Amines, N-C16-22-alkyltrimethylenedi-

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2022

Report date:

2022

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

Amines, N-C16-22-alkyltrimethylenedi-

EC Number:

292-564-1

EC Name:

Amines, N-C16-22-alkyltrimethylenedi-

Cas Number:

90640-45-2

Molecular formula:

R-NH-(CH2)3-NH2 R = alkyl mainly C16-22-(even numbered)-alkyl

IUPAC Name:

Amines, N-C16-22-alkyltrimethylenedi-

Test material form:

solid

Details on test material:

Chemical registery number : 292-564-1
Chemical name : Amines, N-C16-22-alkyltrimethylenedi-

Based on the qualitative and quantitative information on the composition, the sample used are representative of the boundary composition shared and agree by each registrant.

Test animals

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

The animals were housed in a limited access rodent facility. Animal room controls were set to maintain temperature and relative humidity at 22°C 2°C and 55% 15%, respectively; actual conditions were monitored, recorded and the records retained. No relevant deviations from these ranges were recorded during the study. There were approximately 15 to 20 air changes per hour and the rooms were lit by artificial light for 12 hours each day.
From arrival to pairing, animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 38 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least twice a week.
During mating, animals were housed one male to one female in clear polysulfone cages measuring 42.5 26.6 18.5 cm with a stainless steel mesh lid and floor (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Each cage tray held absorbent material which was inspected and changed daily.
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 42.5 26.6 18.5 cm). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
Recovery animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5 38 20 cm (Tecniplast Gazzada S.a.r.l., Buguggiate, Varese). Nesting material was provided inside suitable bedding bags and changed at least 2 times a week.
Drinking water was supplied ad libitum to each cage via water bottles.
A commercially available laboratory rodent diet (4 RF 21, Mucedola S.r.l., Via G. Galilei, 4, 20019 Settimo Milanese (MI), Italy) was offered ad libitum.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on exposure:

All doses were administered at a constant volume of 5 mL/kg body weight.

Details on mating procedure:

Mating was monogamous (one male to one female). Vaginal smears were taken from the day after the start of pairing until positive identification of copulation (sperm identification, vaginal plug in situ or copulation plugs found in the cage tray).
The female was paired with the same male until positive identification of copulation occurred.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis was performed in a separate study in order to validate both the analytical method and the preparation procedure and to verify the stability of the preparations (ERBC Study No. A3848).



The analytical method was validated in the range from 1 to 15 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 80-120%; precision CV <10%). In the same study, 4-hour stability at 37°C was verified in the range from 1 to 15 mg/mL. The proposed preparation procedure for the test item was checked in the range from 1 to 15 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was suitable.
In the present study, samples of the preparations made on two occasions during the study (Day 1 and again towards the end of the study) were analysed to check the homogeneity and concentration. Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. The software used for this activity was Analyst 1.6.2 (ABSciex).
Results of the analyses were within the acceptability limits stated in ERBC validation pro- tocol (80-120% for concentration and CV <10% for homogeneity) and are presented in Addendum 3.

Duration of treatment / exposure:

Main groups (Groups 1 to 4)
Males
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing, through the pairing period and thereafter until the day before necropsy (Days 30 and 31). Thus, males were treated for a total of 29 or 30 days.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight.

Females
Animals were dosed once a day, 7 days a week, for 2 consecutive weeks prior to pairing and thereafter during pairing, post coitum and post partum periods until Day 13 post partum or the day before sacrifice (for at least 51 days). Non-pregnant females (nos. X14000053 - Group 3 and X1400071 - Group 4) and one female that lost the litter (no. X1400059 - Group
3) were dosed up to the day before necropsy.
Dose volumes were adjusted once per week for each animal according to the last recorded body weight up to mating. During the gestation period, dose volumes were calculated according to individual body weight on Days 0, 7, 14 and 20 post coitum and on Days 1, 4, 7 and 13 post partum.



Recovery groups (Groups 5 and 6)

Males: animals were dosed once a day, 7 days a week, for up to 4 consecutive weeks (total of 28 days).
Females: animals were dosed once a day, 7 days a week, for up to 8 consecutive weeks (total of 56 days).
Dose volumes were calculated once per week for each animal according to the last recorded body weight.
No treatment was given during the recovery period (14 days).

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

0 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 4

Dose / conc.:

75 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups: 10 animals/sex/group
Recovery groups: 5 animals/sex/group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on 14-day dose range finding study

Positive control:

Not included

Examinations

Parental animals: Observations and examinations:

Full records were maintained for all measurements and observations.

Mortality
Throughout the study, all animals were checked 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.

Clinical signs
Before commencement of treatment and at least once daily during the study, each animal was observed and any clinical signs were recorded. Observations were performed at the same time interval each day, the interval was selected taking into consideration the presence of post-dose reactions (2-2.5 hour after treatment).
The observations performed before commencement of treatment are not tabulated in this report but will be archived together with all other raw data.

Functional Observation Battery Tests
Once before commencement of treatment and at least once a week from the start of treat- ment until termination, each animal was given a detailed clinical examination. Each animal was removed from the home cage and observed in an open arena. The tests included ob- servation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous sys- tem (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also recorded. All observations were recorded for individual animals.
Animals were examined in an open arena for a minimum of three minutes.

Body weight
Main groups
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.
Recovery groups
Each animal was weighed on the day of allocation to treatment groups, on the day that treatment commenced, weekly thereafter and just prior to necropsy. Body weights of recovery animals on the day of allocation are not tabulated in this report, but will be archived with all raw data.

Food consumption
Main groups
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. Individual food consumption for the females was measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.
Recovery groups
The weight of food consumed by each cage of rats was recorded at weekly intervals starting from Day 1 of dosing.

Blood samples for haematology, clinical chemistry and coagulation were collected, at the end of the treatment period, by random selection from 5 males and 5 females (females with viable litters) of each main group. Further blood samples for haematology, clinical chemistry and coagulation were taken under identical conditions at the end of the recovery period.

Blood sampling
Males
Blood samples for haematological investigations, biochemical tests and hormone determination were collected in condition of food deprivation under isoflurane anaesthesia from the retro-orbital sinus. Blood samples for coagulation test (food available) were collected at necropsy from the vena cava under isoflurane anaesthesia. The order of collection was equalised between groups.
Females
As a part of the sacrificial procedure, blood samples for all determinations were withdrawn from the abdominal vena cava in condition of food deprivation under isoflurane anaes- thesia. The order of collection was equalised between groups.

Urinalysis
Individual overnight urine samples were collected during the last week of treatment from the same male animals selected for the clinical pathology investigations and under the same conditions. Further urine samples were taken under identical conditions at the end of the recovery period.
Before starting urine collection, water bottles were removed from each cage and each an- imal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis

Oestrous cyclicity (parental animals):

Stock females
Oestrous cycle was monitored by vaginal smears in all stock females for 2 weeks before allocation in order to exclude from the study females with irregular cycle. These data are not tabulated in this report, but will be archived with all raw data.

Females allocated to groups
Vaginal smears were taken in the morning from Day 1 of treatment, up to positive identific- ation of mating including not less than 2 weeks before the pairing.

The vaginal smear data were examined to determine the following:
– anomalies of the oestrous cycle;
– pre-coital interval (i.e., the number of nights paired prior to the detection of mating).
Vaginal smears were also taken from all females (both for main and recovery females), before despatch to necropsy.

Sperm parameters (parental animals):

The morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed.

Litter observations:

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.
Pups dying during the lactation period were weighed before the despatch to necropsy. Observations were performed once daily for all litters.
After culling, all pups were sacrificed with the dams on Day 14 post partum.

The anogenital distance (AGD) of each pup was measured on Day 1 post partum. The measure of AGD was normalized to the cube root of the pup’s body weight measured on Day 1 post partum.

The presence of nipples/areolae in male pups was checked on Day 13 post partum (a day before despatch to necropsy).

Postmortem examinations (parental animals):

The clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.

Females
Parental females were examined also for the following:

– number of visible implantation sites (pregnant animals);
– number of corpora lutea (pregnant animals).

Uteri of apparently non-pregnant females were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.

From all animals completing the scheduled test period, the selected organs were dissected free of fat and weighed. For animal no. X1400090 (Group 5), the weights of epididymides (total, left and right) were collected after fixation due to an oversight.
The ratios of organ weight to body weight were calculated for each animal.

Postmortem examinations (offspring):

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 14 post partum were killed and examined for external abnor- malities and sex confirmation by gonadal inspection.
Pups with abnormalities were retained in 10% neutral buffered formalin.

Thyroids were weighed from one male and one female pup selected for blood collection for hormones determination and preserved in 10% neutral buffered formalin.
The thyroid weights were determined after fixation.

Statistics:

Standard deviations were calculated as appropriate. For variables, e.g. body weight, food consumption, clinical pathology parameters and organ weights, 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.
The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non- parametric version of the Williams test. 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.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Males
No clinical signs were observed in low dose animals during the study. Salivation was occasionally observed in mid-dose males and high dose males. Piloerection was observed in one occasion only in one male of the high dose group.

Females
No clinical signs were observed in all treated females before pairing.
During mating period, no clinical signs were observed in females of the low and mid-dose. In three females of the high dose group, salivation was observed once during mating period. In the same occasion, two of these animals showed also cyphosis. Also brown staining on the muzzle or hairloss were noted.
During the post coitum period, no clinical signs were observed in females of the low and mid- dose. Clinical signs observed in one female of the mid-dose such as cyphosis, piloerection, pale appearance and red staining of the vagina was considered related to the difficulty in the delivery. In fact, these animals lost the litter on the same day. Three out of 10 females of the high dose group, showed cyphosis and piloerection for several days during the gestation. One of these animals, showed also rales for a few days.
During the post partum period, no treatment-related clinical signs were observed in all groups.

Recovery groups
In only two treated females of the recovery groups, treatment-related clinical signs such as rales were observed towards the end of treatment period and also during the two weeks of the recovery phase. In addition, cyphosis and/or piloerection and/or swollen abdomen were also observed in one female in the same period.
During the treatment or the recovery phase, no clinical signs were observed in male animals of the recovery groups.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred throughout the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Main groups
A slight decrease in body weight was recorded in high dose males and females starting on Week 2 of treatment up to the end of the study. The decrease in body weight was observed also in mid-dose females on lactation Days 1 and 7 only.



Reduction in body weight gain was seen in all treated males starting from Week 2 of treat- ment up to the end of the study. At the end of the study, no differences were seen between the treated and the control groups.
Body weight gain of treated females was comparable to controls.

Recovery groups
The trend of body weight and body weight gain of recovery animals was similar to the main groups animals. A complete recovery was observed during the treatment free period.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Main groups
No relevant changes were seen in food consumption during the treatment period in males. A reduction in food consumption was noted in mid- and high dose females at the end of the gestation and lactation periods.

Recovery groups
In recovery animals no relevant changes were noted in food consumption during the treatment or recovery period.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematology
Dosing phase - Main groups
Treatment-related changes were represented by a dose-related increase of leucocytes, mostly due to neutrophils increment, recorded in almost all treated animals. This could be due to the stress condition of the animals, as confirmed by the findings observed in thymus and spleen at histopathology.

Recovery phase - Recovery groups
In males, neutrophils showed complete reversibility. Lymphocytes were slightly decreased. In females, neutrophils showed only a partial reversibility, being still higher than controls.

Coagulation

Dosing and recovery phases
No treatment-related changes were recorded in prothrombin time measured during the study.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No treatment-related changes were found at the clinical chemistry investigation performed in the study.

Endocrine findings:

no effects observed

Description (incidence and severity):

No differences between control and treated animals of both sexes were recorded in thyroid hormones determination.

Urinalysis findings:

no effects observed

Description (incidence and severity):

No relevant changes were observed in urine analysis performed in males at the end of the treatment period.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

No relevant alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examinations performed during the study.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Final sacrifice
At the end of the treatment period, treatment-related microscopic observations considered to be associated with the oral administration of Dinoram 42 were present in the spleen, mesenteric lymph node, small intestine (duodenum and/or jejunum and/or ileum) in a set of treated animals of both sexes dosed with 75 (high dose), 50 (mid-dose) and 25 (low dose) mg/kg/day.

Recovery sacrifice
After 2 weeks of recovery period, histopathological changes were still present in the spleen, mesenteric lymph node and small intestine (duodenum and/or jejunum and/or ileum) of high treated animals of both sexes (5 males and 5 females) previously treated with the high dose (75 mg/kg/day), exhibiting similar incidence and severity when compared to the rats given 75 mg/kg/day sacrificed at term.

Other effects:

effects observed, treatment-related

Description (incidence and severity):

The findings observed in thymus and spleen were considered at least partially secondary to the stress induced by the small intestine and mesenteric nodes lesions and therefore not directly related to treatment.

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Description (incidence and severity):

Oestrous cycle, fertility index, reproductive parameters and mating performance were similar between the treated groups and control animals.

Reproductive function: sperm measures:

no effects observed

Description (incidence and severity):

Regular layering in the germinal epithelium was noted in the seminiferous tubules.

Reproductive performance:

no effects observed

Description (incidence and severity):

Oestrous cycle, fertility index, reproductive parameters and mating performance were similar between the treated groups and control animals.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Description (incidence and severity):

Clinical signs in pups, did not reveal any treatment-related effects

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Litter data of treated females (e.g. total litter size, live litter size, pup loss, litter weight and mean pup weight) recorded at birth and on Days 1, 4 and 13 post partum did not show treatment-related effects.

Sexual maturation:

no effects observed

Anogenital distance (AGD):

no effects observed

Description (incidence and severity):

No treatment-related effects were observed in anogenital distance measured in male and female pups of the treated groups.

Nipple retention in male pups:

no effects observed

Description (incidence and severity):

No nipples were found in male pups at Day 13 of lactation

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

No significant differences were noted in thyroid weight between control and pups of treated groups.

Gross pathological findings:

no effects observed

Description (incidence and severity):

No findings noted at necropsy were considered related to treatment

Other effects:

no effects observed

Description (incidence and severity):

At Days 4 and 14 post partum, no differences or treatment-related changes were recorded in thyroid hormones.

Effect levels (F1)

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

sexual maturation

clinical signs

mortality

Overall reproductive toxicity

Reproductive effects observed:

no

Applicant's summary and conclusion

Conclusions:

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 75 mg/kg/day for both males and females parental animals and pups.

Executive summary:

The toxic effects on rats after repeated oral dosing with the registered substance, as well as any effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and early lactation of the offspring were investigated. A 2 week treatment-free period was included in order to assess any delayed toxicity or recovery from any adverse effects observed during the dosing phase.
The vehicle was corn oil. All doses were administered at a constant volume of 5 mL/kg body weight.

Main groups
Males were treated for 2 weeks prior to pairing until the day before necropsy, for a total of 29/30 days.
Females were treated for 2 weeks prior to pairing, during pairing, post coitum and postpartum periods until Day 13 post-partum (for at least 51 days).
The non-pregnant females and one that lost its litter were dosed up to the day before necropsy.
The following investigations were performed: mortality, clinical signs (including neuro- toxicity assessment, motor activity and sensory reactivity to stimuli), body weight, food consumption, oestrous cycle, mating performance, litter data, sex ratios, measurement of anogenital distance, clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis for males only), blood collection for hormone assay (adult animals and pups), macroscopic observations and organ weights. Vaginal smears in all adult female groups were investigated at termination.

Histopathological examination was performed in the first instance only on control and high dose groups (5 animals/sex/group). Since histopathological changes in the small intestine, mesenteric lymph nodes, spleen and thymus (females only) were observed in high dose animals, the examination of the above listed organs was extended to 5 animals/group/sex for low, intermediate and recovery groups.
The identification of the stages of the spermatogenic cycle was also performed in five randomly selected males of the control and high dose groups.
Thyroid hormone determination was performed in all parental animals.
Clinical signs were performed in all pups. Thyroid weight and thyroid hormone determination of each sex/litter (where possible) at Day 4 and 14 post partum were determined. All pups found dead in the cage were examined for external and internal abnormalities. All culled pups sacrificed at Day 4 post partum and those sacrificed on Day 14 post partum were subjected to an external examination and the sex was determined by internal gonads inspection.

Recovery groups
Recovery males were treated for up to 4 consecutive weeks and killed after 2 weeks of recovery (treatment free) period.
Recovery females were treated for up to 8 weeks. The females were sacrificed after 2 weeks of recovery (treatment free) period.
The following parameters were evaluated in these animals: mortality, clinical signs (including neurotoxicity assessment, motor activity, grip strength and sensory reactivity to stimuli), body weight, food consumption, clinical pathology investigations (haematology comprising coagulation, clinical chemistry and urinalysis for males only), macroscopic and microscopic observations and organ weights.

Mortality and fate of females

Mortality
No mortality occurred throughout the study.

Fate of females
One mid-dose female and one high dose female were not pregnant at necropsy. One mid-dose female lost its litter on Day 0 post partum.
The number of females with live pups on Day 14 post partum was: 10 in the control, 10 in the low dose, 8 in the mid-dose and 9 in the high dose group.

Clinical signs

Main groups Males
No clinical signs were observed in low dose animals during the study. Salivation was occasionally observed in mid-dose males and high dose males. Piloerection was observed in one occasion only in one male of the high dose group.

Females
No clinical signs were observed in all treated females before pairing.
During mating period, no clinical signs were observed in females of the low and mid-dose. In three females of the high dose group, salivation was observed once during mating period. In the same occasion, two of these animals showed also cyphosis. Also brown staining on the muzzle or hairloss were noted.
During the post coitum period, no clinical signs were observed in females of the low and mid- dose. Clinical signs observed in one female of the mid-dose such as cyphosis, piloerection, pale appearance and red staining of the vagina was considered related to the difficulty in the delivery. In fact, these animals lost the litter on the same day. Three out of 10 females of the high dose group, showed cyphosis and piloerection for several days during the gestation. One of these animals, showed also rales for a few days.
During the post partum period, no treatment-related clinical signs were observed in all groups.

Recovery groups
In only two treated females of the recovery groups, treatment-related clinical signs such as rales were observed towards the end of treatment period and also during the two weeks of the recovery phase. In addition, cyphosis and/or piloerection and/or swollen abdomen were also observed in one female in the same period.
During the treatment or the recovery phase, no clinical signs were observed in male animals of the recovery groups.

Clinical observations

Main and recovery groups
Observation of animals at removal from the cage and in an open arena (neurotoxicity assessment) did not reveal changes attributable to the test item.

Body weight and body weight gain

Main groups
A slight decrease in body weight was recorded in high dose males and females starting on Week 2 of treatment up to the end of the study. The decrease in body weight was observed also in mid-dose females on lactation Days 1 and 7 only.

Reduction in body weight gain was seen in all treated males starting from Week 2 of treatment up to the end of the study. At the end of the study, no differences were seen between the treated and the control groups.
Body weight gain of treated females was comparable to controls.

Recovery groups
The trend of body weight and body weight gain of recovery animals was similar to the main groups animals. A complete recovery was observed during the treatment free period.

Food consumption
Main groups
No relevant changes were seen in food consumption during the treatment period in males. A reduction in food consumption was noted in mid- and high dose females at the end of the gestation and lactation periods.

Recovery groups
In recovery animals no relevant changes were noted in food consumption during the treatment or recovery period.

Motor activity and sensory reaction to stimuli
Main and Recovery groups
No relevant alterations in motor activity, grip strength or sensory reaction to stimuli were observed in any treatment group at the examinations performed during the study.

Haematology
Dosing phase - Main groups
Treatment-related changes were represented by a dose-related increase of leucocytes, mostly due to neutrophils increment, recorded in almost all treated animals. This could be due to the stress condition of the animals, as confirmed by the findings observed in thymus and spleen at histopathology.

Recovery phase - Recovery groups
In males, neutrophils showed complete reversibility. Lymphocytes were slightly decreased. In females, neutrophils showed only a partial reversibility, being still higher than controls.

Coagulation

Dosing and recovery phases
No treatment-related changes were recorded in prothrombin time measured during the study.

Clinical chemistry

Dosing and recovery phase
No treatment-related changes were found at the clinical chemistry investigation performed in the study.

Urinalysis

Dosing and recovery phases
No relevant changes were observed in urine analysis performed in males at the end of the treatment period.

Thyroid hormones determination

Parental animals
No differences between control and treated animals of both sexes were recorded in thyroid hormones determination.

Pups
At Days 4 and 14 post partum, no differences or treatment-related changes were recorded in thyroid hormones.

Oestrous cycle, reproductive parameters, pairing combination and mat- ing performance
Oestrous cycle, fertility index, reproductive parameters and mating performance were similar between the treated groups and control animals.

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

Implantation, pre-implantation, pre-natal loss and gestation length were unaffected by treatment.

Litter data from birth to Day 13 post partum
Litter data of treated females (e.g. total litter size, live litter size, pup loss, litter weight and mean pup weight) recorded at birth and on Days 1, 4 and 13 post partum did not show treatment-related effects.

Sex ratio of pups

Sex ratios did not show any treatment-related effects.

Clinical signs of pups

Clinical signs in pups, did not reveal any treatment-related effects. No nipples were found in male pups at Day 13 of lactation.

Anogenital distance

No treatment-related effects were observed in anogenital distance measured in male and female pups of the treated groups.

Necropsy findings in pups

No findings noted at necropsy were considered related to treatment.

Pups thyroid weight

No significant differences were noted in thyroid weight between control and pups of treated groups.

Terminal body weight and organ weights

Final sacrifice
A treatment-related decrease in terminal body weight, statistically significant or not, was observed in high dose males and in mid- and high dose females, when compared to the controls.
A statistically significant increase in absolute and/or relative mean spleen weight was observed in treated males of all groups, when compared to the controls. A statistically significant decrease in relative mean thymus weights was recorded in high dose females treated at 75 mg/kg/day compared to the control group.

Recovery sacrifice
At the end of the recovery period no terminal body weight changes were observed in treated animals of both sexes when compared to the controls. A statistically significant increase in absolute and relative mean weight was seen in the spleen of the females previously dosed at 75 mg/kg/day.

Macroscopic observations

Final sacrifice
Treatment-related macroscopic observations were present in the mesenteric lymph node, spleen and thymus. Enlarged mesenteric nodes were observed in 1/10 high dose male, 5/10 high dose females, 3/10 mid-dose females and in 1/10 low dose female; swollen spleen was described in 1/10 high dose male; an increased incidence of reduced thymus was observed

in 7/10 high dose, 5/10 mid-dose, 4/10 low dose and 1/10 control females.

Recovery sacrifice
At the end of the recovery period, enlarged mesenteric lymph node was observed in females previously treated with the high dose.

Microscopic observations

Final sacrifice
At the end of the treatment period, treatment-related microscopic observations considered to be associated with the oral administration of The registered substance were present in the spleen, mesenteric lymph node, small intestine (duodenum and/or jejunum and/or ileum) in a set of treated animals of both sexes dosed with 75 (high dose), 50 (mid-dose) and 25 (low dose) mg/kg/day.

Recovery sacrifice
After 2 weeks of recovery period, histopathological changes were still present in the spleen, mesenteric lymph node and small intestine (duodenum and/or jejunum and/or ileum) of high treated animals of both sexes (5 males and 5 females) previously treated with the high dose (75 mg/kg/day), exhibiting similar incidence and severity when compared to the rats given 75 mg/kg/day sacrificed at term.

Spermatogenic cycle
Regular layering in the germinal epithelium was noted in the seminiferous tubules.

Overall

The findings observed in thymus and spleen were considered at least partially secondary to the stress induced by the small intestine and mesenteric nodes lesions and therefore not directly related to treatment.

Conclusion

Based on the results of the present study, the NOAEL (No Observed Adverse Effect Level) for general toxicity and reproductive and developmental toxicity was considered to be 75 mg/kg/day for both males and females parental animals and pups.