Registration Dossier

Administrative data

Key value for chemical safety assessment

Effects on fertility

Description of key information

An OECD 421 study is available to assess toxicity on reproduction; the results were as follows:
NOAEL: reproductive performance and fertility = 200 mg/kg bw/day (male/female)
NOAEL: developmental toxicity = 200 mg/kg bw/day (male/female)
NOAEL: systemic toxicity = 50 mg/kg bw/day (male)



Link to relevant study records

Referenceopen allclose all

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
20. Apr 1999 - 28. Jun 1999
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
GLP compliance:
yes
Limit test:
no
Specific details on test material used for the study:

- Lot/batch No.: Mixture from January 27, 1999
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Boehringer Ingelheim, Pharma KG, Biberach/Riss
- Age at study initiation: 89-90 days
- Weight at study initiation: Males: 399.7 (365.8 - 430.9) g; Females: 262.5 (235.6 - 291.5) g
- Housing: individually in type DK 111 stainless steel wire mesh cages.
- Diet: Kliba Iaboratory diet rat/mouse/hamster ad libitum
- Water: tap water ad libitum
- Acclimation period: 13 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24 °C
- Humidity (%): 30 - 70 %
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): The preparations were prepared twice a week, every 96 hours latest.


Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: maximum 2 weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The test substance solutions were analyzed by gas chromatography.
Duration of treatment / exposure:
males: 28 days
females: 46-56 days
Frequency of treatment:
daily
Dose / conc.:
10 mg/kg bw/day (nominal)
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
10 mg/kg body weight/day: as the expected no observed adverse effect level.
50 mg/kg body weight/day: as the intermediate dose level.
200 mg/kg body weight/day: as the dose level with possible toxic effects.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily. The nesting, littering, and lactation behavior of the dams was generally evaluated in the mornings in connection with the daily clinical inspection of the dams.


DETAILED CLINICAL OBSERVATIONS: No data


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

Sperm parameters (parental animals):
Parameters examined in P male parental generations:
testis weight, epididymis weight

Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead.
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals after themating period.
- Maternal animals: All surviving animals after the last litter of each generation was weaned.


GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.

Postmortem examinations (offspring):
All surviving pups, all stillborn pups andthose pups that died before schedule, were examined externally, eviscerated and their organs were assessed macroscopically.
Statistics:
DUNNETT-test, FISHER'S EXACT test, WILCOXON-test
Clinical signs:
no effects observed
Description (incidence and severity):
see " Details on results"
Dermal irritation (if dermal study):
not examined
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:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
see " Details on results"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
see " Details on results"
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no substance-related mortalities in any of the male and female F0 parental animals in any of the groups. One high dose male animal showed piloerection on study day 2. Another male animal of this test group had respiratory sounds between days 11-23.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
The food consumption of the high dose F0 male animals was statistically significantly reduced during study weeks 0 - 1 and remained slightly reduced for the whole study period without attaining statistical significance. This was in-line with Iowered mean body weight and impaired body weight gain.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
With the exception of two F0 parental females all mated females of test groups 0 - 3 (0, 10, 50, 200 mg/kg body weight/day) became pregnant. Therefore, the fertility index varied between 90 and 100 %. This difference concerning the female fertility index was regarded to be spontaneous in nature and not associated with the treatment of the animals.

ORGAN WEIGHTS (PARENTAL ANIMALS)
The mean absolute and relative weight parameters did not show significant differences when compared with the control group.

GROSS PATHOLOGY (PARENTAL ANIMALS)
A few gross lesions were noted in the forestomach (erosion/ulcer and thickening of wall) in a high dose male; liver (small yellow focus: in a control male, two males of the mid and one of the high dose group; a mid sized necrosis in a mid dose female rat; lungs (atelectasis, diffuse red discoloration or large hematoma in three different males of the high dose group); kidneys (clay colored discoloration or focal unilateral contraction in the cortex of two different high dose males), and spleen (focal grey white deposition in a low dose group male).

Two animals were not pregnant. In one control animal a dilation of the uterine horns may have contributed to this situation.

HISTOPATHOLOGY (PARENTAL ANIMALS)
With the exception of the clay colored discoloration of the kidneys of one animal (male, high dose group), all gross lesions could be correlated with a meaningful microscopic finding. Histopathology of testes, epididymides and ovaries did not reveal any treatment related microscopic finding.
Dose descriptor:
NOAEL
Remarks:
reproductive performance and fertility
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
reproductive performance
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
50 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: body weight; food consumption
Clinical signs:
no effects observed
Description (incidence and severity):
see " Details on results"
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
not examined
Gross pathological findings:
no effects observed
Description (incidence and severity):
see " Details on results"
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
not examined
VIABILITY (OFFSPRING)
There were no substance-related differences concerning pup viability and mortality.

CLINICAL SIGNS (OFFSPRING)
The F1 pups did not show any clinical signs which could be attributed to the treatment. An unilateral microphthalmia was the only clinical observations which occurred in one high dose pup. However, this finding was considered to be spontaneous in nature in respect to the known historical background data of the rat strain investigated in the present study.

BODY WEIGHT (OFFSPRING)
Mean body weights/body weight gains were not influenced by the test substance administration.

GROSS PATHOLOGY (OFFSPRING)
The macroscopic examination of all pups did not reveal any differences between the test groups neither in the type nor in the number of pup necropsy observations.
OnIy spontaneous findings were seen at necropsy (e.g. post mortem autolysis, dilated renal pelvis and microphthalmia) in a few of the large number of examined F1 pups of all groups including the controls generally without a dose-response relationship.
Key result
Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
200 mg/kg bw/day (nominal)
Sex:
male/female
Basis for effect level:
viability
mortality
Reproductive effects observed:
not specified

Under the conditions of this reproduction/developmental toxicity screening test, the oral administration by gavage of 3-Dimethylaminopropyl- amin had no adverse effects on reproductive performance or fertility of the F0 parental animals of all substance-treated groups. There were no signs of developmental toxicity in the progeny of the F0 parents up to and including the high dose group (200 mg/kg body weight/day).

Signs of general, systemic toxicity in the F0 parental animals were confined to the male rats of the 200 mg/kg body weight/day group. Toxicity was characterized by decreased food consumption.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
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 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
not specified
Specific details on test material used for the study:
High purity Ethylenediamine (99,9% ) was used to synthesize ethylenediammoniumchloride (EDA*2HCl). Analysis by chromatographic and spectrophotometric procedures showed the salt to be free from impurities.
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc., Portage, Michigan.
- Age at study initiation: (P) 43 wks;
- Weight at study initiation: (P) Males: 112-141 g; Females: 85-111 g;
- Housing: Two or three males or three or five females were housed per cage prior to mating;
- Diet : ad libitum, Purina Certified Rodent Chow 5002.
- Water :ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22
- Humidity (%): 55
- Photoperiod (hrs dark / hrs light): 12 hr light cycle
Route of administration:
oral: feed
Details on exposure:
Fresh diet was prepared biweekly with the percentage of EDA*2HCl in the diet adjusted to maintain a constant dosage level in grams per kilogram
for each sex according to the average animal body weight gain and diet consumption. At monthly intervals two diet samples were analysed.
Details on mating procedure:
The F0 parents were mated afier having received dietary EDA * 2HCl for 100 days. Twenty-six females and 13 males (randomly selected from 25 males) were mated per treated group while 52 females and 26 males (randomly selected from 50 males) were mated for the control group. During mating, two females were placed in a cage with one male; at this time all rats received EDA * 2HCl at the concentration in the diet for the respective female groups. Fifteen days after cohabitation, mating was discontinued and the females were placed in individual shoebox cages fitted with wire rod metal tops.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
At monthly intervals two diet samples were analysed.
Duration of treatment / exposure:
for two generations
Frequency of treatment:
daily
Details on study schedule:
At each dose level 13 male and 26 female rats were mated in both F0 and F1 generation (control group: 26 male and 52 female rats each); continuous treatment starting 100 days prior to cohabitation of F0 rats until weaning of F2 rats.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Dose / conc.:
500 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
13 male and 26 female. Control group: 26 male and 52 female rats
Control animals:
yes, concurrent no treatment
Parental animals: Observations and examinations:

BODY WEIGHT: Yes, measured weekly
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Food consumption determined on each cage of animals every 2 week. Dosage adjusted to keep a constant dosage level.
Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
no effects observed
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day
Sex:
not specified
Basis for effect level:
other: Body weight; food consumption; organ weights; (Recalculated to EDA base)
Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
no effects observed
Sexual maturation:
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed
Key result
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
227 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: Body weight; food consumption; organ weights; histopathology: Hepatocellular pleomorphism; (Recalculated to EDA base)
Reproductive effects observed:
not specified

Significant reduction in parental body weight gain of female rats in the intermediate and high dose group of the F0 generation, in the high dose group of the F1 generation and of male rats in the high dose group of both F0 and F1 generations.

No substance-related parental deaths in the F0 or F1 generation.

Significant decrease of absolute liver weight in male rats of the high dose F1 generation.

No macroscopic or histopathologic findings except a significant higher incidence of hepatocellular pleomorphism in both sexes of the high dose group of the F1 generation (6/10 male, 10/10 female; control: 0/20 each) and a significant decreased prevalence of kidney tubular mineralization in female rats of the high dose group of the F1 generation (0/10 female; control: 10/20).

In conclusion there was no evidence of fertility impairment or embryotoxic effect at dose levels that show maternal or paternal toxicity.

REPRODUCTIVE INDICES        
         
  Dosage (g/kg,lday)
  0.50 0.15 0.05 0
  F0 → F1
Fertility index (%)        
Male 92 100 92 92
Female 81 77 73 77
Gestation index (%)  100 100 100 100
Gestation survival index (%)  99.0 ± 3.4  100.0 ± 0.0  97.2 ± 11.8  99.2 ± 5.2
0- to 4-Day survival index (%)  90.0 ± 30.0  100.0 ± 0.0  94.4 ± 23.6  93.2 ± 22.4
4- to 14-day survival indexb (%)  99.4 ± 2.5  100.0 ± 0.0  94.1 ± 24.3  100.0 ± 0.0
4- to 2I-day survival indexb (%)  99.4 ± 2.5  100.0 ± 0.0  94.1 ± 24.3  99.7 ± 2.0
Pups born alive/litter  6.8 ± 3.5  8.6 ± 2.8  6.2 ± 3.9  8.4 ± 3.0
Days from first mating to parturition 28.3 ± 3.8 29.6 ± 2.0  29.8 ± 3.0  28.1 ± 3.0
  F1 → F2
Fertility index (%)        
Male  100 92 92 100
Female  92 88 73 88
Gestation index (%)  100 100 100 100
Gestation survival index (%)  98.6 ± 6.7  99.1 ± 3.1  99.4 ± 2.5  99.5 ± 2.0
0- to 4-Day survival index (%) 98.0 ± 4.1  99.3 ± 3.5 98.6 ± 3.3  98.5 ± 4.1
4- to 14-day survival index (%) 100.0 ± 0.0  98.7 ± 6.3 100.0 ± 0.0  99.6 ± 3.0
4- to 21-day survival index (%) 100.0 ± 0.0  98.3 ± 8.3 100.0 ± 0.0  99.6 ± 3.0
Pups born alive/litter 8.5 ± 3.0  8.8 ± 2.2 9.4 ± 2.8 10.0 ± 2.5
Days from first mating to parturition 28.5 ± 3.2  28.8 ± 3.3 27.3 ± 3.6  28.4 ± 3.8
Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
GLP compliant OECD Guideline study.
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

A Reproduction/Development Toxicity Screening Test of the test substance by oral administration to Sprague-Dawley Rats according OECD method 421 was carried out with four groups of 10 male and 10 female rats each (BASF SE, 1999, reliability score: 1). The rats received 0 (control), 10, 50 or 200 mg/kg bw/day; males: 14 days prior to mating and during the 14-day mating period, females: 14 days prior to mating and during the mating period, pregnancy and lactation period. Examinations were carried out on mortality, clinical signs, body weight, food consumption, reproduction, macroscopic post mortem findings/organ weights of parent animals and histopathology. Under the conditions the oral administration of the test item by gavage to rats had no adverse effects on reproductive performance or fertility of the F0 parenteral animals of all substance-treated groups. Mating behavior, conception, gestation, parturition and lactation as well as the determined sexual organ weights, gross and histopathological findings of these organs were similar between the substance-treated animals and the corresponding controls. With the exception of each one male and female F0 parental rat in the control group and mid dose group all F0 parental rats proved to be fertile. Thus, the observable difference was regarded to be incidental in nature and not of toxicological or biological concern. Signs of general, systemic toxicity in the F0 parental animals were confined to the male rats of the 200 mg/kg bw/day group. Toxicity was characterized by decreased food consumption. If calculated for the whole treatment period, it was about 6 % below the concurrent control value. The high dose F0 parental males showed also impairments in body weight gain during the whole treatment period. If calculated for the entire treatment period (weeks 0 - 4) body weight gain was about 35 % lower than the concurrent control value although there was a statistically significant increase in body weight gain during weeks 2 - 3. Indications for a slight impairment in the general state of health were noted in two F0 male animals at 200 mg/kg bw/day. At this dose level piloerection and transient respiratory sounds were observed in each one of the F0 male animals. However, the transiently observed respiratory sounds are considered to be predominantly related to the gavage application procedure 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. No compound-related adverse effects were observed in female rats up to the highest dose level of 200 mg/kg bw/day. There were no signs of developmental toxicity in the progeny of the F0 parents up to and including the high dose group (200 mg/kg bw/day). Observed differences did not show any biological relevance between the substance-treated groups and the control group. Thus, the NOAEL (no observed adverse effect level) for reproductive performance and fertility was 200 mg/kg bw/day for the F0 parental rats. The NOAEL for general, systemic toxicity of the test substance was 200 mg/kg bw/day for the F0 parental females, while it was 50 mg/kg bw/day for the F0 parental males. The NOAEL for developmental toxicity could be fixed at 200 mg/kg bw/day for the F1 progeny.

With the structurally related ethylenediammonium dichloride (EDA*2HCL) there is two-generation reproduction toxicity study available (Yang et al., 1984, reliability score: 2, see section 13, read across justification).Male and female Fischer 344 rats were fed diets containing 0, 50, 150 or 500 mg/kg bw/day of EDA*2HC continuously, starting 100 days prior to cohabitation of F0 rats until weaning of F2 rats. At each dose level 13 male and 26 female rats were mated in both F0 and F1 generation. Body weight was recoded weekly and food consumption and substance intake at two-weekly intervals.Complete necropsies were performed on 5 weanlings from both F1 and F2 generation at each dose group, on 10 adults/sex/dose group of F1 generation and on 20 rats/sex of the control group. Necropsies were performed on all dead pups.In the high dose group there was a decrease in body weight gain in both F0 and F1 generations, a higher incidence of hepatocellular pleomorphism in F1 generation, and decreased liver weights in male rats in the F1 generation. In the intermediate dose group, female rats of F0 generation showed a decreased body weight gain. No evidence of impaired fertility or embryotoxicity was noted at any dose level. The NOAEL for parental or systemic toxicity was 50 mg/kg bw/day forEDA*2HCL(corresponding to 23 mg/kg bw/day of EDA). The NOAEL for reproductive and developmental toxicity was 500 mg/kg bw/day for EDA*2HCL(corresponding to 227 mg/kg bw/day of EDA).


Effects on developmental toxicity

Description of key information

The screening test conducted with DMAPA according to the OECD 421 (please refer to chapter 7.8.1) revealed a NOAEL for developmental toxicity of 200 mg/kg bw/day since no adverse effects could be evidenced at this dose level which was the highest tested. Prenatal developmental toxicity studies with the structurally related ethylenediammonium dichloride (EDA*2HCL) are available in rats and rabbits. In rats, The NOAEL for maternal toxicity was 50 mg/kg bw/day of EDA*2HCl (corresponding to 22 mg/kg bw/day of EDA). The NOAEL for developmental toxicity including was 250 mg/kg bw/day of EDA*2HCl (corresonding to 114 mg/kg bw/day of EDA). There was no indication for a selective teratogenic effect at any dose level investigated. In rabbits, the NOAEL for EDA for maternal as well as for developmental toxicity including teratogenicity was 80 mg/kg bw/day, the highest dose level investigated.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 August 2015 -- 16 September 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 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 10-11 weeks old and had a mean body weight of 305 g (range: 270 g to 336 g).
- Fasting period before study: no
- Housing: polycarbonate cages
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: at least 4 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: 31 August 2015 to 16 September 2015
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Type of formulation
(visual observation): Solution in the vehicle

Preparation procedure: According to the validation study describing the preparation procedure (homogeneity and stability testing) for a range of concentrations covering the lowest and highest used in this study.
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 30 35%, TraceSELECT® Ultra, for ultratrace analysis. 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: Test item dose formulations
Based on test item dose formulation stability and vehicle expiry
Vehicle preparation
Based on the CiToxLAB France in-house procedures (frequency documented in study raw data)
Storage condition
(control dose formulation(s)): At room temperature
Storage conditions
(test item dose formulations): At room temperature and protected from light
Delivery conditions: At room temperature and protected from light
Vehicle
Name: Drinking water treated by reverse osmosis using ELIX 5 (Millipore SA)
Used for preparation of test item dose formulations and administered to control group (control dose formulation)
Preparation: By CiToxLAB France Pharmacy
Analytical verification of doses or concentrations:
no
Details on analytical verification of doses or concentrations:
No chemical analysis was performed.
Details on mating procedure:
- Proof of pregnancy: vaginal plug, referred to as Day 0 post-coitum
Duration of treatment / exposure:
Day 6 to Day 20 post-coitum
Frequency of treatment:
Daily
Duration of test:
21 days
No. of animals per sex per dose:
8 time-mated females
Control animals:
yes, concurrent vehicle
Details on study design:
The dose-levels were selected in agreement with the Sponsor, following the results of a previous study performed in the same species.
In the previous dose-range finding study by the oral route (gavage), male and female Sprague-Dawley rats received Diethylaminopropylamine (as pH-neutralized dose formulations) daily by gavage at dose-levels of 100, 300 or 1000 mg/kg bw/day for 2 weeks. At 1000 mg/kg bw/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 of 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 bw/day.

Thus, based on these available data, the selected dose-levels of the present study are: 100, 300 and 1000 mg/kg bw/day.
Maternal examinations:
MORBIDITY AND MORTALITY:
- Time schedule: at least twice a day during the treatment period, including weekends.

CLINICAL OBSERVATIONS:
- Time schedule: once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT (GAIN):
- Time schedule: on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c., and prior to premature sacrifice.

FOOD CONSUMPTION:
- Time schedule: on Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c..

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on Day 21 p.c.,
- Organs examined: the principal thoracic and abdominal organs and the weight of the gravid uterus .
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
- number of corpora lutea,
- number and distribution of dead and live fetuses,
- number and distribution of early and late resorptions,
- number and distribution of uterine scars,
- number and distribution of implantation sites.

The following classification was used to record:
- uterine scar: uterine implantation without implant,
- early resorption: evidence of implant without recognizable embryo,
- late resorption: dead embryo or fetus with external degenerative changes,
- dead fetus: non live fetus with no external degenerative changes.
Fetal examinations:
Each fetus was subjected to a detailed external examination, which included the observation of all visible structures, surfaces and orifices. All fetuses were then discarded.
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Indices:
% of pre-implantation loss =
Number of corpora lutea - Number of implantations
_______________________________________________________________ x 100
Number of corpora lutea

% of post-implantation loss =
Number of implantation sites - Number of live fetuses
__________________________________________________________________________ x 100
Number of implantation sites
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
One female given 1000 mg/kg bw/day was not pregnant.
All other mated females were pregnant.
One female given 1000 mg/kg bw/day was sacrificed prematurely on Day 15 p.c. for ethical reasons. Prior to sacrifice, signs of poor clinical condition were observed (i.e. piloerection, round back, pallor of extremities, hypoactivity, half-closed eyes and reddish vaginal discharge). A body weight loss of 66 g was recorded between Days 6 and 15 p.c. together with reduced food consumption.
At necropsy, thymus was reduced in size, whitish nodules were seen in the forestomach, blackish contents were observed in the cecum and reddish contents were noted in the vagina. Seventeen corpora lutea and fourteen implantation sites were recorded.
No other unscheduled deaths occurred in any other groups.
Ptyalism was observed in all surviving females given 1000 mg/kg/day during the first and/or the second weeks of treatment for 1 to 6 days duration. This was considered to be of minor toxicological significance.
Piloerection in 1/7 females given 1000 mg/kg bw/day was noted on Day 14 p.c. only.
Reflux at dosing was observed on two occasions in 2/8 and 1/7 females at 300 and 1000 mg/kg/day, respectively.
One palpable mass, measuring 2 cm in diameter, was seen on mammary gland in 1/8 females at 300 mg/kg/day from Day 18 p.c.. This was not considered to be test item-related as it was of isolated occurrence and not dose-related (Table 1).
At 1000 mg/kg bw/day and when compared with controls, mean body weight loss was noted over the first 3 days of treatment (-13 g vs. +13 g), followed by a lower mean body weight gain until Day 15 p.c. (+12 to +14 g vs. +15 to +25 g). This trend was accentuated by the body weight loss (-50 g) recorded in the prematurely sacrificed female between Days 9 and 15 p.c.. A statistically significantly lower body weight gain was therefore observed over the whole study period (+105 g vs. +140 g).
At 300 mg/kg bw/day and when compared with controls, a lower mean body weight gain was noted over the first 3 days (+9 g vs. +13 g).
At 100 mg/kg bw/day and when compared with controls, there were no effects on mean body weight or mean body weight change (Table 2).
At 1000 mg/kg bw/day and when compared with the controls, lower mean food consumption (-14 to -52%) was recorded between Days 6 and 15 p.c.. This effect was associated with reduced food consumption recorded in the prematurely sacrificed female between Days 9 and 15 p.c..
At 300 mg/kg bw/day, a comparable same trend was observed over the first three days of the study, but to a lesser extent (-11% vs. controls). These differences correlated with the mean body weight loss or the lower mean body weight gain also observed at 300 mg/kg bw/day.
At 100 mg/kg bw/day, there were no effects of treatment with the test item.
When compared with the control group, dams given 1000 mg/kg bw/day had a lower gravid uterus weight observed in high-dose dams (-9% compared to controls). Lower net weight change (+16 g vs. +41 g in controls) was considered to be a consequence of the lower body weight gain in these females.
Gestation parameters were not impacted by the test item treatment at any dose-levels.
Key result
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
300 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Abnormalities:
effects observed, non-treatment-related
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
There was no effect on the fetal body weight and on the percentage of male fetuses (sex ratio).
There were no treatment-related fetal external malformations or variations in any groups.
Key result
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Abnormalities:
no effects observed
Developmental effects observed:
no

Table 1: clinical signs

Dose-level (mg/kg bw/day)

0

100

300

1000

Piloerection

0

0

0

1

Ptyalism

0

0

0

7

Reflux at dosing

0

0

2

1

Mass on mammary gland

0

0

1

0

Total affected animals

0/8

0/8

3/8

7/7

Table 2: mean body weights and body weight changes

Dose-level (mg/kg bw/day)

0

100

300

1000

Body weight (g)

 

 

 

 

Day 6 p.c.

306

-

304

(-1)

304

(-1)

304

(-1)

Day 21 p.c.

445

-

444

(0)

452

(+2)

410

(-8)

Body weight change (g)

 

 

 

 

Days 6 - 9 p.c.

+13

+14

+9

-13#

Days 9-12 p.c.

+25

+24

+25

+14

Days 12-15 p.c.

+15

+16

+19

+12

Days 6 - 21 p.c.

+140

+140

+148

+105**

Statistical significance; **: p<0.01 and #: p<0.001. p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage difference vs. controls.

Table 3: Mean food consumption changes (expressed in g/animal/day) 

Dose-level (mg/kg bw/day)

0

100

300

1000

Days 6 - 9p.c.

27

-

27

(0)

24

(-11)

13#

(-52)

Days 9-12p.c.

28

-

27

(-4)

28

(0)

16#

(-43)

Days 12-15p.c.

29

-

29

(0)

30

(+3)

25

(-14)

Statistical significance:#: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

Table 4: mean carcass, net change and gravid uterus weights (a) (g)

Dose-level (mg/kg bw/day)

0

100

300

1000

Gravid uterus weight

98

-

95

(-4)

109

(+11)

89

(-9)

Carcass weight

347

-

350

(+1)

343

(-1)

321

(-7)

Net weight change from Day 6.p.c.

+41

+45

+39

+16#

Statistical significance:#: p<0.001.

( ): in brackets, percentage differencevs.controls.

p.c.:post-coitum.

(a): weights are rounded values.

-: not applicable.

Table 5: Hysterectomy data 

Dose-level (mg/kg bw/day)

0

100

300

1000

Number of females with live fetuses at term

8

8

8

6

Mean number of corpora lutea

16.5

14.3

14.9

15.0

Mean number of implantation sites

13.4

13.0

14.3

13.3

Mean pre-implantation loss (%)

17.0

8.8

4.2

8.9

Mean number of fetuses

12.5

11.8

13.9

12.0

Percentage of dead fetuses (%)

0

0

0

0

Mean number of uterine scars + resorptions

0.9

1.3

0.4

1.3

Mean number of early resorptions

0.8

1.3

0.4

1.3

Mean number of late resorptions

0.1

0.0

0.0

0.0

Mean post-implantation loss (%)

6.3

10.4

2.7

10.2

  

Table 6: Fetal data 

Dose-level (mg/kg bw/day)

0

100

300

1000

Mean fetal body weight (g)

5.69

-

5.77

(+1.4)

5.72

(+0.5)

5.36

(-5.8%)

Mean percentage of male fetuses (%)

49.5

53.7

47.4

40.8

( ): in brackets, percentage differencevs.controls.

-: not applicable.

Endpoint:
developmental toxicity
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 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. certificate)
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 test animals and environmental conditions:
TEST ANIMALS
- Source: Janvier, le Genest-Saint-Isle, France.
- Age at study initiation: 10-11 weeks old
- Weight at study initiation: 308 g (range: 256 g to 373 g)
- Fasting period before study: no
- Housing: individually housed in polycarbonate cages
- Diet: SSNIFF R/M-H pelleted maintenance diet, ad libitum
- Water: tap water (filtered with a 0.22 µm filter), ad libitum
- Acclimation period: 4 or 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): 12
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
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 [30 35%, TraceSELECT® Ultra, for ultratrace analysis (96208 Fluka)]. The volume of HCl added was recorded for each concentration
The final pH was checked and recorded by using a pH-meter

VEHICLE
- Concentration in vehicle: 10, 50 and 150 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Gas Chromatography with FID detection (GC-FID)
Once on Days 6, 13 and 19 p.c.
Details on mating procedure:
The females were mated at the breeder's facilities. The day of confirmed mating (detection of a vaginal plug) was designated as Day 0 p.c..
Duration of treatment / exposure:
From Day 6 to Day 20 p.c., inclusive.
Frequency of treatment:
Daily
No. of animals per sex per dose:
24
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected based on a preliminary study for effects on embryo-fetal development by the oral route in rats (Papineau, 2016b; CiToxLAB France/Study No. 42761 RSR).
Maternal examinations:
CAGE SIDE OBSERVATIONS:
Each animal was checked for mortality and morbidity once a day before the treatment period and at least twice a day during the treatment period, including weekends and public holidays.

DETAILED CLINICAL OBSERVATIONS:
From arrival, each animal was observed once a day as part of the routine examinations.
From the start of the treatment period, each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

BODY WEIGHT:
The body weight of each female was recorded on Days 2, 4, 6, 9, 12, 15, 18 and 21 p.c.

FOOD CONSUMPTION:
The quantity of food consumed by each female was recorded for the following intervals:
Days 2-4, 4-6, 6-9, 9-12, 12-15, 15-18 and 18-21 p.c.

WATER CONSUMPTION: No

POST-MORTEM EXAMINATIONS: Yes / No / No data
- Sacrifice on gestation day 21
- Organs examined: principal thoracic and abdominal organs.
- The weight of the gravid uterus was recorded for each pregnant female (with at least one live fetus) at hysterectomy
Ovaries and uterine content:
The ovaries and uterus of the females were examined to determine:
. number of corpora lutea,
. number and distribution of dead and live fetuses,
. number and distribution of early and late resorptions,
. number and distribution of uterine scars,
. number and distribution of implantation sites.

The following classification was used to record:
. uterine scar: uterine implantation without implant,
. early resorption: evidence of implant without recognizable embryo,
. late resorption: dead embryo or fetus with external degenerative changes,
. dead fetus: non-live fetus with no external degenerative changes.

For apparently non-pregnant females, the presence of implantation scars on the uterus was checked using the ammonium sulphide staining technique (Salewski, 1964).

A gross evaluation of placentas was also undertaken.
Fetal examinations:
- External examinations: Yes: half per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No
Statistics:
Data were compared by one-way analysis of variances and Dunnett test (mean values being considered as normally distributed, variances being considered as homogenous) or by Fischer exact probability test (proportions).
Indices:
- For each pregnant female:
. body weight change for different intervals
. net body weight (presented as carcass weight)
. net body weight change
- For each litter:
. total number of resorptions
. total number of dead fetuses
. % of dead fetuses per litter
. total number of live fetuses
. % of live fetuses per litter
. % of pre-implantation loss
. % of post-implantation loss
. average fetal body weight
- For each group:
. mean and standard deviation: body weight, body weight change, food consumption, number of corpora lutea, number of implantation sites, number of fetuses and fetal body weight with litter as the experimental unit,
. number of animals affected: clinical signs, necropsy findings, fetal observations (number of fetuses, number of litters),
. % of animals affected: fetal observations (% of fetuses, % of litters),
. mean % per litter of fetuses affected,
. % of pre-implantation loss relative to the number of corpora lutea (mean of pre-implantation loss per litter),
. % of live fetuses and % of dead fetuses (relative to total number of fetuses),
. % of male fetuses (relative to total number of live fetuses),
. mean, standard deviation and % relative to the number of implantation sites: resorptions plus uterine scars, uterine scars, early resorptions, late resorptions,
. % of post-implantation loss relative to the number of implantation sites (mean of post-implantation loss per litter), % of dams affected.
Historical control data:
Available
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
CLINICAL EXAMINATIONS
- Pregnancy status: Table 1
- Mortality
There were no unscheduled deaths in control, 50 and 250 mg/kg bw/day groups.
At 750 mg/kg bw/day, one female was found dead on Day 20 p.c.. Prior to death, this female showed piloerection, round back, emaciated appearance, dyspnea and chromorhinorrhea. A body weight loss of 39 g was recorded between Days 6 and 18 p.c. together with reduced food consumption. At macroscopic post-mortem examination, no abnormalities were observed. This female was pregnant (1 early resorption and 9 dead fetuses for 13 corpora lutea).
This unscheduled death was considered to be test item treatment related.
- Clinical signs
Ptyalism was observed in one female given 250 mg/kg bw/day and in most of the surviving females given 750 mg/kg bw/day from Day 9 p.c. up to Day 20 p.c.. Sneezing, chromorhinorrhea and/or dacryhorrhea were transiently noted on some occasions at all dose-levels. Reddish vaginal discharge observed at 50 mg/kg bw/day was isolated and a relationship to the test item treatment was considered to be unlikely. These signs were considered to be of minor toxicological significance.
From 250 mg/kg bw/day, test item-related clinical signs were mainly observed from Day 11 or 12 p.c and consisted of round back, emaciated appearance, piloerection, loud breathing and/or reddish vaginal discharge. These clinical signs had dose-related increased incidence and were considered to be adverse.
- Body weight and body weight change: Table 2
At 750 mg/kg bw/day, when compared with controls, mean body weight loss was noted over the first 3 days of treatment (-16 g vs. +15 g, p<0.001), followed with a lower mean body weight gain until the end of the treatment period. (+11 to +44 g vs. +20 to +51 g) resulting in a lower body weight gain over the whole dosing period (+97g vs. +149 g in controls, p<0.001).
Effects on mean body weight change at 750 mg/kg bw/day contributed to a lower terminal mean body weight gain (-12% vs. controls, p<0.001). These effects were attributed to the test item treatment and correlated with reduced mean food consumption between Days 6 and 18 p.c. (see below). They were therefore considered to be
adverse.
At 250 mg/kg bw/day, when compared with controls, low mean body weight gain was noted over the first 3 days of treatment (+5 g vs. +15 g, p<0.01) with a return to control values thereafter.
At 50 mg/kg bw/day, there were no effects on mean body weight or mean body weight change.
- Food consumption: Table 3
At 750 mg/kg bw/day, when compared to controls, a low mean food consumption (down to -63% on Days 6 to 9 p.c., p<0.001) was recorded between Days 6 and 15 p.c..
At 250 mg/kg bw/day, a comparable trend was observed over the first three days of the study, but to a lesser extent (-21% vs. controls during the period of Days 6 to 9 p.c., p<0.001; with a return to control values thereafter).
These differences correlated with the lower mean body weight gain and/or the mean body weight loss also observed at 250 or 750 mg/kg bw/day, respectively. Therefore, they were considered to be adverse at 750 mg/kg/day.
At 50 mg/kg bw/day there were no effects of treatment with the test item.
- Maternal necropsy findings
Spleen enlargement, raised focus on the spleen and colored focus on the stomach wall were observed only in females given the high dose-level. Therefore a relationship to the test item treatment was not excluded.
All other macroscopic observations (i.e. granular surface of the spleen, absence of kidney, ureter, ovary and uterine horn, serous cyst on ovary, dilation of uterine horn, uterine horn with colored or serous content, enlarged cervix, colored content in cervix and/or transverse vaginal septum) were not attributed to the test item treatment as they were reported both in control and test item-treated animals at comparable incidences and/or are findings commonly observed in rats of this strain and age.

MATERNAL TERMINAL EXAMINATIONS
- Net weight change: Table 4
At 750 mg/kg bw/day, when compared with controls, females had a low mean gravid uterus weight ( 18%, p<0.05) together with a low net weight change (+9 g vs. +42 g in controls, p<0.001). This was associated with a lower mean carcass weight (-10%, p<0.001) and correlated with the lower mean female body weight gain, number of fetuses and fetal body weight recorded at this dose-level.
These observations confirmed that there was a toxicologically significant effect of the test item on the maternal body weight and the gravid uterus weight.
At 250 mg/kg bw/day, when compared with controls, females had a slightly lower mean gravid uterus weight ( 9%). This was associated with the lower mean number of fetuses recorded at this dose-level.
There were no test item-related effects at 50 mg/kg bw/day.
- Hysterectomy data: Table 5
At 250 and 750 mg/kg bw/day, one female in each group had total resorption. At these dose levels and when compared with controls, there were higher mean post-implantation loss (15.5% and 20.0% vs. 4.6%, respectively, with p<0.05 at 750 mg/kg bw/day) and lower mean number of live fetuses (11.4 and 11.2 vs. 13.5, respectively). In addition all values were outside the limits of the Historical Control Data. A higher mean number of early resorptions were also recorded at 750 mg/kg bw/day (2.1 vs. 0.6 in controls, p<0.001).
Taking into account these differences which were also dose-related, these findings were considered to be test item treatment-related and adverse
At 50 mg/kg bw/day there were no test item treatment-related effects.
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
50 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
other: dose-related increases in the incidences of skeletal abnormalities from 250 mg/kg bw/day (and external abnormalities at 750 mg/kg bw/day), in a context of marked to severe fetal toxicity, involving mainly the axial skeleton
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
uterus
Description (incidence and severity):
lower mean gravid uterus-weight
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Body weight and sex: Table 6
In the 750 mg/kg bw/day group, when compared with controls, mean fetal body weight was lower ( 5%, p<0.05). This finding was attributed to the test item treatment since mean value was below the lower limit of the HCD and correlated with the lower mean gravid uterus weight recorded at this dose-level. As this difference was minimal, this was considered to be of minor toxicological importance.
In the 50 and 250 mg/kg bw/day groups there were no effects on mean fetal body weight.
The sex ratio was unaffected by the test item treatment at any dose-level.

- Fetal external examination
. Variations: Table 7
There was no test-item treatment related increase of the frequencies of external variations.
Malrotated paw was observed at comparable incidence at 750 mg/kg bw/day and at the upper limit of the historical control data, and malrotated hindlimb was also observed in one control fetus. Therefore, these findings were considered to be unrelated to the test item treatment.
. Malformations: Table 8
There were no fetal external malformations in the 0, 50 and 250 mg/kg/day groups.
In the 750 mg/kg/day group, two fetuses from two different litters had external malformations:
. one fetus (fetal body weight: 4.13 g): anasarca, cleft palate, short trunk (absent cervical vertebra(e) at skeletal examination) and short tail,
. one fetus (fetal body weight: 3.58 g): anal atresia and acaudia.
These findings affecting the trunk/anus and/or tail were observed in two multi-malformed fetuses from two different litters with body weights significantly lower than the other fetuses from the same litter and sex. If a test item treatment related effect cannot be ruled out, it is most probably secondary to the fetal toxicity in a context of severe maternal toxicity.

- Fetal soft tissue examination
. Variations: Table 9
In the 750 mg/kg/day group, the incidences of absent innominate artery (3.9% and 16.7%, fetal and litter incidences respectively) were higher than the upper limit of the HCD (0.8% and 5.0%, respectively). Taking into account the amplitude of the incidences (litter and fetal) of this non-adverse finding at 750 mg/kg/day, a test item treatment related effect cannot be excluded.
When compared with controls and HCD, there were tendencies toward increased fetal and litter incidences of dilated renal pelvis and thymus with reddish foci at 750 mg/kg/day and dilated ureter in all test item treated-groups. However as these variations were not dose-related, within and/or closed to HCD values, they were considered not to be test-item treatment related.
There were no test-item treatment related increase of the frequencies of soft tissue variations in the 0, 50 and 250 mg/kg/day groups.
Other soft tissue variations (i.e. kidney with paleness and short innominate artery) observed at comparable incidence in control and test item-treated groups were considered to be unrelated to the test item treatment.
. Malformations: Table 10
There was no test item treatment-related increase in the frequencies of soft tissue malformations.

- Fetal skeletal examination
. Cartilage: Table 11
In the 750 mg/kg bw/day group and when compared with controls, there were higher litter and fetal incidence of fetuses with cartilage of cervical vertebra(e) present. This finding associated with a delayed centrum ossification as described in the § Variations (Cervical vertebra(e): incomplete ossification of centrum) was considered to be test item treatment-related but non-adverse (the cartilage/bone structure still present).
. Variations: Table 12
Up to 250 mg/kg bw/day, there were no test-item treatment related skeletal variations.
In the 750 mg/kg bw/day group and when compared with controls, there were higher litter and fetal incidences of fetuses with incomplete ossification of cervical vertebra(e) centrum. This finding associated with higher litter and fetal incidence of cervical vertebra(e) with cartilage (see § Cartilage) was therefore considered to be test item treatment-related but non-adverse (ossification delay).
When compared with controls and HCD, the fetal and litter incidences of caudal vertebra(e) with unossified centrum (1.8% and 5.6%, respectively) were higher than control (0% and 0%, respectively) and HCD (2.0% and 7.1%, respectively). Therefore finding was considered to be test-item treatment related but non-adverse.
. Malformations: Table 13
In the 750 mg/kg bw/day, two litters had a malformed fetus:one with absent cervical vertebra, fused sternebrae, absent rib(s) and fused metacarpal bones and the other with absent thoracic vertebra(e) and rib(s).
In the 250 mg/kg bw/day group, three litters had one malformed fetus: one with absent thoracic vertebra and absent rib(s), one with absent lumbar vertebra and one with supernumerary lumbar vertebra.
In the 50 mg/kg bw/day group, one litter had 5 malformed fetuses with absent lumbar vertebra and two with absent rib.
In the control group, one litter had one malformed fetus with absent lumbar vertebra(e).
Taking into account the number and percentage of fetus and litter affected in all groups (including controls), a test-item treatment related effect cannot be demonstrated.
Key result
Dose descriptor:
NOEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: teratogenicity
Key result
Abnormalities:
effects observed, non-treatment-related
Key result
Developmental effects observed:
no

Table 1: Pregnancy status

Dose-level(mg/kg bw/day)

0

50

250

750

Number of females

24

24

24

24

Non-pregnant females

0

3

0

4

Females with total resorption

0

0

1

1

Females with live fetuses at term

24

21

23

18

Table 2: Mean body weight and body weight changes (g)

Dose-level (mg/kg bw/day)

0

50

250

750

Body weight (g)

 

 

 

 

Day 6p.c.

310

310

309

307

 

-

(0)

(0)

(-1)

Day 9p.c.

326

323

315

292#

 

-

(-1)

(-3)

(-10)

Day 12p.c.

346

346

333

303#

 

-

(0)

(-4)

(-12)

Day 15p.c.

365

366

353

319#

 

-

(0)

(-3)

(-13)

Day 18p.c.

408

410

394

355#

 

-

(0)

(-3)

(-13)

Day 21p.c.

459

459

442

404#

 

-

(0)

(-4)

(-12)

Body weight change (g)

 

 

 

 

Days 6 - 9p.c.

+15

+13

+5**

-16#

Days 9 - 12p.c.

+20

+23

+19

+11**

Days 12 - 15p.c.

+20

+20

+20

+16

Days 15 - 18p.c.

+43

+44

+40

+36

Days 18 - 21p.c.

+51

+49

+48

+44

Days 6 - 21p.c.

+149

+149

+132

+97#

 

-

(0)

(-11)

(-35)

Statistical significance; **: p<0.01 and#: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

 

Table 3: Mean food consumption (g/animal/day)

Dose-level (mg/kg bw/day)

0

50

250

750

. Days 6 - 9p.c.

24

24

19#

9#

 

-

(0)

(-21)

(-63)

. Days 9 - 12p.c.

27

27

24

17#

 

-

(0)

(-11)

(-37)

. Days 12 - 15p.c.

28

29

27

23#

 

-

(+4)

(-4)

(-18)

. Days 15 - 18p.c.

32

-

33

(+3)

31

(-3)

29

(-9)

Statistical significance; #: p<0.001.

p.c.:post-coitum.

-: not applicable.

( ): in brackets, percentage differencevs.controls.

 

Table 4: Mean carcass, net change and gravid uterus weights (a) (g)

Dose-level (mg/kg/day)

0

50

250

750

Gravid uterus weight

107

-

103

(-4)

97

(-9)

88*

(-18)

Carcass weight

352

-

356

(+1)

345

(-2)

316#

(-10)

Net weight change from Day 6.p.c.

+42

+46

+36

+9#

Statistical significance:*: p<0.05 and#: p<0.001.

( ): in brackets, percentage differencevs.controls.

p.c.:post-coitum.

(a): weights are rounded values.

-: not applicable.

 

Table 5: Hysterectomy data 

Dose-level (mg/kg/day)

0

50

250

750

HCD

Number of pregnant females at hysterectomy

24

21

24

19

194

Number of females with live fetuses at termination

24

21

23

18

185

Number of females with total resorption

0

0

1

1

93

Mean number ofcorpora lutea

15.4

14.8

14.9

15.1

[14.4; 16.6]

Mean number of implantation sites

14.2

13.4

13.2

14.1

[12.9; 14.6]

Mean pre-implantation loss (%)

6.9

8.2

11.8

6.7

[6.5; 12.7]

Mean number of live fetuses

13.5

12.7

11.4

11.2

[12.5; 14.0]

Dead fetuses (%)

0.0

0.0

0.0

0.0

[0.00; 0.10]

Mean number of implantation scars

0.0

0.0

0.5

0.7

/

Mean number of early resorptions

0.6

0.7

0.9

2.1#

/

Mean number of late resorptions

0.1

0.0

0.3

0.0

/

Mean post-implantation loss (%)

4.6

6.0

15.5

20.0*

[1.5; 6.2]

Statistically significant *: p<0.05, #: p<0.001.

HCD: Historical Control Data (control data collected from nine studies covering a period ranging from March 2013 to June 2014), [min.; max.].

/: not reported in HCD.

 

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP, no guideline
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were administerd a dietary dose on gestation day 6- 15.. A pair-fed control group received the same amount of feed as the dosed group.
GLP compliance:
not specified
Limit test:
yes
Specific details on test material used for the study:
Ethylenediamine dihydrochloride was prepared from high purity ethylenediamine (99,9%). The salt was analysed by chromatography and spectrophotometric methods, and found to be free of impurities.
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY).
- Age at study initiation: 100 days
- Housing: stainless-steel cages
- Diet : Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): Yes
Route of administration:
oral: feed
Details on exposure:
To determine whether arterial defects observed in a conventional teratology study were the result of reduced feed intake, a pair-feeding study was performed in which EDA.2HCl was fed on gestation days 6 through 15 at 1000 mg/kg/day. A pair-fed control group received the same
amount of diet consumed by the EDA*2HCl-treated rats. An untreated control group was fed ad libitum.
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
Gestation day 6 - 15
Frequency of treatment:
daily
Duration of test:
cesarean section on gestation day 21
No. of animals per sex per dose:
20
Control animals:
yes, concurrent no treatment
Details on study design:
A pair-fed control group received the same amount of diet consumed by the EDA*2HCl-treated rats. An untreated control group was fed ad libitum.
Maternal examinations:
Body weight and diet consumption.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Reduced body weight and diet consumption on gestations days 6 to 15.
Key result
Dose descriptor:
LOAEL
Effect level:
454 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes
Key result
Dose descriptor:
LOAEL
Effect level:
454 mg/kg bw/day
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Significant reduced mean body weight, mean crown-rump length and mean length of innominate artery of fetuses. In both treatment group and pair-fed control group there were 2 fetuses each with missing innominate artery.


Number of pups affected:

 

  Dosage level (mg/kg/day)     

   Control Pair-fed control   1000
Fetal weight, males (g) 4.4±0.3 4.2±0.3 4.0±0.3
Fetal weight, females (g) 4.2±0.2 4.0±0.2 3.8±0.2
Fetal length, males (mm)    39±2 39±2 38±2
Fetal length, females (mm)     39±2 38±2  37±2
Missing innominate artery  (fetuses/litter)  0/0  2/2  2/2
Shortened innominate artery,males (mm) 1.07±0.29   1.08±0.28    0.75±0.31  

Shortened innominate artery, females (mm)

 1.03±0.24    1.02±0.28   0.78±0.33  
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: pre-GLP
Reason / purpose:
reference to other study
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rats were dosed via diet during gestation day 6-15. Standard endpoints for teratogenicity were evaluated.
GLP compliance:
not specified
Specific details on test material used for the study:
Ethylenediamine dihydrochloride was prepared from high purity ethylenediamine (99,9%). The salt was analysed by chromatography and spectrophotometric methods, and found to be free of impurities.
Species:
rat
Strain:
Fischer 344
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. (Kingston, NY).
- Age at study initiation: 100 days
- Housing: stainless-steel cages
- Diet : Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): Yes
Route of administration:
oral: feed
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis indicated that the test diet preparations were accurate and homogeneous. The stability of the test diets was also confirmed. Substance was added to diet based on established diet consumption data and an established growth curve to produce the dosage goals.
Details on mating procedure:
At approximately 100 days of age, female rats were introduced into the male rats' cages. The rats were mated one male to one female to achieve a goal of approximately 20 pregnant females per treatment group, and 40 in the negative control group. Expelled vaginal plugs were used as evidence that mating had occurred
Duration of treatment / exposure:
Gestation day 6 - 15
Frequency of treatment:
daily
Duration of test:
cesarean section on gestation day 21
No. of animals per sex per dose:
20 and 40 in control group.
Control animals:
yes, concurrent no treatment
Details on maternal toxic effects:
Maternal toxic effects:yes
Key result
Dose descriptor:
NOAEL
Effect level:
23 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
LOAEL
Effect level:
114 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
114 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Key result
Dose descriptor:
LOAEL
Effect level:
454 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: fetotoxicity
Abnormalities:
not specified
Developmental effects observed:
not specified

Significant reduction in maternal body weight gain in the intermediate dose group during gestation day 6 - 15 and in the high

dose group during gestation day 6 - 21; significant decreased diet consumption in the intermediate and high dose group during gestation day 6 - 15; significant increased number of resorptions/litter and significant decreased mean fetal body weight and reduced fetal crown-rump length in the high dose group; significant higher incidence of a shortened mandible, edematous eye bulge, shortened or missing innominate artery, unossified sternebrae in fetuses of the high dose group.

              number of pubs affected
              Dosage level (mg/kg/day)
     Control  50  250  1000
 No. of litters    40  23  21  24
 No. of pubs    379  232  201  242
           
Pup body weight, males (g)     4.5 +0.3   4.5 +0.2  4.5 +0.2   4.1 +0.3 
      4.2 +0.2   4.2 +0.2  4.2 +0.3    3.8 +0.3
           
Fetal crown rump lenght, males (mm)     40 +2  40 +2    40 +2   39 +2 
      38 +1  39 +2    39 +2    37 +2 
           
Slightly edematous eye bulge           
   F  0  0  0  0
   L 0  0  0  0
 Shortened mandible          
   F  0  0  0  18
   L  0  0  0  4
 Missing innominate artery          
   F  0
   L  0
Shortened innominate artery           
   F  2  27
   L  14

  F = fetuses L = litters

The first artery to branch off of the aorta is the brachiocephalic which becomes the innominate after the left carotid branches off.  The innominate then branches into the right subclavian and right carotid artery.  When the authors stated it was missing, they really mean that the right and left carotid branch off of the brachiocephalic artery at the same time.  Thus there is no innominate.  However, this would not affect blood supply to areas served by these arteries.                                                                                                                           

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
weight of evidence
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: No guideline followed, no information on GLP
Qualifier:
no guideline followed
Principles of method if other than guideline:
Rabbits were dosed during gestation days 6 - 19. On day 21 uterine contents are examined, and the foetuses are evaluated for externally visible anomalies and for soft tissue and skeletal changes.
GLP compliance:
yes
Species:
rabbit
Strain:
New Zealand White
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Hazelton Research Products Inc., Denver, PA
- Age at study initiation: 6 months
- Weight at study initiation: 2500-3830 g
- Housing: Stainless steel cages with mesh flooring
- Diet : Purina Certified Rabbit Chow ad libitum
- Water : filtered water ad libitum
- Acclimation period: 14 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19
- Humidity (%): 52
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Artificaially inseminated New Zealand rabbits were fed EDA dihydrochloride salt solution by gavage during gestation day 6-19.
Details on analytical verification of doses or concentrations:
Analysed concentrations within 91-104% of theoretical concentration.
Duration of treatment / exposure:
gestation days 6-19
Frequency of treatment:
Daily
Duration of test:
cesarean section on gestation day 21
No. of animals per sex per dose:
26
Control animals:
yes, concurrent vehicle
Maternal examinations:
Fetal growth, viability and morphological development were examined.
Details on maternal toxic effects:
Maternal toxic effects:no effects
Key result
Dose descriptor:
NOAEL
Effect level:
>= 80 mg/kg bw/day (actual dose received)
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects
Remarks on result:
not measured/tested
Abnormalities:
not specified
Developmental effects observed:
not specified

There were no treatment-related maternal deaths in this study, and no characteristic clinical signs of toxicity in EDA-treated does. At scheduled necropsy, 19-22 pregnancies per group were confirmed. There were no statistically significant effects of EDA on maternal food intake, body weight, weight gain, liver or kidney weight (absolute or relative), or gravid uterine weight. Uterine examination on gd 30 revealed no adverse effects of EDA upon prenatal viability, litter size, fetal weight or fetal morphology.
In conclusion, the maternal and developmental NOAEL for EDA in the New Zealand White rabbit exposed during major organogenesis is >=80 mg/kg/day. Higher doses were not evaluated in this study due to the observation of >=20% maternal mortality at >=100 mg/kg/day in a preliminary investigation (NTP, 1991).

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the study conducted with DMAPA according to the OECD 421, there were no signs of developmental toxicity in the progeny of the F0 parents up to and including the high dose group (200 mg/kg bw/day).

In rats, there is a prenatal developmental toxicity study with the structurally related ethylenediammonium dichloride (EDA*2HCL) available (DePass et al., 1987, reliability score: 2, see read across justification section 13). Fischer 344 rats were fed EDA*2HCl on gestation day 6-15. Groups of 20 rats were fed doses 50, 250, or 1000 mg/kg bw/day. Two control groups were used. Standard endpoints for teratogenicity were evaluated. Reduction in maternal body weight gain was noted in the high and intermediate dose group. Reduced diet consumption was also noted in those groups. In the high dose group there was an increased number of resorptions/litter, decreased mean fetal weight and length, higher incidence of shortened mandible, edematous eye bulge, shortened or missing sternebrae, delayed ossification and missing, or shortened innominate arteries. The fetal alterations were either of questionable teratologic significance or indicative of growth retardation, possibly as a result of reduced food consumption. The most important finding was an increase in the incidence of missing and shortened innominate artery. Further testing was performed to determine, whether this was an effect of malnutrition from reduced food consumption (see paragraph below). The NOAEL for maternal toxicity was 50 mg/kg bw/day of EDA*2HCl (corresponding to 22 mg/kg bw/day of EDA). The NOAEL for developmental toxicity including was 250 mg/kg bw/day of EDA*2HCl (corresponding to 114 mg/kg bw/day EDA).

The additional prenatal developmental toxicity study with EDA*2HCL was performed to determine whether arterial defects observed in the above study were the result of reduced feed intake (DePass et al., 1987, reliability score: 2, see read across justification section 13). This study was carried out as a pair-feeding study in which EDA*2HCl was fed on gestation days 6 through 15 at 1000 mg/kg bw/day. The pair-fed control group received the same amount of diet consumed by the EDA*2HCl-treated rats. An untreated control group was fed ad libitum. Maternal toxicity occurred in the form of reduced body weights and feed consumption during gestation days 6 to 15. At this maternal toxic dose of 1000 mg/kg bw/day, male and female fetal weights and crown-rump lengths were significantly lower than the negative control group and the pair-fed control group. In addition, the length of the innominate artery in male and female pups was shorter than for the pair-fed control and negative control groups. However, both the EDA*2HCl and the pair-fed control group had two fetuses from different litters with missing innominate artery. The authors concluded that the shortened innominate artery from pups fed EDA*2HCl is not a teratogenic effect, since it would result in no functional deficit and is not an irreversible change. It is considered to be part of the overall growth retardation along with reduced fetal weight. Since the incidence of missing innominate artery was the same in the EDA*2HCl-treated group as in the pair-fed controls, the results of the pair-feeding study provide no evidence for an EDA*2HCl-related increase in the incidence of missing innominate artery. Finally, the results of the above and this study indicated that EDA*2HCl was not teratogenic in the Fischer 344 rat but led to growth retardation at the maternal toxic dose level of 1000 mg/kg bw/day of EDA*2HCl (corresponding to 454 mg/kg bw/day of EDA).

In rabbits, there is a prenatal developmental toxicity study with the structurally related EDA*2HCL available (Price te al., 1993, reliability score: 2, see read across justification section 13). In order to avoid the irritant/corrosive properties of the EDA base, the test substance was administered as the dihydrochloride salt (EDA*2HCI). Artificially-inseminated New Zealand White rabbits (26/group) were administered EDA at doses of 0, 10, 40 or 80 mg/kg bw/day (equivalent to 0, 22, 89 or 178 mg/kg bw/day of EDA*2HCI) by gavage on gestational days 6 through 19. Higher doses were not evaluated in this study as in a preliminary study dose levels of 100 mg/kg bw/day and above led to 20% or higher percentages of maternal deaths. At termination on gestation day 30, the uterus was removed and examined to determine pregnancy status and to evaluate the number of resorption and dead or live fetuses. Dead or live fetuses were weighed, and live fetuses examined for external, visceral and skeletal defects. There were no treatment-related maternal deaths and no no substance- or treatment-related clinical signs of toxicity in EDA-treated does at any dose level. At scheduled necropsy, 19-22 pregnancies per group were confirmed. There were no statistically significant effects of EDA on maternal food intake, body weight, weight gain, liver or kidney weight (absolute or relative), or gravid uterine weight. Uterine examination on gestational day 30 revealed no adverse effects of EDA upon prenatal viability, litter size, fetal weight or fetal morphology at any dose level. Thus, the NOAEL for EDA for maternal as well as for developmental toxicity including teratogenicity was 80 mg/kg bw/day, the highest dose level investigated.

The potential toxic effects of the test item, diethylaminopropylamine, on the pregnant female and on embryonic and fetal development was evaluated in a range-finding study following daily oral administration (gavage) to pregnant female rats from implantation to the day prior to the scheduled hysterectomy (Day 6 to Day 20 post-coitum inclusive; Consortium of Alkylamines 42761), in order to select dose-levels for a further main study.

Three groups of eight time-mated female Sprague-Dawley rats were given the test item at 100, 300 or 1000 mg/kg bw/day by oral gavage once daily from Days 6 to 20 p.c. Another group of eight time-mated rats received the vehicle, drinking water treated by reverse osmosis, under the same experimental conditions and acted as a control. On Day 21 p.c., all control females and females given 100 or 300 mg/kg bw/day and 6/8 females given 1000 mg/kg/day were pregnant with live conception. Unscheduled mortality occurred in 1/8 females at 1000 mg/kg bw/day. At final sacrifice, transient ptyalism was observed in all females given 1000 mg/kg bw/day associated with piloerection in 1/7 females. Reflux at dosing was noted on rare occasions in 2/8 and 1/7 females given the test item at 300 or 1000 mg/kg bw/day, respectively.  A body weight loss was observed in females given 1000 mg/kg bw/day at the beginning of the treatment period, whereas slight lower body weight gain was recorded in females given 300 mg/kg bw/day; this was followed with lower body weight gain until Day 15p.c.at 1000 mg/kg/day leading to a statistically significantly lower body weight gain throughout the whole study period (+105 g vs. +140 g in controls). Body weight gain of females given 300 mg/kg bw/day returned to control values from Day 9 p.c. These differences were accompanied with reduced food consumption. There were no effects of treatment with the test item at 100 mg/kg bw/day. At necropsy on terminal sacrifice, there were no test item-related gross findings. When compared with the control group, dams given 1000 mg/kg bw/day had a lower gravid uterus weight observed in high-dose dams. Lower net body weight change was considered to be a consequence of the lower body weight gain in these females. Gestation parameters were not impacted by the test item treatment at any dose-levels. There was no effect on the fetal body weight and on the percentage of male fetuses (sex ratio). There were no treatment-related fetal external malformations or variations in any groups. Based on these findings, 750 mg/kg bw/day was selected as a suitable high dose-level for the main prenatal developmental toxicity study with the test item.

The main embryo-fetal developmental GLP study was carried out according to OECD TG 414. The potential toxic effects of the source substance DEAPA on pregnant female and on embryonic and fetal development was evaluated in a standard prenatal developmental toxicity study. Three groups of 24 time-mated female Sprague-Dawley rats received the DEAPA as pH-neutralized dose formulations by gavage, at dose-levels of 50, 250 or 750 mg/kg bw/d, once daily from day 6 until day 20 p.c. Another group of 24 time-mated rats received the vehicle only under the same experimental conditions and acted as a control group. At study termination on day 21 p.c., there were 24, 21, 23 and 18 females with live fetuses in the groups given 0, 50, 250 or 750 mg/kg bw/d, respectively. On day 20 p.c., one pregnant female at 750 mg/kg bw/d was found dead. From 250 mg/kg bw/d, ptyalism, sneezing, chromorhinorrhea and/or dacryhorrhea were considered to be test item treatment-related but of minor toxicological importance. At 250 and 750 mg/kg bw/d, 4/24 and 19/23 dams, respectively, showed clinical signs, which were considered to be test item treatment-related and adverse at 750 mg/kg bw/d. An adverse body weight loss was observed in females given 750 mg/kg bw/d at the beginning of the treatment period, which resulted in a lower body weight gain until the end of the treatment period. None adverse effects on body weight gain were noted at 250 mg/kg bw/d. Lower mean food consumption was considered to be adverse at 750 mg/kg bw/d. Spleen enlargement, raised focus on the spleen and colored focus on the stomach wall were observed only in females given the high dose-level. At 750 mg/kg bw/d, statistically significantly lower mean net body weight change and lower carcass weight were noted. At 250 and 750 mg/kg bw/d, mean gravid uterus weights were 9% and 18% lower than controls, respectively. This correlated with a lower mean number of live fetuses at both dose levels and with a lower mean fetal body weight at 750 mg/kg bw/d. At 250 and 750 mg/kg bw/d, total resorption in one female at each dose level and higher mean postimplantation loss were also observed. At 750 mg/kg bw/d, higher mean number of early resorptions was noted, as along with lower mean net body weight change and lower carcass weight. At 750 mg/kg bw/d, the lower mean fetal body weights were statistically significant. The sex ratio was unaffected by the test item treatment at any dose-level. There were no test item treatment-related variations at external/oral cavity examination of the fetuses. At 750 mg/kg bw/d, two fetuses, from two different litters had anasarca, cleft palate, short trunk, short tail and/or anal atresia and acaudia. There were no test item-related variations and malformations at soft tissue examination. At 750 mg/kg bw/d, there were higher litter and fetal incidences of fetuses with incomplete ossification of cervical vertebrae centrum.

The NOAEL for maternal toxicity was 50 mg/kg bw/d (based on clinical signs: reductions in food consumption, body weight, net body weight change and/or gravid uterus weight from 250 mg/kg bw/d and mortality at 750 mg/kg bw/d). The NOAEL for developmental toxicity and teratogenicity was 50 and 250 mg/kg bw/d, respectively. Thus, developmental toxicity including teratogenicity (high dose only) were only noted at dose levels of marked (250 mg/kg bw/d) or severe (750 mg/kg bw/d) maternal toxicity, including premature deaths (Consortium of Alkylamines 42762).

 

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.