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Description of key information

In a reliable subacute screening study (gavage study with DCH, a representative member of the amine heads category) with rats various effects were seen at the highest dose group tested, therfore leading to a NOAEL of 150 mg/kg bw/day (i.e. the most conservative NOAEL observed in any oral  toxicity study performed with any of the category members). In reliable inhalation studies with rats the most conservative LOAEC identified based on local effects on   the upper respiratory tract was established to be 9.2 cubic metres. Based on these effects single target organ toxicity after single inhalation exposure was determined.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable subacute screening study for the submission substance (Klimisch score = 1) is available. Two further subacute studies conducted with members of the category approach and a subchronic study with a mixture of the members of this category category are at hand (all Klimisch score = 2). Overall the quality of the database is high.

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEC
9.2 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
One reliable subacute study for the submission substance (Klimisch score = 2) is available. Another subacute study conducted with a member of the category approach and a subchronic study with another member of the same category are at hand (both Klimisch score = 2). Overall the quality of the database is high.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Additional information

Oral:

In a OECD 422 study (combined 28 day repeated dose toxicity study with reproductive/developmental toxicity) in rats the orally administered test material (DCH) induced significant 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 (e.g. decreased eosinophils, ALT and AST activity increased), macroscopic and microscopic examinations (liver, lung and adrenal glands), which correlated with changes in organ weights.

Furthermore the following effects were observed:

- at 500 mg/kg bw/day (highest dose tested): reduced number of living pups, reduced gestation index.

- at all dose levels (500, 150, 50 mg/kg bw/day), but not in controls: increased postnatal loss, and, in conclusion, decreased viability index. This postnatal loss was obviously caused by cannibalism in the treatment groups.

- Reduction in viability index was however not dose-dependent.

From the results presented above a No Observed Adverse Effect Level (NOAEL) for DCH of 150 mg/kg bw/day was established.

There is one key study in which subacute toxicity of MPMD was tested under the conditions set out in OECD guideline test 407. Male and female rats were fed diets containing target concentrations of 300, 3000, or 10000 ppm of test material for approximately 28 days. According to the authors the estimated mean daily intake of the test item for the 300, 3000, and 10000 ppm dose groups - over the course of the study - was 24.3, 238.5, and 745.2 mg of test item/kg body weight/day, for male rats; in the female groups, the values were 27.5, 275.7, and 791 mg of test item/kg body weight/day, respectively. Taking into consideration the fact that the test item was identified to be slightly unstable under exposure conditions (i.e. room temperature) at concentrations of 3000 ppm and above and as it is not obvious from the study if the study authors considered this fact, the most conservative assumption would lead to the following values for the mean daily intake after recalculation: for male rats 24.3, 167, and 581.3 mg of test item/kg body weight/day and for females rats 27.5, 193, and 617 mg of test item/kg body weight/day, respectively (i.e. at 3000 ppm: 70 -78% test item recovery; at 10000 ppm: 78 -82% test item recovery).

Significant depression of body weight gain was observed in female rats from the 10000 ppm dose group during the exposure period but was comparable with control group within the recovery period. Due to the fact that food consumption during the study was lower for females of this dose group, as well as the body weight itself was only slightly depressed (minus 5.3% compared to control animals at the end of the study), these effects on bw gain were not assessed as adverse. No other effects on body weight were observed. The hematologic, clinical chemical, and urinalysis parameters measured in this study were not adversely affected. There were no significant compound-related differences in organ weight data and there were no compound-related changes detected either grossly or microscopically in tissues. Under the conditions of this study, the no-observable-adverse-effect level (NOAEL) for MPMD was 10000 ppm (i.e. 581.3 mg/kg bw/day in male rats and 617 mg/kg bw/day in female rats).

In a repeated-dose oral toxicity study male and female F344/N rats were exposed for 2 weeks to 1,6 -hexanediamine dihydrochloride (HDDC) in drinking water at concentrations of 0, 0.75, 1.5, 3.0, 4.5 and 6.0 mg/mL for males (96, 187, 357, 449, 545 mg/kg bw/d) and 0, 0.83, 1.7, 3.3, 5.0, 6.7 mg/L (126, 263, 422, 517, 634 mg/kg bw/d) for females. Performing oral toxicity studies with HDDC instead of Hexamethylene diamine (HMD) seems appropriate considering that HMD solutions (nonneutralized) are highly basic and, as such, are extremely caustic and have a pungent odor and would be unpalatable in drinking water.

Body weights were recorded on the day before dosing began and on Days 8 and 15 of the study. A complete necropsy was performed on all treated and control animals. No changes in survival or body weight, no gross or microscopic pathologic changes and no clinical abnormalities related to HDDC consumption were measured. Significant decreases in absolute and relative liver were recorded at 1.7 mg/L and at both highest doses in females while in the males, significant decrease was observed in the mean liver weight only at the 3.0 mg/L dose. These effects were not accompanied to microscopic pathologic changes. Therefore, these effects should be considered as side effects to dehydration induced by the decrease water intake due to the poor palatability of the dosed water at the highest HDDC concentrations.

This sub-acute oral study is considered as acceptable. It does satisfy the guideline requirement for a 2-week preliminary oral study for a OECD 408 guideline in the rat (limited assay parameters were measured).

Because no relevant toxic effects were noted in this study and because higher concentrations of HDDC would probably have caused ill effects resulting from decreased water consumption, no drinking water studies with longer exposure periods were conducted.

Under the test conditions, no effects were observed at HDDC doses tested up to the highest dose (545 mg/kg b.w./d for males and 634 mg/kg b.w./d for females) corresponding to 335 mg HMD/kg bw/day in males and 390 mg HMD/kg bw/day in females I.e. NOEL = no-effect level).

Furthermore male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the a mixture called amine heads (containing approx 85% diamines, of which 55% are the substances of the amine heads category, which is subject in this registration).

The test article, amine heads in a vehicle of deionized water, was administered daily by oral gavage to Charles River CD rats at dosage levels of 10, 30 and 125 mg/kg bw/day. Control rats received the vehicle on a comparable regimen. Each group comprised 15 rats/sex (male and female animals were tested). Criteria evaluated for treatment effect included clinical signs of toxicity, body weights, food consumption, hematological, biochemical and urological determinations (10/sex/group) at 13 weeks, ophthalmological examinations (pretest and at 13 weeks), necropsy examinations, organ weights, and the microscopic evaluation of tissues (full tissues for the control and high dose group as well as animals died in extremis; liver, kidney, lung and gross lesions for the low- and mid-dose groups).

With the possible and argueable exception of respiratory rales seen in a few males and females at the high-dose level, no signs of toxicity associated with treatment were observed. There were no test article related effects noted in this study, therefore, the no-effect level was 125 mg/kg/day, thus supporting the findings of the key study as well as all studies performed with any of the category members.

Inhalation:

Exposure via inhalation to any of the category members causes local effects on the upper respiratory tract. This ws shown in the studeis described below and finally led to classification (specific target organ toxicity after single exposure category 3).

In a subacute inhalation toxicity study male rats were exposed for 6 hours per day and overall 10 times within two weeks to an aerosol/vapour mixture of MPMD. Concentrations used were 0, 9.2, 59 and 250 mg/m3 (analytical).  

The effects observed in rats exposed to MPMD ranged from severe (including death) in the 250 mg/cubicmeter group to minimal in the 9.2 mg/m3 group. Two rats in the 250 mg/cubic metre group died, one on the 8th day of the exposure period and one on the 2nd day of the recovery period. The body weights of the rats in the 250 mg/cubic metre group were significantly lower than those of the control rats during the exposure period and in the first week of the recovery period. Lung weights were increased in this group immediately after the 2-week exposure period and after the 2-week recovery period.

Clinical laboratory results for rats in the 250 mg/cubic metre suggested the presence of dehydration/haemoconcentration indicated by increases in relative numbers of red blood cells, in haemoglobin concentration, and in haematocrit percentage, and by decreases in urine volume and increases in urine osmolality. In addition, there was a decrease in lymphocytes which was considered to be generalized reaction to stress.

There was no evidence of dehydration/haemoconcentration or lymphopenia following the 14-day recovery period.

Gross examination at necropsy showed multifocal areas of discoloration present in the lungs of the rats exposed to 250 mg/cubic metre immediately after the 2-week exposure and 2 weeks later.

Microscopically, exposure-related lesions were confined to the respiratory tract and were dose related. In the 59 and 250 mg/cubic metre groups, lesions were observed in the nose, trachea, larynx/pharynx, and lung. These lesions were mainly minimal to mild in the 50 mg/cubic metre group and mild to moderate in the 250 mg/cubic metre group. Nasal lesions consisted of inflammation, necrosis, regeneration, and squamous metaplasia of the olfactory and respiratory epithelium; laryngeal/pharyngeal lesions consisted of subacute inflammation; tracheal lesions observed were hyperpla, hypertrophy, and necrosis of the respiratory epithelium; pulmonary lesions consisted of hypertrophy and hyperplasia of bronchial and bronchiolar epithelium. After the 2 -week recovery period, the above nasal and pulmonary lesions were diminished in severity and the tracheal and laryngeal/pharyngeal lesions were no longer present. At this time, there was a slight increase in fibrous connective tissue (collagen formation) associated with pulmonary inflammatory lesions in the 250 mg/cubic metre group and in one rat in the 59 mg/cubic metre group.

In rats exposed at 9.2 mg/cubic metre only the nose was affected. Minimal to mild inflammation of the nasal epithelium was observed in this group immediately after the 2 -week exposure period. After a 2 -week recovery period the nasal lesions in this group were diminished in severity and were only seen in 2 of 5 rats.

Due to the fact that effects on the respiratory tract were seen in all concentration groups tested and these effects were clearly dose related a no observed adverse effect level could not be determined. The LOAEC of this study was established to be 9.2 mg/cubic metre.

 

In another subacute inhalation toxicity study with the same study regimen (6 hours per day and overall 10 times within two weeks)

male rats were exposed for to an aerosol/vapour mixture of DCH. Concentrations used were 0, 10, 49 and 240 mg/m3 (analytical).

There were no adverse effects observed in any of the test groups with respect to the haematologic, clinical chemical, urinalysis, and body weight parameters measured. As expected from its chemical basicity, inhaled test material produced respiratory tract irritation in exposed rats. However, the lesions (inflammation/necrosis) observed occurred only in the upper respiratory tract (mainly the nose, larynx, and pharynx), were moderate at the highest concentration tested, and were decreased in severity at the lower tested concentrations. Effects observed in the 49 mg/m3 group were mainly mild and those in the 10 mg/m3 group were minimal. The observed effects were completely reversible after a two-week recovery period even at the 240 mg/m3 concentration. Although a no-adverse-effect concentration was not determined in this study, the respiratory tract effects observed at the lowest tested concentration (10 mg/m3 ) were considered minimal and reversible. The LOAEC of this study was established to be 10 mg/m3.

 

Summarised the effects observed in Crl:CD®BR rats exposed to MPMD ranged from severe (including death) in the 250 mg/m3 group to minimal in the 10 mg/m3 group. Determined effects diminished in severity over time, but were only partially reversible within the 2 week observation period. Lesions observed after exposure to DCH were less severe, confined to the upper respiratory tract and were fully reversible within the 14 day observation period.

 

Except for these subacute toxicity studies a 13 -week study of the toxicity of the dihydrochloride salt of HMD (HDDC) was conducted in male and female Fischer 344/N rats using whole body inhalation exposure (NTP, 1993; Hébert. 1993). The method followed was comparable to OECD 413 and was conducted according to the US-EPA GLP which were similar to the EU-GLP. Under the test conditions, the NOAEC (HDDC) = 16 mg/m3/day for local respiratory damage (nasal respiratory epithelium degeneration), corresponding to a NOAEC (HMD) = 10 mg/m3/day. However this study could not be used to assess the local effects caused by the category members as only the dihydrochloride salt was used instead of the alkaline and therefore corrosive substance itself.

In a repeated- inhalation toxicity study (Ben-Dyke, 1981), 60 S-D Rats/sex were exposed to aqueous Hexamethylene Diamine (HMD) aerosol for six hours per day, five days per week at mean analytical concentrations of 0, 12.8, 51 and 215 mg/m³ for thirteen weeks. Under the test conditions, no systemic effects were observed related to the treatment with HMD. The significant local irritation of respiratory tract, inducing clinical signs, was observed at the two highest concentrations tested. The NOAEC in male and female rats exposed by whole-body inhalation was 12.8 mg HMD/m³.

As in all of these studies the main effect observed after repeated inhalation is a local effect on the respiratory tract. No adverse systemic effects were noted at concentrations were the local effects occured. The type and the severity of the local effect observed are the same whatever the time duration the exposure is (2 weeks or 13 weeks). Therefore, the local effect observed appears to be not driven by the time of exposure.

Apart from rather systemic effects most probably due to ageneralized reaction to stress only local irritating effects were observed after repeated inhalation exposure to the members of the amine heads category. And the local effects observed are clearly due to the corrosivity of the substances. Since corrosivity is the underlying cause the classification for their corrosive properties and for STOT single exposure category 3 is considered sufficient in order to protect from long term inhalation effects - together with the risk characterisation based on a DNEL derived from the long-term inhalation NOAEC identified in the sub-chronic study.

In the ‘Guidance on the application of the CLP criteria, page 376, paragraph 3.9.2.5.1, section irritating/corrosive substances, the following sentences were written:

‘Substance classified as corrosive may cause severe toxicological effects following repeated exposure, especially in the lungs following inhalation exposure. In such cases, it has to evaluated whether the severe effect is a reflection of true repeated exposure toxicity or whether it is in fact just acute toxicity (i.e. corrosivity). One way to distinguish between these possibilities is to consider the dose level which causes the toxicity. If the dose is more than an order of magnitude lower than that mediating the evident acute toxicity (corrosivity) then it could be considered to be repeated-dose effect distinct from the acute toxicity. In this case, classification as specific target organ (repeated exposure) would be warranted even if the substance is also classified as acutely toxic and/or corrosive.’

Since results observed in the repeated inhalation studies are clearly due to the corrosivity of the substance, we propose not to classify the members of the Amine head category as STOT RE cat 1. Instead classification of the submission substance as Corr. Cat. 1A, H314 and STOT SE Cat 3, H335is considered suitable to protect from repeated inhalation effects.

Dermal:

No studies for the dermal path are available.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Reliable study (reliability 1) with the most conservative effect dose observed after repeated oral exposure, read-across based on grouping of substances (category approach)

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
In reliable studies the most relevant effects were local effetcs on the upper respiratory tract.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Reliable study (reliability 2) which reveals the most relevant effects, read-across based on grouping of substances (category approach)

Justification for classification or non-classification

Organ-specific effects observed after repeated inhalation and oral exposure are confined to the respiratory tract. The most probable cause of the effects is the corrosive activity of the substance, which is expected to occur even after short-term exposure. Therefore, classification with respect to specific target organ toxicity after short-term exposure is proposed:

STOT SE Cat 3, H335, together with Corr. Cat 1

Based on findings in the oral repeated dose studies, a classificationfor specific target organ toxicity after repeated dose exposure is not necessary according to Regulation (EC) No 1272/2008.

After repeated inhalation exposure, the effects noted in rats are clearly due to the corrosivity of the members of the amine heads category. Accordingly, the classification for their corrosive properties and STOT single exposure Cat 3 is sufficient.

No classification for effects after repeated dose exposure is warranted according toCouncil Directive 67/548/EEC.