Registration Dossier

Toxicological information

Toxicity to reproduction

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Administrative data

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.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2021
Report date:
2021

Materials and methods

Test guidelineopen allclose all
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)

Test material

Constituent 1
Chemical structure
Reference substance name:
Ethyldimethylamine
EC Number:
209-940-8
EC Name:
Ethyldimethylamine
Cas Number:
598-56-1
Molecular formula:
C4H11N
IUPAC Name:
ethyldimethylamine
Test material form:
liquid: volatile
Details on test material:
Name of test material (as cited in study report): Dimethylethylamine
- Source (i.e. manufacturer or supplier):Arkema
- Lot/batch number of test material: J093973621
- Expiry date: 03 April 2021
- Purity :99.63 %
- Volatile: Yes, vapour pressure: 527 hPa at 20¿
- Specific gravity / density: 660 Kg/ m3 at 20¿

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: In refrigerator (2-8°C)
- Stability and homogeneity of the test material in the vehicle/solvent under test conditions (e.g. in the exposure medium) and during storage: Stability for at least 4 hours at room temperature under normal laboratory light conditions and stability for at least 10 days in the refrigerator confirmed over the concentration range 1 to 125 mg/mL (solutions), Project 20240947

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: the test substance was kept cold in a refrigerator or in an ice bath during the preparation of formulations

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.

Test animals

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

Administration / exposure

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
Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Effect levels (P0)

open allclose all
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.

Target system / organ toxicity (P0)

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

Results: F1 generation

General toxicity (F1)

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.

Effect levels (F1)

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.

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

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


 


 

Applicant's summary and conclusion

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).