N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Sep 2017 to 13 Mar 2018

Reliability:

1 (reliable without restriction)

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Report date:

2018

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

EC Number:

247-361-2

EC Name:

N'-(ethylcarbonimidoyl)-N,N-dimethylpropane-1,3-diamine monohydrochloride

Cas Number:

25952-53-8

Molecular formula:

C8H17N3.ClH

IUPAC Name:

N-[3-(dimethylamino)propyl]-N'-ethylcarbodiimide hydrochloride

Test material form:

solid

Specific details on test material used for the study:

Appearance: White crystalline powder
Batch: AAN0680
Purity/Composition: 99.9 %
Test item storage: In refrigerator (2-8°C)
Stable under storage conditions until: 30 August 2018 (expiry date)
Purity/Composition correction factor: No correction factor required
CAS number: 25952-53-8

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han)

Sex:

male/female

Details on test animals or test system and environmental conditions:

On 27 Sep 2017, female Crl: WI(Han) rats were received and on 11 Oct 2017, male Crl:WI(Han) rats were received from Charles River Deutschland, Sulzfeld, Germany. At initiation of dosing, males were 11 weeks old and weighed between 263 and 306 g and females were 13 weeks old and weighed between 195 and 225 g. A health inspection was performed before the initiation of dosing.

Prior to start of the pre-test period (females) or treatment period (males), each animal was identified using earmark and tattoo. Prior to the pre-test period, reserve females were numbered R1 through R8 at random by indelible marker. Pups were identified on postnatal day (PND) 1. They were randomized per litter and individually identified by means of subcutaneous injection of Indian ink. When general hair growth blurred the identification, the pups were identified by tattoo on the feet.

The animals were allowed to acclimate to the Test Facility toxicology accommodation for 7 days prior to start of the pre-test period (females) or 7 days before the commencement of dosing (males).

On arrival and following the pre-test (females only) and pre-mating period, animals were group housed (up to 5 animals of the same sex and same dosing group together) in polycarbonate cages (Macrolon, MIV type, height 18 cm). During the mating phase, males and females were cohabitated on a 1:1 basis in Macrolon plastic cages (MIII type, height 18 cm). During the post-mating phase, males were housed in their home cage (Macrolon plastic cages, MIV type, height 18 cm) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type, height 18 cm).
During the lactation phase, females were housed in Macrolon plastic cages (MIII type, height 18 cm). Pups were housed with the dam, except during locomotor activity monitoring of the dams, when the pups were kept warm in their home cage using bottles filled with warm water. In order to avoid hypothermia of pups, pups were not left without their dam or a bottle filled with warm water for longer than 30-40 minutes.
During locomotor activity monitoring, animals were housed individually in a Hi-temp polycarbonate cage (Ancare corp., USA; dimensions: 48.3 x 26.7 x 20.3 cm) without cage-enrichment, bedding material, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaier & Söhne GmbH + CO. KG, Rosenberg, Germany) and were equipped with water bottles. The rooms in which the animals were kept were documented in the study records.
Animals were separated during designated procedures/activities. Each cage was clearly labeled with a color-coded cage card indicating Test Facility Study No., group, animal number(s), and sex.

Environmental Conditions
Target temperatures of 18 to 24°C with a relative target humidity of 40 to 70% were maintained. The actual daily mean temperature during the study period was 22°C with an actual daily mean relative humidity of 38 to 57% (see deviations in Appendix 8). A 12 hour light/12 hour dark cycle was maintained, except when interrupted for designated procedures. Ten or greater air changes per hour with 100% fresh air (no air recirculation) were maintained in the animal rooms.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

Groups 1-3: The test item and vehicle were administered to the appropriate animals by once daily oral gavage 7 days a week for a minimum of 28 days. Males were treated for 29 days, i.e. 2 weeks prior to mating, during mating, and up to and including the day before scheduled necropsy. Females that delivered were treated for 50-56 days, i.e. 14 days prior to mating (with the objective to cover at least two complete estrous cycles), the variable time to conception, the duration of pregnancy and 13 or 15 days after delivery, up to and including the day before scheduled necropsy. Females without offspring (not pregnant) were treated for 41 days.
Group 4: Due to severe toxicity, high dose animals were treated for 7-9 days. Six Group 4 animals died or were euthanized in extremis on Days 7-8, the 14 survivors were sacrificed on Day 10.
The first day of dosing was designated as Day 1.
Female nos. 47 (Group 1), 51, 53, 54, 55, 58, 59, 60 (Group 2) and 65 (Group 3), were not dosed on one occasion as these females were littering at the time of dosing. The omission of one day of dosing over a period of several weeks was not considered to affect the toxicological evaluation.
Animals were dosed approximately at the same time each day with a maximum of 6 hours difference between the earliest and latest dose. The dose volume for each animal was based on the most recent body weight measurement. The doses were given using a plastic feeding tube.
The dosing formulations were stirred continuously during dose administration.
A dose control system (DCS) was used as additional check to verify the dosing procedure according to Standard Operating Procedures.
Pups were not treated directly but were potentially exposed to the test item in utero, via maternal milk, or from exposure to maternal urine/feces.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were performed using a validated analytical procedure (Analytical work instruction AWI 4296, entitled: 1-(3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (TS208713) in formulations using LC-MS/MS, validated in ABL validation study no. 17251).

Concentration Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Concentration results were considered acceptable if mean sample concentration results were within or equal to ± 10% for solutions of target concentration.

Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homogeneity results were considered acceptable if the coefficient of variation (CV) of concentrations was +/-10%.

Stability Analysis
Stability analyses performed previously in conjunction with the method development and validation study (ABL No. 17251) demonstrated that the test item is stable in the vehicle when prepared and stored under the same conditions at concentrations bracketing those used in the present study. Stability data have been retained in the study records for ABL No. 17251.

Duration of treatment / exposure:

28 days (Males were treated for 29 days)

Frequency of treatment:

7 days a week

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males/10 females

Control animals:

yes, concurrent vehicle

Details on study design:

The oral route of administration was selected because this is a possible route of human exposure during manufacture, handling or use of the test item.
The dose levels were selected based on the results of a dose range finder with oral administration of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride in rats.
The animals were randomly allocated to treatment groups using a randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Positive control:

none

Examinations

Observations and examinations performed and frequency:

Throughout the study, animals were observed for general health/mortality and moribundity twice daily, in the morning and at the end of the working day. Animals were not removed from cage during observation, unless necessary for identification or confirmation of possible findings.
Animals showing pain, distress or discomfort which was considered not transient in nature or is likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The circumstances of any death were recorded in detail
Clinical observations were performed once daily, beginning during the first administration of the test item and lasting throughout the dosing periods up to the day prior to necropsy.
During the dosing period, these observations were performed after dosing at no specific time point, but within a similar time period after dosing for the respective animals (no peak effect of occurrence of clinical signs was observed in the dose range finder. The time of onset, grade and duration of any observed sign was recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Arena Observations – F0-Generation
Clinical observations were conducted in a standard arena beginning before the first administration of the test item and then once weekly throughout treatment (except in Weeks 6 and 7
Animals were weighed individually on the first day of treatment (prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
In order to monitor the health status, Animal Nos. 32, 72, 75 and 79 were also weighed on Day 7 of the treatment period
Terminal body weights were recorded on the day of necropsy (fasted for males, non-fasted for females).

Food consumption was quantitatively measured weekly, except for males and females which were housed together for mating and for females without evidence of mating. Food consumption of mated females was measured on Days 0, 4, 7, 11, 14, 17, and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.

Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.
For Groups 1-3, functional tests were performed on the selected 5 males during Week 4 of treatment and the selected 5 females during the last week of lactation (i.e. PND 6-13). These tests were performed after dosing, after completion of clinical observations.
The following tests were performed (abbreviations mentioned in the respective tables are indicated between brackets):
• Hearing ability (HEARING) (Score 0 = normal/present, score 1 = abnormal/absent).
• Pupillary reflex (PUPIL L/R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Static righting reflex (STATIC R) (Score 0 = normal/present, score 1 = abnormal/absent).
• Fore- and hind-limb grip strength, recorded as the mean of three measurements per animal (
• Locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA).

Sacrifice and pathology:

Animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination.
Scheduled necropsies were conducted on the following days:
Males: Following completion of the mating period (a minimum of 28 days of administration).
Females which delivered: PND 14 or 16.
Females which failed to deliver
(Nos. 46, 57 and 63): With evidence of mating: Post-coitum Day 25-26
All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0- females were not fasted

Sample Collection
For Groups 1-3, blood of F0-animals was collected on the day of scheduled necropsy. Samples were collected, between 7.00 and 10.30 a.m., from the retro-orbital sinus under anesthesia using isoflurane in the animal facility. Due to clotting of non-serum samples of individual animals, additional blood samples were obtained in the necropsy room. After collection, all samples were transferred to the appropriate laboratory for analysis.
Group 1-3 F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Blood of F1-animals was collected on PND 4 and PND 14-16, if possible. This was performed in the necropsy room

HAEMATOLOGY: Yes
- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals: all surviving animals from each test and control group at the end of the study
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices - mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count - neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time
(APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11
mol/L).

CLINICAL CHEMISTRY: Yes
-- Time schedule for collection of blood: day 28
- Anaesthetic used for blood collection: Not specified
- Animals fasted: No
- How many animals:
all surviving animals from each test and control group at the end of the study
The following parameters were measured on plasma from blood collected into tubes
containing lithium heparin anti-coagulant:
Urea, Inorganic phosphorus (P)
Glucose, Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.), Alanine aminotransferase (ALAT)
Albumin, Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation), Creatinine (Creat)
Sodium (Na+), Triglycerides (Trigs/Tri)
Potassium (K+), Total cholesterol (Chol)
Chloride (Cl-), Total bilirubin (Bili)
Calcium (Ca++), Bile acids

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
Prior to the start of treatment and on Days 6, 13, 20 and 27 all animals were observed for
signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different
stimuli. Observations were carried out from approximately two hours after dosing on each
occasion.
- Battery of functions tested: sensory activity / grip strength / motor activity / other: behavior assessment

IMMUNOLOGY: No

Other examinations:

Organ Weights
The following organs, removed from animals that were killed at the end of the dosing period were dissected free from fat and weighed before fixation:
Brain
Cervixa
Epididymisb
Gland, adrenalb
Gland, coagulationb, c
Gland, parathyroidd
Gland, prostate
Gland, seminal vesicleb
Gland, thyroid
Heart
Kidneyb
Liver
Ovariesb
Spleen
Testesb
Thymus
Uterus
a Weighed together with the uterus.
b Paired organ weight.
c Weighed together with the seminal vesicles.
d Weighed together with the thyroid.

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Adrenals, Ovaries
Aorta (thoracic), Pancreas
Bone & bone marrow (femur including stifle joint), Pituitary
Bone & bone marrow (sternum), Prostate
Brain (including cerebrum, cerebellum and pons), Rectum
Salivary glands (submaxillary)
Caecum, Sciatic nerve
Colon, Seminal vesicles (with coagulating glands and fluids)
Duodenum
Epididymides ♦ Skin
Esophagus Spinal cord (cervical, mid thoracic and lumbar)
Eyes *
Gross lesions, Spleen
Heart, Stomach
Ileum, Testes ♦
Jejunum, Thymus
Kidneys, Thyroid/Parathyroid
Liver, Trachea
Lungs (with bronchi)#, Urinary bladder
Lymph nodes (mandibular and mesenteric), Uterus & Cervix
Mammary gland , Vagina
Muscle (skeletal)

* Eyes fixed in Davidson’s fluid Preserved in modified Davidson’s fluid

Statistics:

All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% and 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE when deemed appropriate) were reported whenever possible. Inferential statistics were performed according to the comparison matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1

Parametric
Datasets with at least 3 groups (the designated control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test).

Non-Parametric
Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test).
The motor activity data set was compared using an overall Kruskal-Wallis.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs in the 300 mg/kg animals that died or were euthanized prematurely are described in the section on mortality
Findings in the remaining animals of the 300 mg/kg group (all sacrificed at study Day 10) and in animals of the lower dose groups are described below.
Rales occurred in females of the 300 mg/kg group, mostly at Days 7-8.
Salivation was noted in all animals of the 300 mg/kg group (at Days 7-8) and in most females of the 100 mg/kg group (on several days in treatment Weeks 1, 2 and 6). This salivation was considered to be a physiological response rather than a sign of systemic toxicity considering its slight severity and the time of occurrence (i.e. after dosing), and may be related to the irritancy of the test item.
No additional clinical signs were noted during the weekly arena observations.
Any other clinical signs noted incidentally occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and showed no dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

Mortality:

mortality observed, treatment-related

Description (incidence):

At 300 mg/kg there were six premature decedents (three males and three females) which were considered to be related to treatment with the test item. After six or seven days of treatment, one male (no. 39) was found dead (necropsy on the same day) and five animals were euthanized for humane reasons (males nos. 32 and 38 and females nos. 72, 75 and 78; female no. 78 was necropsied at study Day 8, the other animals at Day 7).
All decedents showed respiratory difficulties (gasping and/or rales) at treatment Days 6 and/or 7. The females showed body weight loss in the week prior to death (8-13% of their initial weight). Male no. 32 showed normal weight gain prior to sacrifice (no data on weight gain were available for the other two males).

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In females at 300 mg/kg, body weight loss occurred in the three females which were euthanized at Days 7-8 (8-13% of their initial weight) and, to a lesser extent, in 5/7 females sacrificed at Day 10 (2-6% of their initial weight). Body weight gain of the 300 mg/kg males sacrificed at Day 10 was normal, except in male no. 34 which lost 1% of its initial weight. No weight gain data were available for 2/3 males that died at Day 7 (the third male gained some weight). Mean body weights at 300 mg/kg did not differ significantly from those of controls.
No treatment-related changes in body weight (gain) were observed in males and females treated up to 100 mg/kg. The slightly lower mean body weight gain noted in 100 mg/kg females at Day 13 of the lactation period was due to lower weight gain or initial weight loss followed by normal growth in three animals which occurred in the absence of signs of toxicity. Moreover, the difference from controls was not statistically significant. Therefore, this finding was not attributed to treatment.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males and females at 300 mg/kg consumed about 20% less food than controls during their short treatment period.
No treatment-related changes in food consumption before or after correction for body weight were noted in rats treated up to 100 mg/kg.

Food efficiency:

not examined

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

no effects observed

Description (incidence and severity):

Subjective appraisal was maintained during the study, but no quantitative investigation was introduced as no effect was suspected.

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg was not associated with changes in red blood cell parameters, white blood cell parameters or number of platelets.
An isolated, statistically significant difference noted in females (higher mean corpuscular hemoglobin concentration at 30 mg/kg) was considered to be unrelated to treatment due to the lack of a dose-related trend.
Coagulation parameters were considered not to be affected by treatment up to 100 mg/kg.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg was not associated with changes in clinical chemistry parameters.
An isolated, statistically significant difference noted in females (higher mean calcium concentration at 30 mg/kg) was considered to be unrelated to treatment due to the lack of a dose-related trend.

Thyroid hormone analyses:
Serum levels of T4 in F0 males were considered not to be affected by treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No treatment-related findings were noted for functional observations up to 100 mg/kg.
Hearing ability, pupillary reflex and static righting reflex were normal in all examined animals. Grip strength was not affected by treatment.
The variation in motor activity did not indicate a relation with treatment. All examined groups showed a similar habituation profile with a decreasing trend in activity over the duration of the test period. Higher (females) or lower (males) mean values for total movements and ambulations were noted at 30 mg/kg. As the differences from control values were not statistically significant and showed no dose-related trend, they were regarded as unrelated to treatment.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Treatment up to 100 mg/kg was not associated with changes in organ weights.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related gross observations in animals treated up to 100 mg/kg. Macroscopic findings in animals treated at 300 mg/kg are described in the section on mortality
All of the recorded macroscopic findings were within the range of background gross observations encountered in rats of this age and strain. These findings were therefore considered to be unrelated to treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

There were no test item-related microscopic observations in animals treated up to 100 mg/kg. At 300 mg/kg, findings were mainly present in the gastro-intestinal tract for the six premature decedents
no microscopic examination was performed for the remaining Group 4 animals.
All of the recorded microscopic findings at 30 and 100 mg/kg were within the range of background pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations

Histopathological findings: neoplastic:

not examined

Description (incidence and severity):

There were no test item-related microscopic observations in animals treated up to 100 mg/kg. At 300 mg/kg, findings were mainly present in the gastro-intestinal tract for the six premature decedents
no microscopic examination was performed for the remaining Group 4 animals. All of the recorded microscopic findings at 30 and 100 mg/kg were within the range of background
pathology encountered in rats of this age and strain. There was no test item related alteration in the prevalence, severity, or histologic character of those incidental tissue alterations
Principal macroscopic and microscopic findings in the decedents in the 300mg/kg group consisted of: Stomach:
- Reddish foci/focus (microscopic correlate: slight hemorrhages).
- Thickened limiting ridge (microscopic correlates: ulcerations/erosions (up to marked) and/or inflammation (moderate) and/or edema (slight) of the glandular stomach).
- Distended with gas (no microscopic correlate). Intestines (all parts except for the rectum):
- Distended with gas (no microscopic correlate).
Mesenteric lymph node:
- Dark red discoloration (microscopic correlates: erythrophagocytosis and/or sinusoidal erythrocytes).
Trachea:
- Necrosis (up to massive) and/or inflammation (marked) in several animals and perforation of the trachea in one of them (male no. 39). The perforation may suggest that the tracheal lesions were related to the dosing procedure (oral gavage), either directly or indirectly by reflux. It seems unlikely that the tracheal lesions were caused directly by the test item.
All remaining animals of the 300 mg/kg group were euthanized for humane reasons after nine days of treatment (necropsy at study Day 10) as severe gastrointestinal effects were anticipated for these animals based on the findings in the decedents. This was confirmed at macroscopic examination (these animals were not subjected to microscopic examination).

These animals had similar macroscopic changes in the stomach (reddish foci and/or thickened limiting ridge) and mesenteric lymph nodes (dark red discoloration) as the decedents. Additional findings included thickened wall of the duodenum and/or jejunum (both sexes) and enlarged axillary and/or mesenteric lymph nodes (males only). Female no. 48 of the control group died at the scheduled necropsy date when she was under anesthesia for blood collection.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no effects seen in the 100mg/kg dose group

Target system / organ toxicity

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: toxicity seen in the highest dose group at days 6-7. Respiratory difficulty, weight loss

Organ:

intestine

mesenteric lymph node

stomach

trachea

other: Highest dose group of 300mg/kg were euthanised due to mortaility and for humane resaons

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

no

Applicant's summary and conclusion

Conclusions:

Severe effects seen at the higest test group of 300 mg/kg/day at days 6-7 in both sexes with mortality, body weight loss and severe clinical effects observed. As a result all animals were euthanised for humane reasons. As a result the substance is classifed as STOT RE Cat 2 as per the CLP 1272/2008 requirements

Executive summary:

The objectives of this study were to determine the potential toxic effects of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride when given orally by gavage for a minimum of 28 days to Wistar Han rats, and to evaluate the potential to affect male and female reproductive performance such as gonadal function, mating behaviour, conception, parturition and early postnatal development.

In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.

The dose levels in this study were selected to be 0, 30, 100 and 300 mg/kg/day, based on the results of the dose range finder.

Chemical analyses of formulations were conducted once during the study to assess accuracy and homogeneity.

The following parental parameters were evaluated in this study: mortality/moribundity, clinical signs,functional tests,body weight, food consumption, estrous cycle length and regularity,clinical pathology,serum level of thyroid hormone T4 (F₀-males), gross necropsy findings, organ weights and histopathologic examination. Group 4 (300 mg/kg) was terminated on study Day 10 after nine days of treatment due to severe toxicity (including mortality). Group 4 animals were not subjected to functional tests and clinical pathology examinations. At necropsy on Day 10, no organs were weighed and only gross lesions were preserved for possible future histopathological evaluation. A full histopathological examination was conducted on the six Group 4 animals that died or were euthanized for humane reasons on Day 7 or 8 (their organs were not weighed).   

The following reproduction/developmental parameters were determined for Groups 1-3: mating and fertility indices, precoital time, number of implantation sites, gestation index and duration, parturition, maternal care, sex ratio and early postnatal pup development (mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, serum level of thyroid hormone T4 in PND 14-16 pups, and macroscopy).

Formulation analysis showed that formulations were prepared accurately and homogeneously.

Parental results

At 300 mg/kg, three males and three females became moribund, resulting in premature death or euthanasia after six or seven days of treatment. These animals showed respiratory difficulties (gasping and/or rales) and the females had lost weight. Macroscopic findings consisted of reddish foci (slight hemorrhages) and thickened limiting ridge in the stomach (microscopic correlates: up to marked ulcerations/erosions, moderate inflammation and/or slight edema), gas distended intestines, red discolored mesenteric lymph nodes (microscopic correlates: erythrophagocytosis and/or sinusoidal erythrocytes), and, in one animal, a perforation in the trachea (microscopic correlate: necrosis, which also occurred in two decedents which showed no perforation at necropsy). Necropsy findings in the remaining animals (sacrificed on Day 10) included similar changes in the stomach and mesenteric lymph nodes as the decedents, and, additionally, a thickened wall of the duodenum and/or jejunum in both sexes and enlarged lymph nodes (mostly the axillary lymph nodes) in males. 

Other findings at 300 mg/kg consisted of slight salivation after dosing (regarded as a physiological response rather than a sign of systemic toxicity) and reduced body weight gain (females) and food consumption (both sexes). 

Test item-related findings in animals treated up to 100 mg/kg were limited to slight salivation after dosing among 100 mg/kg females. This was regarded as a physiological response, related to the irritant properties of the test item, rather than a sign of systemic toxicity. In conclusion, based on the results of this combined 28-day repeated dose toxicity study with the reproduction/developmental toxicity screening test, the following No Observed Adverse Effect Levels (NOAELs) of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were established:

Parental NOAEL:      100 mg/kg, based on moribundity/mortality due to adverse changes in the stomach (ulcerations/erosions and inflammation) and trachea (necrosis and inflammation)