Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

No adverse effects were observed in an EOGRTS with basic test design up to the limit dose of 1000 mg/kg bw/day.

Link to relevant study records
Reference
Endpoint:
extended one-generation reproductive toxicity - basic test design (Cohorts 1A, and 1B without extension)
Type of information:
experimental study
Adequacy of study:
key study
Study period:
study currently in draft
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
according to guideline
Guideline:
OECD Guideline 443 (Extended One-Generation Reproductive Toxicity Study)
Version / remarks:
25 June 2018
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
SPECIFICATION OF STUDY DESIGN FOR EXTENDED ONE-GENERATION REPRODUCTION TOXICITY STUDY WITH JUSTIFICATIONS
- Premating exposure duration for parental (P0) animals : 10 weeks as required by ECHA, because there is no substance specific information available supporting shorter premating exposure duration as advised in the ECHA Guidance on information requirements and chemical safety assessment Chapter R.7a, Section R.7.6 (version 6.0, July 2017).
- Basis for dose level selection : Dose levels of 100, 300, and 1000 mg/kg bw/d were selected in consultation with the Sponsor based on information from a previous GLP compliant study (OECD TG 421, which is also included in the dossier), in which after 2 weeks of treatment in juvenile rats no systemic toxicity effects were noted. The dose levels selection took into consideration all available information, including the dosing regimens from previous studies performed for the test item. Moreover, the highest dose level was chosen with the aim to induce some systemic toxicity, without death or severe suffering of the animals.
- Inclusion/exclusion of extension of Cohort 1B: conditions to include the extension of Cohort 1B are not met.
- Inclusion/exclusion of developmental neurotoxicity Cohorts 2A and 2B: No particular concern for (developmental) neurotoxicity was identified for Cohorts 2A and 2B.
- Inclusion/exclusion of developmental immunotoxicity Cohort 3: No particular concern for (developmental) immunotoxicity was identified for Cohort 3.
- Route of administration : ECHA considers that the oral route is the most appropriate route of administration for substances except gases to focus on the detection of hazardous properties on reproduction as indicated in ECHA Guidance on information requirements and chemical safety assessment (version 6.0, July 2017) Chapter R.7a, Section R.7.6.2.3.2. Since the substance to be tested is a liquid, ECHA concludes that testing should be performed by the oral route.
- Other considerations, e.g. on choice of species, strain, vehicle and number of animals: According to the test method EU B.56./ OECD TG 443, the rat is the preferred species. On the basis of this default assumption, ECHA considers that testing should be performed in rats.



Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: P: 7-8 wks
- Weight at study initiation: (P) Males: 266 - 341 g; Females: 173 -243 g
- Housing: From arrival to pairing: up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm.
During mating (P generation): one male to one female in clear polysulfone cages measuring approximately 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor.
After mating: males were re-caged as they were before mating. The females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm).
- Diet (e.g. ad libitum): laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019, SettimoMilanese (MI), Italy); ad libitum (except during the fasting procedure necessary for clinical pathology investigations)
- Water (e.g. ad libitum): ad libitum (except in the case of urinalysis investigations)
- Acclimation period: 18 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±2°C
- Humidity (%): 55±15%
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of the test item was suspended in the vehicle. The preparations were made daily. Concentrations were calculated and expressed in terms of test item as supplied. The preparation of the test item included a very slow addition of the vehicle to the test item and a manual mixing followed by a magnetic mixing for at least 1 hour. The very slow addition of the vehicle and a low stirring speed of the preparations were necessary conditions in order to minimize the formation of air bubbles.

VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg body weight
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of pregnancy
- If mating had not occurred after 14 days of cohabitation the animals were separated, without further opportunity for mating.
- After successful mating each pregnant female was caged in individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5x26.6x18.5 cm).
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in the range from 10 to 100mg/mL (r > 0.98; accuracy 85-115%; precision CV < 10%).
6 hour stability at room temperature was verified in the range from 10 to 100mg/mL.
Duration of treatment / exposure:
Males: 96-100 days (10 weeks prior to pairing, during mating period and thereafter until the day before necropsy)
Females: at least 85 days (at least 10 weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum, the day before sacrifice)
The not pregnant females, humanely killed females, females with total litter loss and females that did not give birth were dosed up to the day before necropsy.

Cohort 1A: Males and females were treated starting from the day of the selection, post-natal Day 21 (PND 21), up to the day before necropsy (approximately 13 weeks of age).
Cohort 1B: Males and females were treated starting from the day of the selection post-natal Day 21 (PND 21) up to the day before necropsy (approximately 14 weeks of age).
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals in the study: 17-18 weeks (P generation)
F1 animals were not mated to produce F2


Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25 (P generation)
20 (Cohorts 1A and 1B)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected in consultation with the Sponsor based on information from a previous GLP compliant study (OECD TG 421).

Positive control:
n.a.
Parental animals: Observations and examinations:
Animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar procedure was followed except that the final check was carried out at approximately mid-day.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment (P generation only) and at least once a week from the start of treatment (Day 21 or 22 of age for Cohorts 1A and 1B) until termination
- Signs recorded included, but were not limited to, changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in gait, posture, response to handling, as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling) or bizarre behaviour (e.g. self-mutilation, walking backwards) were also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations:
males: day of allocation (Day 21 of age for Cohorts 1A and 1B) and then at approximately weekly intervals from the first day of treatment
females: day of allocation (Day 21 of age for Cohorts 1A and 1B) and then approximatelyweekly from the first day of treatment until termination or until positive identification of mating (P generation)
Females of P generation after mating were also weighed on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7, 14 and 21 post partum.
Body weight was also recorded in Cohorts 1A and 1B on the day when they attained puberty (completion of preputial separation or vaginal patency).
All animals were weighed on the day of sacrifice.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
at weekly intervals starting from Day 1 of dosing and up to pairing for P generation and starting from nominal Day 28 for F1 generation
P males: at weekly intervals from the end of the mating period to termination
P females: on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7, 14 and 21 post partum starting from Day 1 post partum.

WATER CONSUMPTION: No

OTHER:
Urinalysis (10 (random) animals/sex/group): P generation and Cohort 1A
- during last week of treatment, individual overnight urine samples were collected from 10 males and 10 females (for P generation females with viable litters), all randomly selected from each group (the same animals selected for clinical pathology investigation under the same condition) Before starting urine collection, water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage.
- parameters: Appearance, Volume (manually recorded), Specific gravity, pH, Protein, Glucose, Ketones, Bilirubin, Urobilinogen, Blood, Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

Blood clotting time (10 randomly selected animals/sex/group): P generation
During the last week of treatment, a measure of blood clotting time was performed once from 10 parental males and 10 parental females (females with viable litters), all randomly selected from each group.

Clinical pathology investigations: P generation and Cohort 1A
Males: Once during the last week of treatment, samples of blood for haematology and clinical chemistry were collected from the retro-orbital sinus under isoflurane anaesthesia, from 10 males randomly selected from each group, under condition of food deprivation.
Females: As a part of the sacrificial procedure, and under condition of food deprivation, blood samples for haematology and clinical chemistry were withdrawn under isoflurane anaesthesia from the abdominal vena cava, of 10 females, all randomly selected from each group.
Parameters:
Haematology: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets
Coagulation: Prothrombin time, Activated partial thromboplastin time
Clinical chemistry: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma glutamyltransferase, Bile acids, Urea, Creatinine, Glucose, Triglycerides, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride, Phosphorus

Thyroid hormone determination (T3, T4 and TSH) (10 animals/sex/group): P generation and F1 pups
Males: Once during the last week of treatment, samples of blood for thyroid hormone determination were collected from the retro-orbital sinus under isoflurane anaesthesia, from 10 males randomly selected from each group, under condition of food deprivation. The timing of the blood collection for thyroid hormone determination was as close as possible between animals (within a time window of 1-4 hours) and at the same time of the day (within the same time window of 1-4 hours) in case of sampling on different days.
Females: As a part of the sacrificial procedure, and under condition of food deprivation, blood samples were withdrawn under isoflurane anaesthesia from the abdominal vena cava (for P generation females, with viable litters, if possible) of all females. The timing of the blood collection for thyroid hormone determination was as close as possible between animals (within a time window of 1-4 hours) and at the same time of the day (within the same time window of 1-4 hours) in case of sampling on different Days.
F1 pups (Days 4 and 21 or 22 post partum): On Day 4 post partum, as part of the necropsy procedure, blood samples (approximately 0.2 mL) were taken from 2 of the non selected pups (1 male and 1 female, if possible) of each litter per group if the number in the litters was sufficient. Blood samples were withdrawn under light ether anaesthesia from the heart by intracardiac puncture. Samples from different pups per sex was pooled if necessary. On Day 21 or 22 post partum, as part of the necropsy procedure, blood samples (approximately 0.5 mL) were taken from 2 pups (1 male and 1 female, if possible) if the number in the litters was sufficient.

Toxicokinetic studies:
Blood collection for detection of test item: P generation and F1 pups
Males: Blood samples (approximately 0.3 mL) were taken from the tail vein of 5 males/group during the last day of treatment at 2 time points (1 and 4 hours post-dose).
Females: Blood samples (approximately 0.3 mL) were taken from the tail vein of 5 females/group on Day 18 post coitum and Day 21 post partum at 2 time points (1 and 4 hours post-dose).

Milk collection for detection of test item: Dams
Milk samples were collected (at approximately 1 hour after dosing) from 5 females of each group, on Day 21 post partum (the same females selected for blood collection). On the day of collection oxytocin was administered to the selected females. Milk samples were collected under isoflurane anaesthesia. The samples were transferred into tubes and were frozen at -80°C, pending further information from the Sponsor.

Oestrous cyclicity (parental animals):
The assessment of oestrous cycles, with vaginal smears, was performed once daily from allocation to dosing. Oestrous cycles were also monitored for at least 2 weeks before pairing up to positive identification of mating. The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle;
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were taken also on the day of necropsy.
Sperm parameters (parental animals):
Parameters examined in all P generation and Cohort 1A males:
testis weight, epididymis weight, enumeration of cauda epididymal sperm reserve, sperm motility, sperm morphology

Staging of spermatogenic cycle - P generation and Cohorts 1A and 1B males
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- The size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, 5 males and 5 females per litter. Selective elimination of pups, e.g. based upon body weight was not performed. Whenever the number of male or female pups prevented to have five of each sex per litter, partial adjustment (for example, six males and four females) was performed.

PARAMETERS EXAMINED
The following parameters were examined in offspring:
number and sex of pups (live and dead), stillbirths, live births, and the presence of gross anomalies (i.e. externally visible abnormalities, abnormal skin colour or texture; lack of milk in stomach; presence of dried secretions) and a qualitative assessment of body temperature (presence of cold pups), state of activity and reaction to handling
All live pups were individually weighed on Days 1, 4, 7, 14 and 21 post partum. Observations were performed once daily for all litters.
The anogenital distance (AGD) of each pup was measured on PND 1. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum (F1 generation).
The presence of nipples/areolae was checked on PND 13 (males, F1 generation).
Testes descent was checked on Day 21 post partum (F1 generation).
The onset of vaginal opening was monitored starting from Day 28 of age until 100% occurrence. On the day of occurrence the body weight was recorded.
The cleavage of the balano-preputial skinfolds separation of males was checked once daily from Day 35 of age until 100% occurrence. On the day of occurrence the body weight was recorded.

GROSS EXAMINATION OF DEAD PUPS:
Pups found dead on PND 0 or at a later time were subjected to necropsy (external and internal examination) for the identification of possible defects and cause of death. All pups found dead or sacrificed for humane reasons were necropsied with the exception of those excessively cannibalised or autolysed.



Postmortem examinations (parental animals):
SACRIFICE
- Male animals: All surviving animals (Males of the P generation were killed after the weaning of the majority of F1 females.)
- Maternal animals: All surviving animals (P generation: Females with live pups were killed on Day 22 post partum. The females with total litter loss were killed on the day of the occurrence of total litter loss or shortly after. The females showing no evidence of copulation were killed after 25 days of the last day of the mating session. The females which did not gave birth 25 days after positive identification of mating were killed shortly after.)

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

Dams and litters - of P generation
All dams with litters were sacrificed on Day 22 post partum. The uteri and ovaries of each female which gave birth were inspected for the following:
1. Number of corpora lutea
2. Number of implantation sites

HISTOPATHOLOGY / ORGAN WEIGHTS
The examination was performed as detailed below:
i Tissues specified in table 6.9.13 and 6.9.14 from all animals in the control and high dose groups
ii Tissues specified in table 6.9.13 and 6.9.14 from all animals in the low and medium dose groups killed or dying during the treatment period
iii Reproductive organs of animals suspected of reduced fertility (e.g. that failed to mate or conceive)
iv All abnormalities in all groups

Bone marrow collection - P generation and Cohort 1A
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all animals (Parental generation and Cohort 1A).
The evaluation of bone marrow was performed in 10 male and 10 female animals per group, all randomly selected. A differential count was performed including calculation of the myeloid/erythroid cell ratio.

Organ weights - P and Cohorts 1A and 1B animals
- all animals completing the scheduled test period
- see table 6.9.13

Enumeration of ovarian follicles - P generation and Cohort 1A and 1B females: all animals in the control and high dose groups


Postmortem examinations (offspring):
SACRIFICE
Cohort 1A animals were killed at approximately 13 weeks of age.
Cohort 1B animals were killed at approximately 14 weeks of age.
Culled pups (not selected for blood collection), pups in extremis or killed for humane reasons were euthanised by intraperitoneal injection of Sodium Thiopenthal.
Pups selected for blood collection at PND 4 were sacrificed by exsanguination under ether anaesthesia.
Pups selected for blood collection at PND 21 or 22 collection were sacrificed under isoflurane anaesthesia.
Pups non selected for blood collection were killed by carbon dioxide asphyxiation.

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

Pups 0-20 PND of P Generation
All pups were examined externally and internally. Pups that were humanely killed in a moribund condition and dead pups were examined externally and internally for possible defects and/or cause of death and preserved.

HISTOPATHOLOGY / ORGAN WEIGHTS
The examination was performed as detailed below:
i Tissues specified in table 6.9.13 and 6.9.14 from all animals in the control and high dose groups
ii Tissues specified in table 6.9.13 and 6.9.14 from all animals in the low and medium dose groups killed or dying during the treatment period
iii Reproductive organs of animals suspected of reduced fertility (e.g. that failed to mate or conceive)
iv All abnormalities in all groups

Bone marrow collection - P generation and Cohort 1A
During the necropsy procedure, shortly after the death of each animal (except for those found dead), bone marrow samples were obtained from the femur of all animals (Parental generation and Cohort 1A).
The evaluation of bone marrow was performed in 10 male and 10 female animals per group, all randomly selected. A differential count was performed including calculation of the myeloid/erythroid cell ratio.

Lymph nodes weight and splenic lymphocyte subpopulation - Cohort 1A
From 10 males and 10 females of Cohort 1A of each treatment group (1 male or 1 female per litter; all litters represented by at least 1 pup; all randomly selected), mesenteric lymph nodes were weighed and using one half of the spleen were subject to spleen lymphocyte subpopulation analysis (CD4+ and CD8+ T lymphocytes, B lymphocytes, and natural killer cells). The other half of the spleen was preserved for histopathological evaluation.

Organ weights - P and Cohorts 1A and 1B animals
- all animals completing the scheduled test period
- see table 6.9.13

Organweight and tissue preservation - Pups at PND22 - 10 pups/sex/per group
The pups of parental generation (P generation) not selected for cohorts, were sacrificed after weaning (on PND 22). All pups were subjected to gross necropsy (external and internal examination)

Enumeration of ovarian follicles - P generation and Cohort 1A and 1B females: all animals in the control and high dose groups


Statistics:
Standard deviations were calculated as appropriate. For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n>5.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters.
Intergroup differences between the control and treated groups was assessed by the nonparametric version of theWilliams test. Details of all tests used and the data to which they were applied are included in the study report. The criterion for statistical significance was p<0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
Copulatory Indexof males (%)
Fertility Index (%) males
Copulatory Indexof females (%)
Fertility Index (%) females
Pre Coital Interval
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Males
At daily clinical examination, no adverse clinical signs were observed in treated males.
In the control group, one male (no. X0180032) showed swollen ear and was isolated in cage for approximately 2 weeks.
In the low dose group, hunched posture, dyspnoea and piloerection were noted in one male (no. X0180064) found dead after 97 days of treatment. Scabs and scabs with hairloss were recorded in two males from the same group (nos. X0180082 and X0180084).
In the mid-dose group, hairloss (no. X0180120), tooth cut or broken (nos. X0180130 and X0180132), scabs (no. X0180136) and swollen ear (no. X0180140) were observed.
In the high dose group, salivation was recorded in all males starting from approximately 9 weeks of treatment. Other minor clinical signs, such as rales, damaged tail, scabs and hairloss were occasionally noted in few animals.

Females
At daily clinical examination, no adverse clinical signs were observed in treated females.
Kyphosis and/or pallor were recorded in one control female (no. X0180033) and one high dose female (no. X1080181) sacrificed on Days 19 and 26 post coitum, respectively.
The other observed minor clinical signs, such as hairloss, scabs, staining and damaged ear were of low incidence and sporadically distributed among the control, low and mid-dose groups.
In the high dose group, salivation was observed in most females after approximately 9 weeks of treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, non-treatment-related
Description (incidence):
Males
One male (animal no. X1080064) of the low dose group was found dead on Day 27 of the mating phase (Day 97 of treatment). At post mortem examination, this animal showed a single firm mass in the thymus, enlarged and swollen spleen, dark and/or red colour of epididymides, jejunum and left testis. The histopathological evaluation revealed a malignant leukemia with metastases in the heart, kidneys, liver, lungs, prostate, spleen and thymus. The factor contributory to the death of this animal could be attributed to malignant leukemia.
Females
Two females were sacrificed for humane reasons: one female (no. X1080033) from control group and one female (no. X1080181) from high dose group.
Female no. X1080033 (control) was sacrificed on Day 19 post coitum (pregnant). At macroscopic observations, a single dark firmsubcutaneous mass associated with a single scab of the skin was observed. At histopathology, the most relevant changes were an adenocarcinoma in the mammary glands (which correlated to the subcutaneous mass observed during necropsy) and scab formation of the skin. These findings could be considered the factors contributory to the poor conditions of this animal.
Female no. X1080181 (high dose) was sacrificed on Day 26 post coitum with clinical signs of piloerection and pallor. At post mortem examination, brown staining of the muzzle and presence of dead foetuses in uterus were observed. The luminal dilatation of the uterus observed at histopathology was probably due to this condition. The presence of dead foetuses in uterus was considered the factor contributing to the illness status of this animal, suggesting that the animals had problem with the parturition.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
Means of body weight of treated males and female were, in general, comparable to the control group throughout the study. The occasionally statistically significant increases noted in high dose females at the end of the mating phase and in mid-dose females during the post partum period were not adverse.
Means of body weight gain of treated males and females were, in general, comparable to the control group. The statistically significant increase or decrease noted occasionally was considered not adverse.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption in treated males and females was comparable to the control group during the whole study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The statistically significant decrease of monocytes (38% below controls) recorded in males dosed at 100 mg/kg/day (low dose group) was not dose-related, therefore it was considered to be incidental. In addition, lymphocytes were increased in females receiving 100 mg/kg/day (46% above controls).
Concerning females, those receiving 1000 mg/kg/day (high dose group) showed an increase of leucocytes. Compared with controls, the change was 72% and comprised almost all sub-populations. Due to the low severity and the absence of other related changes, the above finding was considered to be not adverse.
No relevant changes were recorded. The statistically significant increase of prothrombin time (4% above controls) recorded in females dosed at 300 mg/kg/day (Group 3) was not dose-related, therefore it was considered to be incidental. Means of blood clotting time of treated and control animals were comparable.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant fluctuations of some biochemical parameters were recorded between control and animals dosed at 1000mg/kg/day, such as: increase of alanine aminotransferase ( 79%) and sodium (1%) and decrease of potassium (6%) in males, decrease of creatinine (14%) and sodium (1%) and increase of calcium (7%) in females. These changes were insufficient in magnitude to represent an organ/tissue injury, therefore the above findings were considered to be not adverse. Furthermore, the observed changes in liver enzymes were not accompanied with liver weight changes and were without histopathological correlate.
In addition, alanine aminotransferase, urea and creatinine were decreased in females receiving 100 mg/kg/day (23%, 19% and 12% below controls, respectively). Due to the absence of dose-relation, these findings were considered to be unrelated to treatment. Compared with controls, other sporadic changes were recorded, such as: a relevant increase of alanine and aspartate aminotransferases recorded in one female dosed at 1000 mg/kg/day (no. X1080195, 3.2 and 8.0 fold, respectively) and an increase of triglycerides in one other female from the same group (no. X080161, 9.6 fold).
Due to the minimal incidence, the above findings were not attributed to treatment.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were recorded between treated and control animals.
Immunological findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) were comparable to controls.
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Parental generation, Cohorts 1A and 1B
Following histopathological evaluation, no treatment-related changes were noted. All reported changes were considered spontaneous and incidental, having a comparable incidence in control and treated groups, and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age.

Parental generation and Cohorts 1A
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Cohort 1B
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone determination:
Compared with controls, males dosed at 1000 mg/kg/day showed statistically significant increases of both triiodothyronine and thyroxine (19% and 20%, respectively) and statistically significant decrease of thyroid stimulating hormone (42%). Statistically significant increases of T3 and T4 were also recorded in males receiving 300 mg/kg/day (38% and 40%, respectively) and in those treated at 100 mg/kg/day (T4 only, 18%). Concerning mean group data in females, those receiving 1000 mg/kg/day showed a slight but statistically significant increase of thyroxine (14% above controls), while T3 and TSH mean values did not significantly differ from controls. Hormone values were outside the range of the lab’s historical data in few animals of all treated groups. In addition, only three animals showed
simultaneous changes of two or three parameters (e.g. high T4 and low TSH or low T3/T4 and high TSH), such as: one male dosed at 300 mg/kg/day (no. X1080130) and two females treated at 1000 mg/kg/day (nos. X1080157 and X1080175) showed T3 and T4 levels above the high limit (95% percentile); female no. X1080175 (1000 mg/kg/day) also showed TSH below the low limit (5% percentile).
The currently available historical control data for the thyroid hormone panel mainly origin from studies performed according to the testing guidelines OECD 421 and OECD 422 while the availability of such data from OECD 443 studies is rather limited. Since the treatment period for the latter is considerably
different, some thyroid hormone values measured in the current study fall outside the range of the overall historical control data just due to the difference in the reference values and hence do not have toxicological relevance.
In conclusion, concurrent changes of two or three hormone values in individual animals were sporadic. There was no indication of a dose relationship in mean group values of thyroid hormones. Thyroid weights of treated animals did not differ from controls and no histopathological changes were recorded. Thus, the findings were considered unrelated to treatment.

Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
Oestrous cycle and pre-coital interval of treated femaleswere comparable to control females.

Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
Sperm motility, sperm concentration and sperm morphology did not show differences between control and treated groups.

Spermatogenic cycle
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Three females were found not pregnant at necropsy: one in the control group (no. X1080001), one in the low dose group (no. X1080075) and one in the mid-dose group (no. X1080115). One female (no. X1080179) in the high dose group had unilateral implantation with live pups on Day 22 post partum.
Mating was not detected in one female (no. X0180127) in the mid-dose group. However, this female gave birth and had live pups on Day 22 post partum.
Two females did not give birth after 25 days post coitum and were sacrificed on Day 28 post coitum: one in the control group (no. X1080009), which showed dead foetuses in uterus at necropsy suggesting problem with the parturition, although no sign of sofference were detected, and one in the low dose group (no. X1080097), which showed total resorption at necropsy.
Four females lost their litters: one in the control group (no. X1080027) onDay 1 post partum, one in the low dose group (no. X1080065) on Day 0 post partum and two in the high dose group (nos. X1080153 and X1080199) on Days 1 and 3 post partum, respectively. All these cases were considered incidental.
The number of females with live pups on Days 22 post partum was: 21 in the control, 22 in the low dose (100 mg/kg/day), 24 in the mid-dose (300 mg/kg/day) and 22 in the high dose (1000 mg/kg/day) groups.

All females had a spermpositive identification between 1-5 days of cohabitation.
Pre-coital interval for female no. X1080127 was not identified since the sperm positive identification was not detected (female with mating not detected).
Copulatory index was 100% both for males and females in control and treated groups.
Fertility index was 96% in control, low and mid-dose groups and 100% in the high dose group, both for males and females.

Number of implantation sites, total and live litter size at birth and gestation length, as well as the percentage of pre-natal and post-natal loss were comparable between treated and control females.

Litter data at birth were similar between the control and the treated groups.
No differences in total and live litter size, pup loss litter weights and mean pup weight were observed among treated and control females at birth and on Days 1, 4, 7 and 21 post partum.
On Day 21 of lactation (the day of selection of Cohorts 1A and 1B), litter size and mean pup weight were comparable between the control and the treated groups.
Sex ratio was comparable between treated and control groups.

Enumeration of ovarian follicles
Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females of parental generation, when compared to the control.
As reported in the validation of the analytical method study, a fast hydrolysis of the test item in plasma was observed during the feasibility study and consequently, in agreement with the Sponsor, it was decided
to monitor the presence of the test item in plasma via the quantification of Bis(2-hydroxyethyl)dimethylammonium (named Core 134).
Levels of Core 134 were quantified from rat plasma of 5 dams of each treatment group, on Day 18 post coitum and Day 21 post partum, at 1 and 4 hours after treatment and on Day 21 post partum at necropsy from 1 pup/sex/litter of the above females (at approximately 24hours after treatment of the respective dams).
Individual plasma levels of Core 134 are reported in Addendum 9; mean plasma levels are presented in the text table below.
No levels of Core 134 were found in the plasma samples of the control group of females and pups.
Plasma levels from animal no. X1080171 (Group 4) at 4 hours was excluded from mean group calculation.

Group Dose (mg/kg/day) Concentration (ng/mL) Dams Pups
Day 18 post coitum Day 21 post partum Day 21 post partum
1h 4h 1h 4h
2 100 128.2±19.4 178.7±70.1 225.2±49.8 159.9±75.1 BLOQ
3 300 274.9±130.4 561.4±174.5 386.9±121.9 471.0±207.5 16.2±2.3
4 1000 401.9±163.2 603.7±160.3 368.7±93.2 623.1±140.1 40.6±16.7

Dams
On Day 18 post coitum the exposure increased with the dose with higher values at 4 hours compared to 1 hour post-dose.
OnDay 21 post partum a greater variability was observed. Exposure at 1 hour from treatment increased from Group 2 to Group 3, but not from Group 3 to Group 4. The exposure increased with the dose from Group 2 to Group 4 at 4 hours after treatment, with higher values at 4 hours compared to 1 hour with the exception of Group 2.
Pups
During lactation (Day 21 post partum), no exposure to Core 134 was detected in pups of Group 2, while low levels were detected in Groups 3 and 4, although with an increase with the dose of the dams.
In conclusion, exposure to Core 134 was demonstrated in all treated dams on Day 18 post coitum and Day 21 post partum and in pups of Groups 3 and 4 during lactation, on Day 21 post partum. No exposure to Core 134 was detected in pups of Group 2.
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no toxicologically relevant adverse effects observed
Critical effects observed:
no
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Small appearance and/or apparently no food intake and/or pallor were the main clinical signs noted in pups from control and treated groups. Found dead or missing pups were also observed both in control and treated groups with similar incidence. All the findings were considered not adverse since are commonly observed in the litters during the lactation period.

Clinical signs of Cohort 1A animals
At daily clinical examination, not adverse clinical signs were observed in treated males and females.
In the high dose group, salivation was recorded in the majority of males and females starting from approximately 7weeks of treatment. Other minor clinical signs, such as hairloss, scabs and damaged eye were occasionally recorded in few animals. One female in the low dose group (no. X1080249) with marked to moderate abrasion and scabs of the neck was isolated in cage.

Clinical signs of Cohort 1B animals
At daily clinical examination, no clinical signs of toxicological relevance were observed in treated males and females. In the high dose group, salivation was recorded in the majority of males and females. No signs were noted in mid-dose animals of both sexes. Two males in the low dose group (nos. X1080424 and X1080430) with marked to moderate abrasion and scabs of the neck were isolated in cage.
Other minor clinical signs, such as hairloss, scabs and damaged eye were occasionally recorded in few animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No mortality occurred in Cohort 1A or Cohort 1B animals.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Cohort 1A
No differences of toxicological relevance were recorded in body weight and body weight gain of both sexes.
The statistically significant increases occasionally recorded in low and high dose groups compared to the controls were considered incidental and not adverse.

Cohorts 1B
Body weight of treated males and female were unaffected by treatment. The statistically significant increase noted in low dose males was considered incidental. No differences considered treatment related were recorded in body weight gain of both sexes. The statistically significant increases occasionally recorded in low and mid-dose males compared to the controls were considered incidental.
Terminal body weight of treated animals of both sexes of the three groups (Groups 2, 3 and 4) was comparable to the concurrent control group (Group 1).
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Cohort 1A and Cohort 1B
No treatment related differences were recorded in food consumption of both sexes.
The statistically significant increases occasionally recorded in mid- and high dose groups compared to the controls were considered incidental.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Description (incidence and severity):
No changes were recorded in the haematological parameters between treated and control animals.
No changes were recorded in the coagulation parameters between treated and control animals.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
No changes were recorded between treated and control animals.
The statistically significant increase of potassium (8% above controls) recorded in females dosed at 300 mg/kg/day was not dose-related, therefore it was considered to be incidental.
Urinalysis findings:
no effects observed
Description (incidence and severity):
No changes were recorded between treated and control animals.
Sexual maturation:
no effects observed
Description (incidence and severity):
Cohort 1A and Cohort 1B
The mean values of the the age in which vaginal opening and the first oestrous occurred were comparable between treated and control groups.
The mean values of the the age in which the balano-preputial separation occurred were comparable between treated and control groups.
Oestrous cycle interval of treated females (Cohort 1A) was comparable to controls.
Spermanalysis of Cohort 1A males: Spermmotility, concentration and morphology in the control and all treated groups did not show differences between treated and control males.

Spermatogenic cycle (Cohort 1B): Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. As regular layering in the germinal epithelium was noted, there was no treatment-related effect on the spermatogenic cycle.
Enumeration of ovarian follicles (Cohort 1B): Differential follicle and corpora lutea counts per ovary did not reveal any relevant differences in all high dose females, when compared to the control.
Anogenital distance (AGD):
no effects observed
Description (incidence and severity):
The anogenital distance (normalised to the cube root of the body weight) determined in all live pups at Day 1 of lactation was comparable between pups from control and treated groups, both for males and females.
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were retained in male pups on Day 22 post partum, since they were not observed.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
Cohort 1A
Statistically significant increase in absolute spleen weight and relative thymus weight was noted in females of Group 4 when compared to the control group. Due to the limited severity and/or the absence of dose relation, the above findings were considered not adverse.

The organ weight variations between control and treated animals were considered incidental as unrelated to the dose, as all individual values were within the physiological range of variability. In particular,
the statistically significant decrease, unrelated to the dose (-16% for low dose group, -12% for mid-dose group and -14% for high dose group) of mean relative thyroid weight was considered incidental and not treatment-related due to:
– Lack of dose response relantionship
– Minimal severity
– Individual values in the range of variability and similar to thyroid weight of parental animals
– No histopathological correlation

Cohort 1B
No relevant changes were reported in the absolute and relative organ weights of treatment groups of both sexes, when compared to control data, with the exception of the statistically significant decrease of
relative (to bodyweight) testesweight (-7%) of the high dose male group. However, since the histopathological evaluation of testes was comparable to control animals, no toxicological significance could be attributed to this finding.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Decedent pups
Autolysed or cannibalized thoracic and/or abdominal organs were observed in the majority of decedent pups both in control and treated groups, without any possible macroscopic examination of internal organs.

Pups sacrificed on Days 4 and 22 post partum
No abnormalities were recorded at the external and internal examination in pups sacrificed on Days 4 and 22 post partum. Only tip of tail missing was noted in one pup of the control group.

No gross pathology treatment-related changes were noted. The macroscopic findings were considered spontaneous and incidental, having a comparable incidence in control and treated groups and/or are characteristically seen in untreated Sprague Dawley SD rats of the same age
Histopathological findings:
no effects observed
Description (incidence and severity):
Cohorts 1A and 1B
No treatment-related changes were noted in animals sacrificed at the end of the treatment period.

Cohort 1A
No treatment-related changes were noted in animals sacrificed at the end of the treatment period. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Cohort 1B
Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormone determination (Cohort 1A):
Compared with controls, males dosed at 100 mg/kg/day showed a statistically significant decrease of thriiodothyronine (18%). Due to the absence of dose-relation, this finding was considered to be unrelated to treatment. Looking at individual data, most of the values were within the ERBC historical data. Singular instances of values outside this range was observed for T3, T4 and TSH in few animals of all groups, including control animals; in addition, simultaneous changes of two parameters (high T4 and low TSH values) were only recorded in one female control animal (X1080227), one male and one female dosed at 100 mg/kg/day (nos. X1080250 and X1080279, respectively) and one female dosed at 300 mg/kg/day (no. X1080283), therefore the above changes were considered to have no pathological significance.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Cohort 1A and 1B
Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) were comparable to the controls.
Developmental immunotoxicity:
no effects observed
Description (incidence and severity):
Bone marrow of Cohort 1A
No relevant differences between control and treated animals were recorded. Male no. X1080274 (100 mg/kg/day) and female no. X1080347 (1000 mg/kg/day) showed an increase of the erythroid cells, with no relevant changes of the maturation of erythroid or myeloid lineages. On the other hand, an increase of the myeloid lineage was recorded in male no. X1080260 (100 mg/kg/day) and female nos. X1080297 and X1080303 (both dosed at 300 mg/kg/day) Due to the minimal incidence and the absence of dose-relation, these findings were considered to be unrelated to treatment.

Lymphnodes weight and splenic lymphocyte subpopulation of Cohort 1A animals
No statistically significant differences were observed in the frequencies of all cell populations in any treatment group of both sexes. Expression of surface markers was similar for all groups for both genders. In conclusion, using this protocol of analysis, no sign of alteration in the immune cell distribution was observed in splenocytes of animals treated with the test item at any dose level, when compared to controls and to historical control data.
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1A)
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Dose descriptor:
NOAEL
Generation:
F1 (cohort 1B)
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects observed
Critical effects observed:
no
Reproductive effects observed:
no

The results are attached as pdf files below.

 

Conclusions:
In conclusion, the dosage of 1000 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in Parental generation and for general and reproductive toxicity in Cohort 1A and 1B.
Executive summary:

In an extended one generation reproduction toxicity study in accordance with OECD TG 443 (adopted on 25 June 2018) the pre- and post-natal effects of MDEA-Esterquat C16-18 and C18 unsatd. on development, as well as a thorough evaluation of systemic toxicity were investigated in male and female rats of the parental generation treated for 10 weeks before pairing and during gestation and lactation period.

40 male and 40 female pups/group, from different litters,were selected and assigned to Cohort 1 A and Cohort 1 B in order to follow the toxicity effect in the second generation administered from weaning (Day 21 of age) up to 13 weeks.

In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems.

The following parameters were considered: gonadal function, oestrous cycle, epididymal sperm maturation, mating behaviour conception, pregnancy, parturition and lactation.

The dose levels were 0 (control), 100, 300, and 1000 mg/kg bw/d.

 

Parental generation

Males were treated for 10 weeks prior to pairing, during pairing with females until the day before necropsy, for a total of 96-100 days. Females were treated for 10 weeks prior to pairing, during pairing and throughout the gestation and lactation periods until Day 21 post partum, for at least 85 days. The following parameters were evaluated in parental animals: mortality check, clinical signs (including neurotoxicity assessment), body weight, food consumption, oestrous cycle, mating performance, clinical pathology investigations (haematology/coagulation and clinical chemistry), blood collection for thyroid hormone determination and detection of test item (adult animals and pups), anogenital distance, litter data, macroscopic observations and organ weights. Measurement of body weight, clinical signs and macroscopic observations of pups including nipple check/count were also performed, as well as sperm parameters examination in all adult male groups and vaginal smears in all adult female groups. Organ weights were recorded for at least 10 pups per sex per group. Histopathological examination was performed in all control and high dose animals. Bone marrow was evaluated in 10 male and 10 female animals per group. In addition, the identification of the stages of the spermatogenic cycle and the enumeration of ovarian follicles were performed in all control and high dose group males and females. The following parameters were evaluated in F0 dams which littered: gestation length, sex ratios, pre- and post-natal loss and litter data.

 

Cohort 1A

Males and females were treated starting from Day 21 of age up to approximately 13 weeks of age and then sacrificed. The following parameters were evaluated: mortality check, clinical signs (including neurotoxicity assessment), bodyweight, food consumption, oestrous cycle, vaginal opening, testis descent and balano-preputial separation, thyroid hormone determination, macroscopic observations and organ weights. Sperm analysis was performed in all males, determining the motility, morphology and concentration of sperm. Histopathological examination, the identification of the stages of the spermatogenic cycle and the enumeration of ovarian follicles were performed only on control and high dose group animals. Bone marrow was evaluated from 10 male and 10 female animals per group. In addition, from 10 males and 10 females of each treatment group (1 male or 1 female per litter), lymph nodes (mesenteric) were weighed and one half of the spleen was used for the splenic lymphocyte subpopulation analysis (CD4+and CD8+T lymphocytes, B lymphocytes, and natural killer cells).

 

Cohort 1B

Males and females were treated starting from Day 21 of age up to approximately 14 weeks of age and then sacrificed. The following parameters were evaluated in Cohort 1B animals: mortality check, clinical signs (including neurotoxicity assessment), body weight, food consumption, oestrous cycle,

vaginal opening, testis descent and balano-preputial separation, macroscopic observations, organ weights, spermatogenic cycle and enumeration of ovarian follicles.

 

Results

P generation (F0)

Mortality and fate of females in Parental generation

Males

One male of the low dose group was found dead on Day 27 of the mating phase (Day 97 of treatment). The histopathological evaluation revealed a malignant leukemia with metastases in the

heart, kidneys, liver, lungs, prostate, spleen and thymus. The factor contributory to the death of this animal could be attributed to malignant leukemia.

Females

Two females were sacrificed for humane reasons:

one female from control group was sacrificed on Day 19post coitum(pregnant). At macroscopic observations, a single dark firmsubcutaneous mass associated with a single scab of the skin was observed. At histopathology, the most relevant changes were an adenocarcinoma in the mammary glands (which correlated to the subcutaneous mass observed during necropsy) and scab formation of the skin. These findings could be considered the factors contributory to the poor conditions of this animal.

one female from high dose group was sacrificed on Day 26post coitumwith clinical signs of piloerection and pallor. Atpost mortemexamination, brown staining of the muzzle and presence of dead foetuses in uterus were observed and the luminal dilatation of the uterus observed at histopathology was probably due to this condition. The presence of dead foetuses in uterus was considered the factor contributing to the illness status of this animal.

Details on the pregnancy data of females were as follows:

Three females were found not pregnant at necropsy: one in the control group, one in the low dose group and one in the mid-dose group. In addition, one female in the high dose group had unilateral implantation with live pups on Day 22post partum.

Mating was not detected in one female in the mid-dose group. However, this female gave birth and had live pups on Day 22post partum.

Two females did not give birth after 25 dayspost coitumand were sacrificed on Day 28post coitum: one in the control group with dead foetuses in uterus at necropsy and one in the low dose group with total resorption at necropsy.

Four females lost their litters: one in the control group, one in the low dose group and two in the high dose group.

The number of females with live pups on Days 21/22post partumwas: 21 in the control, 22 in the low dose (100 mg/kg/day), 24 in the mid-dose (300 mg/kg/day) and 22 in the high dose (1000 mg/kg/day) groups.

 

Clinical signs of Parental generation

At daily clinical examination, not adverse clinical signs or signs of toxicological relevance were observed in treated males and females.

 

Clinical observations (Functional Observation Battery Tests) of Parental generation

Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) were comparable to controls.

 

Body weight and body weight gain of Parental generation

Body weight and body weight gain of males and females were unaffected by treatment.

 

Food consumption of of Parental generation

Food consumption of males and females was unaffected by treatment.

 

Haematology, Coagulation, Clinical chemistry and Urinalysis of Parental generation

No changes that could be considered adverse were seen in haematology parameters of treated animals compared to controls.

 

Blood clotting time of Parental generation

No treatment related changes were recorded between treated and control animals.

 

Thyroid hormone determination of Parental generation

No changes that could be attributed to treatment were seen in the serum levels of thyroid
hormones of treated animals compared to controls.

 

Blood detection of test item – P generation and F1 pups

A fast hydrolysis of the test item was observed during the feasibility study, so, it was decided to monitor the presence of the test item via the quantification of the Bis(2 hydroxyethyl)dimethylammonium (named Core 134). Exposure to Core 134 was demonstrated in all treated dams on Day 18 post coitum and Day 21 post partum at 1 and 4 hours after treatment and in pups of Groups 3 and 4 during lactation, on Day 21 post partum. No exposure to Core 134 was detected in pups of Group 2.

 

Oestrous cycle, reproductive parameters, pairing combination and mating performance of Parental generation

Oestrous cycle, pre-coital intervals, copulatory index and fertility index did not show any differences that could be related to treatment.

 

Implantation sites, pre-natal loss data and gestation length of Parental generation

Implantations, litter size and pre-natal loss (percentage) were similar in control and treated groups. Gestation periods were similar between treated and control females.

 

Litter data at birth, onDays 1 and 4post partum(before culling) and from Day 7 (after culling) through Day 21post partumof Parental females

Litter data at birth and on Days 1, 4, 7 and 21 post partum did not show treatment related differences.

 

Sex ratios of F1 pups

Sex ratios at birth and on Days 4, 14 and 21 post partum was comparable differences between treated and control groups.

 

Anogenital distance (AGD) of F1 pups

The anogenital distance (normalised to the cube root of the body weight) determined in all live pups at Day 1 of lactation was unaffected by treatment.

 

Clinical signs of F1 pups

No signs of toxicological relevance were observed in the litters during the lactation period.

 

Necropsy findings in decedent pups, in pups sacrificed on Days 4 and 22 post partum and nipple count

Necropsy findings in deceased pups and in pups sacrificed on Days 4 and 22 post partum did not reveal any treatment-related effect.

 

Organ weights of F1 pups on Day 22 post partum

Statistically significant increase in absolute spleen weight and relative thymus weight was noted in females of Group 4 when compared to the control group. Due to the limited severity and/or the absence of dose relation, the above findings were considered not adverse.

 

Sperm analysis of Parental males

No differences were observed at sperm analysis including sperm motility, sperm concentration, sperm morphology and cauda weight, between the control and treated males.

 

Bone marrow of Parental generation

No treatment related differences between control and treated animals were recorded.

 

Terminal body weight and organ weights of Parental generation

No treatment related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

 

Macroscopic observations of Parental generation

Animals that completed the treatment period and were killed at termination did not show relevant macroscopic changes that could be considered treatment-related.

 

Microscopic observations of Parental generation

No treatment-related changes were noted in animals sacrificed at the end of the treatment period.

Spermatogenic cycle

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Enumeration of ovarian follicles

Differential follicle andcorpora luteacounts per ovary did not reveal any relevant differences in all high dose females, when compared to the control.

 

Cohort 1A

Mortality of Cohort 1A animals

No mortality occurred in Cohort 1A animals.

 

Clinical signs of Cohort 1A animals

At daily clinical examination, no clinical signs of toxicological relevance were observed in treated males and females.

 

Clinical observations (Functional Observation Battery Tests) of Cohort 1A animals

Observations of treated animals at removal from the cage and in an open arena (neurotoxicity assessment) were comparable to controls.

 

Body weight and body weight gain of Cohort 1A animals

No differences of toxicological relevance were recorded in body weight and body weight gain of both sexes.

 

Food consumption of Cohort 1A animals

No differences were recorded in food consumption of both sexes.

 

Vaginal opening and first oestrous - Cohort 1A females

No differences were detected in the onset of vaginal opening and first oestrous.

 

Balano-preputial skin folds separation - Cohort 1A males

No differences were seen in balano-preputial separation between treated and control groups.

 

Oestrous cycle of Cohort 1A females

Oestrous cycle interval of treated females was comparable to controls.

 

Haematology, Coagulation, Clinical chemistry and Urinalysis of Cohort 1A animals

No changes were recorded in the haematology, coagulation, clinical chemistry and urinalysis parameters between treated and control animals.

 

Thyroid hormone determination of Cohort 1A animals

No treatment-related differences between control and treated animals were recorded.

 

Bone marrow of Cohort 1A

No treatment-related differences between control and treated animals were recorded.

 

Sperm analysis of Cohort 1A males

Sperm motility, concentration and morphology in the control and all treated groups did not show differences.

 

Lymph nodes and splenic lymphocyte subpopulation of Cohort 1A males

No sign of alteration in the immune cell distribution was observed in splenocytes of animals treated with the test item at any dose level, when compared to controls and to historical control data.

 

Terminal body weight and organ weights of Cohort 1A animals

No treatment related changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

 

Macroscopic observations of Cohort 1A animals

Animals that completed the treatment period and were killed at termination did not show relevant macroscopic changes that could be considered treatment-related.

 

Microscopic observations of Cohort 1A animals

No treatment-related changes were noted in animals sacrificed at the end of the treatment period.

Spermatogenic cycle

Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted.

Enumeration of ovarian follicles

Differential follicle andcorpora luteacounts per ovary did not reveal any relevant differences in all high dose females, when compared to the control.

 

Cohort 1B

Mortality of Cohort 1B animals

No mortality occurred in Cohort 1B animals.

 

Clinical signs of Cohort 1B animals

At daily clinical examination, no clinical signs of toxicological relevance were observed in treated males and females.

 

Clinical observations (Functional Observation Battery Tests) of Cohort 1B animals

Clinical observation performed weekly for neurotoxicity assessment (observation of animals at removal from the cage and in an open arena) did not show differences between treated and control animals.

 

Body weight and body weight gain of Cohort 1B animals

No differences of toxicological relevance were recorded in body weight and body weight gain of both sexes.

 

Food consumption of Cohort 1B animals

Food consumption in treated males and females was comparable to the control group during the whole study.

 

Oestrous cycle of Cohort 1B females

No difference in oestrous cycle intervals was noted between control and treated animals.

 

Vaginal opening and first oestrous - Cohort 1B females

No treatment-related differences were detected in the onset of vaginal opening and first oestrous.

 

Balano-preputial skin folds separation - Cohort 1B males

No treatment-related differences were seen in balano-preputial separation.

 

Terminal body weight and organ weights of Cohort 1B animals

No relevant changes were observed in terminal body weight or absolute and relative organ weights of treated animals of both sexes, when compared to the controls.

 

Macroscopic observations of Cohort 1B animals

Animals that completed the treatment period and were killed at termination did not show relevant macroscopic changes that could be considered treatment-related.

 

Spermatogenic cycle and enumeration of ovarian follicles of Cohort 1B animals

In high dose males of Cohort 1B, seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages; regular layering in the germinal epithelium was noted. In high dose females of Cohort 1B, differential follicle andcorpora luteacounts per ovary did not reveal any relevant differences, when compared to the control.

 

Conclusions

In conclusion, the dosage of 1000 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in Parental generation and for general and reproductive toxicity in Cohort 1A and 1B.

 

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
OECD guideline studies, no deviations, GLP
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available
Additional information

Screening study


The toxic effects on Sprague Dawley rats of both sexes were investigated after repeated dosing with MDEA-Esterquat C16-18 and C18 unsatd. in accordance with OECD TG 421 (adopted on 29 July 2016). Furthermore, effects of the test item on male and female reproductive performance were examined, i.e. gonadal function, mating behaviour, conception, development of conceptus, parturition and early lactation of the offspring.


Groups of 10 males and 10 females received the test item, by gavage, at dosages of 0, 100, 300 and 1000 mg/kg body weight/day. An additional group of 10 males and 10 females received the vehicle alone (softened water) at the dose volume of 10 mL/kg and acted as control.


No effects were observed in the in vivo parameters of parental animals: no relevant clinical signs and no effects on body weight, body weight gain and food consumption were noted at any dose level investigated. Thyroid hormones (T3, T4 and TSH) evaluated in parental males did not show any changes of toxicological relevance. No remarkable differences were noted at post mortem examination including organ weights and no treatment-related macroscopic and microscopic changes were observed in treated animals, when compared to controls. Futhermore, no effects on the spermatogenic cycle were described. No intergroup differences were seen in oestrous cycle, pre-coital intervals, copulatory and fertility indices.


No significant differences were observed in the number of corpora lutea, implantations, total litter size, pre-implantation loss, pre-birth loss and gestation length between control and treated females. Litter data at birth and on Days 1, 4 and 13 post partum and sex ratios were also comparable between treated and control females.


There were neither treatment-related signs at clinical observation of pups, nor at necropsy of deceased pups or those sacrificed after culling or at term. The statistically significant decrease noted in the ano-genital distance of female pups on Day 1 of age in all treated groups cannot be considered as adverse, since it had a positive trend (feminisation effect).


No nipples were observed on Day 13 of age, in male pups at all dose levels. Increases of circulating T3 levels were found in mid- and high dose male pups sacrificed at Day 14 of age. However, no clear consistency of hormones variations, dose and/or sex relation were noted to finally address an adverse effect of the test compound on thyroid function.


Analysis of plasma samples showed that after oral administration of the test item, females were exposed to the test item (detected as Core 134) both on Day 18 post coitum and Day 13 post partum, as well as, via milk, in pups on Day 13 post partum. The exposure increased with the dose and the duration of treatment (Day 18 post coitum corresponds to approximately 33 days of treatment; Day 13 post partum corresponds to approximately 50 days of treatment.


Based on the results obtained in this study, the NOAEL (No Observed Adverse Effect Level) for both general toxicity and reproduction/developmental toxicity was considered to be 1000 mg/kg/day for both males and females.


These results were used to set the dose levels for the subsequent EOGRTS.


 


Extended One Generation Reproduction Toxicity Study


In an extended one generation reproduction toxicity study in accordance with OECD TG 443 (adopted on 25 June 2018) the pre- and post-natal effects of MDEA-Esterquat C16-18 and C18 unsatd. on development, as well as a thorough evaluation of systemic toxicity were investigated in male and female rats of the parental generation treated for 10 weeks before pairing and during gestation and lactation period.


40 male and 40 female pups/group, from different litters, were selected and assigned to Cohort 1 A and Cohort 1 B in order to follow the toxicity effect in the second generation administered from weaning (Day 21 of age) up to 13 weeks.


In addition, the study provided and/or confirmed information about the effects of the test item on the integrity and performance of the adult male and female reproductive systems. The following parameters were considered: gonadal function, oestrous cycle, epididymal sperm maturation, mating behaviour conception, pregnancy, parturition and lactation.


 


Parental generation (F0)


The dose levels of 100, 300 and 1000 mg/kg/day were selected for parental animals and were administered orally by gavage. The control group received softened water.


Males were treated for 10 weeks prior to pairing, through the mating period and thereafter until the day before necropsy, for a total of 196-100 days.


Females were treated for 10 weeks prior to pairing, during mating, gestation and post partum periods until Day 21 post partum, for at least 85 days.


The number of females with live pups on Days 21/22 post partum was: 21 in the control, 22 in the low dose (100 mg/kg/day), 24 in the mid-dose (300 mg/kg/day) and 22 in the high dose (1000 mg/kg/day) groups.


At the daily and weekly clinical observation, no signs considered adverse and no effect in the neurotoxicity assessment were observed.


Body weight, body weight gain and food consumption were unaffected by treatment.


No changes that could be considered adverse were seen in haematology, coagulation and clinical chemistry parameters of treated animals compared to controls.


Changes noted in thyroid hormone levels of treated males and high dose females were considered to be unrelated to treatment since the simultaneous changes of two or three hormones (conditions which could represent a pathological significance) were sporadic and not dose-related, and no histopathological changes were recorded.


Exposure to Core 134 (metabolite used to monitor the presence of the test item in plasma) was demonstrated in all treated dams on Day 18 post coitum and Day 21 post partum at 1 and 4 hours after treatment and in pups of Groups 3 and 4 on Day 21 post partum, after approximately 24 hours of treatment of the respective dams. No exposure to Core 134 was detected in pups of Group 2.


No treatment-related anomalies were noted in the oestrous cycle of the treated females, when compared to controls. Copulatory and fertility indices did not show any treatment-related differences among treated and control groups. Implantation, pre-natal loss, litter data and sex ratio did not show any changes of toxicological relevance. No significant differences in the anogenital distance were seen between control and treated groups both for male and female pups. No nipples were observed in male pups. Clinical signs and findings at necropsy and organs weight did not reveal any treatment-related or adverse effect.


Sperm analysis performed in all treated males was comparable to controls. Enumeration of ovarian follicles performed in control and high dose females did not show any treatment-related effects. No relevant changes were seen in bone marrow evaluation.


No treatment-related changes were noted in animals sacrificed at the end of treatment at organ weight, macroscopic and microscopic examination.


 


Cohorts 1A and 1B


No mortality occurred in Cohort 1A and Cohort 1B animals.


At the daily and weekly clinical observation, no signs of toxicological relevance and no effect in the neurotoxicity assessment were observed in treated males and females of both Cohorts.


Body weight, body weight gain and food consumption of both sexes of both Cohorts were unaffected by treatment.


Oestrous cycle, vaginal opening and balano-preputial skin folds separation did not eveal


differences considered adverse in Cohort 1A and Cohort 1B animals.


No changes were recorded in the haematology, coagulation, clinical chemistry and urinalysis parameters between treated and control animals of Cohort 1A.


The increase in Triiodothyronine level noted in low dose males was considered unrelated to treatment, in the absence of a of dose-relation.


No sign of alteration in the immune cell distribution was observed in splenocytes of treated animals of Cohort 1A.


No treatment-related changes were noted at sperm analysis (in Cohort 1 A), organ weights, gross pathology and the histopathological examination (in Cohort 1 A). Enumeration of ovarian follicles enumeration and the staging of spermatogenic cycle performed did not show any treatment-related effects in Cohort 1A and Cohort 1B.


 


In conclusion, the dosage of 1000 mg/kg/day was considered the NOAEL for general and reproductive toxicity and pups development in Parental generation and for general toxicity


in Cohort 1A and 1B.


 


There are no data gaps for effects on fertility. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.


 

Effects on developmental toxicity

Description of key information
The results of the prenatal developmental toxicity study do not indicate a substance-related effect on the fetus up to the limit dose of 1000 mg/kg bw/day.
Link to relevant study records
Reference
Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1992-10-09 to 1992-11-21
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to same study
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:
- Physical state: liquid
- 10% aqueous dispersion Diethyl ester dimethyl ammonium chloride
Species:
rat
Strain:
other: Rat WIST HanIbm: WIST (SPF)
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: BRL, Biological Research Laboratories Ltd.
- Age at study initiation: No data. (Age at pairing: 12 weeks, minimum).
- Weight at study initiation: No data. (189-228 g at day 0 post coitum).
- Fasting period before study: N/A.
- Housing: individually in Makrolon cages (type-3) with wire mesh tops and standarized granulated softwood bedding (Lignocel, Schill AG, CH 4132 Muttenz/Switzerland).
- Diet (e.g. ad libitum): pelleted standard Kilba 343 rat/mouse maintenance diet ("Kilba", Klingentalmuehle AG, CH 4303 Kaiseraugust/Switzerland) ad libitum. (Batch no. 65-92).
- Water (e.g. ad libitum): tap water in bottles ad libitum.
- Acclimation period: 10 days (minimum).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3 (air-conditioned).
- Humidity (%): 40-70.
- Air changes (per hr): 10-15.
- Photoperiod (hrs dark / hrs light): 12-hrs artificial fluorescent light/12 hours dark.


IN-LIFE DATES: From: 1992-10-09 To: 1992-11-21
Route of administration:
oral: gavage
Vehicle:
other: NOAEL emBe-distelled water, previously adjusted with hydrochloric acid to pH 2.5-2.6
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The mixtures of the test substance and vehicle were prepared daily before administration. The test substance was weighed into a glass beaker on a tared precision balance (Mettler PE 360) and the vehicle added (w/v). The mixtures were prepared using a homogenizer. During the daily administration period, homogeneity was maintained using a magnetic stirrer. All animals received a dose volume of 10 ml/kg bw with a daily adjustment of the individual volume to the actual body weight.


DIET PREPARATION
- Rate of preparation of diet (frequency): N/A.
- Mixing appropriate amounts with (Type of food): N/A.
- Storage temperature of food: N/A.


VEHICLE: Bi-distilled water (previously adjusted with hydrochloric acid to pH 2.5-2.6).
- Justification for use and choice of vehicle (if other than water): N/A.
- Concentration in vehicle: N/A.
- Amount of vehicle (if gavage): 10 ml/kg.
- Lot/batch no. (if required): N/A.
- Purity: N/A.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples of the test substance/vehicle dilutions were taken on two occasions during the dosing period of the study and sent to the Sponsor for analysis (concentration, homogeneity and stability over 2-hrs).
Details on mating procedure:
- Impregnation procedure: cohabitation.
- If cohoused:
- M/F ratio per cage: 1:1.
- Length of cohabitation: overnight
- Further matings after two unsuccessful attempts: no data.
- Verification of same strain and source of both sexes: no data. (The male rats used for mating were in the possession of the testing laboratory. The fertility of these males was proved and was continuously controlled).
- Proof of pregnancy: sperm in vaginal smear/vaginal plug referred to as day 0 post coitum.
- Any other deviations from standard protocol: After mating, female rats were removed and allocated to the test groups.
Duration of treatment / exposure:
Day 6 through 15 post coitum.
Frequency of treatment:
Once daily in the morning.
Duration of test:
Females were sacrificed on day 21 post coitum.
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
25/group.
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dosages were based on the results of the dose range-finding sudy (RCC Project 326158--see cross reference section).
- Rationale for animal assignment (if not random): Mated rats were assigned to the different groups using a computer-generated random algorithm.
- Rationale for method of administration: International guidelines recognize the efficacy of oral administration.
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes.
- Time schedule: The animals were checked at least twice daily for any mortalities. The animals were observed at least twice daily for signs of reaction to treatment and/or symptoms of ill health.


DETAILED CLINICAL OBSERVATIONS: No


BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recordered daily, from day 0 until day 21 post coitum.
- Body weight gains from days 0-6 p.c., 6-9 p.c., 9-12 p.c., 12-16 p.c., 6-16 p.c., 16-21 p.c., and 6-21 p.c. were calculated.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes. Food consumption was recorded for the following periods: days 0-6, 6-9, 9-12, 12-16, and 16-21 post coitum.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food consumed per period/days per period: Yes.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No


POST-MORTEM EXAMINATIONS: Yes. Any female sacrificed or found dead during the study was subjected to gross macroscopic examination of all internal organs.
- Sacrifice on day # 21 post coitum by CO2 asphyxiation. Fetuses were removed by Caesarean section.
- Organs examined: Emphasis on the uterus and its contents, position of the fetuses in the uterus, and number of corpora lutea.
- Specimens of abnormal tissue were fixed in neutral phosphate buffered 4% formaldehyde solution.


Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes.
- Gravid uterus weight: Yes. (The uteri (and contents) of all females with live fetuses were weighed at necropsy on day 21 post coitum to enable the calculation of the corrected body weight gain).
- Number of corpora lutea: Yes.
- Number of implantations: Yes. If no implantation sites were evident, the uterus was placed in an aqueous solution of ammonium sulfide to accentuate possible haemorrhagic areas of implantation sites.
- Number of early resorptions: Yes.
- Number of late resorptions: Yes.
- Other: position of the fetuses was recordered.
Fetal examinations:
The fetuses were removed from the uterus, sexed, weighed individually, examnined for gross external abnormalities and allocated to one of the following procedures: Wilson's slicing technique or skeletal examination.

- External examinations: Yes: all per litter.
- Soft tissue examinations: Yes: half per litter. Wilson's slicing technique for examination of the viscera and brain. One half of the live fetuses from each litter were fixed in a mixture of ethyl alcohol, formol and acetic acid. After examination, the sections were preserved in a solution of ethyl alcohol and glycerine (one fetus/container).
- Skeletal examinations: Yes: half per litter. Fetuses were placed in a solution of potassium hydroxide for clearing and stained with alizarin red S (modified technique). The skeletons were examined and all abnormalities were recordered. The specimens were preserved individually in plastic bags.
- Head examinations: No data.
Statistics:
The following statistical methods were used to analyze body weights, food consumption, reproduction and skeletal examination data: means and standard deviations, univariate one-way analysis of variance, Dunnett-test, Steel-test, Fisher's Exact test for 2x2 tables. Individual values, means, standard deviations and t-statistics were rounded off before printing.
Indices:
N/A.
Historical control data:
N/A.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
In group 4 (1000 mg/kg), one female had bleeding from the vagina on days 15-16 post coitum. At scheduled Caesarean section, this female had one empty implantation site, only. No abnormal findings were noted in the dams of groups 1 (0 mg/kg), 2 (50 mg/kg), or 3 (250 mg/kg) with the exception of the dam in group 2 (50 mg/kg) mentioned above.
Mortality:
mortality observed, non-treatment-related
Description (incidence):
No test substance related deaths were noted. One female in group 2 (50 mg/kg), was found dead in the evening of day 10 post coitum. At first daily inspection in the morning, this female had ruffled fur, ataxia, tremor and bleeding from the vagina. This single finding in the low dose group was considered to be incidental.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
The differences in mean body weights between the control group and any dose group did not reveal any test substance related effect. In all groups the body weight gain and the corrected body weight gain (corrected for uterus weight) were similar. The dams of group 4 (1000 mg/kg), had statistically significantly increased mean body weights on day 5, between days 7-13 and on day 15 post coitum. These findings were considered to be a consequence of the slightly increased initial mean body weight: 213 g compared to 207 g in the control group, on day 0 post coitum.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Up to and including the highest dose level of 1000 mg/kg, food consumption of the dams was similar in all groups. No test substance related effects were noted.
Gross pathological findings:
no effects observed
Description (incidence and severity):
No test substance related abnormal findings were noted at terminal necropsy of the dams in any group. One female in group 2 (50 mg/kg) which was found dead on day 10 post coitum had reddish lungs with dark red foci, reduced right kidney and enlarged left kidney with nodules and calcus in pelvis (both kidneys with pelvic dilation) and in the urinary bladder dark red discoloration of the mucosa and urine with hemorrhagic contents were noted. No abnormal findings were noted in the dams of groups 1 (0 mg/kg), 3 (250 mg/kg), or 4 (1000 mg/kg).
Pre- and post-implantation loss:
effects observed, treatment-related
Description (incidence and severity):
Values for post-implantation loss were: 4.8 and 8.7 in groups 1 (0 mg/kg) and 4 (1000 mg/kg), respectively. The latter value attained statistical significance. Although the post-implantation losses were within the normal range of the historical control data of this strain of animals, two additional females (which were not included in the statistical analysis) had total postimplantation losses (i.e. implantation sites, only). Therefore, the increased post-implantation loss in group 4 (1000 mg/kg) was considered to be a slight effect of treatment with the test substance. In groups 2 (50 mg/kg) and 3 (250 mg/kg), none of the reproduction parameters as assessed by the mean number per dam of corpora lutea and implantation sites, pre- or post-implantation losses, and mean number of fetuses per dam were adversely affected by treatment with the test substance.
A slight (statistically non significant) decrease in the number of corpora lutea and a slight (statistically non significant) increase in pre-implantation losses were observed in the high dose group (17.4 %) as compared to the controls (12.8 %); however, this is not a substance-related effect, since exposure started at gestation day 6, the day of implantation.
Total litter losses by resorption:
effects observed, non-treatment-related
Description (incidence and severity):
At 1000 mg/kg, two females with total post-implantation losses were noted. One of the two females had bleeding from the vagina on days 15-16 post coitum. However these two animals had only two and one corpora lutea, respectively, and were obviously not fit for reproduction.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: maternal toxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: developmental toxicity
Abnormalities:
no effects observed
Fetal body weight changes:
no effects observed
Description (incidence and severity):
The mean fetal body weights were similar in all groups. No test substance related effects were evident.
Changes in sex ratio:
no effects observed
Description (incidence and severity):
In all groups, the sex ratios of the fetuses were not affected by the treatment with the test substance.
External malformations:
no effects observed
Description (incidence and severity):
No test substance related abnormal findings were noted. Neither the type nor the incidences of the abnormal findings listed below indicated test substance related effects: In each group 1 (0 mg/kg) and 3 (250 mg/kg), one fetus with reduced mouth and shortened and tapering lower jaw was noted. In group 2 (50 mg/kg), one runt (fetal weight < 2.5 g) was noted. In group 4 (1000 mg/kg), one fetus with several abnormal findings--generally oedematous, cranioschisis with excencephaly, protusion of the tongue, eventration and both eyes partially missing--was noted.
Skeletal malformations:
no effects observed
Description (incidence and severity):
The incidences of abnormal findings at skeletal examination were as following:
2/145 (=1%) fetuses in 2/25 litters in group 1 (0 mg/kg);
10/141 (=7%) fetuses in 6/24 litters in group 2 (50 mg/kg);
4/136 (=3%) fetuses in 4/25 litters in group 3 (250 mg/kg) and
4/116 (=3%) fetuses in 4/21 litters in group 4 (1000 mg/kg).
Mainly: wavy or fused ribs, abnormally or incompletely ossified sternebrae, dumbbell shaped or hemicentric thoracic vertebral body and in two fetuses in group 2 (50 mg/kg) fused os interparietale and occipitale were noted. Neither the type nor the incidences of these abnormal findings indicated test substance related effects. In all groups, the stage of the skeletal development of the fetuses was similar. The few statistically significant differences (on individual basis, only) between the control group and the dose group 4 (1000 mg/kg) in the ossification grade of the phalangeal nuclei did not indicate any test substance related effect. All values evaluated in this study were in the normal range of the historical control data of this strain of animals.
Visceral malformations:
no effects observed
Description (incidence and severity):
No test substance related abnormal findings were noted at visceral examination of the fetuses. In group 3 (250 mg/kg), in the fetus which had reduced mouth, shortened and tapering lower jaw at external examination a small palatoschisis was additionally observed at visceral examination. No abnormal findings were noted in groups 1 (0 mg/kg), 2 (50 mg/kg), or 4 (1000 mg/kg).
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
EXTERNAL EXAMINATION: No test substance related abnormal findings were noted. Neither the type nor the incidences of the abnormal findings listed below indicated test substance related effects: In each group 1 (0 mg/kg) and 3 (250 mg/kg), one fetus with reduced mouth and shortened and tapering lower jaw was noted. In group 2 (50 mg/kg), one runt (fetal weight < 2.5 g) was noted. In group 4 (1000 mg/kg), one fetus with several abnormal findings--generally oedematous, cranioschisis with excencephaly, protusion of the tongue, eventration and both eyes partially missing--was noted.

SEX RATIOS: In all groups, the sex ratios of the fetuses were not affected by the treatment with the test substance.

BODY WEIGHTS: The mean fetal body weights were similar in all groups. No test substance related effects were evident.

VISCERAL EXAMINATION BY WILSON TECHNIQUE: No test substance related abnormal findings were noted at visceral examination of the fetuses. In group 3 (250 mg/kg), in the fetus which had reduced mouth, shortened and tapering lower jaw at external examination a small palatoschisis was additionally observed at visceral examination. No abnormal findings were noted in groups 1 (0 mg/kg), 2 (50 mg/kg), or 4 (1000 mg/kg).

SKELETAL EXAMINATION: The incidences of abnormal findings at skeletal examination were as following:
2/145 (=1%) fetuses in 2/25 litters in group 1 (0 mg/kg);
10/141 (=7%) fetuses in 6/24 litters in group 2 (50 mg/kg);
4/136 (=3%) fetuses in 4/25 litters in group 3 (250 mg/kg) and
4/116 (=3%) fetuses in 4/21 litters in group 4 (1000 mg/kg).
Mainly: wavy or fused ribs, abnormally or incompletely ossified sternebrae, dumbbell shaped or hemicentric thoracic vertebral body and in two fetuses in group 2 (50 mg/kg) fused os interparietale and occipitale were noted. Neither the type nor the incidences of these abnormal findings indicated test substance related effects. In all groups, the stage of the skeletal development of the fetuses was similar. The few statistically significant differences (on individual basis, only) between the control group and the dose group 4 (1000 mg/kg) in the ossification grade of the phalangeal nuclei did not indicate any test substance related effect. All values evaluated in this study were in the normal range of the historical control data of this strain of animals.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: embryotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: fetotoxicity
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day (nominal)
Basis for effect level:
other: teratogenicity
Abnormalities:
no effects observed
Developmental effects observed:
no

 

 

 

Group 1

0 mg/kg bw/d

Group 2

50 mg/kg bw/d

Group 3

250 mg/kg bw/d

Group 4

1000 mg/kg bw/d

number of mated females

25

25

25

25

number of pregnant females

25

25

25

23

Number of mortalities

0

1

0

0

Number of dams with implantation sited only

0

0

0

2

Number of females with live foetuses at termination

25

24

25

21

Mean number of corpora lutea

SD

13.5

1.3

13.7

1.7

13.6

1.7

14.0

1.4

Pre-implantation loss, mean

SD

%of corpora lutea

Number of dams affected

1.7

1.6

12.8

19

1.6

2.1

11.6

15

2.2

2.2

15.9

19

2.4

2.1

17.4

18

Implantation sites, mean

SD

%of corpora lutea

11.8

2.2

87.2

12.1

2.5

88.4

11.4

2.7

84.1

11.5

2.4

82.6

Post-implantation loss, mean

SD

%of implantation sites

Number of dams affected

0.6

0.9

4.8

8

0.7

1.0

5.9

11

0.8

1.1

7.0

13

1.0

1.4

8.7 #

11

Implantation site scars

0

0

0

0

Embryonic/fetal deaths total

14

17

20

21

Embryonic resorptions, total

mean

SD

%of implantation sites

Number of dams affected

14

0.6

0.9

4.8

8

17

0.7

1.0

5.9

11

16

0.6

0.9

5.6

12

18

0.9

1.2

7.4

10

Fetal resorptions, total

mean

SD

%of implantation sites

Number of dams affected

0

0

4

0.2

0.8

1.4

1

3

0.1

0.4

1.2

3

Foetal data

Total foetuses 

280

273

265

221

%of implantation sites

95.2

94.1

93.0

91.3 #

Mean

11.2

11.4

10.6

10.5

SD

2.7

2.4

2.9

2.7

Live foetuses

280

273

265

221

Dead foetuses

0

0

0

0

Abnormal foetuses

1

1

1

1

% of foetuses

0.4

0.4

0.4

0.5

Sex of foetuses

Total males

139

137

118

94

% of fetuses

49.6

50.2

44.5

42.5

Weight of live foetuses, litter basis, total

N (litters)

25

24

25

21

Mean

4.9

4.9

5.0

4.8

SD

0.3

0.3

0.3

0.4

Weight of live foetuses, litter basis, males

N (litters)

25

24

25

21

Mean

5.0

5.1

5.1

5.0

SD

0.3

0.2

0.3

0.4

Weight of live foetuses, litter basis, females

N (litters)

25

24

25

21

Mean

4.8

4.8

4.9

4.7

SD

0.3

0.3

0.3

0.4

 

# Fisher's exact test, significant at level 5%

Conclusions:
NOAEL = 1000 mg/kg bw/day (general tolerability in the females and for the fetal organism).
NOAEL = 1000 mg/kg bw/day (maternal reproduction).
NOAEL = 1000 mg/kg/day (teratologic effects)
Executive summary:

In the developmental toxicity study (OECD guideline 414), groups of 25 mated female Wistar rats were treated with the test substance MDEA-Esterquat C16-18 and C18 unsatd. orally by gavage once daily from day 6 through 15 post coitum, at dose levels of 0, 50, 250 and 1000 mg/kg bw/day. Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

At 50, 250, and 1000 mg/kg, for the dams no test substance-related deaths or clinical signs as were noted as reaction to treatment. Up to and including the highest dose level of 1000 mg/kg, food consumption and body weight development of the dams were not affected by treatment with the test substance. At necropsy, no test substance-related abnormal findings in the dams were noted in any group.

A slight (statistically non significant) decrease in the number of corpora lutea and a slight (statistically non significant) increase in pre-implantation losses were observed in the high dose group (17.4 %) as compared to the controls (12.8 %); however, this is not a substance-related effect, since exposure started at gestation day 6, the day of implantation.

The slightly but statistically significant increased post-implantation losses at the high dose of (8.7 %) as compared to the controls (4.8 %) resulted in a slightly reduced portion of total fetuses per implantation site (91.3%) and in a reduced mean litter size (10.5 fetuses/litter) as compared to the controls (95.2 %; 11.2 fetuses/litter). The values were within the range of historical control values (3.9 % to 11.6 % for post-implantation losses and 10.2 to 12.2 fetuses/litter).

At 1000 mg/kg, two females with total post-implantation losses were noted. One of the two females had bleeding from the vagina on days 15-16 post coitum. However these two animals had only two and one corpora lutea, respectively, and were obviously not fit for reproduction. A small but significant increase in post-implantation losses was noted for the remaining females; however, the increase was within the historical data of the laboratory. These findings were considered by the authors to be a potentially effect of the test substance.

As the values were well within the range of the historical control values recorded at the same laboratory, it is likely that the effects observed are incidental and therefore not treatment related.

At 50 and 250 mg/kg, no test substance-related effects were noted on the maternal reproductive parameters, assessed by the mean number per dam of corpora lutea and implantation sites, pre- or post-implantation losses, and by the mean number of fetuses per dam.

Up to and including the highest dose level of 1000 mg/kg, no adverse effects on the fetal parameters were recorded. No external, skeletal or soft tissue malformations and no external variations were found. Mean fetal body weights and the sex ratios of the fetuses were comparable in all groups.

The maternal NOAEL is 1000 mg/kg bw/day. 

The developmental NOAEL is 1000 mg/kg bw/day.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat
Quality of whole database:
OECD guideline study, no deviations, GLP
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available
Additional information

In the developmental toxicity study, groups of 25 mated female Wistar rats were treated with the test substance orally by gavage once daily from day 6 through 15 post coitum, at dose levels of 0, 50, 250 and 1000 mg/kg bw/day. Females were sacrificed on day 21 post coitum and the fetuses were removed by Caesarean section.

At 50, 250, and 1000 mg/kg, for the dams no test substance-related deaths or clinical signs as were noted as reaction to treatment. Up to and including the highest dose level of 1000 mg/kg, food consumption and body weight development of the dams were not affected by treatment with the test substance. At necropsy, no test substance-related abnormal findings in the dams were noted in any group.

A slight (statistically non significant) decrease in the number of corpora lutea and a slight (statistically non significant) increase in pre-implantation losses were observed in the high dose group (17.4 %) as compared to the controls (12.8 %); however, this is not a substance-related effect, since exposure started at gestation day 6, the day of implantation.

The slightly but statistically significant increased post-implantation losses at the high dose of (8.7 %) as compared to the controls (4.8 %) resulted in a slightly reduced portion of total fetuses per implantation site (91.3%) and in a reduced mean litter size (10.5 fetuses/litter) as compared to the controls (95.2 %; 11.2 fetuses/litter). The values were within the range of historical control values (3.9 % to 11.6 % for post-implantation losses and 10.2 to 12.2 fetuses/litter).

At 1000 mg/kg, two females with total post-implantation losses were noted. One of the two females had bleeding from the vagina on days 15-16 post coitum. However these two animals had only two and one corpora lutea, respectively, and were obviously not fit for reproduction. A small but significant increase in post-implantation losses was noted for the remaining females; however, the increase was within the historical data of the laboratory. These findings were considered by the authors to be a potentially effect of the test substance. At 50 and 250 mg/kg, no test substance-related effects were noted on the maternal reproductive parameters, assessed by the mean number per dam of corpora lutea and implantation sites, pre- or post-implantation losses, and by the mean number of fetuses per dam.

Up to and including the highest dose level of 1000 mg/kg, no adverse effects on the fetal parameters were recorded. No external, skeletal or soft tissue malformations and no external variations were found. Mean fetal body weights and the sex ratios of the fetuses were comparable in all groups.

There are indications that the slightly increased incidences of post-implantation losses at 1000 mg/kg bw/day are due to some maternal (toxic) effects, which could not be further evaluated because this dose level has not been tested in the repeated dose toxicity studies.

The slightly increased post-implantation losses at the high dose compared to the controls could be caused by some maternal toxicity, incidentally and therefore not treatment relatedor through a direct toxic effect on the fetus. Since two females of the high dose had total post-implantation losses (implantation-sites only) and one of these females showed vaginal bleeding, this may indicate that the post-implantation losses are due to (some) maternal toxicity effects. As the values were well within the range of the historical control values recorded at the same laboratory, it is likely that the effects observed are incidental and therefore not treatment related.Since there was no effect on fetal body weight and no increased incidences of abnormal fetuses, it is assumed that the post-implantation losses are not due to a direct effect on the fetus.

The results of the prenatal developmental toxicity study do not indicate a substance-related effect on the fetus up to the limit dose of 1000 mg/kg bw/day. Therefore, the aspect of prenatal developmental toxicity is sufficiently covered.

There are no data gaps for effects on development. No human data are available. However, there is no reason to believe that these results from rat would not be applicable to humans.


Justification for classification or non-classification

Results of the prenatal developmental toxicity study do not indicate a substance-related effect on the fetus up to the limit dose of 1000 mg/kg bw/day. The slightly increased incidences of post-implantation losses at 1000 mg/kg bw/day are within the range of historical control data and might therefore be accidental and not treatment related. Vaginal bleeding observed in one animal with total post-implantation losses might be due to some maternal (toxic) effects which cannot be further evaluated. Therefore, the aspect of prenatal developmental toxicity is sufficiently covered and the substance does not require to be labelled for developmental toxicity.

Results of the extended one generation reproduction toxicity study do not indicate a substance-related effect to reproduction up to the limit dose of 1000 mg/kg bw/day

In conclusion, results of existing studies indicate that MDEA-Esterquat C16-18 and C18 unsatd.  does not need to be classified for effects on toxicity to reproduction according to GHS Regulation EC No 1272/2008 and therefore labelling is not necessary.

Additional information