Registration Dossier

Administrative data

Description of key information

Repeated dose toxicity: oral
In a 28 days repeated dose oral toxicity study conducted on rats, the NOAEL was 50 mg/kg bw/day and the LOAEL was found to be 250 mg/kg bw.

In a 90 days repeated dose oral toxicity study with the structurally related Diethylaminopropylamine (as pH-neutralized dose formulations) conducted on rats, the NOAEL was established at 750 mg/kg/day in males and 250 mg/kg/day in females.

In a 13-week subchronic study with the structurally related ethylene diamine (EDA) conducted on rats, no NOAEL could be established. The LOAEL was 100 mg/kg bw/day based on the eye effects. In a 3-months subchronic study with the structurally related ethylenediammonium dichloride (EDA*2HCL) on rats, the NOAEL was considered to be approximately 50 mg/kg bw/day for EDA*2HCl (corresponding to 22 mg/kg bw/day of EDA).


In a combined chronic toxicity and carcinogenicity with the structurally related ethylenediammonium dichloride (EDA*2HCL) there was no evidence for a carcinogenic potential ofEDA*2HCLin male and female F344 rats. The NOAEL for chronic toxicity was 20 mg/kg bw/day for EDA*2HCl (corresponding to 9 mg/kg bw/day of EDA).


Repeated dose toxicity: inhalation
In a 6-week subsacute inhalation toxicity study with the structurally related ethylene diamine (EDA) on rats, the NOAEC for subacute inhalation was 59 ppm

(corresponding to 144 mg/m3).


Repeated dose toxicity: dermal
In a dermal carcinogenicity study with the structurally related ethylene diamine (EDA) in mice, there was no evidence for a carcinogenic potential of EDA after life-time

dermal administration. The NOAEL was 8.3 mg/kg bw/day (25 µL 1% solution/per mouse).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:
analytical purity: separate certificate not enclosed
Purity test date: 21.08.1995
Lot/batch No.: 8/95
Expiration date of the lot/batch: 21.08.1996
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hoechst Aktiengesellschaft, Kastengrund, SPF breeding colony
- Age at study initiation: approximately 6 weeks
- Weight at study initiation: male: 108 g (mean), female: 114 g (mean)
- Housing: in groups of 5
- Diet: ssniff R/M-H (V 1534) ad libitum, except for the period in which the animals were kept in diuresis cages.
- Water: tap water in plastic bottles ad libitum, except for the period in which the animais were kept in diuresis cages.
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 50 ± 20 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The determination of the test substance was performed by spectrophotometrical detection (λ = 274 nm).
Duration of treatment / exposure:
28 days
Frequency of treatment:
daily (7x week)
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
10 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: In a dose range finding study groups of 3 male and 3 female Wistar rats received 3-Dimethylaminopropylamin at dose levels of 50 and 250 mg/kg body weight per day over a period of 14 days. The animals of the 50 mg/kg body weight group showed no clinical signs. The animals of the of the 250 mg/kg body weight showed sporadically swollen abdomen during the study period.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: the animals were observed once daily.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weights of all animals were determined before the start of the study and then twice weekly throughout the study.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: weekly
- Dose groups that were examined: all

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the termination of the study
- Anaesthetic used for blood collection: Yes (intrapenitoneal injection of 50-100 mg Ketamin/kg body weight)
- Animals fasted: No data
- How many animals: all
- Parameters examined: Erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), leucocyte count, thrombocyte count, differential leucocyte count and red ceII morphology, reticulocyte count*, heinz bodies*, coagulation time. *These parameters were scored in the control group and high dose group only.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the termination of the study
- Animals fasted: No data
- How many animals: all
- Parameters examined: Sodium, potassium, inorganic phosphorus, uric acid, bilirubin total, creatinine, glucose, urea, calcium, chloride, aspartate aminotransferase (ASAT/GOT), alanine aminotransferase (ALATIGPT), alkaline phosphatase (AP), gamma-glutamyltranspeptidase (GGT), cholesterol, triglycerides, total protein, albumin.

URINALYSIS: Yes
- Time schedule for collection of urine: overnight from day 26 to day 27
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: Appearance, color, pH-Value, hemoglobin, protein, glucose, ketone bodies, bilirubin, urobilinogen, specific weight, sediment, volume.

OTHERS: The animals were examined weekly for neurological disturbances, damage to the oral mucosa and impairment of dental growth.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes. The autopsy included macroscopic examination of the skin, orifices, eyes, teeth, oral mucosa and internal organs.

HISTOPATHOLOGY: Yes. The necropsy included examination of the heart, stomach, liver, jejunum, kidneys, colon, adrenals, uterus, spleen, ovanies, testes, epididymides, lungs.
Statistics:
The following parameters were compared statistically with the control group values at the level of significance p = 0.05.
Body weights at the designated measurement times, hematological data, clinical chemistry parameters, urine analysis, absolute organ weights and organ to body weight ratios.
Details on results:
CLINICAL SIGNS AND MORTALITY
Mortality occurred in four females of the 250 mg/kg body weight group. The animals were found dead on days 11, 18, 22 and 25 of the study.
One male animal of the high dose group showed irregular respiration as well as respiratory sounds at day 11 and 12 of the study. The following clinical signs were observed in female animals of the high dose group sporadically between day 11 and 24 of the study: decreased spontaneous activity, stilted gait, swollen abdomen, respiratory sounds, gasping and panting. The clinical signs were mainly seen in those females which died intercurrently. Behavior and state of health remained unaffected by the administration of the test compound in all other dose groups.
BODY WEIGHT AND WEIGHT GAIN
Body weight development was not impaired by the administration of the test compound and was comparable in all groups.

FOOD CONSUMPTION AND COMPOUND INTAKE
Absolute and relative food consumption remained unaffected by the administration of the test compound throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE
Likewise, the administration of the test compound did not alter the water consumption.

OPHTHALMOSCOPIC EXAMINATION
No abnormalities were observed

HAEMATOLOGY
Hematological examinations revealed statistically significant decreases in erythrocyte counts as weil as in hemoglobine and hematocrit values in males of the intermediate dose group. Haematocrit values were also decreased in males of the high dose group. Leukocyte counts were statistically significantly decreased in males of the intermediate and females of the low dose group. In all cases there was no dose dependency or the values were within the physiological range of rats. Therefore, a compound-related effect is not evident.

CLINICAL CHEMISTRY
Statistical evaluation revealed increases in aspartate aminotransferase and decreases in total protein values in females from the high dose group. lnorganic phosphorus values were decreased in females of the low dose group. Males of the intermediate dose group showed statistically significant decreases in total bilirubin values. In all dose groups of the females the uric acid values were statistically significantly decreased. In all cases there was no dose dependency or the values were within the physiological range of rats. Therefore, a compound-related effect is not evident.

URINALYSIS
Examination of the urine did not reveal any abnormalities.

ORGAN WEIGHTS
In males of the intermediate dose group statistical evaluation of the organ weights revealed increases in relative liver weights. As there was no dose dependency a compound-related effect is not evident.

GROSS PATHOLOGY
In terminally killed animals, no macroscopically visible organ alterations attributable to the compound administration were observed. Dilatation of renal pelvices was found in same male or female animals of all treatment groups and therefore was not considered to be compound-related but to be a strain specific alteration. In the four high dose females which died intercurrently gross findings included discoloration of Iungs with multiple red spots an its surfaces and foamy content. One female also showed a small spleen.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examinations revealed lesions in the four females of the high dose group dying intercurrently. These lesions included congestion of organs, pulmanary hemorrhage, and edema, consistent with cardiorespiratory failure as cause of death. In addition, one of these females exhibited marked loss of lymphatic follicles of the spleen with massive marginal zone and periarteriolar lymphoid sheath atrophy, probably reflecting chronic stress due to treatment. In the one high dose male rat which had shown clinical signs, focal ballooning degeneration of the stratum corneum of the squamous epithellum of the forestomach with granulocytic infiltration of the submucosa was found, most likely due to the locally irritating effect of the compound.

Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no treatment-related adverse effects were seen.
Key result
Dose descriptor:
LOAEL
Effect level:
250 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: 3-Dimethylaminopropylamin caused clinical symptoms and mortality in male and female Wistar rats when administered 28 times at the dose level of 250 mg/kg body weight. The female rats seem to be more sensitive.
Critical effects observed:
not specified
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 June 2015 -- 08 July 2015
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Qualifier:
no guideline followed
Principles of method if other than guideline:
Range-finding study
GLP compliance:
no
Remarks:
not required for a range-finding study
Limit test:
no
Specific details on test material used for the study:

- Lot/batch No.: A27Z0R010101
- Expiry date: 19 February 2017
- Storage condition: at room temperature
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rat, Rj Han: SD, Indemn of Organism Pathogen Specific Han (IOPS Han)
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Age/Weight: at the beginning of the treatment period, the animals were approximately 6 weeks old. The males had a mean body weight of 245 g (range: 228 g to 261 g) and the females, 200 g (range: 191 g to 210 g).
- Fasting period before study: no
- Housing: polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 7 days before the beginning of the study

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h

IN-LIFE DATES: 24 June 2015 to 08 July 2015
Route of administration:
oral: gavage
Vehicle:
other: drinking water treated by reverse osmosis
Details on oral exposure:
Type of formulation
(visual observation): Solution in the vehicle.
Preparation procedure: According to CiToxLAB France in-house procedures, the test item was weighed and mixed with the required quantity of vehicle. The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of hydrochloric acid (HCl 5N, VWR Prolabo®); the volume of HCl added was recorded for each concentration. The final pH was checked and recorded by using a pH-meter.
Frequency of preparation and storage conditions: Test item dose formulations
Fresh dose formulations were prepared by the CiToxLAB France Pharmacy on the day of administration and kept at room temperature prior to administration
Vehicle preparation
Based on CiToxLAB France in-house procedures (frequency documented in study raw data)
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
2 weeks
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 males and 5 females per dose
Control animals:
yes, concurrent vehicle
Details on study design:
Rationale for dose-level selection
The dose-levels were selected in agreement with the Sponsor, following the results of a previous acute toxicity with the same test item: no mortality and no clinical signs were observed after a single oral administration to rats by gavage at 1000 mg/kg, followed by a 7-day observation period.
Consequently, the dose-levels of 100, 300, and 1000 mg/kg/day were selected.

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: twice a day during treatment, including weekends

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: once a day.

BODY WEIGHT:
- Time schedule for examinations: once before group allocation, on the first day of treatment, and then at least twice a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: twice a week.
Sacrifice and pathology:
ORGAN WEIGHTS:
The body weight of each animal was recorded before sacrifice. The organs specified in the Tissue Procedure Table (Table 1) were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all animals.
Statistics:
Citox software was used to perform the statistical analyses of body weight. Kolmogorov-Lilliefors test was performed to test for data distribution normality. For assessment of variance homogeneity between groups Bartlett and Fisher test were performed. For comparison of treated and control groups using untransformed values Dunn test or Mann-Whitney/Wilcoxon test was utilized.
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01). Here, Kruskal-Wallis test was performed to test for group differences and One-way analysis of variance to test for group differences in means.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
No unscheduled deaths occurred during the study.

CLINICAL SIGNS:
Ptyalism was observed in 1/5 males and 4/5 females at 1000 mg/kg/day from the first or the second week of treatment. Ptyalism was considered to be test item-related, but of minor toxicological importance.Scabs in the interscapular region were noted in a single female at 1000 mg/kg/day from Day 8 and was not considered to be related to the test item.

BODY WEIGHT (Table 1):
At 1000 mg/kg/day and when compared with controls, a lower mean body weight gain was recorded in males during the first 3 days of treatment (-46%, statistically significant) as well as in females ( 67%, not statistically significant). This effect was attributed to the test item treatment and was still present in males until the end of the study but in a lesser extent, leading to a lower mean body weight in males on Day 14 (-7%).
The higher mean body weight gain recorded in females between Days 8 and 11 was isolated and considered to be of no toxicological importance.

No relevant effects were observed in the mean body weight change in animals given 100 or 300 mg/kg/day.

FOOD CONSUMPTION (Table 2):
Slightly lower mean food consumption was observed in males and females given 1000 mg/kg/day over the first 3 days of the study. This correlated to the above-described lower mean body weight gain.

There were no test item-related effects on food consumption at 100 and 300 mg/kg/day.

ORGAN WEIGHTS (Table 3):
There was a slight decrease (-9%) of terminal body weight (p<0.05) in males treated at 1000 mg/kg/day that was considered most likely related to treatment with Diethylaminopropylamine.

There was a minimal increase (not statistically significant) of spleen weights in animals of both sexes treated at 1000 mg/kg/day and in one male treated at 300 mg/kg/day (with macroscopically enlarged spleen). In absence of histopathological examination of this organ, a relationship to treatment was considered to be doubtful.

Statistically significantly lower absolute liver weights were observed in males treated at 1000 mg/kg/day (p<0.05). These weight differences were marginal and not observed in females and were most likely related to the lower final body weights observed in males of this group and not related to treatment with Diethylaminopropylamine.

As other weight changes were minimal, not dose-related and/or of opposing trends between the male and female groups, a relationship to treatment was excluded.

GROSS PATHOLOGY:
No macroscopic findings were attributed to treatment with the test item.
One female treated at 1000 mg/kg/day had a white mass of 0.1 cm in diameter within the cranial part of the wall of the forestomach which was not correlated to any microscopic finding.
One male treated at 300 mg/kg/day and one female treated at 1000 mg/kg/day had an enlarged spleen that correlated with higher spleen weights in these animals and with congestion at histology.
None of these macroscopic findings were attributed to treatment with the test item.
The few other gross findings reported were of those commonly observed in the rat of this strain and age and therefore a relationship to treatment was also excluded.

MICROSCOPIC EXAMINATION
Five out of five males and 4/5 females treated at 1000 mg/kg/day had minimal to slight hyperplasia of squamous cells associated with minimal to slight hyperkeratosis within the forestomach compared with control animals (1/5 males and 0/5 females).
These findings were considered to be attributed to treatment with Diethylaminopropylamine but considered to be non-adverse.
All of the other pathological findings encountered were considered incidental changes and/or are common background findings for the species.
Key result
Dose descriptor:
LOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: lower body weight gain, body weight and food consumption and slight hyperplasia of squamous cells together with minimal to slight hyperkeratosis in the forestomach were observed in male and female rats.
Key result
Dose descriptor:
NOAEL
Effect level:
>= 100 - <= 300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no-treatment related effects
Critical effects observed:
not specified

Table 1. Mean body weight change/Mean body weight (expressed in g)

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Body weight change

 

 

 

 

 

 

 

 

. Days 1 to 4

+24

+24

+24

+13**

+9

+5

+9

+3

. Days 4 to 8

+37

+35

+38

+31

+10

+18

+11

+16

. Days 8 to 11

+32

+29

+26

+29

+7

+13

+11

+17**

. Days 11 to 14

+22

+19

+23

+18*

+4

+3

+6

+8

. Days 1 to 14

+115

+107

+111

+90**

+30

+38

+36

+43

% compared to controls

-

-7

-3

-22

-

+27

+20

+43

Body weight

 

 

 

 

 

 

 

 

. Day 1

246

245

243

245

200

203

200

199

. Day 14

361

352

354

335*

230

241

236

242

-: not applicable; *: p<0.05 and **: p<0.01, statistically significant.

Table 2. Mean food consumption (expressed in g/animal/day)

 

Sex

Male

Female

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

. Days 1 to 4

27.2

26.4

26.0

23.3

17.7

19.2

19.0

16.0

% compared to controls

-

-3

-4

-14

-

+8

+7

-10

-: not applicable.

Table 3. Organ weights

 

Sex

Male

Female

Group

2

3

4

2

3

4

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

Exam. animals / Num. of animals

5/5

5/5

5/5

5/5

5/5

5/5

- Final body weight

-2

-2

-9*

+4

+3

+1

- Spleen

. absolute

+3

+11

+13

+9

-7

+16

. relative

+5

+13

+25

+5

-10

+15

- Liver

. absolute

-2

-3

-14*

-1

+10

+4

. relative

0

-2

-5

-5

+7

+3

Statistically significant from controls: *: p<0.05.

The significance concerned the organ weights values and not the percentages.

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
13 August 2015 to 14 October 2016
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:

- Lot/batch No.: A27Z0R010101
- Expiry date: 19 February 2017
- Storage condition: at room temperature
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
Sprague-Dawley rat, Rj Han: SD, Indemn of Organism Pathogen Specific Han (IOPS Han).
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: breeder: Janvier, Le Genest-Saint-Isle, France.
- Females (if applicable) nulliparous and non-pregnant: not specified
- Age at study initiation: on the first day of treatment, the animals were 9 weeks old
- Weight at study initiation: The males had a mean body weight of 425 g (range: 383 g to 479 g) and the females had a mean body weight of 265 g (range: 234 g to 300 g).
- Fasting period before study:no
- Housing:The animals were housed in twos or threes of the same sex and in the same group, in polycarbonate cages with stainless steel lids containing autoclaved sawdust.
Each cage contained a rat hut as environmental enrichment.
The cages were placed in numerical order on the racks. On Day 1 and in Week 9, all the racks were moved clockwise around the room, rack by rack, in order to reduce possible bias caused by placement in the room
- Diet : All animals had free access to SSNIFF R/M-H pelleted maintenance diet, batch Nos. 3413447 and 3044117, which was distributed weekly.
- Water :The animals had free access to bottles containing tap water (filtered with a 0.22 μm filter)
- Acclimation period: the animals were acclimated to the study conditions for a period of 35 days before the beginning of the treatment period. A larger number of animals than necessary were allocated to the study and acclimated, in order to permit the selection and/or replacement of individuals.

ALLOCATION to groups: during the acclimation period, the required number of animals (50 males and 50 females) were selected according to body weight and clinical condition. The animals were allocated to groups (by sex) using a computerized randomization procedure.

DETAILS OF FOOD AND WATER QUALITY:
The batches of diet and sawdust were analyzed by the Suppliers for composition and contaminant levels.
Bacterial and chemical analyses of water are performed regularly by external laboratories. These analyses include the detection of possible contaminants (pesticides and heavy metals). No contaminants were present in the diet, drinking water or sawdust at levels which could have been expected to interfere with, or prejudice, the outcome of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air.
- Photoperiod (hrs dark / hrs light):12h/12h

IN-LIFE DATES: From: 13 August 2015 To: 16 January 2016
Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
Type of formulation
(visual observation): Solution in the vehicle.
Preparation procedure: According to CiToxLAB France in-house procedures, the test item was weighed and mixed with the required quantity of vehicle. The pH of the dose formulation was adjusted to 8.0 (± 0.5) with a solution of hydrochloric acid (HCl 5N, VWR Prolabo®); the volume of HCl added was recorded for each concentration. The final pH was checked and recorded by using a pH-meter.

Frequency of preparation and storage conditions:
Test item dose formulations
Fresh dose formulations were prepared by the CiToxLAB France Pharmacy on the day of administration and kept at room temperature prior to administration
Vehicle preparation
Based on CiToxLAB France in-house procedures (frequency documented in study raw data)
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical method developed and validated at CiToxLAB France (CiToxLAB France/Study No. 42757 VAA) prior to dose formulation analysis.
Once in Weeks 1, 4, 8 and 13
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
15 (control and high dose), 10 (low and mid doses)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels have been selected on the basis of the results of previous toxicology studY (CiToxLAB France/Study Nos. 42759 TSR) performed in the same species.
In CiToxLAB France/Study No. 42759 TSR, male and female Sprague-Dawley rats received Neutralized Diethylaminopropylamine (as pH-neutralized dose formulations) daily by gavage at dose-levels of 100, 300 or 1000 mg/kg/day for 2 weeks. At 1000 mg/kg/day, in-life observations were limited to ptyalism in both sexes and lower body weight gain at treatment initiation resulted in a final lower body weight in males. Histopathology findings consisted in minimal to slight hyperplasia of squamous cells associated with minimal to slight hyperkeratosis within the forestomach in all males and females given 1000 mg/kg/day.
- Post-exposure recovery period in satellite groups: 6 weeks
Observations and examinations performed and frequency:
MORBIDITY/MORTALITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and twice a day during the treatment and treatment-free periods, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule:once a day.

DETAILED CLINICAL EXAMINATION:
- Time schedule: once before the beginning of the treatment period and then once a week until the end of the study.

FUNCTIONAL OBSERVATION BATTERY:
- Time schedule: all animals (except for the last six group 1 and 4 animals per sex) were evaluated once in Week 12.

BODY WEIGHT:
- Time schedule: once before the beginning of the treatment period, on the first day of treatment and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: once a week, over a 7 day period, during the study.

OPHTHALMOSCOPIC EXAMINATION:
- Time schedule: on all animals, before the beginning of the treatment period and on all control and high-dose animals at the end of the treatment period.

MONITORING OF ESTROUS CYCLE
- Time schedule: the estrous cycle stage was determined for each female planned to be sacrificed at the end of the treatment period.

MOTOR ACITIVITY:
- Time schedule: motor activity was measured by automated infra-red sensor equipment over a 60-minute period.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.

- SEMINOLOGY:
At the end of the treatment period, just before sacrifice, each male was deeply anesthetized by an intraperitoneal injection of sodium pentobarbital and the left epididymis was removed. Animals were then sacrificed.
As no treatment-related changes were observed at the end of the treatment period, these examinations were not carried out at the end of the treatment-free period.

Epididymal sperm
Sperm from the cauda epididymis was sampled for motility and morphology investigations.
The cauda of the left epididymis was separated from the corpus using a scalpel and subsequently kept at 20°C pending further investigation.

Epididymal sperm motility
The sperm was evaluated on a slide, after appropriate dilution. The number of motile and immotile spermatozoa from a sample of 200 spermatozoa was evaluated under a microscope using a 40-fold magnification. Results were expressed as the proportion of motile and non-motile spermatozoa.

Epididymal sperm morphology
The morphology was determined from a sperm smear, after eosin staining and counting of 100 spermatozoa per slide. Results were expressed as the proportion of spermatozoa in each of the following categories:
- normal,
- normally shaped head separated from flagellum,
- abnormal head separated from flagellum,
- abnormal head with normal flagellum,
- abnormal head with abnormal flagellum,
- normally shaped head with abnormal flagellum.

Epididymal sperm count
After thawing, the left cauda epididymis was weighed, minced and homogenized in a saline-triton solution using a Polytron.
An aliquot of the suspension was sampled and the number of spermatozoa was counted in a microscope slide counting chamber.
Results were expressed as the number of spermatozoa per cauda and per gram of cauda.

Testicular sperm
At necropsy, the left testis was sampled and frozen at -20°C for further sperm count investigation. After thawing, the left testis was weighed (without the albuginea) and ground. The resulting preparation was diluted and sperm heads resistant to homogeneization (i.e. elongated spermatids and mature spermatozoa) were counted in a microscope slide counting chamber.
Results were expressed as a number of sperm heads per gram of testis and the daily sperm production rate was calculated (a time divisor of 6.10 which represents the duration of spermatogenic cycle of homogenization-resistant testicular spermatids).
Sacrifice and pathology:
ORGAN WEIGHTS: see table below
The body weight of each animal was recorded before sacrifice. The organs specified in the Tissue Procedure Table were weighed wet as soon as possible after dissection.
The ratio of organ weight to body weight (recorded immediately before sacrifice) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals. This included examination of the external surfaces, all orifices, the cranial cavity, the external surfaces of the brain and spinal cord, the thoracic, abdominal and pelvic cavities with their associated organs and tissues and the neck with its associated organs and tissues.

HISTOPATHOLOGY:
 all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period and for animal found dead,
 all macroscopic lesions from all the low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period,
 immunostained kidneys from control and high-dose males (groups 1 and 4) sacrificed at the end of the treatment period.

In addition testicular staging was performed for control and high-dose males (groups 1 and 4). A detailed examination of the testes was performed, using a thorough understanding of tubule development through the different stages of the spermatogenic cycle. Transverse sections of the testes were stained with hematoxylin: PAS in order to detect retained spermatids, missing germ cell layers, multinucleated giant cells or sloughing of spermatogenic cells into the lumen, etc.
Statistics:
Citox software was used to perform the statistical analyses of body weight, food consumption, hematology, blood biochemistry and urinalysis data. Kolmogorov-Lilliefors test was performed to test for data distribution normality. For assessment of variance homogeneity between groups Bartlett and Fisher test were performed. For comparison of treated and control groups using untransformed values Dunn test or Mann-Whitney/Wilcoxon test was utilized.
PathData software was used to perform the statistical analysis of organ weight data (level of significance: 0.05 or 0.01). Here, Kruskal-Wallis test was performed to test for group differences and One-way analysis of variance to test for group differences in means.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Mortality:
mortality observed, treatment-related
Description (incidence):
see "Details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
see "Details on results"
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):
see "Details on results"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see "Details on results"
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
One female from group 4 was prematurely sacrificed on Day 72 (week 11). Prior to sacrifice, thin appearance, piloerection, hunched posture, ptyalism, loud breathing, hypoactivity and cold to the touch were observed. At necropsy, the spleen was enlarged, the stomach was distended with food and the stomach wall had a red discoloration of approximately 0.2 cm in diameter. The thymus showed red discoloration of up to 0.5 cm in diameter. Test item-related microscopic vacuoles were observed in the white matter from the brain (hippocampus, cingulum, cerebral peduncle and cerebellum), in the pars nervosa of the pituitary gland, in the renal tubules, in the choroid plexus, in the spleen and in the GALT.Vacuolation in the white matter from the brain was not seen in the test item-treated animals at the end of the treatment period

At 750 mg/kg bw/day, the following clinical signs were noted:
. ptyalism in 12/15 males and 14/14 females,
. reflux at dosing in 2/15 males and 2/15 females showed on one occasion,
. redd ish color of urine in 1/15 males and in 2/14 females,
. signs of poor clinical condition (loud and/or abdominal breathing, thin appearance, hunched posture, dyspnea, piloerection, bent head, soiled urogenital region and/or reddish abnormal color of the vagina) in 1/15 males and 3/14 females.

At 250 mg/kg bw/day,none-adverse findings in the form of:
.2/10 males loud breathing was observed
.1/15 males and 2/15 females showed ptyalism and reddish abnormal color was observed in the vagina of 1/10 females.

At 50 mg/kg bw/day, non-adverse findings in the form of:
.1/10 females showed hunched posture and piloerection. These signs were not dose-related.

Alopecia, scabs, soiled nose and mouth, soiled head and neck, wound, thinning of hair, opacity of eyes, nodosities, chromodacryorrhea and/or chromorhynorrhea were observed both in contrai and test item-treated animals. Test item treatment-related clinical signs were no longer observed over the treatment-free period.

BODY WEIGHT AND WEIGHT GAIN
When compared with controls, lower mean body weight gain was noted in all test-treated females and in males given 750 mg/kg bw/day over the treatment period.
However, mean body weight of females was unaffected by the test item-treatment whereas a slightly lower mean body weight, not statistically significant, was noted in males given 750 mg/kg bw/day (-6% vs. controls) at the end of the treatment period. During the recovery period, a higher mean body weight gain was observed in males previously treated with 750 mg/kg bw/day, leading to a terminal mean body weight similar to the control animals. At all dose-levels of the test item in females, some instances of lower mean body weight gain, statistically significant on few occasions, were reported compared with controls over the treatment or treatment-free period. As these differences were occasional, not dose-related and/or of minimal amplitude, and did not affect the mean body weight, they were considered to be of no toxicological importance.


FOOD CONSUMPTION
In females, transient statistically significant lower mean food consumption, when compared with controls, was noted in the fourth week of treatment at 250 mg/kg bw/day (-10%) and first week at 750 mg/kg bw/day (-15%). The opposite trend was noted in males given 750 mg/kg bw/day between weeks 6 and 8 (+8% to +9%; p<0.05). As these differences were of isolated occurrence, not dose-related and/or of opposite trends, they were considered to be of no toxicological importance.No relevant effects were observed on food consumption in the other test item-treated animals during the treatment period.Mean food consumption was slightly higher in previously test item-treated animals during the treatment-free period.


HAEMATOLOGY
End of treatment period
At 250 and 750 mg/kg bw day, lower red blood cell parameters including red blood cell count, hemoglobin concentration and packed cell volume were noted (statistically significant at 750 mg/kg/day). Reticulocytosis, was also noted at 750 mg/kg/day and correlated to the decrease in red blood cell mass. Although these differences were mainly due to the contribution of one male (showing blood in urine at urine collection), they were also recorded in the other males but in a lesser extent.
Lower mean eosinophil counts were observed in males and females given 750 mg/kg bw/day whereas higher mean neutrophil counts were recorded in females only.
At 750 mg/kg bw/day, prolonged prothrombin time was recorded in males and in a lesser extent in females.
Other statistically significant differences between control and test item-treated animais, namely in monocyte counts (females only) were observed.

End of recovery period
No effect.

CLINICAL CHEMISTRY
End of treatment period
None-adverse blood biochemistry findings (when compared with mean control values) were observed in animais given 250 or 750 mg/kg bw/day and consisted of:
. lower mean sodium level (females at 250 mg/kg bw/day and males and females at 750 mg/kg bw/day: -1 % vs. controls),
. lower mean chloride level (males at 750 mg/kg bw/day: -2% vs. controls),
. higher mean inorganic phosphorus level (males and females at 250 mg/kg bw/day: +8 and +15% vs. controls, respectively; males and females at 750 mg/kg bw/day: +15 and +12% vs. controls, respectively),
. lower mean creatinine level (males at 750 mg/kg bw/day: -9% vs. controls),
. higher mean total bilirubin level (males at 250 and 750 mg/kg bw/day: x1 .8 and x 4.9 vs. controls, respectively),
. lower mean proteins and albumin levels (males at 750 mg/kg bw/day: -5% vs. controls),
. higher triglyceride levels (females at 250 and 750 mg/kg bw/day: +38 and +58% vs. controls, respectively),
. minimally higher mean aspartate aminotransferase activity (males at 750 mg/kg bw/day: +23% vs. controls),
. minimally higher mean alanine aminotransferase activity (males and females at 750 mg/kg bw/day: x1 .9 and x1 .6 vs. controls, respectively).
Lower mean total bilirubin was also noted in females given 750 mg/kg bw/day (-59% vs. controls).
Other statistically significant differences between control and test item-treated animals, namely in potassium and triglycerides levels (males) were observed.

End of recovery period
No effect.

URINALYSIS
At 750 mg/kg/day, significant hematuria was noted both in males and females at the end of the treatment period. In the absence of hyperglycemia, glucosuria observed in males could be indicative of a tubular resorption problem. Moreover, in one male, the presence of proteins (≥ 3 g/L), bilirubin (high-level), nitrites, urobilinogen(≥ 66 μmol/L) and marked turbid appearance of urine were noted. At the end of the treatment-free period, the lower mean urine volumes in males and females previously treated with the high dose level were not considered to be treatment-related as they were not observed at the end of the treatment period and were not associated with any other changes in the urine parameters.

ORGAN WEIGHTS
at the end of the treatment period, none-adverse findings in the format of:
. at 750 mg/kg bw/day higher relative-to body kidney weights in males and females when compared with controls (partially related to lower terminal body weights; lower absolute and relative-to-body thymus weights in males and females (considered to be related to test item administration and/or stress).

at the end of the treatment-free period, none-adverse findings in the format of:
. at 750 mg/kg bw/day slightly higher absolute and /or relative-to body kidney weights in males and females when compared with controls (not considered not to be related to previous test item administration in males in view of low magnitude, of absence of microscopic correlates and of absence of kidney weight differences at the end of the treatment period);no reversibility of the difference in thymus weights in females

GROSS PATHOLOGY FINDINGS
at the end of treatment period:
.no item-related gross findings (exception:1 cm in diameter tan discoloration of right kidney from 1/10 males ) treated at 750 mg/kg bw/day which correlated with moderate renal tubular vacuolation, and small thymus seen in 1/9 females treated at 750 mg/kg bw/day which correlated with marked lymphoid atrophy

at the end of treatment-free period:
.no test item-related gross findings

MICROSCOPIC EXAMINATIONS
at the end of treatment period, none-adverse findings in the format of:
. at 750 mg/kg/day: minimal to marked vacuoles in the kidneys (tubules and, to a lesser severity, glomeruli), brain (choroid plexus), pars nervosa (pituitary gland), spleen, mesenteric lymph node and GALT (Gut Associated Lymphoid Tissue) in males and females
. at 250 mg/kg/day vacuoles present in the choroid plexus from only 2/10 females
. vacuoles were round, of moderate to large size and devoid of any staining except those recorded in the pituitary gland which were pale and eosinophilic, empty vacuoles in brain, spleen, lymph node and GALT were scattered, multifocal while the vacuoles in the pars nervosa were diffuse
.forestomach: at 250 and 750 mg/kg/day minimal to slight, focal or multifocal orthokeratotic hyperkeratosis in males and females was related with the test item administration
. no test item-related findings in testes

at the end of treatment-free period, none-adverse findings in the format of:
. vacuoles,forestomach,thymus: reversibility of test item-related findings observed

A few other microscopic findings were not considered to be associated with test item administration because these findings were consistent with spontaneous and background findings described in the literature or their appearance was similar to changes found in controls.
.

OTHER FINDINGS
MONITORING OF ESTROUS CYCLE
End of treatment period
Mean estrous cycle data are presented below:
Dose level (mg/kg bw/day) 0 50 250 750
Number of cycles 4.2 3.9 3.8 3.2
Cycle length (days) 4.2 4.3 5.1 5.9
Number of females having a mean 7 6 5 4
average cycle of 4-5 days

There were no statistical significant test item-related changes on mean estrous cycle length and mean number of cycles. However a trend to an increase in mean estrous cycle length was observed in females given 250 or 750 mg/kg/day.

End of recovery period
Mean estrous cycle data are presented below:
Dose level (mg/kg bw/day) 0 750
Number of cycles 1.0 1.0
Cycle length (days) 4.0 4.0
Number of females having a mean average cycle of 4-5 days 2 1

SEMINOLOGY
No effect.
Key result
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
clinical signs
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
clinical signs
Critical effects observed:
no
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Only abstract report available
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were dosed by oral gavage for 90-days.
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Route of administration:
oral: gavage
Vehicle:
water
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
90 days
Frequency of treatment:
5 days/ week
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
400 mg/kg bw/day (actual dose received)
Dose / conc.:
600 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 animals/sex/dose
Control animals:
yes, concurrent vehicle
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not specified
Food efficiency:
not specified
Ophthalmological findings:
effects observed, treatment-related
Haematological findings:
not specified
Clinical biochemistry findings:
not specified
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
LOAEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Eye lesions
Critical effects observed:
not specified

Six male and one female rat from the 1600 mg/kg group died during the in-life phase of the study. All animals in the lower dose groups survived to the end of the study.

Animals in the two highest doses exhibited gasping, sneezing and squinting of both eyes shortly after dose administration. In some animals from the 600 and 800 mg/kg groups, there was a discoloration of the eye while others exhibited a purple color. Subsequently, rats from both dose groups developed white masses in their eyes. Bilateral pupil dilation was noted in all survivng rats receiving 600 or 800 mg/kg during week 11 or 12 of the study. All of these eye abnormalities appeared to be irreversible. Ophthalmoscopic examination revealed bilateral cataracts in 3 of 6 males and 7 of 10 females after receiving 800 mg/kg for 6 weeks. Hemorrhage in the posterior chamber of the eye and debris floating in the anterior chamber was also observed in two other rats from this group. Eight male and eight female rats in the 600 mg/kg group also had bilateral cataracts after 12 weeks.

Retinal atrophy and posterior chamber hemorrhage were also observed in some animals.

Body weight gains were decreased in male and female rats administered 200 - 800 mg/kg. In males, the differential change in body weight ranged from -47% in the 800 mg/kg group to -20% in the 200 mg/kg group. In females, the differential change in body weight ranged from -50% in the 800 mg/kg group to -2.2% in the 200 mg/kg group. Body weight values of the low dose group were comparable to control values. Liver, heart or lung to body weight ratios were unaffected in either sex. Increases in the right kidney, brain and right testicle (male only) to body weight ratios appeared to be the result of lower body weights in the respective dose groups and not the result of any differences between actual organ weights of the dosed groups and controls. Thymus to body weight mean ratios of the dose group decreased as a function of increasing dose at 200 mg/kg in males and 600 mg/kg in females.

At necropsy, cloudy appearing lens were observed in most of the 600 mg/kg and all of the surviving 800 mg/kg rats. In addition, several of the female rats from the 600 and 800mg/kg groups appeared to have smaller uterine horns and female rats from the 800 mg/kg group had small ovaries than controls. Histopathologic changes were noted in the eyes, kidneys and uterus. Eye lesions were present to some degree in every dose group. In the more severe cases the retina was lacking all the normal layers while in less severe cases there was only rosetting and focal cellular losses. In many cases there were ghost-like cells near the lenticular surface of the lens, mineralized debris in the lens and a globular irregular appearance to the lens material. The iris was adherent to the anterior surface of the lens in most affected eyes. Renal tubular lesions were only observed in the 600 and 800 mg/kg groups. These lesions were characterized by degeneration, regeneration and occasional necrosis of the tubular epithelium especially at the corticomedullary junciton. Mineralization of renal tubules in the papillary ducts of Bellini was also observed. Uterine lesions included atrophy of the myometrium and endometrium in the two highest doses. No ovarian lesions were seen in the rats examined microscopically. The thymus did not appear to be affected.

Endpoint:
repeated dose toxicity: oral, other
Remarks:
combined repeated dose and carcinogenicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
study well documented, meets generally accepted scientific principles, acceptable for assessment
Reason / purpose:
reference to same study
Reason / purpose:
reference to other study
Qualifier:
no guideline available
Principles of method if other than guideline:
With the structurally related ethylenediammonium dichloride (EDA*2HCL) a combined chronic toxicity and carcinogenicity study was conducted. Groups of each 100 F344 rats per sex received the test substance orally via the feed at adjusted dose levels of 0, 20, 100, 350 mg/kg bw/day (males) or 0, 20, 100 and 360 mg/kg bw/day (females). Mortality was comparable to controls during the first 18 months of the study. After 22 months, mortality in males and females increased at the high dose and in females at 100 mg/kg bw/day after 24 months.
GLP compliance:
not specified
Specific details on test material used for the study:
High purity EDA was used for synthesizing the chloride salt. Various chromatographic and spectrophotometric analytical methods showed that the obtained EDA di-hydrochloride was of high purity.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Microbiological Associates, Inc. (Walkersville, MD, USA).
- Age at study initiation: 43 days of age at start of study.
- Weight at study initiation: males: 81-141 g , females: 60-112 g,

- Housing: suspended wire-bottom and front stainless-steel cages. Three males or five females were placed in a cage
- Diet: Ground diet (Zeigler Brothers NIH-07 Rat and Mouse Ration, Zeigler Bros., Inc., Gardners, PA, USA) was supplied in 12 oz.
opal glass jars. Prior to use, the diet was evaluated for nutritional content and possible contaminants
- Water : ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Photoperiod (hrs dark / hrs light): 12 h light cycle
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Dosed diet was prepared and offered to the animals every 2 wk. Based on the predicted mean body weight gain and diet consumption data, the concentration of EDA*2HCl in the diet was adjusted to maintain a constant dose level in g/kg.
A HPLC method was used to analyse the dietary concentration of EDA*2HCl. The distribution was homogenous and EDA*2HCl was stable in the diet for 6 months. Random samples during the study confirmed the accuracy of concentrations in diet.
Duration of treatment / exposure:
2 years
Frequency of treatment:
every 2 wk
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
350 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
100
Control animals:
yes, concurrent no treatment
Observations and examinations performed and frequency:
BODY WEIGHT: Yes
- Time schedule for examinations: Bi-weekly
FOOD CONSUMPTION AND COMPOUND INTAKE : Diet consumption was determined on animals from the first 10 cages/sex/group biweekly.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes / No / No data


WATER CONSUMPTION: Two weeks prior to each scheduled sacrifice, water consumption of animals from the first 10 cages/sex/group was measured.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: 1 week prior to sacrifice
- Anaesthetic used for blood collection: Yes methoxyflurane
- How many animals: 10 per sex/group at 6 and 12 month sacrifice, 20 per sex/group at 18 and 24 months sacrifice
- Parameters examined: Red and white blood cell counts, differental white cell counts, measurement of hemoglobin and mean corpuscular volume,
and calculations of hematocrit, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: 1 week prior to sacrifice
- How many animals: 10 per sex/group at 6 and 12 month sacrifice, 20 per sex/group at 18 and 24 months sacrifice
- Parameters examined: Serum concentrations of glucose, urea, nitrogen, aspartate aminotransferase, alanine, aminotransferase, alkaline phosphatase, total protein, albumin, creatinine, bilirubin (conjugated and total) and sorbital dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: 1 week prior to sacrifice
- Metabolism cages used for collection of urine: Yes
- Parameters examined: The measurements and observations included volume, pH, specific gravity, protein, glucose, ketones, occult blood, turbidity, color, microscopic appearance, bilirubin and urobilinogen

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
10 rats/sex/dose and control group were scheduled for sacrifice at 6 and 12 month, 20 rats/sex/dose and control group were scheduled for
sacrifice at 18 month.
Key result
Dose descriptor:
NOAEL
Effect level:
9 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Recalculated to EDA base
Key result
Dose descriptor:
LOAEL
Effect level:
45 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: body weight; erytrocyte count, hemoglobin conc., hematocrit, serum albumin conc. organ weights - liver, kidney, spleen; other: hepatocellular pleomorphism. LOAEL value recalculated to EDA base.
Critical effects observed:
not specified

Doses attained were close to the dose goal, for males, 20, 100 and 350 mg/kg/day, females 20, 100 and 360 mg/kg/day.

A slight increase in diet consumption of EDA treated females between 5 - 15 months, otherwise no significant differences from control.

Increases in water consumption were observed for both males and females from the high dose group at 12 and 18 month and for females from the high dose group at 24 month associated with increased urine volume and decreased urine specific gravity. 

There was a significant reduction in body weight gain in male rats of the high dose group throughout the whole study course and in female rats of the high dose group from the 18th month until termination. A significant increase of body weight gain in female rats of the intermediate dose group from day 21 until the 21st month was noted. 

 

As shown in Tables 1 and 2, mortality for groups ingesting EDA was comparable to control values during the first 18 months of the study. After 22 months, mortality in males and females ingesting EDA were elevated from both control groups. In addition, the mortality rate in female rats ingesting 100 mg/kg/day was increased after 24 months.

Significant reduction in the absolute weights of liver, kidney, spleen (male) and increase of the relative weights of liver, kidney, heart, brain (females) in rats of the high dose group. No substance-related changes in hematologic data, clinical chemistry values and urinalysis except a decrease in erythrocyte count, hemoglobin concentration, hematocrit (male) and serum albumin concentration (female) in rats of the high dose group. Significantly higher incidence of hepatocellular pleomorphism in female rats of the intermediate and the high dose group; rhinitis and tracheitis were seen with greater frequency in high dose males at 12, 18 and 24 months and in high dose females at 18 months; at 24 months, rhinitis persisted at a significantly greater frequency in high dose females while tracheitis did not; lower incidence of pituitary adenomas and testicular interstitial cell adenoma in the high dose group (incidence ratio: 2/4 in comparison to 25/26 and 12/15 in the control groups); all other tumor incidences did not differ significantly from control. 

Table 1: Cumulative % Mortality of Male Rats after 18 Months

 

   Dose Level, mg/kg/day

Month

Control-A

Control-B

20

100

350

18

5.6

5.9

2.5

6.2

12.0

19

9.7

7.3

2.5

7.7

13.4

20

16.4

14.1

5.8

12.9

20.0

21

21.4

17.6

10.9

21.6

29.8

22

26.4

24.4

15.9

33.8

44.5a,a

23

34.8

41.6

27.7

40.8

65.7a,a

24

46.5

67.4-a

54.6

61.7

88.6a,a

25

63.4

78.7

77.3

68.1

95.4a,a

a,a First letter denotes significantly different from control group A and second letter denotes same from control group B (p0.05).

Table 2:Cumulative % Mortality of Female Rats after 18 Months

 

   Dose Level, mg/kg/day

Month

Control-A

Control-B

20

100

350

18

2.1

2.4

6.2

4.6

3.2

19

6.2

3.7

7.7

6.0

7.4

20

7.9

7.1

9.4

12.6

10.6

21

9.6

10.3

17.8

15.9

20.3

22

11.3

12.0

21.1

19.2

25.2a,a

23

14.6

16.8

21.1

27.5

33.3a,a

24

18.0

21.7

26.1

39.0a,a

41.5a,a

25

30.4

24.9

29.4

56.0a,a

55.7a,a

a,a First letter denotes significantly different from control group A and second letter denotes same from control group B (p0.05).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
1983
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
GLP compliance:
no
Remarks:
pre-GLP
Limit test:
no
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Microbiological Associates, Inc., Walkersville, MD
- Age at study initiation: 41 days
- Weight at study initiation: 101-152 g (males) 91-110 g (females)
- Fasting period before study: no info
- Housing: suspended wire-bottom-and-front stainless steel cages. Three males or 5 females were housed in each cage.
- Water (e.g. ad libitum): ad libitum
Route of administration:
oral: feed
Details on oral exposure:
New concentrations of feed were prepared weekly, with the percentage of EDA.2HCl in the diet adjusted to maintain a constant dosage level in mg/kg for each sex according to the average body weight gain and diet consumption.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
A spectrophotometric method was employed for the analysis of EDA.2HCI in the diet.
Duration of treatment / exposure:
3 months
Frequency of treatment:
Daily
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
260 mg/kg bw/day (actual dose received)
Dose / conc.:
1 040 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10 animals per sex
Control animals:
yes, plain diet
Observations and examinations performed and frequency:

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION : Yes
- Time schedule for examinations: Monthly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Immediately before sacrifice
- Parameters c examined: Red and white blood cell counts, measurement of hemoglobin and mean corpuscular volume, calculation of hematocrit, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and determination of differential white blood cell counts

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Immediately before sacrifice
- Animals fasted: No data
- How many animals:
- Parameters examined: Measurement of serum concentrations of glucose, urea nitrogen, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total protein, albumin and creatinine.

URINALYSIS: Yes
- Time schedule for collection of urine: One week before sacrifice

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
All rats were given a complete gross necropsy examination and organ weights were recorded for the brain, liver, kidneys, spleen, heart, adrenals and
testes. Tissues were taken and fixed in 10% neutral buffered formalin. All tissues were processed for paraffin embedding, sectioned at 5 microns and stained with hematoxylin and eosin. Microscopic lesions were graded as to severity, where possible, into 4 categories (mild, moderate, marked or severe)
Clinical signs:
no effects observed
Description (incidence and severity):
see " Details on results"
Mortality:
no mortality observed
Description (incidence):
see " Details on results"
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
see " Details on results"
Histopathological findings: neoplastic:
no effects observed
Description (incidence and severity):
see " Details on results"
Details on results:
CLINICAL SIGNS AND MORTALITY
No deaths or clinical signs of toxicity.

BODY WEIGHT AND WEIGHT GAIN
Body weight gain was depressed markedly for both sexes at the high dosage level

FOOD AND WATER CONSUMPTION
There was marked reduction in diet consumption in the high level dose females and a significant increase in the low level dose females. While water
consumption rates were equivalent in the male test and control animals, there was a dose-related reduction for the female test animals.

CLINICAL CHEMISTRY
Changes in clinical chemistry values included a reduction in serum glucose level and an elevation of alkaline phosphatase activity, AST and ALT activities

HAEMATOLOGY
In male rats, depression of the red blood cell counts and increased mean corpuscular volumes were seen. In the female rats, in addition to the above changes, there were also depression of hematocrit and hemoglobin values and an increase in mean corpuscular hemoglobin.

URINALYSIS
The median urinary pH at the high dosage level was lowered significantly in both sexes

ORGAN WEIGHTS
In the male rats, there was a significant reduction in liver, kidney, spleen and heart weight and a concomitant decrease in some of the relative organ weights. Similarly, in the females, there was a reduction in liver, heart, adrenal and brain weights with an decrease of relative liver weight.

PATHOLOGY
There was no dose-related gross lesions in any animal on the study. The most significant histologic changes were present in the livers of the high dosage level animals, particularly in the females. The liver changes, termed "hepatocellular pleomorphism", consisted of an increase in the size of hepatocytes and hepatocyte nuclei, increased variation in nuclear size and shape, and an increase in the number of multinucleate hepatocytes. Occasional degenerating hepatocytes were also seen.
Key result
Dose descriptor:
NOAEL
Effect level:
22 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Recalculated to EDA base
Key result
Dose descriptor:
LOAEL
Effect level:
114 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Clinical chemistry: elevation of alanine aminotransferase activity in males;  Haematology :Increased mean corpuscular volumes in females. Recalculated to EDA base.
Critical effects observed:
not specified

High dose group: Diet and water consumption significantly reduced in the high dose female rats. Significant reduction in body weight gain of both sexes in the high dose group; significant reduction in absolute weights of liver and heart (both sexes), adrenal and brain (female), kidney and spleen (male) in the high dose group; increase of relative weight of brain (both sexes), spleen and heart (female) and testis in the high dose group. Significant elevation of alkaline phosphatase activity in males and females. Significant elevation of alanine aminotransferase activity in males and females of high dose groups. Increased mean corpuscular volumes in males and females. Significant increase of mean corpuscular hemoglobin and significant depression of hematocrit and hemoglobin values; significant depression of red blood cell counts, serum glucose level and urinary pH (from 6.0 to 5.0) and significant elevation of aspartate aminotransferase activity in both sexes of the high dose group; hepatocellular pleomorphism in 7/10 female and 2/10 male (control: 0/10 of each sex) in high dose group, hepatocellular degeneration and significant increased prevalence of tracheitis in male of the high dose group.

Intermediate dose group:  Water consumption significantly reduced in female rats. Significant elevation of alanine aminotransferase activity in males of intermediate dose groups. Increased mean corpuscular volumes in females.  Low dose group: Water consumption significantly reduced in female rats. Significant elevation of alkaline phosphatase activity in males, this effect was not noted in the intermediate dose group.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
50 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
Please refer to section 13 for read across justification.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP, no guideline
Principles of method if other than guideline:
Four exposure groups of 30 rats (15 female/15 male) were exposed to EDA vapour. Each exposure group had a control group.
GLP compliance:
no
Species:
rat
Strain:
Sherman
Sex:
male/female
Route of administration:
inhalation: vapour
Type of inhalation exposure:
whole body
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chamber air was passed through an acidified glassbead adsorption tube. The captured Ethylenediamine was quantified by titration.
Duration of treatment / exposure:
6 weeks
Frequency of treatment:
7 h/day, 5 days/week
No. of animals per sex per dose:
15 male and 15 female
Control animals:
yes
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEC
Effect level:
59 ppm (analytical)
Sex:
male/female
Basis for effect level:
other: clinical signs; body weight; organ weights;
Key result
Dose descriptor:
NOAEC
Effect level:
144 mg/m³ air (analytical)
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
48 mg/kg bw/day (actual dose received)
Sex:
male/female
Basis for effect level:
other: Recalculated using rat weight 0.425 kg (mean male/female) and inhalation volume 0.33 m3/7h (values from guidance document)
Dose descriptor:
LOAEC
Effect level:
132 ppm
Based on:
test mat.
Sex:
male/female
Dose descriptor:
LOAEC
Effect level:
323 mg/m³ air (analytical)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
107 mg/kg bw/day (actual dose received)
Sex:
male/female
Critical effects observed:
not specified

132 ppm: The death of 4/30 animals was attributed to lung infection (not substance-related); slight depilation; body weight gain and relative weights of liver and kidney were not affected; no substance-related macroscopic or histologic changes.

225 ppm: The death of 16/30 was substance-related and another 10 death were considered not to be substance-related; the 4 rats which survived showed significantly lower weight gain and higher relative weights of liver and kidney; cloudy swelling of the liver and of the loop and convoluted tubules of the kidney; lung congestion was observed in exposed as well as in control rats in similar proportions.
484 ppm: All rats died within 20 days of initial exposure; depilation was first observed on the 6th day of exposure; cloudy swelling of the liver (in 23/28 animals), cloudy swelling and degeneration of convoluted tubules (in 7/28 animals); congestion of the lung (in 17/28 animals) and of the adrenal cortex (in 5/28 animals).
59 ppm: No effect on weight gain or organ weights. No depilation. No significant damage to examined tissues; lung, liver and kidney.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEC
144 mg/m³
Study duration:
subacute
Species:
rat
Quality of whole database:
Pre-GLP, no guideline. Please refer to section 13 for read across justification.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records
Reference
Endpoint:
repeated dose toxicity: dermal, other
Remarks:
carcinogenicity
Type of information:
experimental study
Adequacy of study:
supporting study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No data on guideline and GLP
Qualifier:
no guideline followed
Principles of method if other than guideline:
The maximum tolerated dose (for local effects) was applied to the back of 50 male mice three times weekly throughout their lifespan. Positive (3-methylcholanthrene) and negative (water) control groups (50 mice/group) were included.
GLP compliance:
no
Specific details on test material used for the study:
Substance no 1:
impurity: 0,07% ammonia
Substance no 2:
Impurities:
0.54 % pyrazine,
0.08 % ammonia,
0.03 % water,
0.02 % monomethylamine,
0.02 % ethylamine,
0.02 % N-methyl-piperazine,
0.02 % methylpyrazine,
trace dimethylamine,
trace ethanol,
trace N-ethylpiperazine,
trace ethylpyrazine
Species:
mouse
Strain:
C3H
Sex:
male
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: C3H/HeJ mice from Jackson Laboratories, Bar Harbor, Maine
- Age at study initiation: 74 to 79 days of age
- Housing: Housed individually in stainless steel cages with wire mesh floors. All mice were housed in the same room with
controlled lighting
- Diet (e.g. ad libitum): Ziegler Bros. NIH 07 pellets (Gardners, Pa.)
- Water (e.g. ad libitum): water from an automatic watering system
Vehicle:
water
Details on exposure:
Mice were treated three times weekly for their complete life span with 25 µl per application of each substance. Substances were applied with an Eppendorf pipet to the back of each mouse from which the fur was clipped once weekly.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentrations verified monthly during the study
Duration of treatment / exposure:
complete life span

Frequency of treatment:
3x/wk
Dose / conc.:
1 other: % aqueous solution/application
No. of animals per sex per dose:
Treatment group singly housed: 50 mice
Control animals:
yes, concurrent vehicle
Details on study design:
Control group singly housed: 50 mice received distilled water. Control group housed 5/cage: 40 mice received water
Positive control:
Positive control group housed 5/cage: 40 mice received 0.1% 3-methylcholanthrene in acetone
Observations and examinations performed and frequency:
All mice were examined daily, and the onset and progress of tumor growth were recorded monthly.
Key result
Dose descriptor:
NOAEL
Effect level:
1 other: % solution
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Mild fibrosis suggesting irritation to skin was noted
Remarks on result:
not measured/tested
Remarks:
Effect level not specified (migrated information)
Dose descriptor:
NOAEL
Effect level:
8.3 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: Weight of male mice 0.03 kg
Critical effects observed:
not specified

Mean survival time of the exposure group for substance 2 (598 days) was shorter than that of the singly housed control group (626 days); no treatment-related macroscopic or histopathologic findings; one mouse of the exposure group had a dermal fibrosis at application site and another one had a mammary adenocarcinoma. One sebaceous adenoma of the skin of the thorax was noted in the control group individually housed.

In the exposure group for substance 1, 1 mouse had a myxosarcoma at the base of the tail, and 11 animals had mild to moderate dermal fibrosis, suggesting skin irritation. Survival time did not differ from negative control groups.

In the 3-methylcholanthrene group, 39 of 40 mice had skin tumors including 37 with confirmed squamous cell carcinomas.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
8.3 mg/kg bw/day
Study duration:
chronic
Species:
mouse
Quality of whole database:
Pre-GLP. Please refer to section 13 for read across justification.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Study duration:
chronic
Species:
mouse

Additional information

A 28-day oral toxicity study according to OECD guideline 407 was performed on Wistar rats exposed to the test substance at 0, 10, 50, and 250 mg/kg bw/day. One of five high dose males showed impaired respiration. Four out of ten high dose females died. Decreased spontaneous activity, stilted gait, swollen abdomen, and impaired respiration were observed between days 11 and 24, mainly in the females that died. In the four high-dose females that died, macroscopically discoloration of lungs with multiple red spots on its surfaces and foamy content were observed among other findings. Histopathology revealed lesions, which included congestion of organs, pulmonary hemorrhage, and edema. In addition, one of the females exhibited marked loss of lymphatic follicles of the spleen with massive marginal zone and periarteriolar lymphoid sheath atrophy. The high-dose male rat that exhibited clinical signs had focal ballooning degeneration of the squamous epithelium of the fore-stomach. These data demonstrate that DMAPA induces clinical symptoms, systemic toxicity in male and additionally mortality in female rats when administered 28 times during 29 days at the dose level of 250 mg/kg body weight. The female rats seem to be more sensitive. Although a direct systemic-toxic effect on the respiratory tract cannot be excluded, it can be considered as more likely that the observed findings are related to the oral treatment with this corrosive substance. Due to gavage either local placement in the laryngeal/pharyngeal area or secondary aspiration after reflux from the stomach/esophagus appeared reasonable as noted for other corrosive substances too. The NOAEL was 50 mg/kg bw/day (Hoechst 1996; reliability score: 1).

The potential toxicity of the test item, diethylaminopropylamine(as pH-neutralized dose formulations), was evaluated following daily oral administration (gavage) to rats for 2 weeks in order to assist the selection of dose-levels for a further OECD TG 408 study to be performed in the same species. Three groups of five male and five female Sprague-Dawley rats received the test item, by daily oral administration (gavage) for 14 days, at dose-levels of 100, 300 or 1000 mg/kg bw/day. The test item was administered as a solution in the vehicle (drinking water treated by reverse osmosis) at a constant dosagevolume of 5 mL/kg/day.The pH of the dosing solutions was adjusted to 8.0 (± 0.5) using a solution of hydrochloric acid. A control group of five males and five females received the vehicle alone under the same experimental conditions. No unscheduled death occurred during the study. Ptyalism was observed at 1000 mg/kg bw/day (1/5 males and 4/5 females). Lower body weight gain was observed at 1000 mg/kg bw/day in males and females over the first 3 days of the study together with slight lower food consumption. This effect was present in a lesser extent in males throughout the study and resulted in a lower final body weight. No macroscopic findings were attributed to treatment with the test item. Slight hyperplasia of squamous cells associated with minimal to slight hyperkeratosis was observed at 1000 mg/kg bw/day in the forestomach of 5/5 males and 4/5 females and was considered to be non-adverse. No treatment-related effects were observed at 100 and 300 mg/kg bw/day (Consortium Alkylamines 42759 TSR).

In a standard subchronic toxicity study (OECD TG 408/GLP) with the structurally related DEAPA Sprague-Dawley rats were treated daily by gavage with the test item for 13 weeks. Male and female rats received dietary target doses of 0, 50, 250 or 750 mg/kg bw/d of DEAPA. During the treatment period, three females given 750 mg/kg bw/d showed clinical signs which were considered to be adverse (i.e. hunched posture, dyspnea, abdominal, loud breathing and/or bent head). One female given 750 mg/kg bw/d more severely affected, showed signs of very poor clinical condition and was sacrificed in week 11. Body weight gain was slightly lower but not adversely effected in males given 750 mg/kg bw/d. Food consumption was not affected by the test item treatment. No ophthalmology findings were observed at the end of the treatment period. Estrous cycle was not altered by the test item treatment. The epididymal sperm motility and morphology and the spermatozoa count were unaffected by the test item treatment. At hematology only minimal to very slight alterations were noted what were finally considered as not adverse. The same was true for none adverse findings in clinical chemistry and urinalysis. Moreover, all changes in hematology, clinical chemistry and urinalysis were completely reversible. At the end of the treatment period, only minor and non-adverse findings were noted in isolated organs and/or animals by histopathology at 750 mg/kg bw/d in both sexes and at 250 mg/kg bw/d in single females. Especially in the females, some of them may be considered as stress-related and due to the irritant potential. Finally, the NOAEL for males was considered to be 750 mg/kg bw/d, and for females 250 mg/kg bw/d predominantly due to the severe clinical findings including death (Consortium Alkylamines 42760 TCR).

There is a subchronic study with the structurally related ethylene diamine (EDA) available (Peters, 1982, reliability score: 2). In this 13-week study, rats received the test substance at doses of 0, 100, 200, 400, 600 or 800 mg/kg bw/day by gavage on weekdays only. At the highest dose, 6 male and 1 female of 10 rats/sex died during the study. Decreased body weight gains were noted in 200 - 800 mg/kg bw/day group of males and in the 400 - 800 mg/kg bw/day group of females. These changes ranged from –20% in 200 mg/kg bw/day male group to –47% in 800 mg/kg bw/day male group and –10% in 400 mg/kg bw/day female group to –50% in 800 mg/kg bw/day female group. Males appeared to be more severely affected than females. Thymus to body weight mean ratios of the dose group decreased as a function of increasing dose at 200 mg/kg bw/day in males and 600 mg/kg in females. However, there were no accompanying histopathologic changes. Histopathologic changes were noted in the eyes, kidneys and uterus. Eye lesions were present to some degree in 100-800 mg/kg bw/day rats. In the more severe cases the retina was lacking all the normal layers while in less severe cases there was only rosetting and focal cellular losses. Renal tubular lesions were only observed in the 600 and 800 mg/kg bw/day groups. These lesions were characterized by degeneration, regeneration and occasional necrosis of the tubular epithelium. Hypoplasia of the uterus was noted in the high dose group and was attributed to inanition. No NOAEL could be established in this 90 day rat oral gavage study. The LOAEL was 100 mg/kg bw/day based on the eye effects.

In an additionally subchronic study with the structurally related ethylenediammonium dichloride (EDA*2HCL, Yang et al., 1983, reliability score: 2, see read across justification section 13), male and female rats were received dietary target doses of 0, 50, 250 or 1000 mg/kg bw/day of EDA-2HCl for three months. There were no deaths and no abnormal clinical signs noted during the study. Body weight gains were significantly decreased in the high dose group which affected a number of absolute and relative organ weights in both males and females. Water consumption was comparable to control values at all dose levels in males but was decreased in a dose-response manner in female rats at all 3 dose levels. Slight reductions in serum glucose levels and an elevation of alkaline phosphatase, AST and ALT activities were observed in the high dose group. An elevation of ALT activity was also observed in the intermediate dose male rats. Urinary pH in t he high dose group was decreased in both males and females. There were no dose-related gross lesions in any animal on the study. The most significant histopathologic lesion, hepatocellular pleomorphism, was observed primarily in the high dose female and, to a lesser extent, in male rats. The LOAEL was 250 mg/kg bw/day. Since the water consumption was only slightly decreased at 50 mg/kg bw/day, the NOAEL was considered to be approximately 50 mg/kg bw/day for EDA*2HCl (corresponding to 22 mg/kg bw/day of EDA).

With the structurally related ethylenediammonium dichloride (EDA*2HCL) there is a combined chronic toxicity and carcinogenicity study available (Hermansky et al., 1999, reliability score: 2, see section 13, read across justification). Groups of each 100 F344 rats per sex received the test substance orally via the feed at adjusted dose levels of 0, 20, 100, 350 mg/kg bw/day (males) or 0, 20, 100 and 360 mg/kg bw/day (females). Mortality was comparable to controls during the first 18 months of the study. After 22 months, mortality in males and females increased at the high dose and in females at 100 mg/kg bw/day after 24 months. There was a significant reduction in body weight gain in male rats and female rats of the high dose group. Significant increases of the relative weights of predominantly the liver and kidney were noted in rats of the high dose group. Significantly higher incidence of hepatocellular pleomorphism in female rats of the intermediate and the high dose groups; rhinitis and tracheitis were also observed in high dose males and females.There was no evidence for a carcinogenic potential ofEDA*2HCLin male and female F344 rats, when administered via the diet over the course of 2 years.The NOAEL for chronic toxicity was 20 mg/kg bw/day for EDA*2HCl (corresponding to 9 mg/kg bw/day of EDA).

With the structurally related ethylene diamine (EDA) there is a subacute inhalation toxicity study available (Pozzani and Carpenter, 1954, reliability score: 2, see section 13, read across justification). In this 6 week study four exposure groups of 30 Sherman rats (15 females/15 males) were exposed to EDA vapour for 7 h/day for 5 days per week at concentrations of 59, 182, 225 and 484 ppm. Each exposure group was compared to a control group.After termination, liver and kidney weights were recoded and samples of liver, lung, heart, kidney adrenal gland and spleen were taken for histological examinations. There was a concentration related increase in mortalities, body weight reductions and indication for liver, kidney and lung damage, especially at the highest concentration.The NOAEC for subacute inhalation was 59 ppm (corresponding to 144 mg/m3).

With the structurally related ethylene diamine (EDA) there is a dermal carcinogenicity study available (DePass et al., 1984, reliability score: 2, see section 13, read across justification).Male CH3 mice received 25μl of an 1% aqueous solution of EDA, the maximum tolerated dose for local effects,to the clipped skin on the backthree times weekly for their complete life span. Two EDA samples from different producers were tested on groups of 50 mice. A negative control group was treated with water and a positive control group received 0.1% of 3 -methylcholanthrene in acetone.Ten mice were intermittently sacrificed at 18 months. Complete necropsies were performed on all mice at intermittent and terminal sacrifice.  The dorsal skin from all animals plus all gross lesions of animals at terminal sacrifice as well as all livers, kidneys and lungs from the 18 month sacrifice were examined histopathologically.There were no treatment-related macroscopic or histopathological findings. No skin tumors were observed in the EDA treated animals. In the positive control group, 98% of the animals revealed skin tumors. Thus, there was no evidence for a carcinogenic potential of EDA after life-time dermal administration in male C3H mice. The NOAEL was 8.3 mg/kg bw/day (25 µL 1% solution/per mouse).


Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. As a result, the substance is not considered to be classified under Regulation (EC) No 1272/2008, as amended for the ninth time in Regulation (EU)No 2016/1179.