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Key value for chemical safety assessment

Effects on fertility

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
A two generation study in rats with HMD (Klimisch score = 1) is available - the NOAEL presented is the highest dose tested. Moreover a screening study with DCH in rats is at hand. The overall quality of the database is therefore high.
Effect on fertility: via inhalation route
Endpoint conclusion:
no adverse effect observed
Quality of whole database:
From the 13 week studies with HMD-dihydrochloride conducted with rats and mice also the results from the mating trials were presented (Klimisch score = 2).
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

In a two generation study (Schardein, 1985), 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 starting already 56 days prior to mating (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 lactation 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 HMD was 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. - 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 and 38. 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 decreased food consumption. Therefore, this effect was likely due to the low palatability of HMD.

-Fertility was not adversely affected by the dietary administration of hexamethylenediamine over 2 generations. 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 were 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 ante mortem 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.

In conclusion:

Under the test conditions, there were no adverse effects on reproduction and fertility, therefore:

The NOAEL (Parental) = 500 mg/kg bw/day (transiently decreased body weights of F0 males and F1 male and female animals were likely due to the decrease of food consumption as the worst case)

The NOAEL (Developmental) = 500 mg/kg bw/day (since the slightly decreased pup weights observed at high dose in comparison to the control, could be attributed to the low palatability of HMD thus inducing decrease in food consumption by the F0 and F1 parents).

The NOAEL (Fertility) = 500 mg/kg bw/day (as the significant but slight decrease in litter size in the high dose group is considered to be in the range of the control group and without any other effects on reproduction).

 

 

In another 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. Complete necropsy and investigation of clincal chemistry and haematology data was performed on the base study animals (13 week inhalational exposure).

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.

The same study regimen was applied to a second species, i.e. mouse. In the experiment with mice 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 effects on male and female fertility were observed. A statistically significant increase in the mean gestation length 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 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. Therefore the NOAEC (Parental) > = 100 mg/m3 and the NOAEC (F1) >= 100 mg/m3 (based on overall effects).

 

In a OECD 422 study in rats the orally administered test material (DCH) induced changes only in the highest dose tested; i.e. changes in clinical appearance (slight salivation), functional observations (slight hyperactivity), body weights and food consumption (decreased), clinical laboratory investigations decreased eosinophils, ALT and AST activity increased), macroscopic and microscopic examinations (liver, lung and adrenal glands), which correlated with changes in organ weights. There were also effects related to reproduction (decreased gestation index) and litter observation (reduced average and total number of living pups) that were considered to be an effect of treatment.

From the results presented in this report a No Observed Adverse Effect Level (NOAEL) for parental and reproduction/developmental toxicity for the test item of 150 mg/kg bw/day was established.

No dermal study was available.


Short description of key information:
In a reliable two generation study (feeding study with HMD, a representative member of the amine heads category) no effects on reproduction/fertility were obvious in any of the examined generations. The NOAEL was established at the highest dose tested, i.e. 500 mg/kg bw/day.
In inhalation studies (mating trials) conducted with mice and rats no effects on repoduction was evident after 13 weeks of exposure. Concentrations tested of HMD were up to 100 mg/cubic metre.

Justification for selection of Effect on fertility via oral route:
Only reliable two generation study (reliability 1), read-across based on grouping of substances (category approach)

Effects on developmental toxicity

Description of key information
Under the test conditions (oral gavage administration from GD7 to GD 16 in female rats), maternal toxicity was observed at 300 mg HMD/kg bw/day. In foetuses effects were seen secondary to this maternal toxicity. However, no adverse effect for maternal toxicity was observed at 184 mg HMD/kg bw/day. The NOAEL for developmental effects was established at 300 mg/kg bw/day. 
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:
Two studies are available (developmental toxicity with HMD and screening study with DCH) - the NOAEL presented is the highest dose tested in the developmental toxicity study with HMD. The developmental study has a Klimisch score = 2 and the screening study a Klimisch score=1 (Nevertheless the results from the dev tox study were used as investigations on pups were more detailed than in the screening study). The overall quality of the database is therefore high.
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 Sprague-Dawley rats (22/group) were treated by gavage with different concentrations of diluted 85.8% w/w aqueous solution of HMD at dose levels of 0, 112, 184 and 300 mg/kg bw/day on days 7-16 of gestation.

Prenecropsy observations were carried out such as body weight changes, food consumption, and cage side observation. Dams that survived to day 22 of gestation were killed on that day and necropsy was performed. Finally, fetals examinations were realized. The maternal parameters assessed during the study included body weight, food consumption, clinical signs, corpora lutea, implantations and resorptions. The fetal parameters assessed during the study included litter size, placental weight, gross malformations, fetal crown-rump length, fetal body weight, sex, visceral and skeletal examinations.

One maternal death occurred in each of the 0, 184 and 300 mg/kg b.w./day groups and one animal in the latter group had to be sacrified prior to term because she appeared moribund. Maternal body weight gain was reduced as compared to concurrent controls for animals treated at 300 mg HMD/kg b.w./day throughout the gestation. Other evidence of maternal toxicity at this dosage was a transient decrease in food consumption and clinical observations such as hunching, kemp fur, red stained fur, wheezing and respiratory rattle.

Litter size was comparable in all groups. Fetal body weight was reduced by treating the dams with 300 mg/kg b.w./day of HMD but lower dosages had no effect on the body weights of the fetuses. Sex ratio, crown-rump length and percent dead or resorbed fetuses were unaffected by the treatment. Pre-implantation loss was significantly lower in the high-dose group, but high in the control group.

Treatment with HMD at a dosage of 300 mg/kg b.w./day on days 7-16 of gestation induced 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. At the maternal toxicity dose level of 300 mg HMD/kg b.w./day, the fetuses were slightly retarded in development as evidenced by body weight, limited retardation of skeletal development, and possibly by liver spottiness.

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. 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). Although the delay of ossification observed in the fetuses from the dams treated at the highest dose were secondary to the maternal toxicity, the retardation in skeletal development observed at the middle dose level occurred in the absence of maternal toxicity.

A dosage of 112 mg/kg b.w./day clearly had no effect on the fetuses including poorly ossified cervical vertebral centra frequency.

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.

Historical control data provide another means of characterizing the normal pattern of skeletogenesis, and are extremely important for interpreting delayed ossification. Moreover, the laboratory-specific data are needed to interpret delayed ossification data because the criteria for scoring criteria for certain bones are so detailed that the historical control incidence of delayed ossification often approached 100%. This situation suggests that the lab has not discriminated between the normal range of variation in skeletal ossification and variation beyond the normal range making the usefulness of the observations questionable. Hence, 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.

Hence, the slight ossification retardation observed in the study at both high and mid dose level is considered to be of low level of concern as nonlethal and not detrimental to postnatal survival variations which are generally reversible or transitory.

This teratogenicity study is considered as acceptable. It does satisfy the guideline requirement for a teratogenicity study for an OECD 414 guideline in the rat. Hence, this study can be considered as valid for classification.

Under the test conditions, no adverse effect for maternal toxicity was observed at HMD dose of 184 mg/kg bw/day and no reliable developmental effect was observed as 300 mg/kg bw/d. In conclusion, no classification for HMD is required as demonstrated by the results observed in the developmental study.


Justification for selection of Effect on developmental toxicity: via oral route:
Only reliable developmental toxicity study (Klimisch score = 2), read-across based on grouping of substances (category approach)

Justification for classification or non-classification

Based on the above presented data no classification for reproductive and developmental effects is necessary according to Regulation (EC) No 1272/2008 and Council Directive 67/548/EEC.