Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

-There is a OECD 443 study ongoing at Charles River with the following preliminary results, the following investigation are not yet available (The available results are reported below altough a number of investigation is still under investigations (thyroid hormone analysis F0/F1 females, number of implanation sites F0 females, biochemitry hematology F 1 animals, macroscopic examination F1, organ weight F1, histopathology F0, F1, Splenic Lymphocyte Subpopulation Analysis – F1 (cohort 1A - PND 89-95)). (final report will be available on March 2022). (see justification letter from Charles River Lab for the delay in submitting the full results at time, letter attached in the results section). The update of the REACH dossier will then be updated in June 2022.


-There is an OECD 421 oral screening study for reproductive and developmental effects, performed according to GLP principles (Charles River, 2021), the parental NOAEL for Dimethylethylamine was derived to be 80 mg/kg bw/day, based on adverse stomach findings resulting in mortality at 150 and 225 mg/kg bw/day. As high dose group (225 mg/kg bw/day) was prematurely terminated before mating due to the occurrence of high mortality and clinical signs of toxicity, both the reproduction and the developmental NOAEL were established at 150 mg/kg bw/day based on the absence of adverse effects up to and including 150 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
22 Jul 2020 - 18 Feb 2021
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Justification for type of information:
The objective of this GLP OECD guideline study was to select dose levels for the extended one generation reproductive toxicity study with Dimethylethylamine in rats (Charles River Study No. 20240952).
The potential toxic effects of Dimethylethylamine when given orally by gavage for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated. In addition, parental, reproduction (up to and including implantation) and developmental (from implantation onwards) No Observed Adverse Effect Levels (NOAELs) were evaluated.
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
version 2016
Deviations:
no
Qualifier:
according to guideline
Guideline:
other: EPA OPPTS 870.3550, Reproduction/Developmental Toxicity Screening Test
Version / remarks:
version 2000
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Specific details on test material used for the study:
Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.
Species:
rat
Strain:
Wistar
Remarks:
Crl: WI(Han)
Details on species / strain selection:
The Wistar Han rat was chosen as the animal model for this study as it is an accepted rodent species for toxicity testing by regulatory agencies. Charles River Den Bosch has general and reproduction/developmental historical data in this species from the same strain and source. This animal model has been proven to be susceptible to the effects of reproductive toxicants.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at study initiation: Males:11-12 weeks old, females: 13-14 weeks old
- Weight at study initiation: Males: 292 - 352 g; females: 198 - 242 g
- Fasting period before study: No.
- Housing: 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) with a maximum of 5 males/cage. Females were individually housed in Macrolon plastic cages (MIII type) during pregnancy and lactation phase. Animals were provided with items such as devices for hiding in, paper and/or objects for chewing, except when interrupted by study procedures/activities.
- Diet: pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany), ad libitum.
- Water: municipal tap water, ad libitum.
- Acclimation period: 7 days

The feed was analyzed by the supplier for nutritional components and environmental contaminants. Periodic analysis of the water was performed. It is considered that there were no known contaminants in the feed or water that would interfere with the objectives of the study.


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 47 - 72
- Air changes (per hr): Ten or greater with 100% fresh air (no air recirculation)
- Photoperiod (hrs dark / hrs light): A 12-hour light/12-hour dark cycle was maintained

IN-LIFE DATES: From: 05 Aug 2020 To: 07 Oct 2020
Route of administration:
oral: gavage
Vehicle:
water
Remarks:
(Elix)
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
Dosing formulations (w/w) were prepared at appropriate concentrations to meet dose level requirements using the following method:
water (Elix) was chilled in an ice bath or in the refrigerator before formulating. A stir bar was added to an empty container suitable to hold the final volume with the minimum amount of head space. The scale was tared with the container (plus the seal). The required volume of chilled water (Elix) was transferred to the container. The filled container (plus the seal) was weighed. The test substance was kept cold in a refrigerator or ice bath. A syringe or pipette was used to slowly add the required volume of cold test substance into the chilled water (Elix) under stirring. Part of the required amount of test item (about 70-80%) was added to the vehicle under stirring while the container was in a bath filled with ice water. The container was closed, taken out of the ice water, dried off and placed on the scale to add the remaining amount of test item up to the correct weight. The container was sealed and weighed after this addition. The formulation was stirred magnetically to mix.

The dosing formulations were prepared at least weekly, filled out in daily portions with the use of a syringe or pipette and stored in the refrigerator. The dosing formulations were removed from the refrigerator, stirred at room temperature for at least 30 minutes, and kept at room temperature until dosing. Test substance formulations were dosed within 4 hours after removal from the refrigerator. On the day of dosing, the regular seal was replaced by a seal with a septum / small hole. If practically possible, the dosing formulations and vehicle were continuously stirred until and during dosing. Adjustment was made for specific gravity of the test item. No correction was made for the purity/composition of the test item.

VEHICLE
- Concentration in vehicle: 16, 30, 45 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg

Details on mating procedure:
- M/F ratio per cage: 1:1 mating
- Length of cohabitation: A maximum of 14 days was allowed for mating
- Proof of pregnancy: appearance of intravaginal copulatory plug or by evidence of sperm in vaginal smear, referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: individually
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Accuracy and homogeneity were determined for formulations prepared for use in Week 1, Week 4 and Week 7.

Duplicate samples (approximately 500 mg) taken from the formulations using a pipette, were accurately weighed into volumetric flasks of 50 mL (Group 1 and Group 2) or 100 mL (Group 3 and Group 4). For determination of accuracy, samples were taken at middle position (50% height) or at top, middle and bottom position (90%, 50% and 10% height). The samples taken at 90%, 50% and 10% height were also used for the determination of the homogeneity of the formulations.
Analyses were performed using a validated analytical procedure (Charles River Study No. 20240947).

Concentration results were considered acceptable if mean sample concentration results were within or equal to 85-115% of target concentration. Homogeneity results were considered acceptable if the coefficient of variation was = 10%.

Stability analyses also performed previously in conjunction with the method development and validation study demonstrated that the test item was 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 Charles River Study No. 20240947.
Duration of treatment / exposure:
Males of groups 1-3 were treated for 29 days, up to and including the day before scheduled necropsy. Females of groups 1-3 that delivered were treated for 50-63 days, females of groups 1-3 which failed to deliver were treated for 41 days.

Due to severe toxicity observed in animals treated at 225 mg/kg bw/day, resulting in the preterm sacrifice of 3 females on study day 4 and 3 males and 2 females on study day 6, the remaining Group 4 animals were sacrificed for ethical reasons on study day 6 (last dose on day 5).
Frequency of treatment:
once daily, 7 days a week
Dose / conc.:
80 mg/kg bw/day (actual dose received)
Remarks:
group 2 - low dose
Dose / conc.:
150 mg/kg bw/day (actual dose received)
Remarks:
group 3 - mid dose
Dose / conc.:
225 mg/kg bw/day (actual dose received)
Remarks:
group 4 - high dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The oral route of administration was selected because this is a possible route of human exposure. The dose levels were selected based on previously performed animal studies with oral administration of Dimethylethylamine in Sprague Dawley rats (i.e. acute oral toxicity study, 14-day preliminary toxicity study, and a OECD 414 study).
During the acute oral toxicity study, an LD50 of 594 mg/kg bw (540-650 mg/kg bw) was determined with 95% confidence intervals. Based on this information, 5 male and female rats received 0, 25, 75 or 225 mg/kg bw/day during the 14-day preliminary toxicity study. No test item-related mortality was observed up to 225 mg/kg bw/day and clinical signs were limited to salivation and chin rubbing in high dose animals. Additionally, the males at 225 mg/kg bw/day had a lower body weight gain (30%) over Days 1-15 of treatment and food consumption of these males was lower during the first week of treatment. The hematology investigation revealed an increased reticulocyte count and red cell distribution width in males and females at 225 mg/kg bw/day, however these findings did not affect the condition or function of the animals. There were no findings attributable to the test item at macroscopic examination, but mean adjusted adrenal weights were 22% higher among males receiving 225 mg/kg bw/day when compared with control. No test item-related effects were observed in the animals treated at 25 or 75 mg/kg bw/day. It was therefore concluded that there were no adverse findings that would preclude the use of at least 225 mg/kg bw/day as the highest dose level for future repeat dose studies.
During the dose range finding study of the OECD 414 study, two rats died on day 3 of treatment when exposed to 360 mg/kg bw/day. The high dose was consecutively reduced to 270 mg/kg bw/day, but all remaining animals died one day later. Histopathology confirmed ulceration in the stomach. During the main OECD 414 study, effects on body weight and food consumption were observed at 180 mg/kg bw/day. Considering all information above, dose levels of 0, 80, 150 and 225 mg/kg bw/day were selected in agreement with the Sponsor.

- Fasting period before blood sampling for clinical biochemistry: Adult males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. Females were not fasted overnight.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: During treatment, animals were observed at least once daily, up to the day prior
to necropsy. These clinical observations were conducted after dosing at no specific time point.

BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed individually on the first day of treatment
(prior to dosing), and weekly thereafter. Mated females were weighed on Days 0, 4, 7, 11, 14, 17,
and 20 post-coitum and during lactation on PND 1, 4, 7, and 13.
In order to monitor the health status, a few animals were weighed on a few extra occasions.
A terminal weight was recorded on the day of scheduled necropsy.

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

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

Thyroid hormone analysis:
Blood of F0-animals (except for high dose group animals and animals which were sacrificed in extremis or found dead) was collected on the day of scheduled necropsy. Samples were collected from the retro-orbital sinus under anesthesia using isoflurane.
Blood samples were collected into tubes without anticoagulant, were processed for serum, and serum was analyzed for Thyroxine (T4) and Thyroid Stimulating Hormone (TSH). Measurement of total T4 and TSH was conducted for F0-males. F0-males were fasted overnight with a maximum of 24 hours before blood sampling, but water was available. F0-females were not fasted overnight.
Assessment of T4 and TSH in F0 females was considered not relevant because there were no test item-related changes in T4 in F0 males, or in the weight and morphology of the thyroid in both sexes.

OTHER:
Females were allowed to litter normally. Postnatal day (PND) 1 is defined as the day when a litter is found completed (i.e. membranes and placentas cleaned up, nest built and/or feeding of pups started). The day prior to PND 1 is considered to be the day when the female started to deliver and is defined as PND 0 and used for recording of delivery. Females that were littering were left undisturbed. Cage debris of pregnant females was examined for evidence of premature delivery and pregnant females were examined to detect signs of difficult or prolonged parturition or deficiencies in maternal care.
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 females beginning 14 days prior to treatment (pretest period), the first 14 days of treatment and during mating until evidence of copulation was observed. Vaginal lavage was continued for those females with no evidence of copulation until termination of the mating period. On the day of necropsy, a vaginal lavage was also taken to determine the stage of estrous.
Sperm parameters (parental animals):
Parameters examined in all adult males: testis weight, epididymis weight.
For the testes of all males in the control and mid dose group, and all males that failed to sire or died before mating (except for males in the high dose group), a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

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, physical or behavioural abnormalities, anogenital distance (AGD), pup weight on the day of AGD, presence of nipples/areolae in male pups, other. Particular attention was paid to the external reproductive genitals which was examined for signs of altered development; gross evaluation of external genitalia.
Live pups were weighed individually on PND 1, 4, 7 and 13. Anogenital distance (AGD) was measured for all live pups on PND 1. The AGD was normalized to the cube root of body weight. All male pups in each litter were examined for the number of areola/nipples on PND 13.

Thyroid hormone analysis:
Blood of F1-animals was collected on PND 4 and PND 14-16. On PND 4 at culling, blood was collected from two surplus pups per litter by decapitation and samples were pooled to one sample per litter. On PND 14-16 separate blood samples were collected from two pups per litter. Blood was drawn by aorta puncture under anesthesia. Blood samples were collected into tubes without anticoagulant, were processed for serum, and serum was analyzed for total Thyroxine (T4) and Thyroid Stimulating Hormone (TSH). Measurement of total T4 and TSH was conducted for PND 14-16 pups. Assessment of T4 for PND 4 pups was considered not relevant because no test item-related changes in T4 were noted in pups at PND 14-16.

GROSS EXAMINATION OF DEAD PUPS:
Yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead, if possible: Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for the presence of milk, if possible. If possible, defects or cause of death were evaluated.
Postmortem examinations (parental animals):
SACRIFICE
- Parental animals: All animals surviving until scheduled euthanasia were weighed, and deeply anaesthetized using isoflurane and subsequently exsanguinated and subjected to a full post mortem examination. All males surviving to scheduled necropsy were fasted overnight with a maximum of 24 hours before necropsy. Water was available. F0-females were not fasted. Males (which sired and failed to sire) were euthanized after the completion of the mating period. Females which failed to deliver (with evidence of mating) were euthanized between 25-26 days post-coitum (after confirmation of mating). Females which delivered were euthanized between 14-16 days post partum.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera and the appearance of the tissues and organs was observed macroscopically. All animals were subjected to a full post mortem examination, with special attention being paid to the reproductive organs.
The numbers of former implantation sites were recorded for all paired females. In case no macroscopically visible implantation sites were present, non-gravid uteri were stained using the Salewski technique in order to detect any former implantation sites and the number of corpora lutea.

ORGAN WEIGHTS
At necropsy, for all scheduled euthanasia animals, terminal body weight was measured and the following organs were weighed: epididymis, coagulation gland with seminal vesicle gland, thyroid with parathyroid, prostate, testes, adrenal gland, liver and ovaries. Paired organs were weighed together. In the event of gross abnormalities, in addition to the combined weight, the weight of the aberrant organ was taken and recorded in the raw data. Organ to body weight ratios (using the terminal body weight) were calculated.

-Tissue collection and preservation:
Representative samples of the following tissues were collected from all animals and preserved in 10% neutral buffered formalin: cervix, seminal vesicles including coagulation gland,
mammary gland, thyroid including parathyroid gland, pituitary gland, prostate gland, ovaries, uterus, vagina, adrenal gland, liver, and stomach. Testes and epididymis were preserved in modified Davidson’s fixative and transferred to formalin after fixation for at least 24 hours. Gross lesions or masses were also collected from all animals if found.

HISTOPATHOLOGY
The following tissues were embedded in paraffin, sectioned, mounted on glass slides, and stained with hematoxylin and eosin:
> gross lesions or masses and stomach from all animals, if any
> epididymis, thyroid gland, ovaries, testes, seminal vesicle including coagulation gland, prostate of all animals of the control and mid dose groups and all animals that die spontaneously or are sacrificed in extremis (except for high dose group animals)
> cervix, epididymis, seminal vesicles including coagulation gland, prostate gland, ovaries, uterus,
vagina, testes of all males that failed to sire and females that failed to deliver pups and females with total litter loss.
For the testes of all males in the control and mid dose group, and all males that failed to sire or died before mating (except for high dose group animals) a detailed qualitative examination was made, taking into account the tubular stages of the spermatogenic cycle.
Postmortem examinations (offspring):
SACRIFICE
Pups, younger than 7 days were euthanized by decapitation. On PND 4, the surplus pups were euthanized by decapitation. All remaining pups (PND 14-16) except for the two pups per litter selected for blood collection were euthanized by an intraperitoneal injection of sodium pentobarbital. The pups selected for blood collection on PND 14-16 were anesthetized using isoflurane followed by exsanguination.

GROSS NECROPSY
Pups that died before scheduled termination were examined externally and sexed (both externally and internally). The stomach of pups not surviving to the scheduled necropsy date was examined for
the presence of milk, if possible. If possible, defects or cause of death were evaluated.
For all remaining pups euthanized on PND 14-16, sex was determined both externally and internally.
Descriptions of all external abnormalities were recorded. Particular attention was paid to the external reproductive genitals to examine signs of altered development.
The thyroid from two pups per litter (if possible one male and one female pup) was preserved in 10% buffered formalin. If possible, the pups selected for blood sampling were the same pups as selected for thyroid preservation.
Statistics:
All statistical tests were conducted at the 5% significance level. All pairwise comparisons were conducted using two sided tests and were reported at the 1% or 5% levels.
Numerical data collected on scheduled occasions for the listed variables were analyzed as indicated according to sex and occasion. Descriptive statistics number, mean and standard deviation (or %CV or SE) were reported when possible. Inferential statistics were performed according to the matrix below when possible, but excluded semi-quantitative data, and any group with less than 3 observations.
The following pairwise comparisons were made:
Group 2 vs. Group 1
Group 3 vs. Group 1
Group 4 vs. Group 1
Datasets with at least 3 groups (control group and 2 other groups) were compared using Dunnett-test (many-to-one-t-test). Datasets with at least 3 groups was compared using a Steel-test (many-to-one rank test). An overall Fisher’s exact test was used to compare all groups at the 5% significance level. The above pairwise comparisons were conducted using Fisher’s exact test whenever the overall test is significant.
For clinical pathology data Levene’s test was used to assess the homogeneity of group variances.
The groups were compared using an overall one-way ANOVA F-test if Levene’s test was not significant or the Kruskal-Wallis test if it was significant. If the overall F-test or Kruskal-Wallis test was found to be significant, then pairwise comparisons were conducted using Dunnett’s or Dunn’s test, respectively. The groups were compared using an overall Kruskal-Wallis test. If the overall Kruskal-Wallis test was found to be significant, then the above pairwise comparisons were conducted using Dunn’s test. The data corresponding to a response variable of interest and to a related covariate was submitted to an analysis of covariance (ANCOVA), including only groups with at least three non-missing paired values and if found to be significant, then pairwise comparisons were conducted using Dunnett's test.
Reproductive indices:
Mating index (%): (Number of females mated/Number of females paired) x 100

Precoital time: (Number of days between initiation of cohabitation and confirmation of mating

Fertility index (%): (Number of pregnant females/Number of females mated) x 100

Gestation index (%): (Number of females with living pups on Day 1/Number of pregnant females) x 100

Duration of gestation: Number of days between confirmation of mating and the beginning of parturition
Offspring viability indices:
Post-implantation survival index (%): (Total number of offspring born/Total number of uterine implantation sites) x 100

Live birth index (%): (Number of live offspring on Day 1 after littering/Total number of offspring born) x 100

Percentage live males at First Litter Check (%): (Number of live male pups at First Litter Check/
Number of live pups at First Litter Check) x 100

Percentage live females at First Litter Check (%): (Number of live female pups at First Litter Check/
Number of live pups at First Litter Check) x 100

Viability index (%): (Number of live offspring on Day 4 before culling/Number live offspring on Day 1
after littering) x 100

Lactation index (%): (Number of live offspring on Day 13 after littering/Number live offspring on Day 4 (after culling)) x 100
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 150 and 225 mg/kg bw/day was associated with clinical signs of toxicity (e.g. calm or lethargic behavior, signs of breathing difficulties, hunched posture and/or piloerection).

At 150 mg/kg bw/day, signs of breathing difficulties (i.e. labored/shallow breathing and/or rales) and piloerection (females only) were also observed in several animals that survived up to scheduled necropsy. Rales and piloerection occurred on incidental occasions with a maximum of seven consecutive days during the treatment period, whereas labored and shallow breathing were seen in two animals on one day only.

At 80 mg/kg bw/day, no clinical signs were observed in males. Piloerection and rales were noted
in four and one female, respectively. Both clinical signs were transient and occurred on 1-5 consecutive days during the treatment period. The salivation seen after dosing among treated animals throughout the dosing period was considered not toxicologically relevant, taking into account the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response rather than a sign of systemic toxicity.

Any other clinical signs noted during the treatment period (e.g. fissures, wounds and scabs) 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 did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.
Mortality:
mortality observed, treatment-related
Description (incidence):
Test item-related mortality occurred in all treatment groups.

At 225 mg/kg bw/day, three males and five females were sacrificed in extremis on Study Days 4 or 6 based on severe weight loss (between 10-20%) when compared with Study Day 1. The sacrificed females (except for one female with no findings), showed next to salivation multiple clinical signs of toxicity (i.e. calm and lethargic behavior, difficulties with breathing (rales, labored respiration and/or gasping), hypothermia, pale appearance and/or piloerection) while observations for the males were
limited to slight salivation.
Based on this mortality and as body weight loss was observed for the majority of the remaining animals at 225 mg/kg bw/day as well (though less severe than the animals sacrificed in extremis), remaining animals were sacrificed on Study Day 6 for welfare reasons. The stomach alterations recorded at necropsy, consisting of foci in the glandular mucosa and/or forestomach, thickened limiting ridge and/or forestomach, yellowish discoloration of the glandular mucosa and/or irregular surface of the forestomach, were regarded to be the main cause of the poor condition of these animals. These animals were sacrificed before mating and no histopathology was performed on these animals.

At 150 mg/kg bw/day four males and two females were euthanized between Day 6 and Day 33 of the study. Several of these animals were sacrificed based on moderate to severe weight loss (between 9-14%) when compared with Study Day 1, but two males were sacrificed due to severe signs of breathing difficulties (i.e. rales, gasping and/or labored/shallow breathing). In addition, one male and one female showed multiple clinical signs of toxicity prior to sacrifice (i.e. lethargy, piloerection, hunched posture and/or a pale appearance). A limited list of organs (reproductive organs, thyroid gland and gross findings) were examined histopathologically. Based on the macroscopic and microscopic findings, the main cause of moribundity was regarded glandular erosions and/or inflammatory lesions in the stomach. Stomach alterations included minimal-slight inflammation of the glandular mucosa (correlating to an irregular surface of the glandular mucosa in one male), slight to marked erosion of the glandular mucosa, minimal-moderate hemorrhage (correlating to reddish/dark-red foci on the glandular mucosa in four males and two females) and/or slight edema of the glandular mucosa. In addition, in the forestomach of a few animals at 150 mg/kg bw/day slight-moderate inflammation and/or slight hyperplasia of the squamous epithelium was recorded.

One female at 150 mg/kg bw/day was found dead on Day 41 of the study. This animal had lost 6% body weight between Study Days 40 and 41 and presented with signs of breathing problems (i.e. gasping and rales) and piloerection on Day 41. This female also showed slightly uncoordinated movements on Days 6-7 of treatment and a slightly tilted head on Days 6-8 and 35-41 of treatment. No cause of death could be determined from the tissue sections examined.

At 80 mg/kg bw/day one male was euthanized on Study Day 6, based on severe weight loss (16%). No clinical signs were observed on the days prior to sacrifice. A limited list of organs (reproductive organs, thyroid gland and gross findings) were examined histopathologically. Based on the macroscopic and microscopic findings, the main cause of moribundity was regarded glandular erosions in the stomach. Stomach alterations included minimal erosion of the glandular mucosa and moderate hemorrhage (correlating to reddish/dark-red foci on the glandular mucosa).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Treatment at 225 mg/kg bw/day was associated with severe body weight loss (between 10-20%).

Treatment at 150 mg/kg bw/day was associated with severe body weight loss for two males
and one female that were sacrificed prior to scheduled necropsy. Several remaining males at 150 mg/kg bw/day showed a slight weight loss or a lower body weight gain when compared with controls, mainly during the first week of treatment. However, individual body weight gains of males surviving up to scheduled necropsy were similar to concurrent control during the last two weeks of treatment (except for a single male). Weight loss was observed in some of the remaining females as well, mainly during the first week of treatment. Individual weight gain values were similar to concurrent control values towards the start of the mating period and during the post-coitum period and beginning of the lactation period. However, body weight gain on PND 13 of females surviving up to scheduled necropsy was noticeably lower when compared with concurrent control values (mean weight gain of 9% vs. 18%, respectively).

Weight gain of females at 80 mg/kg bw/day was similar to concurrent control during the premating, post-coitum and beginning of lactation period. However, body weight gain on PND 13 of 3/7 lactating females was noticeably lower when compared with other females of the same group and concurrent control. Individual values of these three females were similar to values determined for females treated at 150 mg/kg bw/day and were therefore considered test item-related. No test item-related changes in body weights and body weight gain were observed in males at 80 mg/kg bw/day that survived up to scheduled necropsy.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Food consumption before or after correction for body weight was lower for males and females treated at 150 mg/kg bw/day during the first week of treatment. Mean relative food consumption was 12 and 13% lower when compared with concurrent control for males and females, respectively (not statistically significant). This could most likely be attributed to the animals that were sacrificed in extremis. In subsequent intervals, food consumption was similar to the control level.

Food consumption before or after correction for body weight was similar to the control level at 80 mg/kg bw/day.
Endocrine findings:
no effects observed
Description (incidence and severity):
No toxicologically relevant changes were noted in serum levels of T4 and TSH in F0-males at any dose level.
In F0-males at 150 mg/kg bw/day, mean TSH concentration was increased when compared with
concurrent control (1.69x). No statistical significance was achieved. The higher mean value was attributed to the relatively high value of a single animal (1.490 mU/L). After excluding this animal, a mean value of 0.0696 mU/L (0.38x of control) was obtained. As this change was observed in absence of a corroborative change in T4, it was considered not biologically relevant.
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Test item-related microscopic stomach findings consisted of an increased incidence and severity of alterations in the glandular mucosa to include mixed cell inflammation, erosions, hemorrhage and/or edema in males at 80 and 150 mg/kg bw/day and females at 150 mg/kg bw/day. In some cases the hemorrhage was associated with the glandular erosions.
In the forestomach, findings were seen in a few animals at 150 mg/kg bw/day and included mixed
cell inflammation, ulceration and/or hyperplasia of the forestomach. In the duodenum, present in the stomach section of a single male at 150 mg/kg bw/day, a marked ulcer of the duodenum was recorded.
There were no other test item-related macroscopic or microscopic changes. The remainder of the recorded findings 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.

Reproductive Performance:
All cohabitated females showed evidence of mating, however, one control female and three females at 80 mg/kg bw/day were not pregnant. The reproductive organs of males that failed to sire (due to early sacrifice or if cohabitated female was not pregnant) and females that failed to deliver healthy pups were examined histopathologically.

There was 1/10 couples of the control group and 2/10 couples of the 80 mg/kg bw/day group without implantation sites. Additionally, a single female at 80 mg/kg bw/day had no implantation sites. No abnormalities were seen in the reproductive organs of these animals, which could account for their lack of offspring. As all females at 150 mg/kg bw/day were pregnant with implantation sites, the incidence of non-pregnant females at 80 mg/kg bw/day was considered not test item-related in absence of a dose-related response.

Several animals we euthanized prior to mating, one male at 80 mg/kg bw/day, and two males and one female at 150 mg/kg bw/day. There were no findings recorded for the reproductive organs of these animals suggestive of infertility. One female at 150 mg/kg bw/day) was euthanized before delivery and showed features of normal pregnancy.

Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Length and regularity of the estrous cycle were considered not to have been affected by treatment with the test item up to 150 mg/kg bw/day.
Most females had regular cycles of 4 to 5 days. An irregular cycle was noted for one female at 80 mg/kg bw/day. Given the incidental nature and absence of a dose-related response, this finding did not indicate a direct relation with the test item.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Stage dependent qualitative evaluation of spermatogenesis in the testis was performed. The testis revealed normal progression of the spermatogenic cycle, and the expected cell associations and proportions in the various stages of spermatogenesis were present.
Reproductive performance:
no effects observed
Description (incidence and severity):
Mating index was not affected by treatment with the test item up to 150 mg/kg bw/day. All cohabitated females showed evidence of mating, resulting in a mating index of 100% for the control, 80 and 150 mg/kg bw/day groups.

Precoital time was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. All females showed evidence of mating within 4 days, except for one female at 150 mg/kg/day that showed evidence of mating on Day 12 of the mating period. As this also occurs in control animals from time to time and it concerns one animal only, it was considered incidental and unrelated to treatment with the test item.

Fertility index was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. The fertility indices were 90, 70 and 100% for the control, 80 and 150 mg/kg bw/day groups, respectively. A total of three females at 80 mg/kg bw/day were not pregnant. Since these cases of nonpregnancy showed no dose-related incidence across the dose groups, and given the absence of any reproductive/developmental toxicity in this study, this was considered not to be related to treatment with the test item.

Gestation index (females with living pups on Day 1 compared to the number of pregnant females) and duration of gestation were not affected by treatment with the test item up to 150 mg/kg bw/day. All surviving females that were pregnant had live offspring.

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.

Number of implantation sites was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
The mean number of implantation sites was significantly lower at 80 and 150 mg/kg bw/day when compared with concurrent control. However, when compared with historical control data (mean: 12.3, P5-P95:6.0-16.0 (n=1511), it was found that the concurrent control mean was actually relatively high. One female of the 80 and 150 mg/kg/day groups each had two and six implantation sites only, respectively, which is also sometimes seen in control females, and the number of implantation sites of other females were within normal limits. The number of implantation sites was therefore considered unaffected by the test item.
Key result
Dose descriptor:
NOAEL
Remarks:
Reproduction toxicity
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other:
Remarks on result:
other: as the 225 mg/kg bw/day group was terminated on Study Day 6, no reproduction data is available for this group.
Key result
Dose descriptor:
NOAEL
Remarks:
Parental Systemic toxicity
Effect level:
80 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
mortality
gross pathology
histopathology: non-neoplastic
Remarks on result:
other: Based on adverse stomach findings resulting in mortality at 150 and 225 mg/kg bw/day.
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
150 mg/kg bw/day (actual dose received)
System:
gastrointestinal tract
Organ:
stomach
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs occurred among pups that were considered to be related to treatment with
the test item up to 150 mg/kg bw/day.
At 150 mg/kg bw/day, one pup had a paralyzed left hindleg from PND 7 onwards and one other pup from another litter had a swollen left frontleg between PND 2-11. At the incidence observed, no relationship with the test item is indicated.
Additionally, one control pup was sacrificed in extremis on PND 4 as it appeared dehydrated and skinny, with a limited amount of milk in its stomach.

The nature and incidence of other clinical signs remained within the range considered normal for pups of this age, and were therefore considered not to be toxicologically relevant.
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
The number of live offspring on Day 1 after littering compared to the total number of offspring born was not affected by treatment with the test item up to 150 mg/kg bw/day.

Live birth index (number of live offspring on PND 1 as percentage of total number of offspring born) was not affected by treatment with the test item up to 150 mg/kg bw/day. The live birth index was 99, 100 and 99% for the control, 80 and 150 mg/kg bw/day groups, respectively.

One pup of the control group and one pup at 150 mg/kg bw/day were found dead at first litter check. No toxicological relevance was attributed to these dead pups since the mortality incidence did not show a dose-related trend and remained within the range considered normal for pups of this age.

The number of live offspring on day 4 before culling compared to the number of offspring on day 1 was not affected by treatment with the test item up to 150 mg/kg bw/day.
Viability index (number of live offspring on PND4 before culling as percentage of number of live offspring on PND1) was not affected by treatment with the test item up to 150 mg/kg bw/day. Viability indices were 99% for the control and 80 mg/kg bw/day groups and 88% for the 150 mg/kg bw/day group. This apparent lower viability index and the statistically significantly higher postnatal loss noted at 150 mg/kg bw/day could mainly be attributed to the death of a single dam and consequently sacrifice of the remaining eight live pups in this litter on day 2 of lactation. When excluding these sacrificed pups, a viability index of 98% is obtained.
Additionally, one pup of the control group, one pup at 80 mg/kg bw/day and two pups at 150 mg/kg bw/day were sacrificed in extremis, found dead or missing on PND 2 or 4. Pups missing were most likely cannibalised. No toxicological relevance was attributed to these dead/missing pups since the mortality incidence remained within the range considered normal for pups of this age.

The number of live offspring on Day 13 after littering compared to the number of live offspring on day 4 (after culling) was unaffected by treatment with the test item up to 150 mg/kg bw/day. No pups were found dead/missing between lactation days 5 and 13, resulting in a lactation index of 100% for all groups.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weights of pups determined between Day 1-13 of lactation were considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
Serum T4 and TSH levels in male and female PND 14-16 pups were considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Male pups of two litters at 80 mg/kg bw/day had relatively high TSH levels when compared with concurrent and historical controls. As this change was observed in absence of a dose-response and a corroborative change in T4, it was considered not related to the test item.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
Anogenital distance (absolute and normalized for body weight) in male and female pups was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
Treatment with the test item up to 150 mg/kg bw/day had no effect on areola/nipple retention. For none of the examined male pups nipples were observed at PND 13.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No macroscopic findings were noted among pups that were considered to be related to treatment with the test item up to 150 mg/kg bw/day.
At 150 mg/kg/day, one pup had a paralyzed left hindleg. At the incidence observed, no relationship with the test item is indicated. The nature and incidence of other macroscopic findings remained within the range considered normal for pups of this age, and were therefore considered not to be related to treatment with the test item.
Other effects:
no effects observed
Description (incidence and severity):
Litter size was considered not affected by treatment with the test item up to 150 mg/kg bw/day. A statistically significantly lower mean number of living pups/litter was recorded at 80 and 150 mg/kg bw/day (10.0 and 10.1 vs. 12.9 in the control group). The higher mean of the concurrent control group was however attributed to the relative high number of implantation sites in this group. As a dose-related response was absent and the mean remained within the range considered normal for rats of this strain and age, the lower litter size at 80 and 150 mg/kg bw/day was considered unrelated to treatment with the test item.

Additionally, sex ratio was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day.

The total number of offspring born compared to the total number of uterine implantations was considered not to be affected by treatment with the test item up to 150 mg/kg bw/day. Post-implantation survival index (total number of offspring born as percentage of total number of uterine implantation sites) was 91, 93 and 85% for the control, 80 and 150 mg/kg bw/day groups, respectively.
The slightly lower post-implantation survival index of females at 150 mg/kg bw/day was considered unrelated to treatment with the test item, all individual values remained within the range considered normal for rats of this strain and age.
Key result
Dose descriptor:
NOAEL
Remarks:
Developmental toxicity
Generation:
F1
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Absence of developmental toxicity effects at dose levels up to and including150 mg/kg bw/ day.
Remarks on result:
other: as the 225 mg/kg bw/day group was terminated on Study Day 6, no developmental data is available for this group.
Key result
Critical effects observed:
no
Key result
Reproductive effects observed:
no

Dose Formulation Analysis:


Accuracy:
A small response at the retention time of the test item was observed in the chromatograms of the Group 1 formulation prepared for use in Weeks 4 and 7. It was considered not to derive from the formulation since a similar response was obtained in the analytical blanks. The maximum contribution to the Group 2 samples was 0.019%. In the formulations of Group 1 for use in Week 1, no test item was detected.
The concentrations analyzed in the formulations of Groups 2, 3 and 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%) except for the Group 2 formulations prepared for use in Week 4 (mean accuracy of 76%). No analytical reason was found for this out of specification result. As there was no indication of structural preparation issues based on the Weeks 1 and 7 results, the lower accuracy found for Group 2 during study Week 4 was considered to be of no negative impact on the study outcome.


Homogeneity:
The formulations of Groups 2 and 4 were homogeneous (i.e. coefficient of variation ≤ 10%).


 


Summary Test Item-Related Macroscopic Stomach Findings - Including (Premature Decedents)














































































































 



Males



Females



Dose level (mg/kg/day):



0



80



150



0



80



150



 



 



 



 



 



 



 



STOMACH a



10



9 (1)



6 (4)



10



10



7 (3)



 



 



 



 



 



 



 



    Foci glandular mucosa



-



1 (1)



1 (4)



-



-



1 (2)



 



 



 



 



 



 



 



     Irregular surface glandular mucosa



-



-



(1)



-



-



 



 



 



 



 



 



 



 



     Irregular surface forestomach



-



-



-



-



-



1



 



 



 



 



 



 



 



    Thickened limiting ridge



-



-



(2)



-



-



(1)



a  =  Number of tissues examined from each group: surviving animals, followed by (..) number of premature decedents


 


Summary Test Item-Related Microscopic Stomach Findings - Including (Premature Decedents)





























































































































































































































































































































 



Males



Females



Dose level (mg/kg/day):



0



80



150



0



80



150



 



 



 



 



 



 



 



STOMACH a



10



9 (1)



6 (4)



10



10



7 (3)



    Inflammation glandular mucosa



 



 



 



 



 



 



        Minimal



1



4



3 (2)



1



3



3



       Slight



-



1



2 (1)



-



-



1 (1)



       Moderate



-



-



1



-



-



1



 



 



 



 



 



 



 



     Erosion glandular mucosa



 



 



 



 



 



 



       Minimal



-



1 (1)



1



-



-



-



       Slight



-



-



-



-



-



1 (1)



       Moderate



-



-



-



-



-



1 (1)



       Marked



-



-



(1)



-



-



-



 



 



 



 



 



 



 



     Hemorrhage glandular mucosa



 



 



 



 



 



 



       Minimal



-



1



(3)



-



-



-



       Slight



-



-



1



-



-



(1)



       Moderate



-



(1)



(1)



-



-



1 (1)



 



 



 



 



 



 



 



       Edema glandular mucosa



 



 



 



 



 



 



       Minimal



-



1



1



-



-



-



       Slight



-



-



(1)



-



-



(1)



 



 



 



 



 



 



 



     Inflammation forestomach



 



 



 



 



 



 



       Slight



-



-



-



-



-



(1)



       Moderate



-



-



(1)



-



-



1



 



 



 



 



 



 



 



     Ulcer forestomach



 



 



 



 



 



 



       Moderate



-



-



-



-



-



1



 



 



 



 



 



 



 



     Hyperplasia squamous epithelium


                                     forestomach



 



 



 



 



 



 



       Minimal



-



-



-



-



-



2



       Slight



-



-



(1)



-



-



-



 



 



 



 



 



 



 



a  =  Number of tissues examined from each group: surviving animals, followed by (..) number of premature decedents


 


 

Conclusions:
In an oral screening study for reproductive and developmental effects, performed according to GLP principles, the parental NOAEL for Dimethylethylamine was derived to be 80 mg/kg bw/day, based on adverse stomach findings resulting in mortality at 150 and 225 mg/kg bw/day. As high dose group (225 mg/kg bw/day) was prematurely terminated before mating due to the occurrence of high mortality and clinical signs of toxicity, both the reproduction and the developmental NOAEL were established at 150 mg/kg bw/day based on the absence of adverse effects up to and including 150 mg/kg bw/day.
Executive summary:

The potential toxic effects of Dimethylethylamine when given orally by gavage at 80, 150 and 225 mg/kg/day for a minimum of 28 days to Wistar Han rats were determined, and the potential to affect male and female reproductive performance such as gonadal function, mating behavior, conception, parturition and early postnatal development was evaluated up to at least PND 13.


At 225 mg/kg/day, mortality and clinical signs of toxicity (e.g. calm or lethargic behavior, signs of breathing difficulties, hypothermia, pale appearance and/or piloerection) were observed. In total, eight animals were sacrificed in extremis between Study Day 4 and 6 based on severe body weight loss and/or severe clinical signs. Due to this mortality and as body weight loss was observed for the majority of the remaining animals at this dose level as well (though less severe than the animals sacrificed in extremis), this group was terminated on Study Day 6 for animal welfare reasons. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals.


At 150 mg/kg/day, mortality and clinical signs of toxicity (e.g. lethargic behavior, signs of breathing difficulties, hunched posture, pale appearance and/or piloerection) were observed. In total, six animals were sacrificed in extremis between Study Day 6 and 33 based on severe body weight loss and/or severe clinical signs. The alterations in the stomach observed at necropsy in all decedents were considered to be test item-related, adverse and the main cause of the poor condition of these animals. Macroscopic findings were correlated microscopically with glandular erosions and/or inflammatory lesions in the stomach. For one female, found dead on Study Day 41, no clear cause of death could be revealed. For several remaining animals at 150 mg/kg/day, slight body weight loss or lower body weight gain was observed mainly during the first week of treatment. Additionally, a lower body weight gain was observed in females on PND 13 when compared with control. Animals treated at 150 mg/kg/day that survived up to scheduled necropsy showed similar adverse stomach alterations as the preterm sacrifices of the 150 and 225 mg/kg/day groups.


Based on the adverse stomach findings, which were considered the cause of the moribundity observed at 150 and 225 mg/kg/day, both dose levels were considered inappropriate as the high dose for the subsequent OECD 443 study.


At 80 mg/kg/day, one male was sacrificed in extremis on Study Day 6 based on severe weight loss. In the stomach of this decedent, moderate hemorrhage associated with a minimal erosion of the glandular mucosa was observed. In males surviving until scheduled necropsy, findings in the glandular stomach were recorded at low severities. Clinical signs (i.e. rales and piloerection) were observed in females only, were transient and occurred during a short period of time only. In 3/7 lactating females, body weight gain on PND 13 was noticeably lower than other females of the same group and concurrent control. Individual values of these three females were similar to values determined for females treated at 150 mg/kg/day and were therefore considered test item-related. However at the incidence observed and absence of effects on pup development, this change was considered not dose-limiting. As no further toxicity was observed except for the single mortality and minimal stomach findings, a dose level of 80 mg/kg/day was considered feasible as high dose for the subsequent OECD 443 study.


No test item-related changes were noted in any of the remaining parameters investigated in this study (i.e. clinical biochemistry (male T4 and TSH thyroid hormone levels) and organ weights) at 80 and 150 mg/kg/day in F0-animals. 


No reproductive or developmental toxicity was observed up to 150 mg/kg/day.


No test item-related changes were noted in any of the reproductive or developmental parameters investigated in this study (i.e. mating and fertility indices, precoital time, number of implantations, estrous cycle, spermatogenic profiling, histopathological examination of reproductive organs, gestation, viability and lactation indices, duration of gestation, parturition, sex ratio, maternal care, litter size and early postnatal pup development consisting of mortality, clinical signs, body weight, anogenital distance, areola/nipple retention, T4 and TSH thyroid hormone levels and macroscopic examination).

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
150 mg/kg bw/day
Study duration:
subchronic
Species:
rat
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

There are two GLP rat and rabbit developmental studies (Covance, 2020) performed with DMEA according to OECD 414.


1-In the rat developmental study (Covance, 2020), the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day which is the ighest dose tested. No treatment related developemental effect were noted at any dose-levels.


2-In the rabbit developmental study (Covance, 2020) performed according to OECD 414, it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development whcih is the highest dose tested.

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental start date (Animal arrival) 22 May 2019
Experimental completion date (Fetal Pathology) 19 August 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test item: Ethyldimethylamine.
Test item identity (including alternative names): Dimethylethylamine
DMEA
N,N-Dimethylethylamine
CAS number: 598-56-1
Intended use: Industrial chemical.
Appearance: Clear-colorless liquid.
Storage conditions: In a dry, cool and well-ventilated place. Storage in hermetically closed bottle in a refrigerator (2-8¿).
Supplier: Sponsor.
Batch number: SAMP181373
Expiry date: 17 May 2020
Purity: 99.57%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 g representative sample was taken from each batch of test item. This sample was placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
Strain/Species New Zealand White rabbit.
Supplier Envigo RMS UK
Number of animals ordered 96 females
Duration of acclimatization 19 days.
Age of the animals at the start of the study (Day 0 of gestation) 18 to 29 weeks old.
Weight range of the animals at the start of the study (Day 0 of gestation) 2.13 to 4.34 kg.

Animal Care and Husbandry
Environmental Control
Multispecies facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.
Air supply Filtered fresh air which was passed to atmosphere and not recirculated.
Temperature and relative humidity Monitored and maintained within the range of 15-21°C and 45-70%.
There were no deviations from these ranges.
Lighting Artificial lighting, 14 hours light : 10 hours dark.
Alarm systems Activated on ventilation failure and when temperature/humidity limits exceeded.
Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Suspended cages fitted with perforated floor panels and mounted in batteries. Undertrays lined with absorbent paper were changed at least three times a week. Cages were also fitted with a plastic resting platform.
Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Number of animals per cage
Acclimatization one female.
During mating one stock male and one female.
Gestation one female.

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.
Stainless steel key ring Attached to the cage.
Cage paper Provided to each cage from Day 20 after mating and replaced as necessary.

Diet Supply
Diet Teklad 2930 Diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.
Availability Restricted (initially 150 g/animal/day during acclimatization up to one week prior to the onset of mating and 200 g/animal/day thereafter).

In addition to this diet, a small supplement of autoclaved hay was given on a daily basis to promote gastric motility and a small amount of chopped fresh vegetables were given twice weekly. Consumption of hay and vegetables were monitored qualitatively but not quantitatively.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals. Water bowls were also provided.
Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet were scrutinized and approved before any batch of diet was released for use. Certificates of analysis are provided by the water supplier.

Certificates of analysis were also received from the suppliers of the Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.

Allocation and Identification
Allocation On the day of mating. Where possible only females mating at least twice were allocated; the following animals were allocated to study after a single mating, all were confirmed to be pregnant:
• Group 1 nos. 2 and 6
• Group 4 no. 75

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treated group and to prevent more than one sibling female in each group, where possible.
Identification of animals Each animal was assigned a number and identified uniquely within the study using a microchip.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Method of preparation The vehicle was chilled in an ice bath/fridge prior to starting formulation.
The required amount of test item was weighed into the smallest practical sealed container ensuring it was kept cold. 50% of the final volume of chilled vehicle was measured in a cylinder and placed to one side. Approximately 10% of the final volume of vehicle was measured into a separate cylinder. The test item was added to the vehicle. The weigh container was rinsed with vehicle which was added to the measuring cylinder and made to 50% of the final volume with vehicle. The mixture was transferred to a sealed container. The measuring cylinder was rinsed with the premeasured chilled purified water previously weighed and this was added to the mixing container. The mixing container was placed in an ice bath and magnetically stirred to mix. The mixture was then split into the final containers, via syringe. All containers were flushed with Nitrogen.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item in order of ascending concentration.

Frequency of preparation Weekly.

Storage of formulation Refrigerated (2-8°C).

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Homogeneity and stability The suitability of the proposed mixing procedure was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix (Covance Study No. CT68FN). In that study, formulations in the concentration range 5 to 200 mg/mL were confirmed to be stable for:
• two hours stored at ambient temperature (15 to 25°C)
• 14 days stored refrigerated (2 to 8°C).
Stability could not be established for formulation at less than 5 mg/mL.

Achieved concentration Samples of each of the first and last formulations prepared for administration were analyzed for achieved concentration of the test item.

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.
Treated at Constant doses in mg/kg/day.
Volume dose Groups 1, 3 and 4: 3 mL/kg/day
Group 2: 1 mL/kg/day
Individual dose volume Calculated from the most recently recorded scheduled body weight.
Control (Group 1) Vehicle at the same volume dose as Groups 3 and 4.
Frequency Females were treated from Day 6 to Day 28 (inclusive) after mating, once daily at approximately the same time each day.
Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.
Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Procedure
The samples were analyzed in accordance with the validated Covance Analytical Procedure
(DFA/M103/18).

The analytical method involved extraction and dilution in acetone followed by liquid
chromatographic analysis with mass spectrometric detection (LC-MS/MS). Sample
concentrations were determined with reference to single bracketing standards (2000 ng/mL).

Procedural recovery samples were prepared concurrently with samples and results were
corrected for the mean recovery value at each level at analysis.

Due to analytical issues experienced in a related study (CT68FN), the chromatographic
conditions were altered and the instrumentation was optimized in order for the analytical
sequences to run. These included changes to the column length, run time and source
temperature.

¿ Phenomenex Kinetex F5, 2.6 µm, 100 × 3 mm column was used from the original
column of Phenomenex Kinetex F5, 2.6 µm, 150 × 3 mm column.
¿ Due to a shift in the retention time, the run time was extended to 10 minutes from the
original run time of 5 minutes to ensure the test item peak was fully integrated.
¿ The source temperature was changed to 400ºC from the original source temperature of
500ºC.

All changes were made in order to improve the chromatography, repeatability, peak area
responses and overall increased sensitivity of the instrumentation. Therefore, this is
considered to have no impact on the integrity of the results.

Concentration of Dose Formulations
The formulations for the First formulation and Last formulation were sampled. For all
groups, 4 × 1 mL (accurately weighed) was sampled from the middle of the formulation by
Pharmacy personnel.

Two samples from each group were analyzed in accordance with the analytical
procedure. For the First formulation, Groups 2, 3 and 4 re-dilutions in duplicate and
contingency samples were analyzed as the original results were out of specification. The
remaining samples were retained for contingency. Samples were disposed of once
satisfactory results were achieved or confirmatory results were obtained.

Major Computerized Systems
Chromatography data handling: Analyst
Sample handling system: Sample Registry System, Covance
Test item management: Pristima, Xybion Medical Systems Corporation
Version numbers of the systems are maintained by Covance.

RESULTS AND CONCLUSION
The mean concentrations of Ethyldimethylamine in test formulations analyzed during the
study and the deviation of the mean result from the nominal value are detailed in Table 1.
During the First formulation, Groups 2 to 4 had low relative mean error (RME) values.
Re-dilutions and contingency analysis was performed. The re-dilutions confirmed the original
results. Therefore, the contingency results have been reported alongside the original results.
The RME values for the four results for Groups 2, 3 and 4 were -32.1%, -39.5% and -39.1%
respectively.

The mean concentrations for the Last formulation were within the applied limits of +10/-15%
of the nominal concentration, confirming the accuracy of formulation, with the exception of
Group 2 where the RME was -15.5%. Rework was not performed in error.

The difference from mean and coefficient of variation for all samples remained within 4%,
confirming precise analysis, with the exception of the First formulation, Groups 2 to 4 and
the Last formulation, Group 2.

The procedural recoveries remained within the validated range, confirming the continued
accuracy of the analytical methodology, with the exception of one recovery prepared during
the First formulation analysis and one recovery prepared during the Last formulation
analysis. As 2 out of 3 recoveries were acceptable for each occasion, the recoveries can be
excluded in line with the SOP
Details on mating procedure:
Male/female ratio 1:1 using identified stock New Zealand White bucks.

Checks Natural mating observed.

After mating Each female was injected intravenously with 25 i.u. luteinizing hormone.
Day 0 of gestation On the day of mating.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females Day 6 to 28 after mating
Frequency of treatment:
Daily
Duration of test:
Day 0-6: Mating
Day 6-28: Treatment
Day 29: Necropsy
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
8 mg/kg bw/day (actual dose received)
Remarks:
volume dose for Group 2 was adjusted to ensure that concentrations were within the established stability range
Dose / conc.:
25 mg/kg bw/day (actual dose received)
Dose / conc.:
70 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
22 females
Control animals:
yes, concurrent vehicle
Details on study design:
Purpose
The purpose of this study was to assess the influence of ethyldimethylamine on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White Rabbit.

Animal Model
The rabbit was chosen as the test species because it is accepted by regulatory agencies. The New Zealand White strain was used because of the historical control data available in this laboratory.

Route of Administration
The oral gavage route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
The doses used in this study (0, 8, 25 and 70 mg/kg/day) were selected in conjunction with the Sponsor.

Dose levels of 15, 45 and 70 mg/kg/day were selected in conjunction with the Sponsor based on the findings in a pilot rabbit study (Study no. FD84BQ) and a preliminary embryo-fetal study in the rabbit (Study no. XG69WJ).

• In the pilot study with non-pregnant rabbits a dose level of 100 mg/kg/day (administered as 33.3 mg/mL at 3mL/kg) was tolerated for the 14 day treatment period however all animals showed reduced food consumption, one throughout treatment, one during the first 3 days of treatment and one during the last six days of treatment. Macroscopic examination revealed severe stomach lesions in all three animals at necropsy. At 50 mg/kg/day (administered as 16.7 mg/mL at 3mL/kg) all animals showed periods (< 5 days) of low food consumption and one out of the three had stomach findings at necropsy -> one female with macroscopic stomach findings namely dark areas in the non-glandular mucosa and depressions in the glandular mucosa.

• In the preliminary embryo-fetal rabbit study the high dose of 45 mg/kg/day (administered as 15 mg/mL at 3mL/kg) was well tolerated with no sign of maternal toxicity or effects on embryo-fetal survival/development.

It was therefore decided to use a higher dose than the 45 mg/kg/day high dose-level used in the preliminary embryo-fetal study; 70 mg/kg/day was selected as the high dose, with the aim to induce some maternal toxicity, as no effects were evident at 45 mg/kg/day. Low and intermediate dose levels of 8 and 25 mg/kg/day were selected to provide an approximate 3 fold dose interval, provide information on dose-response relationship and threshold for any adverse toxicological effect observed .
Maternal examinations:
Serial Observations
Mortality
A viability check was performed near the start and end of each working day. Animals were killed for reasons of animal welfare where necessary.


Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages and cage trays were inspected daily for evidence of animal ill-health amongst the occupant. Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.
During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing
Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

Pre-dose observation
One to two hours after completion of dosing
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 6, 12, 18, 23 and 29 after mating to monitor general health.

Body Weight
The weight of each adult was recorded weekly during acclimatization, on the day of mating and on Days 3 and 6 to 29 after mating.

Food Consumption
The weight of food supplied to each animal, that remaining and an estimate of any spilled was recorded daily from Day 1 after mating.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Intravenous injection of sodium pentobarbitone.
Method of kill for fetuses Subcutaneous injection of sodium pentobarbitone.

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 29 after mating.
Sequence To allow satisfactory inter-group comparison.

Histology
Processing Tissue samples were dehydrated, embedded in paraffin wax and sectioned at a nominal four to five micron thickness. For bilateral organs, sections of both organs were prepared. A single section was prepared from each of the remaining tissues required.
Stomach All adult females.
Routine staining Sections were stained with hematoxylin and eosin.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).
The following were recorded for all animals (including those prematurely sacrificed, where possible):
For each ovary/uterine horn Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).
Apparently non pregnant animals and for apparently empty uterine horns The absence or number of uterine implantation sites was confirmed.
Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus.
Examined externally with abnormalities recorded, sampled as appropriate and retained in appropriate fixative. All fetuses were subject to a gross internal examination of the viscera of the neck, thorax and abdominal cavities and the sex of each fetus was also recorded.

Fixation Nominally one half of fetuses were decapitated; heads were initially stored in Bouin’s fluid.

Remaining fetuses and torsos were eviscerated and fixed in Industrial Methylated Spirit.

Processing Bouin’s fixed fetal heads were subject to free-hand serial sectioning.
Industrial Methylated Spirit fixed fetuses and torsos were processed and double stained with Alizarin Red and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed heads Serial sections were examined for soft tissue abnormalities.
Alizarin Red and Alician Blue stained fetuses and torsos Assessed for skeletal and cartilage development and abnormalities.
Statistics:
Please refer to "Any other information on materials and methods" below
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea - Number of implantations)/ Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations - Number of live fetuses)/ Number of implantations) x 100


All group values and SD were calculated from the individual litter values.
Historical control data:
PLEASE REFER TO THE ATTACHED ANNEX - HISTORICAL CONTROL DATA
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical signs or signs associated with doisng that were considered to be related to treatment at dose levels up to and including 70 mg/kg/day.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
There were four early decedents in this study. One control female (No. 6) was killed for welfare reasons on GD (gestation day) 28 due to red staining in the cage tray, uterine examination revealed two late resorptions and 6 live fetuses but there were no macroscopic or microscopic abnormalitiesand the cause of death was undetermined. One female (No. 41) that received 8 mg/kg/day was found dead on GD 23; additionally, females Nos. 40 (8 mg/kg/day) and 45 (25 mg/kg/day) were killed for welfare reasons on GD 12 and 11, respectively. Macroscopic examination of these animals revealed the presence of adhesions in the thoracic cavity involving multiple organs, dark area on the oesophagus (No. 41) and perforated oesophagus (Nos. 40 and 45). The cause of death of these animals was therefore considered accidental and attributed to the dosing procedure (oral gavage).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
For females receiving 70 mg/kg/day mean body weight loss of 80 g (0.08 kg) was recorded between Days 6 and 8 of gestation (p<0.01) compared with mean body weight stasis in Controls. Thereafter, body weight change from Day 8 of gestation was generally similar to Controls but overall the weight gain (Day 6 to 29 of gestation) was 25 % lower than Controls (p<0.05).

There was no effect of treatment on body weight change at 8 mg/kg/day.

There was no conclusive effect of treatment on mean gravid uterine weight. Mean maternal body weight loss following adjustment for the gravid uterine weight was greater at 25 and 70 mg/kg/day compared with Controls (p<0.05 and p<0.01 respectively); a dose response was apparent .

PLEASE REFER TO ATTACHED TABLE "BODYWEIGHT AND BODYWEIGHT CHANGES - GROUP MEAN VALUES"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 70 mg/kg/day, mean food consumption was moderately lower than Controls on Days 6-9 of gestation (p<0.01) and slightly/marginally low on Days 23-29 of gestation (p<0.05 or p<0.01).
Food consumption at 8 or 25 mg/kg/day was considered to be unaffected by treatment.
PLEASE REFER TO THE ATTACHED TABLE"FOOD CONSUMTPION - GROUP MEAN VALUES"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
The macroscopic examination performed on GD 29 after 3 weeks of treatment revealed the following changes in the stomach.
Stomach
Dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day.

The incidence and distribution of all other findings were considered to be unrelated to treatment.

Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Changes related to treatment with Ethyldimethylamine were seen in the stomach.
Stomach
Microscopic changes findings consistent with edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum were observed in animals that received
70 mg/kg/day. Dilated glands of the glandular mucosa (fundic mucosa or antrum) were also observed at higher incidence and severity in animals at 70 mg/kg/day.

Summary of treatment related findings in the stomach
Group/sex 1F 2F 3F 4F
Dose (mg/kg/day) 0 8 25 70
Dilation, Glands
Minimal 1 1 1 7
Slight 0 0 0 1
Moderate 0 0 0 1
Total 1 1 1 9
Edema
Minimal 0 0 0 2
Slight 0 0 0 2
Moderate 0 0 0 3
Total 0 0 0 7
Hemorrhage
Minimal 0 0 0 1
Slight 0 0 0 10
Total 0 0 0 11
Infiltrate, Inflammatory Cell, Mucosa/Submucosa
Minimal 0 0 0 1
Slight 0 0 0 1
Total 0 0 0 2
Necrosis, Vascular, Mucosa/Submucosa
Slight 0 0 0 1
Total 0 0 0 1
Ulceration
Slight 0 0 0 1
Total 0 0 0 1
Number Examined 21 20 21 22

Incidental Findings
The incidence and distribution of all other findings were considered to be unrelated to treatment.

PLEASE ALSO REFER TO THE ATTACHED TABLE "HISTOPATHOLOGY - GROP DISTRIBUTION FOR FEMALES ON DAY 28"
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Number of abortions:
no effects observed
Pre- and post-implantation loss:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.

PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Early or late resorptions:
no effects observed
Description (incidence and severity):
There was no effect of treatment on mean numbers of implantations or live fetuses, pre and post implantation losses or sex ratio at dose levels up to and including 70 mg/kg/day.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Dead fetuses:
no effects observed
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
25 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
gross pathology
Key result
Abnormalities:
effects observed, treatment-related
Localisation:
other: Stomach
Description (incidence and severity):
dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day and microscopic examination of the stomach revealed microscopic changes including edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum.
Fetal body weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
At 70 mg/kg/day, mean placental and fetal weights were slightly lower than in Controls with the differences for female fetal weights and overall fetal weights attaining statistical significance (p<0.05); these differences were considered to be related to the slightly higher live litter size in this group rather than an effect of treatment.

Placental and fetal weights at 8 and 25 mg/kg/day showed no adverse effects of maternal treatment.
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
not examined
Changes in litter size and weights:
effects observed, non-treatment-related
Description (incidence and severity):
slightly higher live litter size at 70 mg/kg/day
PLEASE REFER TO THE FOLLOWING ATTACHED TABLES "LITTER DATA- GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS"
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
There were no major abnormalities considered to be related to treatment: in all treated groups there were a small number of major abnormalities, they were of low incidence, several are within historical control data (HCD) range and there was no treatment related effect for any particular abnormality.
At 70 mg/kg/day there was a slight increase in incidence of full supernumerary 13th rib with associated 20 thoracolumbar vertebrae and unilateral shift of pelvic girdle compared to concurrent control (all parameters just outside of fetal HCD range but within litter HCD range).

This indicates a very slight shift in rib/vertebral configuration, which, as variants are not considered adverse, but may be associated with treatment with Ethyldimethylamine.
There was also a slight increase in incidence of incompletely ossified metacarpals/phalanges (forepaw digits) compared to concurrent control but within the HCD range and therefore considered unrelated to treatment. Incomplete ossification is a transient stage in fetal development, indicative of fetal immaturity and may be associated with the slight decrease in mean fetal weight seen at this dose level.


PLEASE REFER TO THE ATTACHED TABLES: "FETAL EXAMINATIONS - MAJOR ABNORMALITIES" AND "FETAL EXAMINATIONS - MINOR SKELETAL ABNORMALITIES"
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
skeletal malformations
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: supernumerary rib
skeletal: vertebra
skeletal: pelvic girdle
Key result
Developmental effects observed:
no

Formulation Analysis


Groups 2 to 4 in the first formulation had low relative mean error (RME) values.  Re-dilutions and contingency analysis was performed. The re-dilutions confirmed the original results. Therefore, the contingency results have been reported alongside the original results.  The RME values for the four results for Groups 2, 3 and 4 were -32.1%, -39.5% and -39.1% respectively. 


The mean concentrations for the last formulation were within the applied limits of +10/-15% of the nominal concentration, confirming the accuracy of formulation, with the exception of Group 2 where the RME was -15.5%. Rework was not performed in error. 


The difference from mean and coefficient of variation for all samples remained within 4%, confirming precise analysis, with the exception of the first formulation, Groups 2 to 4 and the last formulation, Group 2.


The procedural recoveries remained within the validated range, confirming the continued accuracy of the analytical methodology, with the exception of one recovery prepared during the First formulation analysis and one recovery prepared during the Last formulation analysis. As 2 out of 3 recoveries were acceptable for each occasion, the recoveries can be excluded in line with the SOP.

Conclusions:
Based on the results of this study it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development.
Executive summary:

Summary


The purpose of this study was to assess the influence of ethyldimethylamine on embryo-fetal survival and development when administered during the organogenesis and fetal growth phases of the New Zealand White Rabbit.


Three groups of 22 females received ethyldimethylamine at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating.  A similarly constituted Control group received the vehicle, purified water, at a volume dose of 3 mL/kg throughout the same duration.  Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded.  Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded.  Microscopic investigations were also undertaken.  All fetuses were examined macroscopically (internal and external) at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.


Results


There were no deaths, clinical signs or dosing signs considered to be related to treatment.


Treatment at 70 mg/kg/day was associated with mean body weight loss of 80 g (0.08 kg) between Days 6 and 8 of gestation compared with mean body weight stasis in Controls (body weight gain during treatment was appproximately 75 % of Controls), greater adjusted mean maternal body weight loss following adjustmnet for the gravid uterine weight and  moderately low food consumption.


In addition, dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day and microscopic examination of the stomach revealed microscopic changes findings including edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum.  Dilated glands of the glandular mucosa (fundic mucosa or antrum) were also observed at higher incidence and severity in animals at 70 mg/kg/day.


Treatment at 25 mg/kg/day was associated with greater adjusted mean maternal body weight loss when compared with Controls; there was no maternal response to treatment at 8 mg/kg/day.


Embryo-fetal survival was unaffected by treatment and developmnet was not considered to be adversley affected at dose levels up to and including 70 mg/kg/day.


Conclusion


Based on the results of this study it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Animal arrival 02 January 2019
Necropsy 29 January to 01 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
other: Japanese Ministry of Agriculture, Forestry and Fisheries, Test Data for Registration of Agricultural Chemicals, 12 Nohsan No. 8147, Agricultural Production Bureau, November 24, 2000.
Deviations:
no
Qualifier:
according to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
Test item: Ethyldimethylamine
Test item identity (including alternative names): Dimethylethylamine
DMEA
N,N-Dimethylethylamine
CAS number: 598-56-1
Intended use: Industrial chemical
Appearance: Clear-colorless liquid
Storage conditions: In a dry, cool and well-ventilated place. Storage in hermetically closed bottle in a refrigerator (2-8°C)
Supplier: Sponsor
Batch number: SAMP181373
Expiry date: 17 May 2019
Purity: 99.57%
Supplier’s responsibilities: Characterization of the test item and the documentation of the methods of synthesis, fabrication or derivation and stability.
Archive sample: A 0.5 mL representative sample was taken, placed in a well closed glass container and stored in the archives under the same conditions as the bulk material.
Species:
rat
Strain:
other: Crl:CD(SD) rat.
Details on test animals or test system and environmental conditions:
Animals
Strain/Species Crl:CD(SD) rat.
Supplier Charles River (UK) Ltd.
Number of animals 90 females.
Spare animals were removed from the study room after treatment commenced.
Duration of acclimatization Six days before commencement of pairing.
Age of the animals at the start of the study (Day 0 of gestation) 70 to 76 days old.
Weight range of the animals at the start of the study (Day 0 of gestation) 210 to 293 g.

Allocation and Identification
Allocation On the day of positive evidence of mating (Day 0). Only females showing at least two copulation plugs were allocated.

Method To group and cage position in the sequence of mating. Females mating on any one day were evenly distributed amongst the groups.

Allocation was controlled to prevent any stock male from providing more than one mated female in each treatment group.

Identification of animals Each animal was assigned a number and identified uniquely within the study by a microchip inserted subcutaneously in the dorsal cervical region.

Identification of cages Each cage label was color-coded according to group and was numbered uniquely with cage and study number, as well as the identity of the occupant(s).

Animal Care and Husbandry
Environmental Control
Rodent facility Limited access - to minimize entry of external biological and chemical agents and to minimize the transference of such agents between rooms.

Air supply Filtered fresh air which was passed to atmosphere and not recirculated.

Temperature and relative humidity Monitored and maintained within the range of 20-24ºC and 40-70%.

There were no deviations from these ranges.

Lighting Artificial lighting, 12 hours light : 12 hours dark.

Electricity supply Public supply with automatic stand-by generators.

Animal Accommodation
Cages Cages comprised of a polycarbonate body with a stainless steel mesh lid; changed at appropriate intervals.

Solid (polycarbonate) bottom cages were used during the acclimatization and gestation periods.
Grid bottomed cages were used during pairing. Cages were suspended above absorbent paper which was changed daily during pairing.

Cage distribution The cages constituting each group were blocked by group and mounted in batteries.

Bedding Solid bottom cages contained softwood based bark-free fiber bedding, which was changed at appropriate intervals each week.

Number of animals per cage
Acclimatization Up to four animals
During pairing One (stock) male and one female
Gestation One female

Environmental Enrichment
Aspen chew block A soft white untreated wood block; provided to each cage throughout the study and replaced when necessary.

Plastic shelter Provided to each cage throughout the study (except during pairing) and replaced at the same time as the cages.

Diet Supply
Diet SDS VRF1 Certified pelleted diet.

The diet contained no added antibiotic or other chemotherapeutic or prophylactic agent.

Availability Non-restricted.

Water Supply
Supply Potable water from the public supply via polycarbonate bottles with sipper tubes. Bottles were changed at appropriate intervals.

Availability Non-restricted.

Supplier Certificates of Analysis
Certificates of analysis for the diet are scrutinized and approved before any batch of diet was released for use. Certificates of analysis were routinely provided by the water supplier.

Certificates of analysis were also received from the suppliers of the softwood based bark-free fiber bedding and Aspen chew blocks.

No specific contaminants were known that may have interfered with or prejudiced the outcome of the study and therefore no special assays were performed.
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
Method of preparation
The vehicle was chilled in an ice bath/fridge prior to starting formulation.

The required amount of test item was weighed into the smallest practical sealed container ensuring it was kept cold. 50% of the final volume of chilled vehicle was measured in a cylinder and placed to one side. Approximately 10% of the final volume of vehicle was measured into a separate cylinder. The test item was added to the vehicle. The weigh container was rinsed with vehicle which was added to the measuring cylinder and made to 50% of the final volume with vehicle. The mixture was transferred to a sealed container. The measuring cylinder was rinsed with the premeasured chilled purified water previously weighed and this was added to the mixing container. The mixing container was placed in an ice bath and magnetically stirred to mix. The mixture was then split into the final containers, via syringe. All containers were flushed with Nitrogen.

A series of formulations at the required concentrations were prepared by dilution of individual weighings of the test item in order of ascending concentration.

Frequency of preparation Weekly, and may have been prepared in advance of the first day of dosing.

Storage of formulation Refrigerated (2-8°C) for up to 14 days and ambient (15 25¿C) for up to two hours.

Test item accounting Detailed records of compound usage were maintained. The amount of test item necessary to prepare the formulations and the amount actually used were determined on each occasion. The difference between these amounts was checked before the formulations were dispensed.

Formulation Analysis
Stability and homogeneity Before commencement of treatment the suitability of the proposed mixing procedure was determined and specimen formulations were analyzed to assess the stability and homogeneity of the test item in the liquid matrix. Formulations in the concentration range 1 to 200 mg/mL were confirmed to be stable for:

• two hours stored at ambient temperature (15 to 25°C)

• 14 days stored refrigerated (2 to 8°C).
Achieved concentration Samples of each of the first and last formulation prepared were analyzed for achieved concentration of the test item.

Administration
Route Oral gavage using a suitably graduated syringe and a rubber catheter inserted via the mouth.

Treated at Constant doses in mg/kg/day.

Volume dose 4 mL/kg/day

Individual dose volume Calculated from the most recently recorded scheduled body weight.

Control (Group 1) Vehicle at the same volume dose as treated groups.

Frequency Females were treated from Day 6 to Day 19 (inclusive) after mating, once daily at approximately the same time each day.

Formulation A daily record of the usage of formulation was maintained based on weights. This balance was compared with the expected usage as a check of correct administration. No significant discrepancy was found.

Formulations were stirred using a magnetic stirrer before and throughout the dosing procedure.


Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Preparation of Calibration Standards
A primary standard solution (500000 ng/mL) was prepared by dissolving an accurately
weighed quantity (ca. 50 mg) of Ethyldimethylamine in acetone (100 mL). A secondary
standard solution (50000 µg/mL) was prepared by appropriate dilution of the primary
standard (1 mL) using acetone (10 mL). A tertiary standard solution (5000 µg/mL) was
prepared by appropriate dilution of the secondary standard (1 mL) using acetone (10 mL).

Solutions for instrument calibration were prepared by appropriate dilution of the tertiary
standard using acetone and contained Ethyldimethylamine at nominal concentrations of
50 ng/mL, 100 ng/mL, 200 ng/mL, 250 ng/mL, 300 ng/mL, 400 ng/mL and 500 ng/mL.

Calibration solutions were injected onto the LC-MS, at the beginning of each sample analysis
sequence as a minimum with a bracketing standard (200 ng/mL) injected in duplicate after
every three samples, using the conditions detailed in the chromatographic section.

Due to a change in response from the LC-MS/MS part way through the study the calibration
range was increased for some runs. Where this happened the calibration range was prepared
at 500 ng/mL, 1000 ng/mL, 2000 ng/mL, 3000 ng/mL, 4000 ng/mL and 5000 ng/mL, these
were based on the peak areas and responses obtained for these concentrations.

Preparation of Test Samples
A representative sample of test formulation (1 mL, accurately weighed) was dissolved using
ultrasonic vibration in a suitable volume of acetone. The extract was diluted using acetone,
where necessary, to provide a solution containing Ethyldimethylamine at an expected
concentration of 200 ng/mL. Where the calibration concentration range was increased
extracts were diluted to an expected concentration of 2000 ng/mL.

The concentration of Ethyldimethylamine in the final solution was quantified by LC-MS as
detailed in the chromatographic section.

Preparation of Recovery Samples
Procedural recoveries were prepared by fortifying samples (1 mL) of control matrix (purified
water) with known amounts of Ethyldimethylamine. The prepared procedural recoveries
were analyzed in accordance with the analytical procedure.

Instrumentation Parameters
These conditions were optimised depending on which instrument was in use.
High performance liquid chromatograph
(HPLC) and mass spectrometer
(LC-MS/MS):
Shimadzu LC-10AD pump, Shimadzu SIL-HTAautosampler, and a Sciex API 3000 equivalent
mass spectrometer
Column: Phenomenex Kinetex F5, 2.6 µm, 150 × 3 mm
Column temperature: 30°C
Sample temperature: Ambient
Mobile Phase A: 0.05% Acetic acid (aq)
Mobile Phase B: 0.05% Acetic acid in acetonitrile
Isocratic conditions: 50% A/50% B
Flow rate: 0.35 mL/minute
Needle wash: ACN/water 50/50 v/v
Ionization: Turboionspray – positive ion mode
Source temperature: +500°C
Collision gas: Nitrogen
Collision energy: 25 eV
Dwell time: 200 ms
Pause time: 5 ms
Ions to be monitored: m/z 74.16 to m/z 46.10 (used for quantitation)
Injection volume: 20 µL
Run time: 5 minutes
Approximate retention time: 2.91 minutes
Details on mating procedure:
Male/female ratio 1:1 with identified stock males.

Daily checks for evidence of mating Ejected copulation plugs in cage tray and vaginal smears were checked for the presence of sperm.

Day 0 of gestation When positive evidence of mating was detected.

A colony of stud males was maintained specifically for the purpose of mating; these animals were not part of the study and were maintained as stock animals.
Duration of treatment / exposure:
Females: Day 6 to 19 after mating
Frequency of treatment:
Daily
Duration of test:
Day 0-6: Mating
Day 6-19: Treatment
Day 20: Necorpsy
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Vehicle
Dose / conc.:
20 mg/kg bw/day (actual dose received)
Dose / conc.:
60 mg/kg bw/day (actual dose received)
Dose / conc.:
180 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 females
Control animals:
yes, concurrent vehicle
Details on study design:
Purpose
The purpose of this study was to assess the influence of Ethyldimethylamine (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis phase and fetal growth phase of pregnancy in the Sprague-Dawley rat.

Animal Model
The rat was chosen as the test species because of the requirement for a rodent species by regulatory agencies. The Sprague-Dawley [Crl:CD(SD)] strain was used because of the historical control data available at this laboratory.

Route of Administration
The oral (gavage) route of administration was chosen to simulate the conditions of possible human exposure.

Rationale for Dose Level Selection
The doses used in this study (0, 20, 60 and 180 mg/kg/day) were selected in conjunction with the Sponsor.

A high dose level of 180 mg/kg/day was selected based on the results of a preliminary embryo-fetal study in the female rat conducted at these laboratories (Covance No. MK81TG).
In that study animals received daily administration of Ethyldimethylamine over Days 6-19 of gestation at 50, 180 and 360 mg/kg/day.

Females receiving 360 mg/kg/day started showing signs of decreased activity, partially closed eyelids, flattened or hunched posture, shallow breathing, cold to touch and piloerection on Day 8 or 9 of gestation. Two females were euthanised for welfare reasons, both on Day 9 of gestation, after more than 15% bodyweight loss, which reached or exceed the moderate severity limit defined in the Home Office project license; other terminal signs included including partially closed eyelids, gasping and piloerection. The high dose level was subsequently reduced to 270 mg/kg/day for the remaining four females.

The females dosed at 270 mg/kg/day were prostrate at 30 minutes post dosing and underactive, hunched with piloerection and had abnormal breathing at 1 hour post dose. Prior to dosing on Day 7 of gestation at the reduced high dose, one female was found dead and a second female had lost body weight, exceeding the moderate severity limit. As the dose level was not tolerated, the remaining females were euthanised for welfare reasons on Day 9 or 10 of gestation.

Treatment at 180 mg/kg/day was tolerated and was associated with reduced weight gain (86% of Controls), reduced adjusted maternal weight gain (60% of Controls) and a slight reduction in food consumption (90% of Controls). One female had depressions on the nonglandular region of the stomach but remained in good clinical condition. Embryo-fetal survival was unaffected by treatment and mean fetal weight was low when compared with the concurrent Control animals.

Therefore a high dose of 180 mg/kg/day was considered suitable as the high dose with 60 and 20 mg/kg/day chosen as the intermediate and low dose levels to allow assessment of any dose response.
Maternal examinations:
Serial Observations
Clinical Observations
Animals were inspected visually at least twice daily for evidence of ill-health or reaction to treatment. Cages were inspected daily for evidence of animal ill-health amongst the occupant(s). Any deviation from normal was recorded at the time in respect of nature and severity, date and time of onset, duration and progress of the observed condition, as appropriate.

During the acclimatization period, observations of the animals and their cages were recorded at least once per day.

Signs Associated with Dosing

Detailed observations were recorded daily during the treatment period at the following times in relation to dose administration:

Pre-dose observation.
One to two hours after completion of dosing.
As late as possible in the working day.

Clinical Signs
A detailed physical examination was performed on each animal on Days 0, 5, 12, 18 and 20 after mating to monitor general health.

Body Weight
The weight of each adult was recorded on Days 0, 3 and 6-20 after mating.

Food Consumption
The weight of food supplied to each adult, that remaining and an estimate of any spilled was recorded for the periods Days 0-2, 3-5, 6-9, 10-13, 14-17 and 18-19 inclusive after mating.

Terminal Investigations
Method of Kill
Method of kill for all adult animals Carbon dioxide asphyxiation.
Method of kill for fetuses Chilling on a cool plate (approximately 0¿C)

Necropsy
All adult animals were subject to a detailed necropsy. After a review of the history of each animal, a full macroscopic examination of the tissues was performed. All external features and orifices were examined visually. Any abnormality in the appearance or size of any organ and tissue (external and cut surface) was recorded and the required tissue samples preserved in appropriate fixative.

Schedule Animals surviving until the end of the scheduled study period were killed on Day 20 after mating.
Sequence To allow satisfactory inter-group comparison.
Ovaries and uterine content:
For females surviving to term, the following was recorded:
Uterus Gravid uterine weight (including cervix and ovaries).

The following were recorded for all animals

For each ovary/uterine horn
Number of: Corpora lutea.
Implantation sites.
Resorption sites (classified as early or late).
Fetuses (live and dead).

For apparently empty uterine horns The number of uterine implantation sites were checked after staining with ammonium sulphide [modification of the Salewski staining technique (Salewski, E, 1964)].

Fetal examinations:
Fetal Examination and Processing
Examination of all viable fetuses and placentae Dissected from the uterus, individually weighed and identified within the litter using a coding system based on their position in the uterus. Examined externally with abnormalities recorded. The sex of each fetus was recorded.

Examination of nominally 50% of fetuses in each litter Sexed internally and eviscerated.

Fixation Fetuses eviscerated were fixed in Industrial Methylated Spirit (IMS).
Remaining fetuses were fixed whole in Bouin’s fluid.

Processing Bouin’s fixed fetuses were subject to free-hand serial sectioning.
IMS fixed fetuses were processed and double stained with Alizarin and Alician Blue.

Fetal Pathology Examination
Bouin’s fixed fetuses Serial sections were examined for visceral abnormalities.
Double stained with Alizarin Red and Alician Blue fetuses, Assessed for skeletal and cartilage development and abnormalities.

Statistics:
Please refer to "Any other comments on materials and methods"
Indices:
Reproductive Assessment
Prenatal losses are separated into pre- and post-implantation phases. Pre-implantation loss was considered to reflect losses due to non-fertilization of ova and failure to implant. It was calculated from the formula:

Pre-implantation loss (%) = ((Number of corpora lutea – Number of implantations)/ Number of corpora lutea) x 100

Where the number of implantations exceeded the number of corpora lutea observed, pre implantation loss was assumed to be zero (i.e. no pre-implantation loss was considered to have occurred).

Post-implantation loss was calculated from the formula:

Post-implantation loss (%) = ((Number of implantations – Number of live fetuses)/ Number of implantations) x 100

All group values and SD (as appropriate) were calculated from the individual litter values.
Historical control data:
PLEASE REFER TO THE ATTACHED ANNEX - "HISTORICAL CONTROL DATA"
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Signs associated with dose administration were restricted to the animals receiving 180 mg/kg/day, with 4/20 exhibiting a low incidence of rales (no. 61 on one occasion; no. 65 on two occasions; no. 67 on two occasions; no 71 on three occasions) and 4/20 females exhibiting increased salivation each on a single occasion.
Signs at routine physical examination were limited to two females at 180 mg/kg/day (nos. 65 and 71) with rales; there were other signs that could be related to treatment.
The clinical condition of animals receiving 20 or 60 mg/kg/day was unaffected by treatment.
PLEASE REFER TO THE ATTACHED TABLES - "SIGNS ASSOCIATED WITH DOSING - GROUP DISTRIBUTION" AND "CLINICAL SIGNS - GROUP DISTRIBUTION"
Dermal irritation (if dermal study):
no effects observed
Mortality:
mortality observed, treatment-related
Description (incidence):
Two females (nos 61 and 67) receiving 180 mg/kg/day were killed for welfare reasons; female no 61 was euthanised on GD14 and female no.67 was euthanised on GD12. Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
From GD9 (Day 4 of treatment) mean body weight gain for females receiving 180 mg/kg/day was low with the difference attaining statistical significance (p<0.01) and overall (GD6-20) the mean body weight change at 180 mg/kg/day was approximately 14% lower than Controls (p<0.01). The resultant mean bodyweight on GD20 for females receiving 180 mg/kg/day was approximately 6% lower than Controls (p<0.05).

Group Body weight GD20 Body weight change GD6-20
(g) % difference from Controls (g) % difference from Controls
1 411 122
2 401 -2 117 -4
3 402 -2 118 -3
4 388* -6 105** -14
(*- p<0.005; ** p<0.01)

The mean gravid uterine weight was essentially similar across the groups. However the maternal weight gain following adjustment for the gravid uterine was statistically significantly low at 180 mg/kg/day, approximately 40% lower than Controls (p<0.01) and the maternal mean weight on GD20 was approximately 6% lower than Controls (p<0.05).

Both the absolute body weight gain and adjusted maternal weight gain of females receiving 20 or 60 mg/kg/day was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE - "BODYWEIGHT AND BODYWEIGHT CHANGE - GROUP MEAN VALUES"
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
At 180 mg/kg/day mean food consumption was low when compared with Controls with the difference attaining statistical significance for GD10-17 (p<0.01; 86-88% of Controls) and GD18 19 (p<0.05; 93 % of Controls). Overall the total food consumption during the treatment period (GD 6 -19) was approximately 10% lower than Controls.

Group Total Food consumption GD6-19
(g) % difference from Controls
1 366
2 354 -3
3 356 -3
4 330 -10

Food consumption at 20 or 60 mg/kg/day was unaffected by treatment.

PLEASE REFER TO THE ATTACHED TABLE - "FOOD CONSUMPTION - GROUP MEAN VALUES"
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Endocrine 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):
Macroscopic examination of females on GD20 did not reveal any findings that could be attributed to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Number of abortions:
no effects observed
Pre- and post-implantation loss:
effects observed, non-treatment-related
Description (incidence and severity):
A higher incidence of post-implantation loss was observed at 180 mg/kg/day (9.3% versus 2.3% in Controls), however the incidence was within the historical control range and there was no impact on the live litter size. This difference was therefore not considered to be related to treatment.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Total litter losses by resorption:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Early or late resorptions:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.

PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Dead fetuses:
no effects observed
Description (incidence and severity):
Litter data as assessed by the number of implantations, resorptions, live young and sex ratio and the extent of the pre- and post-implantation loss was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Changes in pregnancy duration:
no effects observed
Changes in number of pregnant:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
60 mg/kg bw/day (actual dose received)
Based on:
test mat.
Basis for effect level:
body weight and weight gain
food consumption and compound intake
Key result
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
Litter, placental and fetal weight was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Reduction in number of live offspring:
no effects observed
Changes in sex ratio:
no effects observed
Changes in litter size and weights:
no effects observed
Description (incidence and severity):
Litter, placental and fetal weight was unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.
PLEASE REFER TO THE ATTACHED TABLES - "LITTER DATA - GROUP MEAN VALUES" AND "PLACENTAL, LITTER AND FETAL WEIGHTS - GROUP MEAN VALUES"
Anogenital distance of all rodent fetuses:
not examined
Changes in postnatal survival:
not examined
External malformations:
no effects observed
Skeletal malformations:
effects observed, non-treatment-related
Description (incidence and severity):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.

PLEASE REFER TO THE ATTACHED TABES - "FETAL EXAMINATIONS - MAJOR ABNORMALITY FINDINGS" AND "FETAL EXAMINATIONS - MINOR SKELETAL ABNORMALITIES"
Visceral malformations:
no effects observed
Other effects:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
180 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
not specified
Basis for effect level:
other: Embryo-fetal survival, growth and development showed no adverse effects of maternal treatment at all dose levels tested.
Key result
Abnormalities:
effects observed, non-treatment-related
Localisation:
skeletal: sternum
Description (incidence and severity):
At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher incidence of incompletely ossified 1st to 4th sternebrae, compared with concurrent control (12/11 fetuses in 8 litters per group versus 4 fetuses in 4 Control litters); the incidences were within the historical control range of the litters, which is the principal unit of evaluation, with the exception of fetuses at 60 mg/kg/day which exceeded the fetal historical control range. Incomplete ossification is a transient stage in development and is therefore not considered to be adverse.
Key result
Developmental effects observed:
no

Formulation analysis


For the First dose there were significant instrument problems meaning that the samples could not be injected until a substantial amount of time had passed since extraction.  The results for this occasion are all low and this is considered to be a consequence of the time that elapsed between extraction and injection.  These results are reported for information only and it is considered unlikely that they are a true reflection of the accuracy of the dose prepared.


The Last dose samples were also injected some time after extraction, although the time period was shorter than that for the first dose.  Both the analyzed concentration and the procedural recoveries were outside of acceptance criteria for Groups 2 and 3.  When the analyzed concentration was corrected for the appropriate procedural recovery all results were within acceptable limits.  It is thought that the time between extraction and injection meant that the samples had started to degrade.  As the procedural recoveries had also aged at the same rate as the samples this correction was considered an appropriate action.  After correction the mean concentrations were within 10% of the nominal concentration, confirming the accuracy of formulation.  The difference from mean for Groups 2 and 3 remained within 2%, confirming precise analysis. The difference from mean for Group 4 was ±7.24%.  Due to the factors mentioned above this was considered acceptable.


 

Conclusions:
It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo fetal survival and development the NOAEL was considered to be 180 mg/kg/day.
Executive summary:

Summary


The purpose of this study was to assess the influence of Ethyldimethylamine (an industrial chemical) on embryo-fetal survival and development when administered during the organogenesis phase and fetal growth phase of pregnancy in the Sprague-Dawley rat.


Three groups of 20 females received Ethyldimethylamine at doses of 20, 60 or 180 mg/kg/day by oral (gavage) administration, from Day 6 to 19 after mating.  A similarly constituted Control group received the vehicle, purified water, at the same volume dose as treated groups.  Animals were killed on Day 20 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded.  Adult females were examined macroscopically at necropsy on Day 20 after mating and the gravid uterine weight recorded.  All fetuses were examined macroscopically at necropsy and subsequently by detailed internal visceral examination or skeletal examination.


Results


Two females receiving 180 mg/kg/day were killed for welfare reasons on GD12/14.  Terminal signs for both of these females comprised of respiratory distress (gasping) and body weight loss, however no macroscopic abnormality was apparent at necropsy. 


At the high dose (180 mg/kg/day) there was a low incidence of rales and increased salivation that were attributed to treatment. The high dose animals also exhibited low absolute body weight gain, low food consumption and low maternal weight gain following adjustment for the gravid uterine weight at approximately 86%, 90% and 60% of Controls, respectively. 


Clinical condition, body weight and food consumption for animals receiving 20 or 60 mg/kg/day were unaffected by treatment with Ethyldimethylamine.


A higher incidence of post-implantation loss was observed in the high dose group at caesarian section (9.3% versus 2.3% in Controls) but was within the historical control range. Litter data, fetal and placental weights were unaffected by maternal treatment at dose levels up to and including 180 mg/kg/day.


Macroscopic examination of females at necropsy did not reveal any abnormality that could be related to treatment.


The incidence of major and minor abnormalities and skeletal variants showed no relationship to treatment.  At 60 mg/kg/day and 180 mg/kg/day there was a slightly higher increase in incompletely ossified 1st to 4th sternebrae; the incidences were within the historical control range of the litters with the exception of fetuses at 60 mg/kg/day in which outside the fetal historical control range.


 


Conclusion


Oral administration of Ethyldimethylamine to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 20, 60 and 180 mg/kg/day was not tolerated at the high dose with two animals killed for welfare reasons on Gestation Days 12/14.  Terminal signs included respiratory distress (gasping) and body weight loss; no macroscopic abnormality was apparent at necropsy. These deaths were not considered to be incidental and are attributed to administration of Ethyldimethylamine.


At 180 mg/kg/day females that survived to scheduled termination showed a low incidence of rales and increased salivation with low maternal weight gain and low food consumption; these parameters were unaffected by treatment at either 20 or 60 mg/kg/day and no maternal macroscopic abnormality was apparent at dose levels up to and including 180 mg/kg/day.


Embryo-fetal survival, growth and development showed no adverse effects of maternal treatment for all dose levels tested.


It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
70 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Quality of whole database:
GLP studies
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

1- Rat developmental toxicity study (Covance , 2020), GLP-OECD 414 : Oral administration of Ethyldimethylamine to pregnant Sprague-Dawley rats during organogenesis and the fetal growth phase at 20, 60 and 180 mg/kg/day was not tolerated at the high dose with two animals killed for welfare reasons on Gestation Days 12/14.  Terminal signs included respiratory distress (gasping) and body weight loss; no macroscopic abnormality was apparent at necropsy. These deaths were not considered to be incidental and are attributed to administration of Ethyldimethylamine.


At 180 mg/kg/day females that survived to scheduled termination showed a low incidence of rales and increased salivation with low maternal weight gain and low food consumption; these parameters were unaffected by treatment at either 20 or 60 mg/kg/day and no maternal macroscopic abnormality was apparent at dose levels up to and including 180 mg/kg/day.


Embryo-fetal survival, growth and development showed no adverse effects of maternal treatment for all dose levels tested.


It was therefore concluded that the no observed adverse effect level (NOAEL) for maternal toxicity was 60 mg/kg/day based on the effect on maternal body weight and food consumption. For embryo-fetal survival and development the NOAEL was considered to be 180 mg/kg/day.


2-Rabbit developmental toxicity study (Covance, 2020):


Three groups of 22 females received ethyldimethylamine at doses of 8, 25 or 70 mg/kg/day by oral gavage administration, from Day 6 to 28 after mating.  A similarly constituted Control group received the vehicle, purified water, at a volume dose of 3 mL/kg throughout the same duration.  Animals were killed on Day 29 after mating for reproductive assessment and fetal examination.


Clinical observations, body weight and food consumption were recorded.  Adult females were examined macroscopically at necropsy on Day 29 after mating and the gravid uterus weight was recorded.  Microscopic investigations were also undertaken.  All fetuses were examined macroscopically (internal and external) at necropsy and subsequently by detailed internal visceral examination of the head or skeletal examination.


Results


There were no deaths, clinical signs or dosing signs considered to be related to treatment.


Treatment at 70 mg/kg/day was associated with mean body weight loss of 80 g (0.08 kg) between Days 6 and 8 of gestation compared with mean body weight stasis in Controls (body weight gain during treatment was appproximately 75 % of Controls), greater adjusted mean maternal body weight loss following adjustmnet for the gravid uterine weight and  moderately low food consumption.


In addition, dark and/or raised areas were observed on the stomach wall of animals that received Ethyldimethylamine at 70 mg/kg/day and microscopic examination of the stomach revealed microscopic changes findings including edema, hemorrhage, mucosal ulceration, vascular necrosis and inflammatory cell infiltrate in the mucosa and/or submucosa of the fundic region of the stomach or the antrum.  Dilated glands of the glandular mucosa (fundic mucosa or antrum) were also observed at higher incidence and severity in animals at 70 mg/kg/day.


Treatment at 25 mg/kg/day was associated with greater adjusted mean maternal body weight loss when compared with Controls; there was no maternal response to treatment at 8 mg/kg/day.


Embryo-fetal survival was unaffected by treatment and developmnet was not considered to be adversley affected at dose levels up to and including 70 mg/kg/day.


Conclusion


Based on the results of this study it is concluded that the no observed adverse effect level (NOAEL) for maternal toxicity is 25 mg/kg/day and the high dose of 70 mg/kg/day is the NOAEL for embryo-fetal survival, growth and development.

Justification for classification or non-classification

 


Classification for Reproductive Toxicity related to Fertility according to CLP Criteria: waiting for complementary results from ongoing OECD 443 for assessment.


 


Classification for Reproductive Toxicity Hazard related to unborn child according to CLP criteria: No fetal developmental effects were obseved in any of the rat and rabbit main developmental studies at the highest dose-level tested where maternal toxicity was observed.

Additional information