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Toxicity to reproduction

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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
Cross-reference
Reason / purpose for cross-reference:
reference to other study
Reference
Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
22 November 2018 - 04 February 2019
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
adopted on 29 July 2016
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Justification for study design:
this study was performed as a dose range finding study for the OECD TG 443 study
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: 15-16weeks
- Weight at study initiation: males: 436-539 g, females: 249-381 g
- Housing:
From arrival to mating, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5x38x20 cm.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5x26.6x18.5 cm with a stainless steel mesh lid and floor.
After mating, the 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 42.5x26.6x18.5 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
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 42 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:
The required amount of test item was suspended in the vehicle. The preparations were made daily (concentrations of 10, 30 and 100 mg/mL). Concentrations were calculated and expressed in terms of test item as supplied.
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
- After 14 days of unsuccessful pairing replacement of first male by another male with proven fertility.
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged individually
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 100 mg/mL. Linearity, accuracy and precision were within the limits stated in RTC SOPs for suspensions (r > 0.98; accuracy 85-115%; precision CV < 10%).
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable and all results for all levels were within the acceptability limits stated in RTC SOPs for concentration (85-115%) and homogeneity (CV < 10%). In the same study, a 6 hour stability at room temperature was verified in the range from 10 to 100 mg/mL.
In the present study, samples of the preparations prepared on two occasions during the study (Week 1 and again towards the end of the study) were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in RTC SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
Males: 2 consecutive weeks prior to pairing and thereafter through the day before necropsy (Days 35 to 37), for a total of 34-36 days.
Females: 2 weeks prior to pairing, and thereafter during pairing, post coitum and post partum periods until Day 13 post partum (for at least 51 days). One not pregnant female (no. X1070045 - Group 3) was dosed up to Day 25 post coitum, the day before necropsy.
Frequency of treatment:
daily
Details on study schedule:
screening study
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:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected in consultation with the Sponsor based on information from preliminary studies.
Positive control:
n.a.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: early in each working day in the morning and 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

BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed weekly from allocation to termination. Females were weighed weekly from allocation to positive identification of mating and on Days 0, 7, 14 and 20 post coitum. Dams were also weighed on Days 1, 4, 7, 13 post partum and just before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
The weight of food consumed by each cage of males and females was recorded weekly during the pre-mating period starting from Day 1 of dosing up to mating. Individual food consumption for mated females was measured on gestation Days 7, 14 and 20 starting from Day 0 post coitum and on lactation Days 7 and 13 post partum starting from Day 1 post partum.

WATER CONSUMPTION AND COMPOUND INTAKE: No

OTHER:
Blood collection for detection of test material - Dams and pups
Dams
On Day 18 post coitum and Day 13 post partum, blood samples (approximately 0.3 mL) were taken from the tail vein of 5 females/group at 1 time point (1 hour post-dose).
Pups
On Day 13 post partum, blood samples (approximately 0.3 mL) were taken from 1 pup/sex/litter from 5 selected dams of each group (5 pups/sex/group). Blood samples were withdrawn at necropsy under light ether anaesthesia from the heart (intracardiac puncture). Blood samples were collected into tubes containing Li-heparin anticoagulant and frozen at -80°C pending analysis. Analysis of the samples to determine the concentration of the test item was carried out by Analytical Chemistry Department of RTC according to a validated bioanalytical method (RTC Study No. A3404).

Blood collection for thyroid hormone determination (T3, T4 and TSH)
The timing of the blood collection for thyroid hormone determination was as close as possible between animals. It was within a time window of 15-20 minutes for males and approximately 1-3 hours for females and pups and it was at the same time of the day in case of sampling on different days.
Males at termination
Blood samples (approximately 0.8 mL) were collected under isoflurane anaesthesia from the retro-orbital sinus. The order of collection was equalised between groups.
Females
On Day 14 post partum, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal vena cava. The order of collection was equalised between groups.

Oestrous cyclicity (parental animals):
Before allocation
All females ordered for the study (a total of 57) were evaluated pre-exposure for oestrous cyclicity and animals that exhibited anomalies in the oestrous cycle were not allocated to the study. Oestrous cycle was monitored by vaginal smears for at least 2 weeks before allocation. These data are not tabulated in this report, but will be archived with all raw data.
After allocation
Vaginal smears were taken in the morning from Day 1 of dosing 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)
Before despatch to necropsy
Vaginal smears were also taken from all females before despatch to necropsy and the oestrous cycle phase recorded.
Litter observations:
STANDARDISATION OF LITTERS
On Day 4 post partum, the size of each litter was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter. Partial adjustment (for example, 5 males and 3 females) was acceptable. At least one culled male and one culled female were selected for blood collection for thyroid hormone determination.

PARAMETERS EXAMINED
As soon as possible after parturition was considered complete (Day 0 post partum), all pups (live and dead) were counted, sexed and live pups were identified. Live pups were individually weighed on Days 1, 4 and 13 post partum.
Observation was performed once daily for all litters from Day 0 post partum. Pups found dead at birth were examined at necropsy (external and internal examination). Pups dying during the lactation period were also weighed before the despatch to necropsy. After culling all pups were sacrificed with the dams on Day 13 or 14 post partum.

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was/was not determined for pups born or found dead

Anogenital distance (AGD)
The AGD of each pup was measured on Day 1 post partum. The AGD was normalized to the cube root of body weight collected on Day 1 post partum.

Nipple count
On Day 13 post partum, the presence of nipples/areolae in each male pup was checked.

Blood collection for thyroid hormone determination (T3, T4 and TSH)
On Day 4 post partum and on Day 13 or 14 post partum, as part of the necropsy procedure, blood samples (approximately 0.2 mL and 0.5 mL, respectively) were taken from 2 pups (1 male and 1 female, where possible) (see section 4.3.9). Blood samples were withdrawn under light ether anaesthesia fromthe heart (intracardiac puncture). The order of collection was equalised between groups.
Postmortem examinations (parental animals):
SACRIFICE
- The maleswere killed after the mating of all females after a total of 34 or 36 days of treatment.
- The females with live pups were killed on Day 14 post partum. The female which did not give birth was killed on Day 26 post coitum.

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

All females were examined also for the following:
– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).
Uteri of females with no visible implantations were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

HISTOPATHOLOGY / ORGAN WEIGHTS
Tissue processing, see section "Any other information on materials and methods"
Postmortem examinations (offspring):
SACRIFICE
Pups that had completed the scheduled test period (Day 4, Day 13 or Day 14 post partum) were euthanised by intraperitoneal injection of Thiopenthal.

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera
All pups found dead in the cage were examined for external and internal abnormalities.
All culled pups sacrificed on Day 4 post partum were subjected to an external examination.
Sex was determined by internal gonads inspection.
All live pups sacrificed on Day 13 or 14 post partum were killed and examined for external abnormalities and sex confirmation by gonadal inspection.
All pups with abnormalities were retained in 10% neutral buffered formalin.

Nipple retention at Day 14 post partum
No nipples were found in pups to be retained on Day 14 post partum. Data were not tabulated, but will be archived with all raw data.

HISTOPATHOLOGY / ORGAN WEIGTHS
Pups at Day 13 or 14 post partum
Thyroid was weighed from one male and one female pups from each litter and preserved in 10% neutral buffered formalin for possible histopathological examination. The thyroid weight was determined after fixation.
Statistics:
Standard deviations were calculated as appropriate. For continuous variables, such as body weight, food consumption, hormone determination and organ weight, 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. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters and for the evaluation of the hormone thyroid data in pups. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The mean values, standard deviations and statistical analysiswere calculated fromactual values in the computer without rounding off. Statistical analysis of histopathological findings was carried out by means of the nonparametric Kolmogorov-Smirnov test if ‘n’ was more than 5. The criterion for statistical significance was p<0.05.
Reproductive indices:
males: Copulatory Index, Fertility Index
females: Copulatory Index, Fertility Index
Precoital Interval
Offspring viability indices:
Pre-implantation loss, Pre-natal loss on Day 0 post partum, before culling, Post-natal loss at Day 0 post partum, Post-natal loss on Day 4 post partum, before culling, Post-natal loss on Day 13 post partum (after culling)
Clinical signs:
no effects observed
Description (incidence and severity):
No relevant clinical signs were observed throughout the study in treated animals of both sexes.
Salivation, recorded in high dose males for a few days during treatment, was not considered of toxicological relevance.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred in the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
No changes of toxicological relevance in body weight and body weight gain were observed between treated and control animals of both sexes, during the study.
The statistically significant decrease recorded in body weight gain of mid-dose males on Day 8 of treatment and high dose males on Day 15 of treatment was not considered of toxicological relevance, since it was transient and not dose-related.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No effects on food consumption were recorded in both sexes during the study.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
No treatment-related changes were noted.
The sporadic lesions were considered to be an expression of spontaneous and/or incidental pathology seen in this species.
Histopathological findings: neoplastic:
not examined
Other effects:
no effects observed
Description (incidence and severity):
Thyroid hormones
Parental males: No changes were recorded.
Reproductive function: oestrous cycle:
no effects observed
Description (incidence and severity):
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated animals and control group was comparable.
Reproductive function: sperm measures:
no effects observed
Description (incidence and severity):
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:
no effects observed
Description (incidence and severity):
Pre-coital interval, copulatory index and fertility index did not show any treatment-related intergroup differences.
No statistically significant differences were observed for these parameters between treated and control groups, although a slight increase in prenatal loss was observed in the high dose group. Considering that it did not lead to a reduction in litter size and no other parameters were involved, this finding was not considered of toxicological relevance. All pregnant dams gave birth between Days 21 and 23 post coitum.
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 adverse effects observed
Critical effects observed:
no
Clinical signs:
no effects observed
Description (incidence and severity):
Pre-weaning clinical signs were comparable between treated and control groups. Small and pale appearance was the main clinical sign noted in control and treated pups. In addition, found dead and/or missing pups, frequently noted in this type of experimental design, were observed both in control and treated groups, with similar incidence.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
no mortality observed
Description (incidence and severity):
No significant 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 and 13 post partum.
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
effects observed, non-treatment-related
Description (incidence and severity):
In male pups, no differences in the anogenital distance (AGD), performed on Day 1 post partum, were seen between control and treated groups and the mean values were within the range of RTC historical control data. On the contrary, in female pups slight statistically significant decreases in anogenital distance measured on Day 1 post partum (up to 6%) were seen in all treated groups, when compared to controls. However, the above result was
not considered of toxicological relevance, as all values were within RTC historical control data. In addition, considering that the AGD reflects the prenatal androgenic exposure (values physiologically higher in males than in females), the decrease observed in females represents a positive trend (feminisation as reduction in AGD of females) and not an adverse effect (i.e. masculinisation as increase in AGD of females).
Nipple retention in male pups:
no effects observed
Description (incidence and severity):
No nipples were observed in male pups.
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No significant differences were noted in thyroid weight between controls and pups of treated groups.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 13 or 14 post partum did not reveal any treatment-related effect.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
Thyroid hormones
Pups - Day 14 post partum: An increase of thriiodothyronine (T3) was recorded in male pups of Groups 3 and 4 (51% and 69% above controls, respectively), being statistically significant only in Group 3. This change involved only few animals (pup nos. X1070049 8/M, X1070051 1/Mand X1070059 1/Mof Group 3, X1070065 3/Mand X1070073 1/Mof Group 4), and values were outside the range of RTC historical data. However, since two control pups (X1070093 and X1070010 10/F) showed thriiodothyronine value outside the same range, and no related findings of the other hormones were observed in the above mentioned animals, the above increase was not considered to be of toxicological significance.
Dose descriptor:
NOAEL
Generation:
F1
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
Conclusions:
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.
Executive summary:

The toxic effects on Sprague Dawley rats of both sexes were investigated after repeated dosing with MDEA-Esterquat C16-18 and C18 unsatd. 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.

All doses (0, 100, 300 and 1000 mg/kg/day) were administered orally, by gavage. The control group received water softened by reverse osmosis.

Males were treated for 2 weeks prior to pairing, and thereafter during pairing with females until the day before necropsy, for a total of 34 or 36 days.

Pregnant females were treated for 2 weeks prior to pairing, and thereafter during pairing,post coitumandpost partumperiods until Day 13post partum, for at least 51 days. One not pregnant female was dosed up to Day 25post coitum, the day before necropsy.

The following investigations were performed: mortality check, clinical signs, body weight, food consumption,detection of test item in blood and milk(5 females/group), oestrous cycle, mating performance, litter data, sex ratios, macroscopic observations and organ weights.

Histopathological examination was performed on control and high dose groups, on one low dose male thatdid not mate, on onemid-dose male that failed to induce pregnancy and on all abnormalities detected duringpost partumobservation at the end of treatment period. The identification of the stages of the spermatogenic cycle was also performed in all males of the control and high dose groups.

Thyroid hormone determination was performed in all parental males and, together with the Thyroid weight, also in 1 pup/sex/litter (where possible) at Day 13 or 14post partum. Clinical signs and anogenital distance were recorded in all pups. The presence of nipples/areolae was verified in male pups. All culled pups sacrificed at Day 4post partumand all live pups sacrificed at Day 13 or 14post partumwere examined for external abnormalities and sex was confirmed by gonadal inspection.

All pups found dead in the cage were examined for external and internal abnormalities.

 

Mortality and fate of females

No mortality occurred in the study. One female in the mid-dose group was found not pregnant at necropsy. The total number of females with live pups at Day 14post partumwas 10 each in the control, low and high dose groups, and 9 in the mid-dose group.

 

Clinical signs

No significant clinical signs were observed in both sexes during the study.

 

Body weight and body weight gain

No changes of toxicological relevance in body weight and body weight gainbetween treated and control animals of both sexes were observed during the study.

 

Food consumption

No effects on food consumption were recorded in both sexes during the study.

 

Blood detection of test item

Exposure to the test item (analytically followed as the product of physiological hydrolysis, denominated Core 134) was demonstrated in Dams on Day 18post coitumand Day 13post partumat 1 hour post-dose and in pups during lactation, on Day 13post partum. The exposure increased with the dose, although not in proportional manner, and with the duration of treatment.

 

Thyroid hormones

Parental males

No changes were recorded.

 

Pups - Day 14post partum

An increase of thriiodothyronine (T3) was recorded in male pups of Groups 3 and 4 (51% and 69% above controls, respectively), being statistically significant only in Group 3. This change involved only few animals and values were outside the range of RTC historical data. However, since two control pups showed thriiodothyronine (T3) value outside the same range and no related findings of other hormones were observed in the above mentioned animals, the above increase was not considered to be of toxicological significance.

 

Oestrous cycle, reproductive parameters, pairing combination and mating performance

Oestrous cycle, pre-coital interval, copulatory index and fertility index did not show any treatment-related intergroup differences.

 

Implantation sites, pre-implantation loss data, pre-natal loss data and gestation length of females

No significant differences were observed for these parameters between the treated groups and the controls. All pregnant dams gave birth between Days 21 and 23post coitum.

 

Litter data at birth, on Days 1 and 4post partum(before culling) and on Day 13post partum(after culling) and sex ratio of pups

No significant 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 and 13post partum. Sex ratio did not show any significant differences between groups.

 

Clinical signs of pups and nipple observation

Pre-weaning clinical signs were comparable between treated and control groups. In addition, found dead and/or missing pups, frequently noted in this type of experimental design, were observed both in control and treated groups, with similar incidence.

No nipples were found in male pups on Day 13post partum.

 

Anogenital distance

In male pups, no differences in the anogenital distance (AGD), performed on Day 1post partum, were seen between control and treated groups.

The slight statistically significant decrease in anogenital distance noted in female pups of all treated groups was not considered of toxicological relevance, as all values were within the range of RTC historical control data. In addition, considering that the AGD reflects the prenatal androgenic exposure (values physiologically higher in males than in females), the decrease observed in females represents a positive trend (feminisation as reduction in AGD of females) and not an adverse effect (i.e. masculinisation as increase in AGD of females).

 

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 13 or 14post partum

Necropsy findings in decedent pups and in pups sacrificed on Days 4 and 13 or 14post partumdid not reveal any treatment-related effect.

 

Pups thyroid weight on Day 14post partum

No relevant differences were observed in the weight of thyroid of treated pups, when compared to controls.

 

Terminal body weight and organ weights

No relevant changes were observed on terminal body weight, absolute and relative weight of treated animals, when compared to the controls.

 

Macroscopic observations

No remarkable differences were noted atpost mortemexamination in treated animals, when compared to controls.

 

Microscopic observations

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. No treatment-related changes were observed in treated animals, when compared to the

controls.

 

Conclusion

In conclusion, no signs of treatment-related toxicity were observed following treatment with the test item, when administered to rats by oral route at dose levels of 100, 300 and 1000mg/kg/day at any of the dose levels investigated. Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general, reproductive and developmental toxicity was considered to be 1000mg/kg/day for males and females.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2020
Report date:
2020

Materials and methods

Test guideline
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.



Test material

Constituent 1
Reference substance name:
Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides
Cas Number:
1079184-43-2
Molecular formula:
n.a. (UVCB)
IUPAC Name:
Ethanaminium, 2-hydroxy-N-(2-hydroxyethyl)-N,N-dimethyl-, esters with C16-18 and C18-unsatd. fatty acids, chlorides
Test material form:
solid: particulate/powder

Test animals

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

Administration / exposure

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


Doses / concentrationsopen allclose all
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.

Examinations

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

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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 / performance (P0)

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.

Details on results (P0)

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.

Effect levels (P0)

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

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

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.

Developmental neurotoxicity (F1)

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 (F1)

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.

Effect levels (F1)

open allclose all
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

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

The results are attached as pdf files below.

 

Applicant's summary and conclusion

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.