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Toxicity to reproduction

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Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov 2021 to Oct 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From Nov 2021 to Oct 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Specific details on test material used for the study:
TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature
Species:
rat
Strain:
Wistar
Remarks:
Wistar Hannover rat
Details on species / strain selection:
The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.
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: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing 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, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy)
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC in water, softened by reverse osmosis
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days.
Frequency of treatment:
daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Low-level dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Medium-level dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
High-level dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from subacute oral toxicity studies with structurally related pigments. 1000 mg/kg bw is the maximimum dose prescribed in the OECD testing guideline.
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation 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 system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes

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 to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment

IMMUNOLOGY: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- Necropsy: 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
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
- Pups: all pups found dead in the cage were examined for external and internal abnormalities, all culled pups sacrificed at day 4 post partum were subjected to an external examination, all live pups sacrificed on day 14 post partum were examined for external abnormalities, gonads were inspected from all pups in order to confirm the sex previously determined by external examination

ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation

HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed

The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups
Other examinations:
PARTURITION AND GESTATION LENGTH:
- Parturition check from day 20 - 25 post coitum, three rimes a day during working day or twice daily during weekends and Public Holidays
- Females which did not give birth after 25 days of post coitum period were sacrificed shortly after
- Gestation length calculated as the time between the day of successful mating (day 0 post coitum) and the day of birth
- Day of birth was defined as day 0 post partum

PUPS IDENTIFICATION, WEIGHT AND 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, 7 and 13 post partum
- Pups dying during the lactation period were weighed before the despatch to necropsy
- Observation was performed once daily for all litters from day 0 post partum until termination
- After culling, all pups were sacrificed with the dams on Day 14 post partum

ANOGENITAL DISTANCE (AGD):
- For each pup measured on day 1 post partum
- AGD was normalized to the cube root of body weight collected on day 1 post partum

NIPPLE COUNT CHECK:
- presence of nipples/areolae in male pups was checked on day 13 post partum

REPRODUCTIVE INDICES:
- Males: copulation index, fertility index
- Females: copulatory index, fertility index, pre-natal loss, post-natal loss at day 0, 4 and 13 post partum
- Pre coital interval
- Sex ratio of litter
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. 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 nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
Body weight and body weight gain for male and female animals were comparable between treated and control groups through the study. The statistically significant decrease in body weight gain recorded in high dose males and mid-dose females were considered incidental.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both genders during the study.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
he statistically significant differences between contrTol and treated animals (neutrophils in males and platelets in females) were recorded only in animals dosed at 300 mg/kg/day, therefore they were considered to be incidental.
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Description (incidence and severity):
The determination of T4 and TSH was performed on:
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically significantly lower than controls in parental males (31%). This is due to 2 control animals, which showed values above the range of historical data, therefore the differences observed has no biological/toxicological significance.

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.
No differences were recorded in pre-coital interval and copulation plugs between treated and control groups. Copulatory index and fertility index did not show any treatment-related differences.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no treatment-related macroscopic observations at the end of the treatment period. Any macroscopic observations were within the range of occasionally observed and expected spontaneous changes in rats of the same age and therefore considered unrelated to treatment.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed
Other effects:
effects observed, treatment-related
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no
Conclusions:
The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of systemic toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity was set to 1000 mg/kg bw/day.
Executive summary:

Under the conditions of the OECD TG 422 and in compliance with GLP, a combined repeated dose toxicity study with the reproductive/developmental screening test was conducted in male and female Wistar rats. The test substance was administered daily as a solution to groups of 10 male and 10 female Wistar rats (P0 animals) by gavage at dose levels of 0 mg/kg bw/d (control; test group 1), 100 mg/kg bw/d (test group 2), 300 mg/kg bw/d (test group 3) and 1000 mg/kg bw/d (test group 4). 0.5% carboxymethylcellulose served as vehicle, control animals were dosed daily with the vehicle only. All doses were administered at a constant volume of 10 mL/kg body weight. For males, the duration of treatment covered a 2-week premating period and pairing with females until the day before necropsy (a total of 39 days). Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprised from 51 to 55 days.

No test-item related mortality occurred throughout the study and no treatment-related clinical signs were noted during the study. No signs of neurotoxicity (weekly observations at removal from the cage and in an open arena, alterations in motor activity, grip strength and sensory reactivity to stimuli) were observed during the study in parental males and females. No differences in body weight and food consumption were observed in treated animals, compared to the control group. No treatment-related changes were observed in haematological (including coagulation and blood clotting time) or clinical chemistry parameters. Thyroid hormone evaluation in parental animals and pups sacrificed on day 14 post partum did not show treatment-related changes. 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. No treatment-related changes were observed at post mortem macroscopic observations and microscopic evaluation. No treatment-related changes were observed in reproductive and developmental parameters.

Copulatory and fertility indices did not show any treatment related differences among treated and control groups. Parturition, lactation, implantation, litter data and sex ratio did not show changes. No differences in the anogenital distance (normalised value) and no nipple were seen between control and treated groups both for male and female pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect.

Therefore, the no observed adverse effect level (NOAEL) for general systemic toxicity was determined to be 1000 mg/kg bw/day for male and female Wistar rats.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
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)
Version / remarks:
29 July 2016
GLP compliance:
yes (incl. QA statement)
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]
EC Number:
279-356-6
EC Name:
3,3'-[(2-chloro-5-methyl-p-phenylene)bis[imino(1-acetyl-2-oxoethylene)azo]]bis[4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide]
Cas Number:
79953-85-8
Molecular formula:
C55H37Cl5F6N8O8
IUPAC Name:
3,3'-{(2-chloro-5-methyl-1,4-phenylene)bis[imino(1,3-dioxobutane-2,1-diyl)diazene-2,1-diyl]}bis{4-chloro-N-[2-(4-chlorophenoxy)-5-(trifluoromethyl)phenyl]benzamide}
Test material form:
solid: nanoform, no surface treatment
Details on test material:
BET = 71.4 m2/g
Batch 0004079400
Purity 99.4%
Storage stability: The stability of the test substance under storage conditions was guaranteed until 27 Aug 2022 as indicated by the sponsor, and the sponsor holds this responsibility.
Specific details on test material used for the study:
TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature

Test animals

Species:
rat
Strain:
Wistar
Remarks:
Wistar Hannover rat
Details on species / strain selection:
The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities.
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: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing 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, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx. 2 weeks

DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy)

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5% CMC in water, softened by reverse osmosis
Details on exposure:
PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing
Details on mating procedure:
- M/F ratio per cage: monogamous (one male to one female)
- Length of cohabitation: female was paired with the same male until positive identification of mating
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray, day of successful mating was referred to as day 0 post coitum
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: 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, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Duration of treatment / exposure:
Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days.
Frequency of treatment:
daily
Details on study schedule:
- Age at mating of the mated animals (P0 generation) in the study: approx. 13-14 weeks (approx. 11-12 weeks old at start of administration, followed by 2 weeks pre-mating phase)
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1
Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2
Low-level dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3
Medium-level dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4
High-level dose
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from a preliminary non-GLP compliant study
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes

Examinations

Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation 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 system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes

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 to necropsy

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined

SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment

IMMUNOLOGY: No
Oestrous cyclicity (parental animals):
- Vaginal smears were taken in the morning from day 1 of dosing up to positive identification of mating
- The vaginal smear data was 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 and the oestrous cycle phase recorded
Litter observations:
STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups per sex per litter (partial adjustment was possible); excess pups were killed, and at least one culled male and one culled female per litter were selected for hormone determination
- No pups were eliminated when the litter size dropped below the culling target (8 pups per litter in total)
- If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible hormone determination

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
(i) Number and sex of pups
(ii) Stillbirths, live births
(iii) Body weight measured on days 1, 4, 7 and 13 post partum
(iv) Anogenital distance measured on day 1 post partum, AGD was normalized to the cube root of body weight collected on day 1 post partum
(v) Nipple count check examined in male pups on day 13 post partum
(vi) Necropsy

GROSS EXAMINATION OF DEAD PUPS: Yes
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination
- All pups with abnormalities were retained in a 10% neutral buffered formalin

ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No

ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No
Postmortem examinations (parental animals):
SACRIFICE
- Male animals: sacrifice after the mating of all females on day 40 of the study
- Maternal animals: females with live pups were killed on day 14 post partum, females showing no evidence of copulation were killed 25 days after the last day of the mating session

GROSS PATHOLOGY: Yes
- Necropsy: 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
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)

ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal

HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed

The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups
Postmortem examinations (offspring):
SACRIFICE
- Pups (F1 offspring) that had completed the scheduled test period (days 4 or 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal
- Pups selected for blood collection for hormone determination were killed on the day of blood sampling

GROSS PATHOLOGY
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination

ORGAN WEIGHTS
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation
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. 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 nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
Reproductive indices:
MALES:
Copulation index (%) = (no. of males with confirmed mating/no. of males cohabitated) X 100
Fertility index (%) = (no. of males which induced pregnancy/ no. of males cohabitated) X 100

MALES AND FEMALES:
Pre coital Interval = the number of nights paired prior to detection of mating

FEMALES:
Copulation index (%) = (no. of females with confirmed mating/no. of females cohabitated) X 100
Fertility index (%) = (no. of pregnant females/ no. of females cohabitated) X 100
Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations - live litter size at birth/no. of visible implantations) X 100
Pre-natal loss at day 0 post partum was calculated as a percentage from the formula:
(total litter size - live litter size/total litter size) X 100
Post-natal loss at day 4 post partum (before culling) was calculated as a percentage from the formula:
(live litter size at birth - live litter size at day 4 (before culling)/live litter size at birth) X 100
Post-natal loss at day 13 post partum (after culling) was calculated as a percentage from the formula:
(live litter size on day 4 (before culling) - live litter size at day 13/live litter size on day 4 (before culling)) X 100
Offspring viability indices:
Sex ratios was calculated at birth, on day 4 and on day 14 post partum and was presented as the percentage of males per litter.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Mortality:
mortality observed, non-treatment-related
Description (incidence):
One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Food consumption was unaffected by treatment in both genders during the study.
Food efficiency:
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Endocrine findings:
no effects observed
Description (incidence and severity):
The determination of T4 and TSH was performed on
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically si
gnificantly lower than controls in parental males (31%). This is due to 2 control animals, which sh
owed values above the range of historical data, therefore the differences observed has no biological/
toxicological significance.
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.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment.
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):
There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
Histopathological findings: neoplastic:
no effects observed

Reproductive function / performance (P0)

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.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences.
Reproductive performance:
no effects observed
Description (incidence and severity):
Implantation, pre implantation loss, pre-natal loss data and gestation length of females: Mean gestation period was comparable between control and treated animals, being of 22/23 days. Corpora lutea, number of implantations sites and live litter size did not show dose-related or treatment-related differences, compared to controls. Pre-natal loss (percentage) was similar between control and treated groups.

Effect levels (P0)

open allclose all
Dose descriptor:
NOAEL
Remarks:
reproduction toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Dose descriptor:
NOAEL
Remarks:
systemic toxicity
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No treatment-related clinical signs were observed in pups during the 14-day observation period.
Body weight and weight changes:
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Sexual maturation:
not examined
Anogenital distance (AGD):
no effects observed
Nipple retention in male pups:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
No differences were observed in the weight of thyroid in treated pups, when compared to controls.
Histopathological findings:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Effect levels (F1)

Dose descriptor:
NOAEL
Remarks:
developmental toxicity
Generation:
F1
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:
no

Overall reproductive toxicity

Reproductive effects observed:
no

Any other information on results incl. tables

Tab. 5: Summary of female reproduction data (group data)

control 100 mg/kg bw 300 mg/kg bw 1000 mg/kg bw
paired females 10 10 10 10
not pregnant 0 2 0 2
not mated 0 2 0 0
humane kill 1 0 0 0
Conceiving 1-5 days 10 6 9 8
Conceiving 6-14 days 0 0 1 0
Total litter loss 0 0 0 0
Unilateral implantation 0 0 0 0
No of females with live pups on day 14 post partum 9 6 10 8

Applicant's summary and conclusion

Conclusions:
The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of reproduction and developmental toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity, reproductive performance as well as for developmental toxicity was set to 1000 mg/kg bw/day.