Hexamethylenediamine

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

Effects on fertility have been investigated in an oral two-generation feeding study with rats. Additional information is available from two subchronic inhalation studies with rats and mice, which were mated at the end of the 13 -weeks treatment period and observed till delivery. No effects on fertility were observed in any of these studies.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

500 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

sufficient for evaluation

Effect on fertility: via inhalation route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEC

100 mg/m³

Study duration:

subchronic

Species:

other: rats and mice

Quality of whole database:

sufficient for evaluation

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

The reproductive toxicity of Hexamethylenediamine (HMD) was investigated in a two generation study in rats (Schardein, 1985). Males and females rats received 0, 50, 150 and 500 mg/kg bw/day in the diet, daily over two generations. No treatment- related mortality was observed in any of the groups, the weight gain of parents (F0 and F1) and pups was slightly reduced in the high dose group. These decreased in body weight could be attributed to the unpalatability of HMD inducing decrease in food consumption by the F0 and F1 parents. The litter size was slightly reduced at birth in the high dose group, there was no adverse effect on survival during lactation in any of the treated groups. Thus:

- The NOAEL (Parental) = 500 mg/kg bw/day based on overall effects.

- The NOAEL (Developmental) = 500 mg/kg bw/day since slight decreased pups weight observed at high dose could be attributed to the unpalatability of HMD inducing decrease in food consumption by the F0 and F1 parents.

-The NOAEL (Fertility) = 500 mg/kg bw/day based on a slight but significant decreased in litter size without any other effects and any historical control data to check for abnormal responses in the concurrent control group to conclude on the consistency with a chance event.

In conclusion, there were no adverse effects on reproduction and fertility induced by HMD treatment.

In a one-generation reproduction study, 1,6 -hexanediamine Dihydrochloride (HDDC) was administered to rats at dose level of 0, 16, 50 and 160 mg/m3 by whole-body inhalation for 13 weeks. The exposure concentrations corresponded to 0, 10, 31 and 100 mg/m3 of HMD. 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 and pups were killed on lactation Day 21. Adult females were weighed on gestation Days 0 and 20. Adult males were weighed at the end of the mating period. Dams and 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. Necropsies were performed only on mating-trial females selected for breeding and examined for pregnancy 23 days after the conclusion of breeding.

No reproductive toxicity was observed. There was no effect on male or female fertility, body weight or body weights gains, gestation length, litter size, neonatal survival, pup weights, sex ratios or pups, or pup morphology in rats exposed to HDDC.

This one-generation reproduction toxicity study is considered as acceptable. The inhalation route of exposure used in this study is relevant as the exposure scenario is based on this route of exposure. Indeed, the workers are exposed to HDDC only by inhalation.

In a one-generation reproduction study, 1,6 -hexanediamine Dihydrochloride (HDDC) was administered to mice at dose level of 0, 16, 50 and 160 mg/m3 by whole-body inhalation for 13 weeks. 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 and pups were killed on lactation Day 21. Adult females were weighed on gestation Days 0 and 20. Adult males were weighed at the end of the mating period. Dams and 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. Necropsies were performed only on mating-trial females selected for breeding and examined for pregnancy 23 days after the conclusion of breeding.

Three females exposed to 16 mg/m3 and 1 female and 1 male exposed to 50 mg/m3 died before schedules termination but these deaths were not considered compound related.

Reproductive effects of HDDC were minimal. No body weights or body weights gains were recorded for both sexes. No effect on male and female fertility were observed. A statistically significant increase in the mean gestation lenght of mice in the 50 and 160 mg/m3 exposure groups was noted but without biological significance in the absence of other reproductive toxicity. 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 similarto that of controls 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. Therefore the NOAEL (Parental) > = 100 mg/m3 and the NOAEC (F1) >= 100 mg/m3 (based on overall effects).

This one-generation reproduction toxicity study is considered as acceptable. The inhalation route of exposure used in this study is relevant as the exposure scenario is based on this route of exposure. Indeed, the workers are exposed to HDDC only by inhalation.

Effects on developmental toxicity

Description of key information

In a teratology study in rats, treated by gavage on days 7-16 of gestation (Preach, 1979; similar to OECD 414, RL2) maternal toxicity was observed at 300 mg/kg bw/day. No reliable developmental effects were observed up to 184 mg/kg bw/day in the absence of maternal toxicity while the developmental effects observed in the fetuses from dams treated with 300 mg HMD/kg b.w./day, were clearly secondary to the maternal toxicity.

In a guideline OECD 414 developmental toxicity study in rabbits, which were treated from GD 6 to 28 by gavage to 0, 12.5, 25, 50 mg/kg bw/day, no developmental effects were observed up to the highest dose tested, which caused mortality in dams.

Hence, under the test conditions, HMD is not considered as a developmental toxicant and not classified for teratogenicity.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

qualified data base with developmental toxicity studies in rats and rabbits available.

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a study similar to OECD Guideline 414 (Prenatal Developmental Toxicity Study), pregnant female rats were treated by gavage on days 7-16 of gestation with different concentrations of diluted 85.8% w/w aqueous solution of HMD at dosage level of 0, 112, 184 and 300 mg/kg b.w./day.

Treatment with HMD at a dosage of 300 mg/kg b.w./day showed maternal toxicity as evidenced by reduced body weight gains, transiently decreased food consumption, clinical observations, and by the death of approximately 10% of the animals treated at this dosage level. Dosage below 300 mg/kg b.w./day had no statistically significant effects on the dams; however, the initial dosing at the mid dose level caused a transient mean body-weight loss of 2 grams on day eight of gestation.

Fetuses of dams treated with 300 mg/kg b.w./day were slightly retarded in development as evidenced by body weight, limited retardation of skeletal development, and possibly by liver spottiness. A dosage of 112 mg/kg b.w./day clearly had no effect on the fetuses and was considered as a NOEL.

The frequency of occurrence of fetuses with poorly ossified cervical vertebral centra was significantly greater than for concurrent controls in both the 184 and 300 mg HMD/kg b.w./day dosage groups and for the latter group, there were significantly more fetuses in which the sacral and caudal vertebra had unfused components. Although the delayed of ossification observed in the fetuses from the dams treated at the highest dose were clearly secondary to the maternal toxicity, the retardation in skeletal development observed at the middle dose level occurred in the absence of maternal toxicity.

According to the authors, both these observations are consistent with a slight retardation in skeletal development and considered as a 'fingerprint' of a generalized delay in ossification in near term rats (sacrifice on the gestation day 22). In effect, according to a normal pattern ossification of each vertebral centrum begins with two separate ossification centers, which subsequently use to form a single dumbbell shaped ossification center, later becoming more ovoide in shape.

The generalized delay is characterized by reduced ossification of bones that normally exhibit rapid ossification during the last few days of gestation such as the cervical, sacral and caudal vertebral centra. In rodents, while bones such as ribs and long bones of the limbs ossify early, other bones such as thoracic and lumbar vertebral centra are among the regions that ossify rapidly during late gestation. Therefore, in examining delayed ossification, one confounder is that a great deal of ossification occurs during the end of gestation.

Moreover, historical control data provide another means of characterizing the normal pattern of skeletogenesis, and are extremely important for interpreting delayed ossification. In the absence of the historical control data (along with concurrent control) in this study, it should be impossible to determine the designation and the occurrence of the poorly ossified cervical vertebral centra in the context of the background ¿noise¿ of the population on test or at risk.

Another common but questionable practice is to conduct independent statistical analyses on different degrees of ossification for a single bone (e.g., for cervical vertebral centra ¿ unossified, poor ossified, unarticulated or bilobed). Although these distinctions can be identified readily by experienced technicians from a developmental perspective, they are of minimal significance. Problem can arise when these isolated findings are interpreted solely on the basis of statistical significance rather than considering the overall context of closely related variations. In effect, the incidence of incompletely ossified vertebral centra may be increased statistically, yet the unarticulated and bilobed vertebral centra were decreased as demonstrated in this study. Hence, the interpretation based solely on the statistically identified increase has led to the inappropriate conclusion that ossification of the cervical vertebral centra was delayed, when in fact, consideration of the cervical vertebral centra data as a whole would indicate the lack of an adverse effect.

Therefore, the slight ossification retardation observed at both highest dose levels in this study is considered to be of low level of concern as nonlethal and not detrimental to postnatal survival variations which are generally reversible or transitory. In fact, the delays in ossification resolve in time as the growth of the individual reaches a comparable stage to that of the control group to which it is being compared. This conclusion is confirmed by the two-generation reproduction study performed in rats dosed with HMD up to 500 mg/kg bw/day (Schardein, 1985 as a key study) where no-treatment-related mortality was observed in both the F1 and their pup.

 

In conclusion, the NOAEL value of 184 mg/kg b.w./day for maternal toxicity and 300 mg/kg b.w./day for developmental effects are determined under the test conditions of the developmental study.

In a guideline OECD 414 study prenatal developmental toxicity of hexamethylenediamine was investigated in rabbits. Three groups of 24 principal mated female New Zealand White rabbits were administered the test item, hexamethylenediamine, once daily from Day 6 to Day 28p.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). 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.

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. 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 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.

There were no remarkable clinical observations in the surviving animals. There were no test item treatment-related effects on mean body weights and mean body weight changes. There were no toxicologically significant effects on mean food consumption. There were no statistically significant differences in mean urinalysis parameters recorded in satellite surviving dams or on urinary pH recorded at necropsy. The following observations were made at 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.

- 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 1^stmetacarpals (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.

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.

An unexpected high toxicity was observed with the dams in the OECD TG 414 study with rabbits. This occurred in the absence of any signs of developmental toxicity.

In a mechanistic study (see repeated dose toxicity CiToxLab 2018) it was investigated whether use of PBS buffer had an influence on the toxicity observed in dams in this prenatal developmental toxicity in rabbits (CiToxLab 2017). With non-pregnant female rabbits receiving 75, 100, or 150 mg/kg bw/day by gavage for 7 days high toxicity (including mortality) was observed at the highest dose. Signs of (limited) toxicity in the lower doses were minimal histopathological changes in the GIT and urinary tract and haematuria. No marked difference was seen between using water or PBS buffer as vehicle. Urinary tract changes seem to be more likely linked to the high toxicity seen at the highest dose.

Additional studies of lower reliability also did not detect any developmental toxic effects and thereby support the findings of the key studies.

Based on the information obtained in rats and rabbits HMD is not considered as a developmental toxicant.

Mode of Action Analysis / Human Relevance Framework

In the absence of information on species specific toxicity of HMD it is assumed that the available data on reproductive and developmental toxicity, which did not indicate any reproductive or developmental toxicity of HMD, are relevant for humans.

Justification for classification or non-classification

HMD is not classified in annex VI of Regulation (EC) n°1272/2008 for reproductive effect. Based on the above data no additional self-classification is proposed.