHT: Yes (principal and satellite animals)
- Time schedule for examinations: on Days 2, 4, 5, 6, 9, 12, 15, 18, 21, 24, 27 and 29 p.c. and prior to premature sacrifice
FOOD CONSUMPTION: Yes (principal and satellite animals)
- Time schedule: Days 2-4, 4-5, 5-6, 6-9, 9-12, 12-15, 15-18, 18-21, 21-24, 24-27 and 27-29 p.c.
- Food consumption for each animal determined a as g food/day: Yes
WATER CONSUMPTION: No (principal and satellite animals)
POST-MORTEM EXAMINATIONS: Yes (principal animals and prematurely sacrificed animals)
- Sacrifice on gestation day 29
- Organs examined: macroscopic post mortem examinations of the principal thoracic and abdominal organs. Particular attention was paid to the stomach and to the urinary tract, pH of urine measured. Determination of pregnancy status and the numbers of corpora lutea and implantation sites (recorded and classified as live or dead concepti, early or late resorptions or uterine scars)
OTHER:
Urinalysis performed in animals of the satellite group (between Day 25 and 28 p.c.), due to some effects observed in the urinary tract of animals found dead or prematurely sacrificed in the MTD or dose-range finding study
Urines were collected at the end of the treatment period (on Day 26 p.c.) at the following four periods (thymol was used as a preservative):
[-4h; 0h(a)],
[0h(a); 4h],
[4h, 12/14h(b)],
[12/14h(b); 24h]).
(a): 0h corresponds to the time of dosing.
(b): 12/14h means that for organizational purpose, end and start of collection may be between 12 and 14h after dosing.
qualitative parameters:
- appearance, colour
Quantitative parameters determined in urine:
- pH, volume, specific gravity
Semi-quantitative parameters determined in urine:
- proteins, glucose, ketones, bilirubin, nitrites, blood (hemoglobin), urobilinogen, cytology of sediment - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes - Fetal examinations:
- - External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: all per litter (One half of the fetuses was decapitated and the head was fixed in Harrison’s fluid with decalcification. Serial sectioning was performed for evaluation of brain, nasal passages and tongue (and other structures, where appropriate). In the other half of the fetuses, the brain of each fetus was sampled and fixed in Harrison’s fluid. Serial section was made for examination of the organ.) - Statistics:
- Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
- Indices:
- none
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- emaciated appearance, absence of feces/urine, nearly no food consumption and/or abortion in prematurely sacrificed animals; no remarkable clinical observations in the surviving animals
- Dermal irritation (if dermal study):
- not examined
- Mortality:
- mortality observed, treatment-related
- Description (incidence):
- Two of the principal animals and one of the satellite animals of the high dose group were prematurely sacrificed due to bad health conditions. Additionally, one principal animal of the high dose group died on Day 10 p.c. during dose formulation administration, possibly due to technical issues during the gavage procedure.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- effects observed, treatment-related
- Description (incidence and severity):
- Reddish colored area were observed on the stomach in two females treated at 12.5 mg/kg/day, five females treated at 25 mg/kg/day and four females treated at 50 mg/kg/day but not in controls. In one principal high dose female sacrificed on Day 19, an ulcerated focus was seen on the stomach. In control animals, no reddish colored areas of the stomach were observed. Colored foci/deposit(s) and/or area(s) in the stomach were not ulcerated.
Vagina with a liquid brownish colored content, gall bladder with a blackish colored content, dilated ureter, stomach with ulcerated foci and /or gall bladder with dilatation observed in prematurely sacrificed animals
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- not examined
- Other effects:
- not examined
- Details on results:
- There were no remarkable clinical observations in the surviving animals. Single effects observed in treated and control animals were absence of urine, absence of faeces, lought breathing or blood in the bedding.
There were no unscheduled deaths or abortions in control, 12.5 and 25 mg/kg/day groups.
In the 50 mg/kg/day group (principal and satellite animals), there were three prematurely sacrificed females (E32296, E32301 and E32318) and one found dead female (E32300):
female E32296 was sacrificed for human grounds on Day 23 p.c. (emaciated appearance and absence of feces/urine from Day 15 p.c. and blood in the bedding on Day 23 p.c.). This animal lost 15% of body weight from Days 6 to 21 p.c. and had nearly no food consumption on Days 9 to 21 p.c. At necropsy, this female (with 11 corpora lutea and 11 late resorptions) had a vagina with a liquid brownish colored content and a gall bladder with a blackish colored content,
Female E32301 was sacrificed for abortion on Day 11 p.c. (blood in the bedding from Day 10 p.c. and embryos in the bedding on Day 11 p.c.). At necropsy this female (with 11 corpora lutea and 10 uterine scars) had a dilated ureter and 10 placentas in the uterine horns,
Female E32318 (satellite group) was sacrificed for human grounds on Day 19 p.c. (absence of feces from Day 7 p.c., absence of urine on Days 7 to 8 p.c., blood in the bedding on Days 14 to 15 p.c.). This animal lost 13% of body weight from Day 6 to 18 p.c. and had nearly no food consumption on Days 6 to 18 p.c. At necropsy this female was not pregnant and had a stomach with ulcerated foci and a gall bladder with dilatation,
Female E32300 died on Day 10 p.c. during dose formulation administration. At necropsy this female (with 13 corpora lutea and 13 dead implants) had lungs with a reddish diffuse abnormal color, and a stomach with several whitish colored deposit and brownish colored areas in the mucosa.
For females E32296, E32301 and E32318, the deaths were considered to be test item treatment-related while for female E32300 a technical issue during the gavage procedure cannot be excluded.
Macroscopic changes except stomach findings at terminal necropsy of the surviving dams on Day 29 p.c. are commonly observed in this species and strain.
Reddish colored area were observed on the stomach in two females treated at 12.5 mg/kg/day, five females treated at 25 mg/kg/day and four females treated at 50 mg/kg/day but not in controls. These correlated histologically with haemorrhage.
In female E32300 treated at 50 mg/kg/day and found dead on Day 10 p.c. brownish colored foci (without histopathological correlates) and whitish deposits (correlating with increased mucus on surface) were seen on the stomach. Any relationship of these observations with the test item was excluded.
In female E32318 sacrificed on Day 19, an ulcerated focus was seen on the stomach correlating histologically with slight erosion.
In control animals, no reddish colored areas of the stomach were observed.
Colored foci/deposit(s) and/or area(s) in the stomach were not ulcerated. They were observed with dose related increased incidences in surviving females from the test item-treated groups, but not in control animals.
At microscopic examination of the stomachs, minimal to slight mucosal hemorrhages (mainly superficial) were observed in all groups including controls. Although the highest grades were seen in test item-treated animals, the absence of dose-related incidence and severity and the lack of associated changes (e.g. inflammation or pigmented macrophages) suggested these hemorrhages were likely agonal and not induced by the test item.
There were no effects on mean gravid uterus weight, mean carcass weight and net body weight change on Day 29 p.c. from Day 6 p.c..
Urinalysis in satellite animals did not point to any test item related effects. - Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- no effects observed
- Total litter losses by resorption:
- no effects observed
- Early or late resorptions:
- no effects observed
- Dead fetuses:
- no effects observed
- Changes in pregnancy duration:
- no effects observed
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Effects on pregnancy duration" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsMaternalAnimals.MaternalDevelopmentalToxicity.EffectsOnPregnancyDuration): no effects observed - Changes in number of pregnant:
- no effects observed
- Other effects:
- not examined
- Details on maternal toxic effects:
- There were no effects on hysterectomy data in principal surviving dams on Day 29 p.c.
Hysterectomy data in principal females were the following:
Hysterectomy data in principal surviving dams on Day 29 p.c.
Dose-level (mg/kg/day) 0 (vehicle) 12.5 25 50
Number of females with live fetuses
at termination 24 23 23 20
Mean number of corpora lutea per animal 11.7 12.3 12.0 12.1
Mean number of implantations per animal 10.0 10.6 9.7 10.7
Mean pre-implantation loss (%) 14.6 14.6 18.4 11.3
Mean number of fetuses per animal 9.3 9.8 9.2 10.1
Dead fetuses (%) 0.0 0.0 0.4 0.0
Mean number of implantation scars 0.0 0.0 0.0 0.0
Mean number of early resorptions 0.5 0.4 0.4 0.3
Mean number of late resorptions 0.2 0.3 0.1 0.3
Mean post-implantation loss (%) 6.5 8.5 5.9 5.8 - Key result
- Dose descriptor:
- LOAEL
- Effect level:
- 50 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- mortality
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 25 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Basis for effect level:
- mortality
- Key result
- Abnormalities:
- effects observed, treatment-related
- Localisation:
- other: mortality
- Description (incidence and severity):
- three of 24 animal of the high dose group were prematurely sacrificed due to poor health conditions
- Fetal body weight changes:
- no effects observed
- Description (incidence and severity):
- Migrated Data from removed field(s)
Field "Fetal/pup body weight changes" (Path: ENDPOINT_STUDY_RECORD.DevelopmentalToxicityTeratogenicity.ResultsAndDiscussion.ResultsFetuses.FetalPupBodyWeightChanges): no effects observed - Reduction in number of live offspring:
- no effects observed
- Changes in sex ratio:
- no effects observed
- Changes in litter size and weights:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- no effects observed
- Visceral malformations:
- no effects observed
- Other effects:
- not examined
- Details on embryotoxic / teratogenic effects:
- - fetal body weight and sex-ratio: there was a lower mean fetal body weight in the 50 mg/kg/day group which was considered to be not toxicologically significant (less than 10%),
- external examination: at 50 mg/kg/day, there was a higher percentage of litters with fetuses with malrotated paws,
- soft tissues examination: from 25 mg/kg/day there were increases in litter and fetal incidences of fetuses with colored focus on the gall bladder,
- cartilage and skeletal examination: there were higher litter and fetal incidences of fetuses with unossified 1st metacarpals (metacarpal bone cartilages were present; statistically significant only at 50 mg/kg/day;).
All these external, soft tissue and cartilage/skeletal variations were considered to be test item treatment related but not adverse. There were no tendencies towards a specific trend or syndrome in the distribution of external, soft tissues or skeletal malformations.
See attachment for results of fetal examination - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 50 mg/kg bw/day (nominal)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: no adverse effects observed in fetuses
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- The test item did not induce effects on embryo-fetal development of rabbits up to maternal toxic doses under the conditions of the test.
- Executive summary:
The objective of this prenatal developmental toxicity study was to evaluate the potential toxic effects of the test item, hexamethylenediamine, on the pregnant female and on embryonic and fetal developmentand on urinalysis parameters following daily oral administration (gavage) to pregnant female rabbits from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 28 post-coitum(p.c.) inclusive).
Methods
Three groups of 24 principal mated female New Zealand White rabbits were administered the test item, hexamethylenediamine(batch No. 14 266 13), once daily from Day 6 to Day 28 p.c., by gavage, at dosages of 12.5, 25 or 50 mg/kg/day (groups 2 to 4). An additional group of 24 mated females received the vehicle, Phosphate Buffered Saline (PBS 1X pH 7.4), under the same experimental conditions and acted as the control group (group 1). A constant dose volume of 5 mL/kg/day was used.
In order to ascertain whether or not the test item treatment was associated with urinalysis parameters changes, three satellite animals per group were included in the study and urines were collected at the end of the treatment period (between Days 25 and 28p.c.). These satellite animals were administered the test item or the vehicle within the same experimental conditions.
The animals were checked daily for mortality and/or clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29p.c.animals were sacrificed and submitted to macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. Kidneys, ureters and urinary bladder were sampled and kept preserved in a fixative. The fetuses from principal animals were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.
Results
Chemical analysis of dose formulations: the test item concentrations in the administered dose formulations analyzed in Weeks 1 and 5 remained within an acceptable range of variations (-6.8% to +8.6%) when compared with the nominal values (± 10% of the nominal concentrations). In Weeks 1 and 5, test item was observed in the control dose formulations with concentrations below the LOQ (< 2.5 µg/mL). As control dose formulations were diluted 10-fold before injection, the quantity of test item observed was estimated below 25 µg/mL which was considered to be negligible.
Pregnancy status: in the control, 12.5, 25 and 50 mg/kg/day groups, there were 24, 23, 23 and 20 principal females and 3, 2, 3 and 2 satellite females with live fetuses at hysterectomy, respectively.
Mortality/abortion: there were no unscheduled deaths or abortions in control, 12.5 and 25 mg/kg/day groups.
In the 50 mg/kg/day group (principal and satellite animals), three prematurely sacrifices for poor health condition (e.g. emaciated appearance, absence of feces/urine, nearly no food consumption and/or abortion) were considered to be test item treatment-related. At necropsy of prematurely sacrificied animals a range of findings was recorded (e.g. vagina with a liquid brownish colored content, gall bladder with a blackish colored content, dilated ureter, stomach with ulcerated foci and /or gall bladder with dilatation) for which a test item-relationship was not excluded.
Clinical signs: there were no remarkable clinical observations in the surviving animals.
Body weights and body weight changes: there were no test item treatment-related effects on mean body weights and mean body weight changes.
Food consumption: there were no toxicologically significant effects on mean food consumption.
Urinalysis: there were no statistically significant differences in mean urinalysis parameters recorded in satellite surviving dams or on urinary pH recorded at necropsy.
Maternal terminal examination:
. at necropsy, there were non-ulcerated colored foci/deposit(s) and/or area(s) in the stomach of treated animals. These findings were observed with increased incidences in surviving females from the test item-treated groups (without dose response), but not in controls. Histological findings indicate that they are not test item related and therefore not adverse, which is supported by the absence of clinical signs and significant effects on body weight or food consumption,
. there were no effects on mean gravid uterus weight, mean carcass weight and net body weight change on Day 29p.c.from Day 6p.c.
Hysterectomy data: there were no effect on hysterectomy data in principal surviving dams on Day 29p.c.
Fetal examination:
. fetal body weight and sex-ratio: there was a lower mean fetal body weight in the 50 mg/kg/day group which was considered to be not toxicologically significant (less than 10%),
. external examination: at 50 mg/kg/day, there was a higher percentage of litters with fetuses with malrotated paws,
. soft tissues examination: from 25 mg/kg/day there were increases in litter and fetal incidences of fetuses with colored focus on the gall bladder,
. cartilage and skeletal examination: there were higher litter and fetal incidences of fetuses with unossified 1stmetacarpals (metacarpal bone cartilages were present; statistically significant only at 50 mg/kg/day;).
All these external, soft tissue and cartilage/skeletal variations were considered to be test item treatment‑related but not adverse. There were no tendencies towards a specific trend or syndrome in the distribution of external, soft tissues or skeletal malformations.
Pathology:
No test item-related changes were observed in the stomach at any of the dose-levels.
Minimal to slight hemorrhages correlating with reddish colored area were observed at necropsy in several animals treated at 12.5, 25 or 50 mg/kg/day. In the absence of dose-related incidence or severity and of associated changes (e.g inflammation or pigmented macrophages) any relationship to the test item was considered to be unlikely.
Conclusion
The test item, hexamethylenediamine (batch No. 14 266 13), was administered by gavage, once daily, from Days 6 to 28p.c., inclusive, to mated female New Zealand White rabbits at dosages of 12.5, 25 or 50 mg/kg/day.
On the basis of the results obtained in this study:
. the No Observed Adverse Effect Level (NOAEL) for maternal parameters was considered to be 25 mg/kg/day (based on mortality at 50 mg/kg/day),
. the NOAEL for embryo-fetal development was considered to be 50 mg/kg/day based on absence of adverse effects at this dose-level.
Hexamethylenediamine did not elicit a teratogenic potential.
Referenceopen allclose all
For details, see attached documents.
For details, see attached documents.
For details, see attached documents.
The test item concentrations in the administered dose formulations analyzed in Weeks 1 and 5 remained within an acceptable range of variations (-6.8% to +8.6%) when compared with the nominal values (± 10% of the nominal concentrations).
In Weeks 1 and 5, test item was observed in the control dose formulations with concentrations below the LOQ (< 2.5 µg/mL). As control dose formulations were diluted 10-fold before injection, the quantity of test item observed was estimated below 25 µg/mL, which was considered to be negligible.
The interfering peak detected in the control group chromatogram was also detected in the corresponding analytical diluent. Therefore, this interfering peak could be related to analytical process.
Effect on developmental toxicity: via oral route
- Dose descriptor:
- NOAEL
- 250 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
Developmental toxicity studies have been conducted for adipic acid in rats, mice and rabbits, and for 1,6-hexanediamine (or its dihydrochloride) in rats.
Adipic acid was not embryo- or fetotoxic and not teratogenic after administration by gavage to rats (gd 6 - 15; up to 288 mg/kg bw/day), mice (gd 6 - 15 ; up to 263 mg/kg bw/day), and rabbits (gd 6- 18 ; up to 250 mg/kg bw/day). In none of these studies signs of maternal or fetal toxicity have been observed (U.S. Food and Drug Administration, 1973, 1974). NOAELs for rat, mouse and rabbit (maternal/developmental toxicity) are 250 - 288 mg/kg bw/day.
In pregnant Sprague-Dawley rats treated with 112, 184 and 300 mg/kg bw/day of 1,6- hexanediamine by gavage (gd 6 - 15; n = 22) there was a significant decrease of body weight gain in the top dose. Incidence of implantation sites and resorptions was not affected, also fetal sizes were normal. Fetal body weights were reduced in both sexes and slight skeletal retardations were recorded. At 184 mg/kg bw/day, there was numerical reduction (15 %) of maternal body weight gain during treatment and also a numerical reduction in fetal weight (5 %) and an increase in skeletal retardations. On balance, in the light of the pronounced maternal effects, the fetal effects observed do not indicate a selective fetal toxicity. Contrary to the author's conclusions, 112 mg/kg bw/day is a NOAEL for both maternal and fetal toxicity, with 184 mg/kg bw/day being a LOAEL for both endpoints. 300 mg/kg bw/day is a NOAEL for teratogenicity and embryotoxicity (Johannsen and Levinskas, 1987).
Additionally, 1,6-Hexanediamine dihydrochloride was not embryo- or fetotoxic in a limited study in Fischer 344 rats after gavage of up to 200 mg/kg bw/day (gd 0-15). Maternal toxicity (reduced body weight gain) was observed in this study at 200 mg/kg bw/day. Teratogenicity was not investigated (David and Heck, 1983).
In a two-generation study, an impairment of body weight gain of rat pups in the postnatal period was shown for 1,6-diaminohexane at a dose of 500 mg/kg bw/day, in the absence of maternal toxicity. The NOAEL for this effect is 150 mg/kg bw/day (Short et al 1991).
These doses correspond to 1130 mg and 338 mg AH-salt/kg bw/day, resp . However, as no adverse effects on pup body weights occurred on the day of birth (day 0) and on the day 4 after birth, but only on day 21 after birth (less than 10 %; no data presented on days 7/14 after birth) it cannot be excluded that the effect on pup body weight data is a consequence of the food intake rather than lactation of the pups, particularly between days 14-21 after birth .
Therefore, there is insufficient evidence that AH salt may act as developmental toxicant by impairment of body weight gain of progeny during lactation found at non maternally toxic but high doses of 1,6-hexanediamine.
1,6-Hexanediamine was also assessed in a prenatal developmental toxicity study in rabbits. The study was conducted according to OECD guideline 414 under GLP conditions. Three groups of 24 principal mated female New Zealand White rabbits were administered the test item (purity: ≥ 99.9 %) once daily from Day 6 to Day 28 p.c. by gavage at dosages of 12.5, 25 or 50 mg/kg bw/d. The animals were checked daily for mortality and/or clinical signs. Body weight and food consumption were recorded at designated intervals. On Day 29 p.c. animals were sacrificed and submitted to macroscopic post-mortem examination. Hysterectomy was performed and the numbers of corpora lutea, implantations, early and late resorptions, and live and dead fetuses were recorded. The fetuses were weighed, sexed and examined for external, soft tissue and skeletal abnormalities.
In the 50 mg/kg bw/d group, three prematurely sacrifices for poor health condition (e.g. emaciated appearance, absence of feces/urine, nearly no food consumption and/or abortion) were considered to be treatment-related. At necropsy of these animals a range of findings was recorded (e.g. vagina with a liquid brownish colored content, gall bladder with a blackish colored content, dilated ureter, stomach with ulcerated foci and /or gall bladder with dilatation). There were no remarkable clinical observations in the surviving animals. Body weight, food consumption and urinalysis parameters were not affected in all dose groups. Fetal examination revealed a lower mean fetal body weight in the 50 mg/kg bw/d group which was considered to be not toxicologically significant (less than 10 %). At 50 mg/kg bw/d, there was a higher percentage of litters with fetuses with malrotated paws. From 25 mg/kg bw/d there were increases in litter and fetal incidences of fetuses with colored focus on the gall bladder. Furthermore, there were higher litter and fetal incidences of fetuses with unossified 1st metacarpals (metacarpal bone cartilages were present). This finding was statistically significant only at 50 mg/kg bw/d. However, all these external, soft tissue and cartilage/skeletal variations were considered to be test item treatment‑related but not adverse.
On the basis of the results obtained in this study, the No Observed Adverse Effect Level (NOAEL) for maternal toxicity was considered to be 25 mg/kg bw/d (based on mortality at 50 mg/kg bw/d) and the NOAEL for embryo-fetal development was considered to be 50 mg/kg bw/d based on absence of adverse effects at this dose-level. 1,6-Hexanediamine did not elicit a teratogenic potential under the conditions of this study (CiToxLab, 2017).
Justification for classification or non-classification
There is no need to classify AH-Salt for impairment of fertility or teratogenicity according to the Directive 67/548/EC or GHS criteria (Regulation (EC) N° 1907/2006).
Additional information
Information on Registered Substances comes from registration dossiers which have been assigned a registration number. The assignment of a registration number does however not guarantee that the information in the dossier is correct or that the dossier is compliant with Regulation (EC) No 1907/2006 (the REACH Regulation). This information has not been reviewed or verified by the Agency or any other authority. The content is subject to change without prior notice.
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