Reaction mass of 7-azatridecane-1,13-diamine and hexamethylenediamine

Endpoint summary

Description of key information

In a subchronic toxicity study the submission susbtance was administered orally to rats. The NOAEL of this study is 20 mg/kg bw/day as respiratory rales were seen in male and female rats when higher doses were tested. In another subchronic toxicity study rats were exposed to an aerosol containing the submission substance. Based on the lesions seen in the respiratory tract throughout all exposure concentrations the LOAEC of this study is 15.8 mg per cubic metre air. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 14 NOV 1983 to 14 FEB 1984

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no neurobehavioural examinations

GLP compliance:

yes

Remarks:

according to US EPA GLP Standards of May 2, 1984

Limit test:

yes

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, Inc., Portage, Michigan, USA
- Age at study initiation: 42 days
- Weight at study initiation: males: 186-209 g; females: 132-152 g
- Housing: individually housed
- Diet: Certified Rodent chow #5002 (Ralston Purina Company, ST. Louis, Missouri, USA), ad libitum
- Water: ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): mean 23.5
- Humidity (%): mean 51%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Test solution was prepared fresh daily at concentrations of 2,5, and 12 mg test article per ml test solution. The appropriate amount of test article was weighed and mixed with vehicle (deionized water). Additional vehicle was added to yield the required volume of prepared test article solution. Solutions were shaken by hand. Deionized water was dispensed for the control group. Test and control solutions were dispensed daily in capped containers.

- Concentration in vehicle: constant concentrations of 2, 5, and 12 mg/ml,
- Maximum dose volume applied: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing solutions were analyzed at weeks 1, 4, 8 and 12 and were generally within ± 10% of target concentrations.
Stability of the dosing solutions was verified after storage for 1 and 10 days at room temperature.

Duration of treatment / exposure:

13 weeks (i.e. 91 days)

Frequency of treatment:

once daily, 7 days per week

Remarks:

Doses / Concentrations:
0, 20, 50 and 120 mg/kg bw
Basis:
actual ingested

No. of animals per sex per dose:

15

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: based on results of range finding study (for details see SS_Ascend_IRDC_IR-83-152_1983_Repeated dose toxicity: oral 28 days)

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (mortality and clinical signs)
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined: Yes, weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to study initiation and in week 13
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at week 13
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight, i.e. approx. 16 hours)
- How many animals:10 rats/sexe/group
- Parameters checked: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, platelet count, reticulocyte count, differential leukocyte count, corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
- Animals fasted: Yes (overnight, i.e. approx. 16 hours)
- How many animals: 10 rats/sexe/group
- Parameters checked: sodium, potassium, chloride, calcium, phosphorus, alkaline, phosphatase, total bilirubin, gamma glutamyl transpeptidase (GGT), aspartate aminotransferase (AST), alanine aminotransferase (ALT), ornithine carbamoyltransferase (OCT), creatine phosphokinase (CPK), urea nitrogen, creatinine, total protein, albumin, globulin (calculated), total cholesterol and glucose

URINALYSIS: Yes
- Time schedule for collection of urine: during the 16 hours fasting period before blood sampling
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked: dipstick determinations of pH, protein, glucose, occult blood, nitrite, bilirubin, glucose, ketones and urobilinogen; microscopic examination of sediments and observations on color, specific gravity, volume and appearance were also performed.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following list of tissues were taken from all surviving animals and microscopically examined after processing on the control and high dose groups. For the low and mid dose groups, the liver, kidney, lung and gross lesions were sectioned for microscopic examination.
Bone (femur), Bone marrow (femur), Bone marrow smear
Brain (3 levels: fore,mid and hind brain)
Eye (2)
Gastrointestinal tract: esophagus, stomach, duodenum, jejunum, ileum, cecum, colon, rectum
Gonads: ovary (2) or testis with epididymis (2)
Heart
Kidney (2)
Lung with mainstem bronchi (2)
Lymph nodes: mediastinal, mesenteric
Mammary region (females only)
Pancreas
Pituitary
Prostate and seminal vesicle (2, males only)
Salivary gland, mandibular with submandibular lymph node
Sciatic nerve
Skin
Spinal cord (cervical, midthoracic and lumbar)
Spleen
Thymic region
Thyroid - parathyroid complex
Trachea
Urinary bladder
Uterus

Statistics:

Body weight (weekly), organ weight (absolute and relative to body and brain weights at terminal sacrifice), food consumption (weekly) and clinical laboratory (week 13) data were analyzed using Bartlett's test for homogeneity of variances and a one-way analysis of variance (ANOVA). Treatment groups compared to the control group, by sex, using the appropriate t-statistics (for unequal and equal variances) as described by Steel and Torrie. Dunnett's multiple comparison tables were used to assess significant differences at p < 0.05. Total bilirubin, GGT OCT, chloride and specific gravity were analyzed using a nonparametric approach, by transforming the data to ranks prior to analysis, as described by Conover and Iman.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

One male and one female of the high-dose group died; one male at the mid-dose died. Resppiratory rales in several animals from high- and mid-dose group.

Mortality:

mortality observed, treatment-related

Description (incidence):

One male and one female of the high-dose group died; one male at the mid-dose died. Resppiratory rales in several animals from high- and mid-dose group.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in high-dose females body weight decreased compared to control group

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

slightly decreased food consumption noted in high-dose males (minus 3.5%), when food consumption was calculated on a g/kg/day basis

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Segmented neutrophils were increased in females and males of the high-dose group compared to controls, but mean values for these groups were within the normal range of historical control values for this species in this laboratory.

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

related to the test item

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

related to the test item

Details on results:

CLINICAL SIGNS AND MORTALITY
One male (week 2) and one female (week 5) in the high-dose group died and one male (week 7) at the mid-dose died during the treatment period.
With the possible exception of respiratory rales seen in a few males (4/15) and females (2/15) at the high-dose level and mid-dose females (3/15),
no clinical signs of toxicity associated with treatment were observed.

BODY WEIGHT AND WEIGHT GAIN was impaired in high-dose females during the last two-thirds of the study (statistically significant at weeks 6 and 7, but maximum difference control versus high dose females minus 5%, see als table in "any other information on results")

FOOD CONSUMPTION: slightly decreased food consumption noted in high-dose males (minus 3.5%), when food consumption was calculated on a g/kg/day basis. On a g/animal/day basis, the values were comparable between the groups throughout the study

HAEMATOLOGY
Segmented neutrophils were statistically significantly increased in females of the high-dose group compared to controls. Male values in the high-dose group were also increased when compared to control values although statistical significance was not seen. In each case, mean values for these groups were within the normal range of historical control values for this species in this laboratory.

ORGAN WEIGHTS
Statistically significant variations occurred in the mean absolute weight of adrenals in male rats at 120 mg/kg/day dosage level and in the mean relative weight (to body weight) of female liver at 50 mg/kg/day dosage level. There were no test item related microscopic changes in these organs and therefore, these variations were considered incidental.

HISTOPATHOLOGY: NON-NEOPLASTIC
No test item related changes were observed in any of the tissues from male and female rats evaluated histopathologically from the treatment groups. All changes seen were considered spontaneous or incidental in nature and unrelated to the administration of the test item. A frequently observed lesion in this study was interstitial pneumonia (this was also the cause of death of the animal in the mid-dose group). Other non-treatment related changes occurred in eye, heart, kidney, liver, pituitary, prostate, testis, trachea, urinary bladder, adrenal, brain, ovary and uterus. Interstitial pneumonia was slightly increased in treatment groups but was considered non-specific and unrelated to treatment.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: 3 animals (female) with respiratory effects were noted at 50 mg/kg bw/day

Critical effects observed:

not specified

Body weights at study termination

	Group mean body weights in g (% difference from vehicle control)
Dosage level (mg/kg/day)	males	females
0 (control)	504	281
20	491 (minus 2.6)	286 (plus 1.8)
50	494 (minus 2.0)	276 (minus 1.9)
120	498 (minus 1.2)	267 (minus 5.0)

Conclusions:

The test material was subjected to test subchronic toxicity in rats. For 13 weeks (7days/week) the test substance was applied orally (gavage) to rats (15 males and 15 females/dose) in doses of 0, 20, 50, and 120 mg/kg bw/day. Based on the respiratory effects noted in 3 animals at 50 mg/kg bw/day the NOAEL in this study was conservatively set to be 20 mg/kg bw.

Executive summary:

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance. Doses of 0, 20, 50, and 120 mg/kg bw/day (15 males and 15 females /dose) were applied orally via gavage at 7days/week for 13 weeks.

One male and one female in the high-dose group died and one male at the mid-dose died during the treatment period. With the possible exception of respiratory rales seen in a few males and females at the high-dose level and mid-dose females, no clinical signs of toxicity associated with treatment were observed. The high-dose females showed a slight decrease in body weights in comparison to controls (minus 5% only). No significant weight differences were measured in males at any dose level or females at the low- and mid-dose levels. Some evidence of reduced food consumption was noted for high-dose males (on the basis of mg/kg/day). Ophthalmoscopic examination did not reveal any effects of treatment on the eye. With the exception of an increase in segmented neutrophils in high-dose males and females, there was no evidence of treatment-related changes in the hematologic, clinical chemistry or urinalysis examinations. Nevertheless the values were within the historical control range for this species in this laboratory. The incidental findings from gross necropsy did not appear to be related to treatment. Males at the high-dose level had decreased absolute adrenal weights and the liver weight relative to body weight for mid-dose females was elevated. These changes were not correlated with any microscopic findings and were considered unrelated to treatment. No other significant organ weight differences between control and treatment groups were noted. The microscopic lesions observed were generally comparable in treated and control group animals and were considered to be of spontaneous origin. Interstitial pneumonia was slightly increased in treatment groups but was considered non-specific and unrelated to treatment. Therefore, treatment did not result in histopathologic changes in any organ or tissue.

CONCLUSIONS Administration of the submission substance at dose levels of 20 mg/kg bw/day did not produce any toxicological signs which were attributable to treatment. At 50 and 120 mg/kg bw/day, respiratory rales were seen in males and females. At 120 mg/kg bw/day the females had slightly decreased body weights and both males and females showed increases in segmented neutrophils which may have been treatment-related but the values were in the normal range of historical control data. Overall the no-observable adverse effect level was considered to be 20 mg/kg bw/day for both sexes.

As the only organ showing macroscopic and microscopic findings related to the substance was the lung it is possible that the effects were consecutive to the inhalation route (false route perhaps du to the viscosity of the substance).

Based on this study the HMD heavies oral systemic toxicity appears to be low.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

One reliable subchronic oral toxicity study for the submission substance (Klimisch score = 2) is available. Furthermore the subacute dose range finding study conducted with the same testing material is at hand (Kimisch score = 3). Overall the quality of the database is high.

As explained in detail below, the only effectst seen in these studies were in the upper and lower respiratory tract. Based on this study the oral toxicity of the submission substance appears to be low and the effects observed in rats seems to be consecutive to the inhalation route (false route perhaps du to the viscosity of the substance).

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

sub-chronic toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From 8 AUG 1985 to 8 NOV 1985

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Basic data given: comparable to guidelines

Reason / purpose for cross-reference:

reference to other study

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)

Deviations:

yes

Remarks:

but only minor deviations e.g. slightly elevated humidity and temperature during exposure phase in comparison to recommendations given in the technical guidance, some examinations were not performed (urinalysis, food and water consumption)

GLP compliance:

yes

Remarks:

according to US EPA GLP Standards

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, Inc., Portage, Michigan, USA
- Age at study initiation: 54 days
- Weight two days before study initiation: males: 227-337 g; females: 136-199 g
- Housing: individually housed
- Diet: Pzrina Laboratory Certified Rodent chow #5002 (Ralston Purina Company, St. Louis, Missouri, USA), ad libitum
- Water: community tap water, ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24.5
- Humidity (%): 35-60%
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

inhalation: aerosol

Type of inhalation exposure:

whole body

Vehicle:

air

Remarks on MMAD:

MMAD / GSD: MMAD: 2.36-6.32 micrometers (with only two of the 15 values being above 3.96)
% of particles with less than 10 micrometers: 64.8-96.9 (with only two of the values being below 82.9)

Details on inhalation exposure:

GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: four chambers of 1.75 cubic meter New York University-type glass and stainless steel chambers with pyramidal tops and bottoms
- Method of holding animals in test chamber: individually in wire mesh cages suspended within the chamber (weekly rotation of position in order to guarantee that all animals receive similar exposure concentrations)
- System of generating aerosols: This was achieved by delivering a metered rate of test material (diluted in water) via a Harvard Apparatus Compact infusion pump through a plastic capillary tube into a nebulizer.
- Temperature, humidity, pressure in air chamber: 22.8-28.4 °C, 29.3-82.3%, normal pressure
- Air flow rate: 340 l/min
- Method of particle size determination: gravimetrically via a non-viable, nine-stage Andersen Impactor (Andersen Samplers Inc., 4215-C Wendell Dr., Atlanta, Georgia 30336)

TEST ATMOSPHERE
- Brief description of analytical method used: Test material concentrations in air were measured three to four times daily for each treatment chamber by collection on impingers containing 2-propanol. Analytical determinations of BHMT in the 2-propanol samples were done by gas chromatography.
- Samples taken from breathing zone: no, but chamber distribution measurements were also performed at periodic intervals.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Stability analyses of the test material were performed before, during, and after the study. These were done by comparing peak areas of gas chromatograms obtained from the test material and analytical grade BHMT (approximately 99% pure) that was kept frozen in the dark.

Duration of treatment / exposure:

6 hours/day

Frequency of treatment:

once daily at 5 days/week for 13 weeks (i.e. totals 65 exposures)

Remarks:

Doses / Concentrations:
0, 10, 31 and 62 mg/m³
Basis:
other: analytical concentration of BHMT (which accounts for 63.25% of the test material)

Remarks:

Doses / Concentrations:
0, 15.8, 49 and 98 mg/m³
Basis:
other: calculated to the submission substance (i.e. 100% )

No. of animals per sex per dose:

12

Control animals:

yes

Details on study design:

- Dose selection rationale: based on results of range finding study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (before and after exposure and at comparable times on non-exposure days)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before exposure began and at study week 12 (exposure day 58)
- Dose groups that were examined: in the beginning all animals were examined, at day 58 only control and high-dose animals were examined

HAEMATOLOGY: Yes
- Time schedule for collection of blood: end of study, before sacrifice
- Anaesthetic used for blood collection: No data
- Animals fasted: Yes (overnight, water available)
- How many animals: all
- Parameters checked: total leukocyte count, erythrocyte count, hemoglobin, hematocrit, differential leukocyte count, mean corpuscular volume (MCV), mean
corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets and reticulocytes.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: end of study, before sacrifice
- Animals fasted: Yes (overnight, water available)
- How many animals:
- Parameters checked: alkaline phosphatase, total bilirubin, aspartate aminotransferase (AST), alanine aminotransferase (ALT), urea nitrogen, total protein, glucose, albumin, globulin, creatinine, cholesterol and serum electrolytes (i.e. sodium, potassium, phosphorus, calcium and chloride).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes
The following list of tissues were taken from all surviving animals and microscopically examined after processing on the control and high dose groups. For the low and mid dose groups, nasal passages, trachea and lungs were also examined microscopically.
Abdominal Aorta
Adrenals
Bone (femur)
Bone marrow (femur)
Brain (longitudinal section)
Diaphragm
Esophagus
Eyes (with optic nerve)
Gonades (Ovaries, Testes with epididymides)
Heart
Intestine (Duodenum, Colon, Ileum)
Kidneys (2)
Liver (2 sections, at least 2 lobes and mainstem bronchi)
Lymph nodes (mesenteric & thymic)
Mammary gland
Nasal passages (3 sections)
Pancreas
Pituitary
Prostate
Salivary gland (submandibular)
Sciatic nerve
Skeletal muscle
Skin
Spinal cord (thoracolumbar)
Spleen
Stomach (longitudinal)
Thymus
Thyroid/parathyroid
Trachea
Urinary bladder
Uterus (corpus and cervix)
Vagina
Gross lesions

Other examinations:

The following organ weights were determined at the end of the study: adrenals (together), brain, heart, kidneys (together), liver, spleen, testes with epididymides (together)

Statistics:

Body weight (weekly) and organ weight (absolute and relative to body and brain weights at terminal sacrifice) data were analyzed using a one-way analysis of variance (ANOVA) and Dunnett's (two-tailed) test to detect significant differences between means of treatment groups compared to the respective control group.
Clinical chemistry data were analyzed using Dunnett's test only. Bartlett's test was used to assess the variability of these data.
Statistical analysis of organ-to-body weight ratios were performed using the Mann-Whitney Test with Bonferroni's Inequality Procedure. Differences in the frequency of the lesions were determined using Fisher's exact test with Bonferroni's Inequality Procedure.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

no animals died; high dose animals showed respiratory wheezing, and mostly in females at all exposure levels a discolouration of the fur was noted

Mortality:

mortality observed, treatment-related

Description (incidence):

no animals died; high dose animals showed respiratory wheezing, and mostly in females at all exposure levels a discolouration of the fur was noted

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

in males of the high exposure level group the mean body weights were significantly reduced beginning in study week 2 (approx. 9-17%), in females also reduced starting from week 2, but differences to control group were not always signifcant (approx. 5-9%),

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

in the high dose females: elevated red blood cell counts with associated increases in hemoglobin and hematocrit

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

in high dose males: ALT, AST and phosphorous were elevated significantly; in high dose females: lowered serum glucose levels

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

tendency toward decreased absolute and increased rel. organ weights (particularly in high dose males) were attributed to decreaed body weights rather than a direct compound effect on the organs

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

dose related increase in incidence of pulmonary emphysema

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

related to the test item

Histopathological findings: neoplastic:

no effects observed

Description (incidence and severity):

related to the test item

Details on results:

Body weight:
- high dose group: In males the mean body weights were significantly reduced beginning in study week 2 (approx. 9-17%). In females mean body weights were also reduced starting from week 2, but differences to control group were not always signifcant (approx. 5-9%).
- mid dose group: In males the body weights were slightly lower than those of the control group. In females the mean body weights were comparable to the control.
- low dose group: No effects seen.

Clinical chemistry:
Effects seen in high dose males were mostly attributed to only a few animals of the group (i.e. in case of ALT: 2 animals, AST and phosphorous: 3 animals each).
Moreover all the effects seen were not substantiated by pathological data.
Other than the above mentioned differences in clinical chemistry seen at the low and mid-dose level (i.e. decreases blood urea nitrogen, increased sodium), were within normal ranges and were not considered to be treatment-related as they were not detected in the high dose animals.

Gross pathology/Histopathology:
The pulmonary emphysema noted at gross necropsy was related to the histopathological changes and may have resulted in hypoxia and associated increases in red blood cell production.

Lesions associated with treatment were restricted to the respiratory tract. Three target organs were affected;
the nasal passages, trachea and lungs
all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium. The incidence and severity of the lesions was increased as a function of higher exposure levels, being most notable in the two highest dose groups and much less severe at the low dose level.

Dose descriptor:

LOAEC

Effect level:

15.8 mg/m³ air (analytical)

Based on:

other: submission substance

Sex:

male/female

Basis for effect level:

other: lesions of the respiratory tract (nasal passages, trachea and lungs all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium)

Critical effects observed:

not specified

Results from pathological section affecting the respiratory tract

1) Gross necropsy

males (n=12/dose group)           females (n= 12/dose group)    control low dose  mid dose  high dose   control low dose  mid dose  high dose   lung emphysema 0  2  6  10  0  1  9  10

2) microscopic findings

			males (n=12/dose group)				females (n=12/dose group)
			control	low dose	mid dose	high dose	control	low dose	mid dose	high dose
lung		peribronchial lymphoid hyperplasia/ cuffing	1	2	2	0	-	-	-	-
		interstitial pneumonia	1	0	1	2	1	2	2	1
		accumulation of alveolar macrophages	1	0	9*	9*	0	0	7*	12*
		emphysema	0	2	7*	12*	0	0	7*	12*
		bronchiointerstitial pneumonia, chronic	0	0	4	10*	0	0	3	12*
		squamous metaplasia (bronchi or bronchioles)	0	6	11*	10*	0	3	6	11*
		bronchioal ulceration, chronic	0	0	1	9*	0	0	0	4
		haemorrhage, acute	1	1	0	1	2	0	0	1
		hypertrophy with vacuolar change, bronochial epithelium	0	9*	10*	7*	0	11*	12*	11*
		bronchiolitis obliterans	0	0	1	1	0	0	0	1
		foamy basophilic material within bronchi/bronchioles/alveoli	0	1	6	7*	0	0	5	11*
nose/ turbinates		inflammation, mixed mucosal to submucosal	2	6	8	10*	-	-	-	-
		squamous metaplasia	5	8	12*	12*	1	5	10*	12*
		epithelial hyperplasia	0	6	12*	9*	0	3	10*	10*
		hypertrophy with vacuolar change/respiratory epithelium	0	11*	12*	12*	0	9*	12*	12*
		eosinophilic granularity, olfactory epithelium	0	12*	11*	12*	0	10*	12*	12*
		ulceration	0	0	0	9*	0	0	0	9*
trachea		squamous metaplasia	0	0	9*	11*	0	1	3	11*
		hypertrophy/vacuolar change, epithelial	0	11*	12*	5	0	6	11*	9*
		inflammation, chronic, mononuclear, submucosa	0	0	1	4	1	0	0	1
		hyperplasia, submucosal gland	0	0	1	1	0	0	0	1
		foamy basophilic material in lumen	0	0	1	0	0	0	0	1

* significantly different from control using fishers exact test with bonferroni inequality

Conclusions:

The submission substance was subjected to test subchronic toxicity in rats (equivalent to OECD 413, GLP). For 13 weeks (5 days/week, 6 h/d) the rats (12 males and 12 females/group) were exposed to aerosol containing BHMT at analytical concentrations of 0, 10, 31, and 62 mg/m³/day. This holds for 0, 15.8, 49 and 98 mg of the submission substance per m³ air.
Based on the fact that the lesions of the respiratory tract were seen throughout all exposure concentrations tested in this study the lowest observable adverse effect concentration is 15.8 mg of submission substance per cubic metre (LOAEC).

Executive summary:

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance being given as aerosol for 13 weeks (5 d/week, 6 h/d; equivalent to OECD 413, GLP). Mean analytical values for the three expsoure levels were 10, 31 and 62 mg BHMT/m3. Recalculating this BHMT exposure levels to the submission substance concentrations leads to 0, 15.8, 49 and 98 mg submission substance per cubic metre. The nominal to analytical ratios ranged from 3.2 to 5.9 which resulted from substantial deposition of test material on chamber surfaces; however chamber distribution data indicated acceptable dispersion of the aerosol. Generally, greater than 90% of the test material particles were in the respirable size range. No animals died on study. Clinical signs of toxicity were confined to respiratory wheezing in high dose animals and discoloration of fur in females. High dose animals exhibited significantly decreased body weight in comparison to control animals. Elevated red blood cell counts with associated increases in hemoglobin and hematocrit were observed in the high dose females (maybe as compensatory response to hypoxia which was secondary to the pulmonary changes/emphysema). ALT, AST and phosphorous were elevated in high dose males; high dose females had lowered serum glucose levels. Other differences in clinical chemistry seen at the low and mid-dose level were within normal ranges and were not considered to be treatment-related. In the high-dose animals, several organs showed reduced absolute weights and increased relative weights which was attributed to overall body weight reductions. Dose related increases in the incidence of pulmonary emphysema were noted at gross necropsy. Lesions associated with treatment were restricted to the respiratory tract. Three target organs were affected; the nasal passages, trachea and lungs all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium. The incidence and severity of the lesions was increased as a function of higher exposure levels, being most notable in the two highest dose groups and much less severe at the low dose level. Hypertrophy and eosinophilic granularity of the olfactory epithelium was noted at all levels of treatment and was considered to be an age-related change exacerbated by treatment. Hypertrophy, hyperplasia, squamous metaplasia and ulceration were found in the nasal passages, trachae and lungs with secondary inflammation of the ciliary epithelium. These responses were consistent with those expected for a corrosive substance; there was no evidence that these changes represented a pre-neoplastic condition. The pulmonary emphysema noted at gross necropsy was related to the histopathological changes and may have resulted in hypoxia and associated increases in red blood cell production.

CONCLUSION: Exposure of rats to the submission substance at 98 mg/m3 for thirteen weeks resulted in pathological lesions of the respiratory tract, reduced body weight, increased RBC count and pulmonary emphysema. Lesions of the respiratory tract were observed at the 15.8 and 49 mg/m3 treatment level, although with much lesser incidence and severity at the low-dose level. Therefore a no-effect level could not be determined for this study.The LOAEC is 15.8 mg/m3.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEC

15.8 mg/m³

Study duration:

subchronic

Species:

rat

Quality of whole database:

One reliable subchronic toxicity study after inhalation exposure of the submission substance (Klimisch score = 2) is available. Furthermore the subacute dose range finding study conducted with the same testing material is at hand (Kimisch score = 3). 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 exposure

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance in a reliable study conducted similar to OECD testing guideline 408. Doses of 0, 20, 50, and 120 mg/kg bw/day (15 males and 15 females /dose) were applied orally via gavage at 7days/week for 13 weeks.

One male and one female in the high-dose group died and one male at the mid-dose died during the treatment period. With the possible exception of respiratory rales seen in a few males and females at the high-dose level and mid-dose females, no clinical signs of toxicity associated with treatment were observed. The high-dose females showed a slight decrease in body weights in comparison to controls (minus 5% only). No significant weight differences were measured in males at any dose level or females at the low- and mid-dose levels. Some evidence of reduced food consumption was noted for high-dose males (on the basis of mg/kg/day). Ophthalmoscopic examination did not reveal any effects of treatment on the eye. With the exception of an increase in segmented neutrophils in high-dose males and females, there was no evidence of treatment-related changes in the hematologic, clinical chemistry or urinalysis examinations. Nevertheless the values were within the historical control range for this species in this laboratory. The incidental findings from gross necropsy did not appear to be related to treatment. Males at the high-dose level had decreased absolute adrenal weights and the liver weight relative to body weight for mid-dose females was elevated. These changes were not correlated with any microscopic findings and were considered unrelated to treatment. No other significant organ weight differences between control and treatment groups were noted. The microscopic lesions observed were generally comparable in treated and control group animals and were considered to be of spontaneous origin. Interstitial pneumonia was slightly increased in treatment groups but was considered non-specific and unrelated to treatment. Therefore, treatment did not result in histopathologic changes in any organ or tissue.

CONCLUSIONS Administration of the submission substance at dose levels of 20 mg/kg/day did not produce any toxicological signs which were attributable to treatment. At 50 and 120 mg/kg/day, respiratory rales were seen in males and females. At 120 mg/kg/day the females had slightly decreased body weights and both males and females showed increases in segmented neutrophils which may have been treatment-related but the values were in the normal range of historical control data. Overall the no-observable adverse effect level was considered to be 20 mg/kg/day for both sexes on the basis of the respiratory effects observed at higher doses.

Moreover there are data from the range finding oral toxicity study. Therefore the submission substance was subjected to test subacute toxicity in rats. For 4 weeks (7days/week) the test substance was applied orally (gavage) to Charles River CD rats (15 males and 15 females/dose) in doses of 0, 20, 40, 80, 160, and 320 mg/kg bw/day. Deaths occurred at levels of 160 mg/kg bw/day and above. Rats receiving up to 80 mg/kg bw/day were essentially normal throughout the study period, therefore the no-obeserved effect level (NOEL) in this study is 80 mg/kg bw/day.

Interpretation of effects observed after oral exposure

Based on the two oral studies the range finding one and the 90 days study, the main effects observed in males and females rats were rales, laboured breathing and mortality. The few other effects were noted only in one or two rats and/or were not dose-dependant.

In the 4 weeks range finding study rats were given 0, 20, 40, 80, 160 and 320 mg/kg bw/day of HMD heavies. No statistically significant differences compared to the control group were noted at 20, 40 and 80 mg/kg bw/day dose levels.

The effects observed at the two higher doses were the following:

 

320 mg/kg bw/day

Mortality:4/10 males and 2/10 females

Rales: 5/10 males and 6/10 females

Laboured breathing: 3/10 males and 3/10 females

 

160 mg/kg bw/day

Mortality:2/10 females

Rales: 2/10 females

Laboured breathing: 2/10 females

 

Based on the data there is no correlation between the mortality and the clinical signs observed in exposed animals (rales and laboured respiratory). Furthermore, without pathological examination (range finding study) no conclusion can be drawn on the link between the corrosivity of the substance and the effects observed after oral repeated exposure.

 

In the main study (90 days study) rats were given 0, 20, 50 and 120 mg/kg bw/day of HMD heavies. The same effects as observed in the range finding study were noted excepted laboured breathing. The effects were the following.

 

120 mg/kg bw/day

Mortality:1/15 males and 1/15 females

Rales: 4/15 males and 2/15 females

 

50 mg/kg bw/day

Mortality:1/15 females

Rales: 3/15 females

 

No specific effects appeared at the two lower exposed doses (20 and 50 mg/kg bw/day).

 

Other parameters such as body weight, food consumption, ophtalmoscopic examination, haematology, biochemistry, urinalysis and macroscopic change did not show treatment-related changes after exposure. Moreover , the organ weight change noted in liver and adrenals were observed without microscopic findings.

 

After microscopic examination only lung lesions were noted such as congestion and interstitial pneumonia. Lung congestion was observed only at the higher tested dose and in very few animals (1/15 male and 1/15 female). Interstitial pneumonia was seen at all treated doses and in the control group.

 

The males and the females found dead at 120 mg/kg bw/day presented lung congestion. It is probably the explanation of the death. Surprisingly no effect on esophagus, intestine and stomach occurred after HMD heavies oral exposure whatever the doses.

In our point of view and since lungs are only impacted, the effects noted in rats after oral long term exposure appeared to be indirectly linked to the inhalation and not directly linked to the oral route. Furthermore based on the nature of the effects, these latest seem to be due to the corrosity of the substance.

According to the study authors, specific moderate interstitial pneumonia is the cause of the mortality of one female at 50 mg/kg bw/day. Interstitial pneumonia have been also noted in the control group with less severity, trace or mid instead of moderate for the female died at 50 mg/kg bw/day. Therefore such effect does not appear to be related to the substance.

 

In conclusion based on the 90 days oral study the main effects observed were mortality and rales. No macroscopic and microscopic finding related to the substance was noted except lung congestion.

Only effects with impact on the upper and lower respiratory tract were observed in this oral long term study (rales and lung congestion lead to the death of the two rats at the higher dose). Based on this study the oral toxicity of the submission substance appears to be low and the effects observed in rats seems to be consecutive to the inhalation route.

Therefore finally no classification for oral long term repeated exposure is needed and the derivation of NOAEL/LOAEL appears to be not necessary. Effects observed in this study will be covered by the LOAEC identified in the inhalation long term study.

 

Inhalation exposure

Inhalation toxicity was tested in a reliable study conducted similar to an OECD guiedline study 413.

Male and female Sprague-Dawley rats were subjected to test subchronic toxicity of the submission substance being given as aerosol for 13 weeks (5 d/week, 6 h/d).Mean analytical values for the three expsoure levels were 10, 31 and 62 mg BHMT/m3. Recalculating this BHMT exposure levels to the submission substance concentrations leads to 0, 15.8, 49 and 98 mg submission substance per cubic metre. The nominal to analytical ratios ranged from 3.2 to 5.9 which resulted from substantial deposition of test material on chamber surfaces; however chamber distribution data indicated acceptable dispersion of the aerosol. Generally, greater than 90% of the test material particles were in the respirable size range. No animals died on study. Clinical signs of toxicity were confined to respiratory wheezing in high dose animals and discoloration of fur in females. High dose animals exhibited significantly decreased body weight in comparison to control animals. Elevated red blood cell counts with associated increases in hemoglobin and hematocrit were observed in the high dose females (maybe as compensatory response to hypoxia which was secondary to the pulmonary changes/emphysema). ALT, AST and phosphorous were elevated in high dose males; high dose females had lowered serum glucose levels. Other differences in clinical chemistry seen at the low and mid-dose level were within normal ranges and were not considered to be treatment-related. In the high-dose animals, several organs showed reduced absolute weights and increased relative weights which was attributed to overall body weight reductions. Dose related increases in the incidence of pulmonary emphysema were noted at gross necropsy. Lesions associated with treatment were restricted to the respiratory tract. Three target organs were affected; the nasal passages, trachea and lungs all showed graduated progression of responses from hypertrophy to squamous metaplasia hyperplasia to ulceration of the ciliated respiratory epithelium. The incidence and severity of the lesions was increased as a function of higher exposure levels, being most notable in the two highest dose groups and much less severe at the low dose level. Hypertrophy and eosinophilic granularity of the olfactory epithelium was noted at all levels of treatment and was considered to be an age-related change exacerbated by treatment. Hypertrophy, hyperplasia, squamous metaplasia and ulceration were found in the nasal passages, trachae and lungs with secondary inflammation of the ciliary epithelium. These responses were consistent with those expected for a moderate irritant; there was no evidence that these changes represented a pre-neoplastic condition. The pulmonary emphysema noted at gross necropsy was related to the histopathological changes and may have resulted in hypoxia and associated increases in red blood cell production.

CONCLUSION: Exposure of rats to the submission substance at 98 mg/m3 for thirteen weeks resulted in pathological lesions of the respiratory tract, reduced body weight, increased RBC count and pulmonary emphysema. Lesions of the respiratory tract were observed at the 15.8 and 49 mg/m3 treatment level, although with much lesser incidence and severity at the low-dose level. Therefore a no-effect level could not be determined for this study.The LOAEC is 15.8 mg/m3.

The submission substance was subjected to test subacute toxicity in Sprague-Dawley rats. The animals (5males and females/group) were expsoed to aerosol for 2 weeks (5 d/w; 6 h/d) containing the submission substance at analytical concentrations of 0, 57, 174 and 506 mg/cubic metre (recalculated from the exposure levels analysed for BHMT with 0, 36, 110, and 320 mg BHMT per cubic metre air). All animals from the high dose group died or were sacrificed in extremis. Two male animals from the mid-level were found dead. Body weights were depressed in all exposure levels (at least minus 7,5%; significance was reached in all exposure groups expect female low-concentration at termination). Morover clinical signs and gross necropsy findings indicate the toxicity of the submission substance. Based on these findings the LOAEC of this study is 57 mg/m3 air.

Additional studies on hexane-1,6-diamine (HMD), one main constituent of the reaction mass were reported in the IUCLID file. These studies showed also that the main effect observed after repeated inhalation is a local effect on the respiratory tract. No adverse systemic effects were noted. The type and the severity of the effect observed are the same whatever the time duration (9 days, 2 weeks and 13 weeks). Therefore, the local effect observed appears to be not driven by the time of exposure.

Interpretation of effects observed in inhalation studies

The two available repeated inhalation studies (the range finding and the main study) showed two types of effects, rather systemic effect (decrease of body weight) and local effects (lower and upper tract – lung, nose and trachea). The local effects observed after repeated inhalation exposure is clearly due to the corrosivity of the substance.

 

Since the body weight reduction is due to the corrosivity of the substance and since the LOAEL identified for this effect is higher than the one established for the impact of the respiratory tract the only LOAEL to retain is LOAEL = 15.8 mg/m3.

Additional studies on HMD, one main constituent of the submission substance, verified the fact that local irritating effects on the upper respiratory tract are the main adverse effects after repeated inhalation exposure of rats and mice.

Since the effects observed in rats and mice after repeated inhalation exposure are due to the corrosivity of the substances the classification for its corrosive properties and for STOT single exposure 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 two repeated inhalation studies are clearly due to the corrosivity of the substance, we propose not to classify HMD heavies as STOT RE cat 1. Instead classification of the submission substance as Corr. Cat. 1 , H314 and STOT SE Cat 3, H335 is considered suitable to protect from repeated inhalation effects. 

 

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Study with the longest duration (90 days) and the lowest NOAEL was chosen (key study).

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
A subchronic and a subacute toxicity study with inhalation exposure to the submission substance are available. The critical effects observed in these studies were local irritating effetcs on various sections of the respiratory tract (i.e. nose, trachea, lung).

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Study with the longest duration (90 days) and the lowest effect level (i.e. LOAEC) was chosen (key study). Most critical effects were found to affect various sections of the respiratory tract (i.e. nose, trachea, lung).

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Study not necessarily needed according to tonnage driven data requirements information shall be provided for at least one appropriate route. In this case oral as well as inhalation subchronic toxicity data are available.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
Study not necessarily needed according to tonnage driven data requirements information shall be provided for at least one appropriate route. In this case oral as well as inhalation subchronic toxicity data are available. Moreover local dermal effects are already covered by qualitative assessment of the known corrosive properties of the substance.

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-terrm 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 two oral repeated dose studies, a STOT classification for oral long-term effect is not needed. Indeed the only effects observed after oral repeated exposure appeared to be consecutive to intake by the inhalation route.

After repeated inhalation exposure, the effects noted in rats are clearly due to the corrosivity of the substance. Accordingly, the classification of HMD heavies for its corrosive properties and with regard to STOT single exposure is sufficient.