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

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

The objectives of the OECD 422 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. 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.

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).

No reproductive or developmental toxicity was observed up to 100 mg/kg resulting in a Reproductive and Developmental NOAEL of 100mg/kg. Note: Reproductive performance and developmental endpoints could not be evaluated at 300 mg/kg due to the test item-related early termination of this dose group.

Link to relevant study records

Reference

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

04 Sep 2017 to 13 Mar 2018

Reliability:

1 (reliable without restriction)

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

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

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, ma les 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 hyp
othermia 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 mat
erial, food and water. The cages contained appropriate bedding (Lignocel S 8-15, JRS - J.Rettenmaie
r & 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.

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on exposure:

Groups 1-3: The test item and vehicle were administered to the appropriate animals by once daily ora
l 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 off
spring (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 r
ecent 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. Con
centration results were considered acceptable if mean sample concentration results were within or e
qual to ± 10% for solutions of target concentration.
Homogeneity Analysis
Duplicate sets of samples (approximately 500 mg) were sent to the analytical laboratory. Homo
geneity 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

Dose / conc.:

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

Dose / conc.:

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

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

Oestrous cyclicity (parental animals):

Estrous cycles were evaluated by examining the vaginal cytology of samples obtained by vaginal
lavage.Daily vaginal lavage was performed for all (surviving) females beginning 14 days prior to treatment
(pre-test period), the first 14 days of treatment and during mating until evidence of copulation was
observed. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrus.
This was done for all females, except for females that did not survive until planned necropsy and
non-pregnant females

Sperm parameters (parental animals):

none

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 anima
ls 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.

Dermal irritation (if dermal study):

not examined

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 necrops
ied 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):

not examined

Description (incidence and severity):

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

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

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-re
lated 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.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

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

Reproductive function: oestrous cycle:

effects observed, non-treatment-related

Description (incidence and severity):

Length and regularity of the estrous cycle were considered not be affected by treatment up to 100 mg/kg.
Most females at 30 and 100 mg/kg had regular cycles of four days. One 100 mg/kg female had an irregular cycle (no. 65) and another 100 mg/kg female (no. 67) was acyclic. Both females had normal precoital intervals (they showed evidence of mating after one or two days of cohabitation) and delivered normal litters. Moreover, there were no treatment-related changes in the morphology of the female reproductive tract. Therefore, these incidental findings at 100 mg/kg were considered not to reflect an effect of the test item on estrous cyclicity.
At 300 mg/kg, 7/10 females had regular cycles (mostly of four days) during the pre-mating period, suggesting that estrus cyclicity was not affected at 300 mg/kg. However, there were no corroborative mating data (precoital time, mating index) to confirm this, and during their short treatment period (9 days) only one complete cycle was covered. This was considered insufficient evidence to substantiate any conclusion for this parameter at 300 mg/kg.
For three females at 300 mg/kg (nos. 72, 75 and 78, euthanized on study Days 7 or 8) cycle length during treatment could not be determined.
Gestation index and duration of gestation were not affected by treatment up to 100 mg/kg. All pregnant females had live offspring (gestation index 100% for all groups examined).

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, non-treatment-related

Description (incidence and severity):

Three couples had no offspring (females were not pregnant despite evidence of mating): female/male nos. 46/6 (control), 57/17 (30 mg/kg) and 63/23 (100 mg/kg). No abnormalities were seen in the reproductive organs which could account for these non-pregnancies.
There were no morphological findings in the reproductive organs of either sex which could be attributed to the test item, and stage aware evaluation of the testes did not show any indication for abnormal spermatogenesis.
Fertility index was not affected by treatment up to 100 mg/kg.
Except for one control female and one female at 30 and 100 mg/kg (nos. 46, 57 and 63, respectively), all mated females were pregnant.
These cases of non-pregnancy, without related histopathology changes in reproductive organs, were considered to be unrelated to treatment due to their incidental occurrence and lack of a dose-related trend. No signs of difficult or prolonged parturition were noted among the pregnant females. Examination of cage debris of pregnant females revealed no signs of abortion or premature birth. No deficiencies in maternal care were observed.
Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 92% for the control group and 87% for the 30 and 100 mg/kg groups. Although the index for both treated groups was slightly lower than that for the control group, this difference was not regarded as test item-related since the indices in the treated groups showed no dose-related trend and remained in the normal range.

Only data of the control and 30 and 100 mg/kg groups were available for evaluation of the reproduction endpoints reported below (except for estrous cyclicity). The 300 mg/kg group was terminated early (during the pre-mating period) due to severe toxicity.
Mating index was not affected by treatment up to 100 mg/kg. All females showed evidence of mating (mating index 100% for all groups examined).
Precoital time was not affected by treatment up to 100 mg/kg. All paired females showed evidence of mating within four days.
Number of implantation sites was considered not to be affected by treatment.
One female of the 100 mg/kg group had only three implantation sites. Such a low number of implantation sites occasionally occurs in untreated controls. All other 100 mg/kg females had normal numbers of implantation sites. Therefore, the low number of implantation sites in female no. 64 was regarded as unrelated to treatment

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

Clinical signs:

no effects observed

Description (incidence and severity):

No clinical signs occurred among pups that were considered to be related to treatment.
The clinical signs observed incidentally remained within the range considered normal for pups of this age, and were therefore considered to be unrelated to treatment.

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, non-treatment-related

Description (incidence and severity):

Viability index (number of live offspring on PND 4 before culling as percentage of number of live offspring on PND 1) was not affected by treatment. The viability indices were 99% for the control and 100 mg/kg groups and 98% for the 30 mg/kg group.
One pup of the control group (litter no. 45), two pups of the 30 mg/kg group (litter no. 52) and one pup of the 100 mg/kg group (litter no. 69) went missing at PND 2 (presumably cannibalized). This postnatal loss was regarded as unrelated to treatment as it was incidental and showed no dose-related trend.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

no effects observed

Description (incidence and severity):

Lactation index (number of live offspring on PND 13 as percentage of number of live offspring after culling on PND 4) was not affected by treatment up to 100 mg/kg. No pups died after PND 4, resulting in a lactation index of 100% for all groups.

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

not examined

Ophthalmological findings:

not examined

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Serum T4 levels in male and female PND 14-16 pups were not affected by treatment up to 100 mg/kg

Urinalysis findings:

not examined

Sexual maturation:

no effects observed

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

Only data of the control and 30 and 100 mg/kg groups were available for evaluation of the developmental endpoints reported below. The 300 mg/kg group was terminated early (during the pre-mating period) due to severe toxicity.

Dose descriptor:

NOAEL

Generation:

F1

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 up to 100 mg/kg

Remarks on result:

not determinable due to absence of adverse toxic effects

Reproductive effects observed:

no

Lowest effective dose / conc.:

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

Treatment related:

no

Conclusions:

Wistar Han rats were treated with 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride by daily oral gavage at dose levels of 30, 100 and 300 mg/kg (10 rats/sex/dose level). Concurrent controls (10 rats/sex) received the vehicle, propylene glycol, alone. Males of Groups 1-3 (control, 10 and 30 mg/kg, respectively) were treated for 2 weeks prior to mating, during mating, and up to termination (for 29 days). Females of Groups 1-3 that delivered offspring were treated for 2 weeks prior to mating, during mating, during post-coitum, and 13 or 15 days of lactation (for 50-56 days). Females of Groups 1-3 that had no offspring (not pregnant) were treated for 41 days. The (surviving) animals of Group 4 (300 mg/kg) were sacrificed after 9 days of treatment due to severe toxicity.
No reproduction toxicity was observed up to 100 mg/kg.
At 30 and 100 mg/kg, no treatment-related changes were noted the reproductive parameters examined (i.e. mating and fertility indices, precoital time, number of implantation sites, estrous cycle, spermatogenic profiling, and histopathological examination of reproductive organs).

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

Reproductive and developmental results

No reproductive or developmental toxicity was observed up to 100 mg/kg.

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:

Reproduction NOAEL:          100 mg/kg.

Developmental NOAEL:      100 mg/kg.

Note: Reproductive performance and developmental endpoints could not be evaluated at 300 mg/kg due to the test item-related early termination of this dose group.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The whole data base is conclusive and of high quality.

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Effects on developmental toxicity

Description of key information

No developmental toxicity was observed up to the highest dose level tested (100 mg/kg).

No treatment-related changes were noted in the developmental parameters examined (i.e. gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, litter

size, clinical signs, body weight, anogenital distance (PND 1), areola/nipple retention (PND 13 males), T4 thyroid hormone levels (PND 14-16) and macroscopic examination).  

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available