5-[[4-chloro-6-[(3-sulphophenyl)amino]-1,3,5-triazin-2-yl]amino]-4-hydroxy-3-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]naphthalene-2,7-disulphonic acid, sodium salt

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

No teratogenic or developmental effects were noted in reproductive studies with structural analogues. The only effects noted were unspecific toxic effects or salt effects resulting in higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day in studies for reproduction with structural analogues. The relation to test substance administration is questionable and/or effects are not relevant for humans.

Link to relevant study records

Reference

Reference 1

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

28 April 2011 to 23 June 2011

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)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Species and strain: Wistar RJHan:WI rats
Source: Laboratoire Elevage Janvier, B.P. 4105, Route des Chênes Secs, 53940 Le Genest-St-Isle CEDEX France
Hygienic level: SPF at arrival; standard laboratory conditions during the study
Justification of species/strain: The rat is regarded as suitable species for toxicology and reproduction studies. Wistar rat was selected due to experience with this strain of rat in toxicity and reproduction toxicity studies and known fertility.
Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group
At the completion of the study, the spare animals were returned to CiToxLAB Hungary Ltd. spare colony, as their use was not required (no replacements with spare animals were performed)
Age of animals: Young adult rats, approximately 10-11 weeks old at starting and 12-13 weeks at mating. The age range within the study was kept to the minimum practicable.
Body weight range: Males: 366 g – 407 g, Females: 219 g - 249 g; did not exceed ± 20% of the mean weight for each sex at onset of treatment
Acclimation period: At least 7 days (5 days from animal arrival to pre-treatment ophthalmoscopy examination, 7 days from animal arrival to onset of treatment)
Animal health: Only healthy animals were used for the test, as certified by the veterinarian. Females were nulliparous and non-pregnant.
Room number: 524, 505
Cage type: Type II and/or III polypropylene/polycarbonate
Bedding: Lignocel® Hygienic Animal Bedding produced by J. Rettenmaier & Söhne GmbH+Co.KG (Holzmühle 1, D-73494 Rosenberg, Germany). Details of bedding quality are reported.
Light: 12 hours daily, from 6.00 a.m. to 6.00 p.m.
Temperature: 19.9 – 24.2°C
Relative humidity: 33 - 69%
Ventilation: 15-20 air exchanges/hour
Housing/Enrichment: Rodents were group-housed, up to 5 animals of the same sex and dose group/cage, with the exception of the mating and gestation/delivery period, when they were paired or individually housed, respectively. Group housing allowed social interaction and the deep wood sawdust bedding allowed digging and other normal rodent activities (i.e. nesting).

The temperature and humidity were measured twice daily; no deviations from the target ranges were noted during the study.

Food and water supply: Animals received ssniff® SM R/M-Z+H "Autoclavable complete feed for rats and mice – breeding and Maintenance" produced by ssniff Spezialdiäten GmbH, D-59494 Soest Germany ad libitum, and tap water from municipal supply, as for human consumption from 500 ml bottle ad libitum. Water quality control analysis is performed once every three months and microbiological assessment is performed monthly by Veszprém County Institute of State Public Health and Medical Officer Service (ÁNTSZ, H-8201 Veszprém, József A.u.36., Hungary).

The food and water are considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study.

Animal identification: Each parental/adult animal (P Generation) was identified by a number unique within the study, written with indelible ink on the tail and cross-referenced to the Animal Master File at CiToxLAB Hungary Ltd.
This number consisted of 4 digits, the first digit being the group number, the second, 0 for the males and 5 for the females, and the last 2, the animal number within the group, as indicated in the Experimental design section. The boxes were arranged in such a way that possible effects due to cage placement were minimized and were identified by cards showing the study code, sex, dose group, cage number and individual animal numbers, date of mating and delivery. The new-borns (Offspring, F1 Generation) were identified by cutting off digit-tips up to one day after birth.

Randomization: All parental/adult (P) male and female animals were sorted according to body weight by computer and divided to weight ranges. An equal number of animals from each weight group was randomly assigned to each dose group to ensure that test animals were as nearly as practicable of a uniform weight. The grouping was controlled by SPSS/PC software according to the actual body weight, verifying the homogeneity/variability between/within the groups and cages. Males and females were randomized separately.

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

Vehicle
Name: Distilled, sterile water for injection, PhEUR
Lot No.: 8490910, 0110111
Manufacturer: TEVA Pharmaceutical Corporation
Expiry Date: September 2013, January 2014 respectively
Storage: Room temperature

Details on mating procedure:

Main and Positive Control MNT animals: Mating began after the animals have attained full sexual maturity, 2 weeks after the initiation of treatment, with one female and one male of the same dose group (1:1 mating) in a single cage. Females remained with the same male until copulation occurred, for up to 6 days. A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines). Sperm positive females were caged individually. Mating pairs were clearly identified in the data, mating of siblings was avoided.

Recovery animals: Recovery animals were not mated and, consequently, were not used for the assessment of reproduction/developmental toxicity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The samples were evaluated by UV-HPLC method

Duration of treatment / exposure:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period).
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing).

Frequency of treatment:

Test item or Control (water)-treated Groups 1-4 Main animals were administered the dosing solutions daily on a 7 days/week basis.

Details on study schedule:

Males were dosed for 28 days (14 days pre-mating and 14 days mating/post-mating period), then were euthanized and subjected to necropsy examination, as no additional mating was considered required.
Females were dosed for 14 days pre-mating, for up to 5 days mating period, through gestation and up to the day of necropsy (at least 4 days post-partum dosing). The day of birth (viz. when parturition was complete) was defined as Day 0 post-partum. Females showing no-evidence of copulation were sacrificed as practical, 26-27 days after the end of the mating period.

Dose / conc.:

62.5 mg/kg bw/day (actual dose received)

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Number of animals: Main groups: 48 male, 48 female rats, 12 animals/sex/group, 4 groups; a sufficient number of at least 8 pregnant females/group was achieved.
Recovery groups: 10 male, 10 female rats, 5 animals/sex/group, 2 groups, Control and High dose
Positive Control MNT group: 12 male and 12 female rats, 1 group

Control animals:

yes, concurrent vehicle

Details on study design:

Rationale for dose selection and route of administration: The dose levels and the vehicle were selected by the Sponsor in consultation with the Study Director based on available data, formulation and analytical trials and information from previous experimental work, including the results of an acute oral toxicity study (CiToxLAB Hungary Ltd. study code 11/008-001P) and a repeated dose range finding study in the rat (CiToxLAB Hungary Ltd. study code 11/008-220PE), with the aim of inducing toxic effects but ideally no death or suffering at the highest dose and a NOAEL at the lowest dose. The oral route was selected as it is a possible route of exposure to the test item in humans.

Positive control:

A Positive Control group for the Mammalian Erythrocyte Micronucleus Test (MNT) was added; the animals were mated and females allowed to deliver similarly to the Main animals, then treated once with 20 mg/kg bw Cyclophosphamide, administered by intraperitoneal injection approximately 24 h prior to scheduled necropsy (males, on Day 27 for necropsy on Day 28; females, on PPD4 for necropsy on PPD5).

Parental animals: Observations and examinations:

Clinical observations and functional observation battery (FOB)
All animals: Animals were inspected for signs of morbidity and mortality twice daily, at the beginning and end of the working day. General clinical observations were performed daily, after treatment at approximately the same time with minor variations, or in the afternoon (pm) as practical during the working day, as no peak period of effects was noted after dosing during the first days of treatment. During Recovery period, the animals were similarly observed daily as practical.
All animals were monitored for pertinent behavioural changes, signs of difficult or prolonged parturition and all signs of toxicity including mortality. Any changes were recorded including onset, degree and duration of signs as applicable.
More detailed examinations were made once before the first exposure (to allow for within-subject comparisons), then at least weekly, in the morning (am) or before treatment. These observations were made outside the home cage in a standard arena, at similar times as practical. The animals were monitored for changes in skin, fur, eyes, mucous membranes, occurrence of secretions and excretions, and autonomic activity (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), or changes in gait, posture and response to handling as well as the presence of clonic or tonic movements, stereotypies (e.g. excessive grooming, repetitive circling), difficult or prolonged parturition or bizarre behaviour (e.g. self-mutilation, walking backwards); special attention were directed towards the observation of tremors, convulsions, salivation, diarrhoea, lethargy, sleep and coma. No such clinical signs were observed during the study.

Main animals, 5 males and 5 females/group, “subgroup A”:
Assessment of any potential test item related neurotoxicity was performed during the last exposure week (males, on Day 26 am, females, on PPD 3 am). In order to avoid hyperthermia of pups, dams were removed from the pups for not more than approximately 30-40 minutes. Selected animal were subjected to the functional observation battery, including qualitative assessment of the grip strength, and to measurements of the landing foot splay and fore/hind grip strength.
To measure the landing foot splay, the fore/hind paws of the rat were painted with ink and the rats were dropped from a horizontal position onto the appropriate record sheet covering the examination table. The distance between the two resulting ink spots was measured.
Fore/hind grip strength measurements were conducted using a grip strength meter (Model GS3, Bioseb, Chaville, France), an instrument designed to quantify objectively rodent muscular strength, in order to identify and assess quantitatively any potential effect of test item. The rats were held appropriately such that the fore limbs were allowed to grip the support bar and gently pulled back until they released the bar; the device measured the maximum grip strength. This was performed 3 times for each animal on each test day. The procedure was repeated with the hind limbs with the appropriate grip support; results are tabulated with individual and mean data.
Sensory reactivity to different type of stimuli (e.g. auditory, visual and proprioceptive), assessment of grip strength and motor activity were conducted and the general physical condition and behaviour of animals were tested. A modified Irwin test was performed. Parameters such as, but not limited to body position, locomotor activity, respiration rate, respiration type, piloerection, head searching compulsive biting or licking, circling, upright walking, retropulsion, jumping, exophthalmos, twitches, clonic convulsions, tonic convulsions, tremor, startle, transfer arousal, spatial locomotion, gait, posture, limb position, finger approach, finger withdrawal, touch escape response, diarrhoea, diuresis, visual placing, grip strength, body tone, corneal reflex, pinna, toe pinch, grasping reflex, positional struggle, skin, mucous membrane colour, salivation, palpebral closure, lachrymation, limb tone, abdominal tone, tail pinch, righting reflex, and/or vocalisation were evaluated.

Recovery animals: Neurotoxicity evaluation was similarly conducted in the Recovery animals towards the end of the Recovery period, on Day 54, for necropsy on Day 56.

Body weight measurement : All adult Main and Recovery animals were weighed with accuracy of 1 g for randomization purposes, then on Day 0, afterwards at least weekly and at termination. Parent females were weighed on gestation Days GD0, 7, 14 and 20 and on postpartal Days PPD0 (within 24 hours after parturition) and PPD4 (before termination). Body weights of the female animals were additionally weighed on gestational Days GD10 and 17 in order to give accurate treatment volumes, but these data were not evaluated statistically.

Food consumption measurement: Animal food consumption was determined by re-weighing the non-consumed diet with a precision of 1 g on Day 7 then at least weekly.

Ophthalmology: The fundus of eyes of all animals was examined before treatment. Five male and 5 female Control and High dose animals (“subgroup C”) randomly selected from groups 1 and 4, during the last week of treatment prior to necropsy (males, Day 26 pm, females, PPD 3 pm). Mydriasis was produced after instillation of a mydriatic agent (eye drops "Mydrum") into the conjunctival sac. The examination was performed using a Gowlland ophthalmoscope. As no ophthalmoscopic alterations were found, no additional examination was performed in other animals.

Observation of the delivery process, offspring and nursing instinct: Main Females were allowed to litter and rear their offspring. Delivery process was observed as carefully as possible. All observations were recorded as applicable. No evidence of abnormal deliveries was recorded. The duration of gestation was recorded and was calculated from Day 0 of pregnancy. Dams were observed to record whether they form a nest from the bedding material and cover their new-borns or not. The efficiency of suckling was observed by the presence of milk in the pups' stomach. All observations were recorded as applicable.

Observations are reported individually for each adult animal. In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes.

CLINICAL PATHOLOGY
All animals selected for blood sampling were fasted (overnight period of food deprivation).

For terminal blood sampling of Recovery animals, 3 samples were taken from each animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood), one for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.

For Day 14 blood sampling of Main animals selected (subgroup B), 2 samples were taken from each scheduled animal: one for haematology (1.2 mL blood, in tubes with K3-EDTA as anticoagulant, 1.6 mg/mL blood) and one to obtain serum (approximately 1 mL blood as practical, in tubes with no anticoagulant) for clinical chemistry.

For terminal blood sampling of Main animals selected (subgroup B), one sample for blood clotting times (1.4 mL blood for APTT and PT measurements, in tubes with sodium citrate as anticoagulant) were taken from scheduled animals.
For urine collection, the selected animals (Main subgroup B and Recovery) were placed in metabolic cages for approximately 16 hours and food and water deprived, then water were provided at libitum for at least approximately 2 hours prior to necropsy and organ weight measurements.

Main animals, 5 males and 5 females/group, “subgroup B”:
Laboratory examinations for haematology and clinical chemistry evaluation were conducted at the end of pre-mating period, on blood samples collected from the sublingual vein, prior to the start of mating on Day 14 from 5 animals/sex/group randomly selected (“subgroup B”).
Coagulation evaluation (APTT and PT) was performed at the completion of the treatment, on blood samples collected by cardiac puncture from subgroup B animals under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

Urine sampling (approximately 16 hours sampling period) was performed prior to necropsy from the same subgroup B (urinalysis on Day 28-males, PND 5-females).

Recovery animals: Haematology, coagulation and clinical chemistry investigations were conducted at the completion of the Recovery period, 14 days after the first scheduled euthanasia of Main dams. Blood samples were collected by cardiac puncture under pentobarbital anaesthesia, immediately prior to scheduled necropsy.

Urine sampling (approximately 16 hours sampling period) was performed from the Recovery animals prior to necropsy (urinalysis on the day of necropsy, conducted 14 days after the first scheduled euthanasia of the Main dams).

Oestrous cyclicity (parental animals):

A vaginal smear were prepared daily during the mating period and stained with 1% aqueous methylene blue solution. The smears were examined with a light microscope, the presence of vaginal plug or sperm in the vaginal smear was considered as evidence of copulation (Day 0 of pregnancy as defined by the relevant guidelines).

Sperm parameters (parental animals):

For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery) and all macroscopic findings (abnormalities) from all animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure.

Litter observations:

Each litter was examined as soon as possible after delivery to establish the number and sex of pups, stillbirths, live births, runts (pups that are significantly smaller than normal pups) and the presence of gross abnormalities.
In addition to the observations on parent animals, the pups (offspring) were monitored for any behavioural changes. Live pups were counted, sexed, weighed individually within 24 hours of parturition (ex. Day 0 or 1 post-partum, PND0 or 1) and on PND4, with accuracy of 0.01g. All the litters were checked and recorded daily for the number of viable and dead pups. The pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination in order to identify the possible cause of death. All observed abnormalities were recorded.

Postmortem examinations (parental animals):

Pathology
Gross necropsy was performed on all animals. Terminally, after completion of the treatment or Recovery periods as applicable, animals were sacrificed under pentobarbital anaesthesia followed by exsanguination. After exsanguination the external appearance was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs were observed macroscopically. Any abnormality was recorded with details of the location, colour, shape and size, as appropriate.

Special attention was paid to the organs of the reproductive system. The number of implantation sites and of corpora lutea was recorded in the Main females as applicable.

At the time of termination, body weight and weight of the following organs of all parental animals were determined:
- With a precision of 0.01 g: uterus (with and without cervix), vagina, testes, epididymides (total and cauda), prostate, seminal vesicles with coagulating glands, brain
- With a precision of 0.001 g: ovaries, pituitary

The weighed organs and all organs showing macroscopic lesions of all adult animals were preserved. The eyes with the optic nerve were retained in modified Davidson’s fixative. Testes and epididymides were preserved in Bouin’s solution, all other organs in 10% buffered formalin solution.

From subgroup B Main animals and Recovery animals, the following organs were weighed in addition to the ones previously mentioned:
- With a precision of 0.01 g: heart, kidneys, liver, spleen and thymus
- With a precision of 0.001 g: adrenals.
For all organs, paired organs were weighed individually. Individual and/or paired absolute organ weight are reported for each animal and adjusted for the body and brain weights. Paired organ weights as applicable were summarised. Relative organ weight (to body and brain weight) were calculated and reported.

Histopathological examination was performed on the selected list of specified tissues from the Control and High Dose Main and Recovery rats and all macroscopic lesions from all toxicology animals. Special attention was paid to evaluation of the stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure. Detailed histological examination of the ovaries covered the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma.

Additionally selected organs/tissues for 5 animals/sex/group (identified as subgroup B Main) were microscopically evaluated from the Control and High dose Main animals, and kidneys and stomach samples from the Subgroup B Low and Mid Dose animals were examined, due to the test item-related microscopic findings noted at the first instance in the animals examined.

The retained tissues and organs were embedded in paraffin wax, sections were cut at 4-6µm by microtome and transferred to slides. Tissue sections were stained with haematoxylin-eosin/phloxine and examined by light microscope.

Postmortem examinations (offspring):

Pups euthanized at PND4 were carefully examined at least externally for gross abnormalities. Any pups showing abnormalities in structure or behaviour, including the pups found dead and intact (not cannibalized) were subjected to necropsy with macroscopic examination, in order to identify the probable cause of death if possible.

Statistics:

Data were recorded on the appropriate forms from the relevant SOPs of CiToxLAB Hungary Ltd., and then tabulated using the Microsoft Office Word and/or Excel, as appropriate. Numerical data obtained during the conduct of the study were subjected as appropriate to calculation of group means and standard deviations.

The statistical evaluation of appropriate data (marked † below) was performed with the statistical program package SPSS PC+4.0. The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected, a one-way analysis of variance (ANOVA) was carried out. If the obtained result was significant, Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed as feasible.

Reproductive indices:

Formulae for Calculation of Mating and Fertility Indices are included in pdf attached under background information.

Offspring viability indices:

Formulae for Calculation of Pups’ Mortality and Sex Ratio Indices are included in pdf attached under background information.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs at up to and including 250 mg/kg bw/day or in the Control animals. At 1000 mg/kg bw/day, dark red discoloration of faeces and urine was noted. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration and of the urine, until completion of the recovery phase and termination. Slight to moderate dark pink discoloration of the skin was also noted in the High dose animals until completion of the study during the Recovery period.
These changes were ascribed to elimination of the test substance (red dye) or its metabolites through faeces or urine and an expected staining effect.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

No adverse effects or test item related changes were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels of up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, attaining statistical significance when evaluated for the whole period of treatment between Days 0 and 28 in males or during the gestation period between GD0 and GD20 in females. In the Recovery animals, the body weights remained minimally lower than controls, however, no statistically significant differences occurred in the body weight gain of the Recovery animals.

Food consumption and compound intake (if feeding study):

no effects observed

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, WBC increases considered possibly related to test substance administration were noted in the Main animals and recovery males. This could be related to the local irritating effects in the stomach.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An apparent increase in total bilirubin T BIL was noted at 1000 mg/kg bw/day and bile acids at 250 and 1000 mg/kg bw/day. In addition, a apparently lower albumin/globulin ratio was seen at 1000 mg/kg bw/day. These effects were considered to be related to a spectral interference with the analytical method (colorimetric test) caused by the discolouration of the serum by the test item and not to reflect an adverse effect. The minimally lower ALT values at 1000 mg/kg bw/day are without toxicological and biological relevance.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Pink urine was noted in all animals at 250 mg/kg bw/day group and red urine at 1000 mg/kg bw/day. These changes were ascribed to elimination of the test substance or its metabolites through urine and an expected staining effect.

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to mild degeneration/regeneration were seen in the cortical tubules of the kidneys of most animals at 1000 mg/kg bw/day and minimal tubular degeneration in single males at 250 mg/kg bw/day. These effects are considered to be caused by adaptive changes in order to absorb, catabolise and eliminate the dye substance and not cytotoxic effects on kidney cells. Furthermore, minimal to mild degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach were seen in animals dosed 1000 mg/kg bw/day and to a lesser degree in single animals at 250 mg/kg bw/day. These effects are considered to be caused by a high salt load administered directly to the stomach mucosa by gavage via a stomach tube and reflects a local irritation effect of and is hence without relevance for humans.
All effects seen, showed to be reversible in the recovery groups, as the effects seen in main animals in kidneys and stomach were found with a much lower incidence and severity in recovery animals and led to an almost complete regression of the lesions in the stomach.

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed. The mean duration of pregnancy was similar in the control and test item treated groups. All the parturitions were normal. The number of corpora lutea and implantation sites were comparable in the treated groups with the mean value recorded in the Control group.
There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control, however, only the post-natal mortality attaining statistical significance. The origin of this effect is not quite clear, as a high individual variability was observed is some females.

Mortality: There was no unscheduled mortality during the study.
Clinical observation: There were no clinical signs at up to and including 250 mg/kg bw/day or in the Control animals. At 1000 mg/kg bw/day, dark red discoloration of faeces and urine was noted. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration and of the urine, until completion of the recovery phase and termination. Slight to moderate dark pink discoloration of the skin was also noted in the High dose animals until completion of the study during the Recovery period.
These changes were ascribed to elimination of the test substance (red dye) or its metabolites through faeces or urine and an expected staining effect.
Neurological assessment: There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment or Recovery periods.
Ophthalmology: No test item related changes compared to pre-treatment were noted during ophthalmoscopy examination of 5 male and 5 female Control and High dose Main animals during the last week of treatment prior to necropsy, thus no additional evaluation was required in other dose groups or Recovery animals.
Body weight and body weight gain: No adverse effects or test item related changes were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels of up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, attaining statistical significance when evaluated for the whole period of treatment between Days 0 and 28 in males or during the gestation period between GD0 and GD20 in females. In the Recovery animals, the body weights remained minimally lower than controls, however, no statistically significant differences occurred in the body weight gain of the Recovery animals.
Food consumption: There were no test item-related differences to Control in the mean daily food consumption in any test-item treated Main or Recovery group when compared to the Control.
Clinical pathology
Haematology: No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, WBC increases considered possibly related to test substance administration were noted in the Main animals and recovery males.
Clinical chemistry: An apparent increase in total bilirubin T BIL was noted at 1000 mg/kg bw/day and bile acids at 250 and 1000 mg/kg bw/day. In addition, a apparently lower albumin/globulin ratio was seen at 1000 mg/kg bw/day. These effects were considered to be related to a spectral interference with the analytical method (colorimetric test) caused by the discolouration of the serum by the test item and not to reflect an adverse effect. The minimally lower ALT values at 1000 mg/kg bw/day are without toxicological and biological relevance.
Urinalysis: Pink urine was noted in all animals at 250 mg/kg bw/day group and red urine at 1000 mg/kg bw/day. These changes were ascribed to elimination of the test substance or its metabolites through urine and an expected staining effect.
Organ weights: There were no toxicologically relevant changes in the organ weight values, or effects considered adverse or related to test substance administration at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, minimal increases in the kidney weights were noted in both males and females, attaining statistical significance only for relative kidney weights in male rats.
Other absolute organ weights or relative to the body and/or brain weights were similar in the control and test item treated groups, or showed minor variations, ascribed to biological variability.
Macroscopic Findings: At 1000 mg/kg bw/day, pink discoloration was observed in numerous organs/tissues including the stomach, small and large intestine, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular) at necropsy.
At 250 mg/kg bw/day, pink discoloration of the stomach, kidneys or trachea was also seen with low incidence in single animals.
These changes were considered due to the expected colouring effect of the test item (dye). All other macroscopic changes were regarded as incidental.

Microscopic Findings: Test item-related findings microscopically observed in the kidneys and stomach and were in correlation with the observations noted at necropsy. The changes were characterized as minimal to mild degeneration/regeneration of the cortical tubules in the kidneys of most animals at 1000 mg/kg bw/day and minimal tubular degeneration in single males at 250 mg/kg bw/day. These effects are considered to be caused by adaptive changes in order to absorb, catabolise and eliminate the dye substance and not cytotoxic effects on kidney cells. Furthermore, minimal to mild degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach were seen in animals dosed 1000 mg/kg bw/day and to a lesser degree in single animals at 250 mg/kg bw/day. These effects are considered to be caused by a high salt load administered directly to the stomach mucosa by gavage via a stomach tube and reflects a local irritation effect of and is hence without relevance for humans.
All effects seen, showed to be reversible in the recovery groups, as the effects seen in main animals in kidneys and stomach were found with a much lower incidence and severity in recovery animals and led to an almost complete regression of the lesions in the stomach.
There was no evidence of treatment-related histopathological findings in reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

Oestrus cycle, reproductive ability assessment and indices: There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant.
In all groups, the mating indices were 100%, the fertility indices, 83%, 100%, 92% and 92% due to 2, 0, 1 and 1/12 non-pregnant females, respectively, in Groups 1, 2, 3 and 4, and the gestation indices, 100%, with the exception of the High dose group in which the gestation index was incidentally 91%, due to 1/11 pregnant, but not delivered females in this group, values which are comparable with concurrent control data in Wistar rats.
Test item administration was considered to have no impact on the duration of the mating period. Successful coitus (sperm positive vaginal smears and/or vaginal plugs) generally occurred within up to 6 days of pairing (cohabitation).

Evaluation of the gestation, parturition and post-partal period: No test item effect on the duration of pregnancy or abnormalities in the gestation outcome ascribed to the treatment were observed. The mean duration of pregnancy was similar in the control and test item treated groups. All the parturitions were normal. The number of corpora lutea and implantation sites were comparable in the treated groups with the mean value recorded in the Control group.
There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control, however, only the post-natal mortality attaining statistical significance. The origin of this effect is not quite clear, as a high
individual variability was observed is some females.

Dose descriptor:

NOAEL

Remarks:

(reproductive toxicity)

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Remarks:

(reproductive toxicity)

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

reproductive performance

Dose descriptor:

NOAEL

Remarks:

general toxicity

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Clinical signs:

no effects observed

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

The test substance administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level.
At 1000 mg/kg bw/day, the mean number of viable pups was minimally lower than control on PND0 and PND4, only attaining statistical significance when evaluated for the total number of pups. Hence, survival index was minimally lower on PND0 and PND4, although without attaining statistical significance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

There were no effects considered adverse on the offspring weight or weight gain at 62.5 and 250 mg/kg bw/day. At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4. When evaluated for all pups, the mean body weights were statistically significant, minimally lower than control on PND0 and on PND4. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained.

Sexual maturation:

not examined

Gross pathological findings:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.

Mortality and clinical observations: The test substance administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level.
At 1000 mg/kg bw/day, the mean number of viable pups was minimally lower than control on PND0 and PND4, only attaining statistical significance when evaluated for the total number of pups. Hence, survival index was minimally lower on PND0 and PND4, although without attaining statistical significance.
The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically. No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect.
In the Control group (131 pups examined), 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), and another pup was found dead on PND2 without displaying any clinical signs prior to their death.
In the 62.5 mg/kg bw/day Low dose group with 155 pups examined, 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), 4 were not suckled on PND0, then survived with no clinical signs until the scheduled necropsy on PND4, and another pup was found dead on PND3 without displaying any clinical signs prior to their death.
In the 250 mg/kg bw/day Mid dose group, with 141 pups examined, 4 pups were found dead on PND0, with negative floating test (possibly stillborn), one was found dead and had positive pulmonary floating test, 5 were cannibalized and 2 cold or not suckled.
In the 1000 mg/kg bw/day High dose group with 113 pups examined, 7 pups were found dead on PND0, with negative floating test (possibly stillborn), 2 were found dead on PND0, with positive pulmonary floating test, 1 was found dead on PND1, and 6 were cold, not suckled and/or cannibalized.
There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.
Body weight and body weight gain: There were no effects considered adverse on the offspring weight or weight gain at 62.5 and 250 mg/kg bw/day. When evaluated per litter basis or for all pups, the mean litter weights on PND 0 and 4, pups body weight and/or body weight gain evaluated on PND 0 and 4 showed no differences compared to controls in the F1 generation that could be considered toxicologically relevant or related to test item administration.
At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4. When evaluated for all pups, the mean body weights were statistically significant, minimally lower than control on PND0 and on PND4. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained.
Pathology evaluation
FOUND DEAD
F1 Generation: Twenty three pups were found dead between Days 0-3 post partum. Necropsy was performed on 4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups, respectively.
Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups. Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND 0 (no floating test was required on the remaining pups which died between PND1-3). White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 offsprings from the Mid and High Dose groups. A cause of death of these pups could not be determined.

TERMINAL (MAIN)
F1 Generation: Macroscopic Findings:
No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Dose descriptor:

NOEL

Generation:

F1

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

body weight and weight gain

Critical effects observed:

no

Reproductive effects observed:

no

Measured concentrations of the dosing solutions

Analytical occasion	Nominal concentration mg/mL	Measured concentrations with the 95% confidence intervals, mg/mL	Measure concentration in the percentage of the nominal
29 April 2011 (first week of treatment)	6.25	6.18±0.52	99
	25	25.7±0.66	103
	100	102±2.83	102
20 May 2011 (mid term)	6.25	6.09±0.19	97
	25	25.6±1.19	102
	100	107±1.03	107
09 June 2011 (last week of treatment)	6.25	6.07±0.12	97
	25	23.9±1.23	95
	100	99.8±5.38	100

 

Selected Clinical Chemistry Parameters, Main animals

		P (Parent) Male – Main				P (Parent) Female – Main
DOSE GROUP		ALT U/L	T-BILμmol/L	A/G	Bile acidsμmol/L	ALT U/L	T-BILμmol/L	A/G	Bile acidsμmol/L
Control (1)	MEAN	51.40	8.48	1.28	16.68	57	7	2	13
	SD	5.03	4.05	0.08	5.34	8	4	0	7
	n	5	5	5	5	5	5	5	5
62.5 mg/kg bw/day (2)	MEAN	48.00	6.36	1.22	15.96	55	8	1	21
	SD	10.02	2.88	0.11	2.18	7	2	0	4
	n	5	5	5	5	5	5	5	5
	±%	-7	-25	-5	-4	-2	14	-8	61
250 mg/kg bw/day (3)	MEAN	54.00	7.44	1.24	23.78	49	8	2	22
	SD	8.15	0.95	0.09	2.44	7	2	0	9
	n	5	5	5	5	5	5	5	5
	±%	5	-12	-3	43	-14	10	1	71
1000 mg/kg bw/day (4)	MEAN	41.00	23.20	1.06	34.20	44	19	1	36
	SD	3.94	6.47	0.05	9.59	8	3	0	10
	n	5	5	5	5	5	5	5	5
	±%	-20	174	-17	105	-23	166	-20	180
		*	*	**	**	*	**	**	**
		DN	U	DN	U	DN	DN	DN	DN

REMARKS:

±% = Percent Deviation Versus Control

NS = Not Significant

* = p<0.05

** = p<0.01

U = Mann-Whitney U – test Versus Control

DN = Duncan’s multiple range test

 

Selected Clinical Chemistry Parameters, Recovery Main animals

		Male – Recovery				Female – Recovery
DOSE GROUP		AST U/L	T-BILμmol/L	A/G	Bile acidsμmol/L	AST U/L	T-BILμmol/L	A/G	Bile acidsμmol/L
Control (1)	MEAN	142.60	4.92	0.94	9.44	122	6	1	8
	SD	15.13	0.77	0.05	1.97	28	1	0	2
	n	5	5	5	5	5	5	5	5
1000 mg/kg bw/day (4)	MEAN	117.00	6.70	1.10	9.04	98	7	1	8
	SD	21.68	0.35	0.10	2.15	14	0	0	2
	n	5	5	5	5	5	5	5	5
	±%	-18	36.2	17	-4	-20	20	13	6
		NS	**	*	NS	NS	*	NS	NS

REMARKS:

±% = Percent Deviation Versus Control

NS = Not Significant

* = p<0.05

** = p<0.01

T - test versus control

TABULAR SUMMARY REPORT

	Dose (mg/kg bw/day)
	Control	52.5	250	1000
Pairs started (N)	12	12	12	12
Surviving females showing evidence of copulation (N)	12/12	12/12	12/12	12/12
Females achieving pregnancy (N)	10/12	12/12	11/12	11/12
Conceiving days 1 – 5 (N)	12	12	11	12
Conceiving days 6 (N)	0	0	1	0
Pregnancy≤21 days (N)	0	0	0	0
Pregnancy = 22 days (N)	10	10	6	10
Pregnancy≥23 days (N)	0	2	5	1
Dams with live young born (N)	10/10	12/12	11/11	10/11
Dams with live young at PN4 (N)	10/10	12/12	11/11	9/11
Corpora lutea/dam (mean)	19.40	20.92	22.36	19.45
Implantations/dam (mean)	14.50	14.17	14.00	13.36
Live pups/dam at birth (mean)	12.80	12.67	12.45	9.64
Live pups/dam at day 4 (mean)	12.80	12.58	12.00	8.91
Sex ratio at birth (mean)	42.87	53.95	50.14	47.05
Sex ratio at PN4 (mean)	42.87	53.72	49.27	34.24
Litter weight at birth (mean)	90.1	85.9	91.9	65.7
Litter weight at day 4 (mean)	140.1	134.2	144.4	112.9
Pup weight at birth (litter mean)	6.90	6.74	7.18	6.54
Pup weight at day 4 (litter mean)	11.12	10.85	12.21	10.12
STRUCTURALLY ABNORMAL PUPS
Dams with 0/ Dams with live born	10/10	12/12	11/11	10/11
Dams with 1 or≥2	0	0	0	0
LOSS OF OFFSPRING
Pre-implantation (corpora lutea minus implantations)
Females with 0	2/11#	1/12	1/11	2/11@
Females with 1	2/11#	1/12	0/11	2/11@
Females with 2	1/11#	0/12	0/11	1/11@
Females with≥3	6/11#	10/12	10/11	6/11@
Pre-natal/post-implantation (implantation’s minus live births) (intrauterine)
Females with 0	1/10#	3/12	4/11	4/11@
Females with 1	4/10#	5/12	3/11	1/11@
Females with 2	3/10#	2/12	2/11	3/11@
Females with≥3	2/10#	2/12	2/11	3/11@
Post-natal (live births minus alive at post-natal day 4)
Females with 0	9/10	11/12	9/11	7/11@
Females with 1	1/10	1/12	1/11	2/11@
Females with 2	0/10	0/12	0/11	0/11@
Females with≥3	0/10	0/12	1/11	2/11@

# females 1502 had incidentally total pre-implantation mortality (14 corpora lutea and no implantation sites), excluded from statistics.

@ female 4501 has 21 corpora lueta, 14 implantation sites and 1 stillborn.

Conclusions:

In conclusion, under the conditions of this study, for reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250 mg/kg bw/day for F1 offspring, based on a possible test substance-related slight effect on pups survival and weight at 1000 mg/kg bw/day. As the effects seen are considered to be secondary effects caused by the inflammation of the stomach mucosa due to gavage effects, they were not considered to be relevant to humans. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study. The NOAEL is considered to be >= 1000 mg/kg bw/day.

Executive summary:

A study has been performed in accordance with the Study Plan, OECD Guidelines for Testing of Chemicals No. 422 (22^ndMarch 1996) and the Principles of Good Laboratory Practice. The purpose of this study was to obtain information on the possible toxic effects of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum.

In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5 Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56. Parameters measured during the study included signs of morbidity and mortality twice daily, daily or detailed weekly observation of clinical signs, neurological and ophthalmoscopic assessment, weekly body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery), all macroscopic findings (abnormalities) from all animals and kidney and stomach samples from the Mid and Low dose groups, based on the results observed at the initial evaluation of the High dose group.

Analysis of formulations (concentration, homogeneity) and assessment of test item stability in the vehicle indicated 1 day stability at room temperature and up to 7 days, while stored refrigerated at 2-8ºC in the dark, with a recovery range of 91%-105%, which lies within the acceptance range of 100 ± 10%. Concentration and homogeneity of formulations varied between 95% and 107% of the nominal concentrations. No test item was detected in the Control solution samples. These results were considered suitable for the study purposes.

No mortality or systemic adverse effects occurred during the study.

 

Mortality: There was no unscheduled mortality during the study.

Clinical observation: There were no clinical signs at up to and including 250 mg/kg bw/day or in the Control animals. At 1000 mg/kg bw/day, dark red discoloration of faeces and urine was noted. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration and of the urine, until completion of the recovery phase and termination. Slight to moderate dark pink discoloration of the skin was also noted in the High dose animals until completion of the study during the Recovery period.

These changes were ascribed to elimination of the test substance (red dye) or its metabolites through faeces or urine and an expected staining effect.

Neurological assessment: There were no toxicologically significant changes in the animal behaviour, general physical condition, in the reactions to different type of stimuli, grip strength or motor activity in the control or treated groups, at the evaluation performed towards the end of the treatment or Recovery periods.

Ophthalmology: No test item related changes compared to pre-treatment were noted during ophthalmoscopy examination of 5 male and 5 female Control and High dose Main animals during the last week of treatment prior to necropsy, thus no additional evaluation was required in other dose groups or Recovery animals.

Body weight and body weight gain: No adverse effects or test item related changes were noted on the mean body weight and body weight gain values following daily administration of the test substance at dose levels of up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, slightly lower than control body weight gain values were noted, attaining statistical significance when evaluated for the whole period of treatment between Days 0 and 28 in males or during the gestation period between GD0 and GD20 in females. In the Recovery animals, the body weights remained minimally lower than controls, however, no statistically significant differences occurred in the body weight gain of the Recovery animals.

Food consumption: There were no test item-related differences to Control in the mean daily food consumption in any test-item treated Main or Recovery group when compared to the Control.

Clinical pathology

Haematology: No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters evaluated in the Main animals at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, WBC increases considered possibly related to test substance administration were noted in the Main animals and recovery males.

Clinical chemistry: An apparent increase in total bilirubin T BIL was noted at 1000 mg/kg bw/day and bile acids at 250 and 1000 mg/kg bw/day. In addition, a apparently lower albumin/globulin ratio was seen at 1000 mg/kg bw/day. These effects were considered to be related to a spectral interference with the analytical method (colorimetric test) caused by the discolouration of the serum by the test item and not to reflect an adverse effect. The minimally lower ALT values at 1000 mg/kg bw/day are without toxicological and biological relevance.

Urinalysis: Pink urine was noted in all animals at 250 mg/kg bw/day group and red urine at 1000 mg/kg bw/day. These changes were ascribed to elimination of the test substance or its metabolites through urine and an expected staining effect.

Organ weights: There were no toxicologically relevant changes in the organ weight values, or effects considered adverse or related to test substance administration at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, minimal increases in the kidney weights were noted in both males and females, attaining statistical significance only for relative kidney weights in male rats.

Other absolute organ weights or relative to the body and/or brain weights were similar in the control and test item treated groups, or showed minor variations, ascribed to biological variability.

Macroscopic Findings: At 1000 mg/kg bw/day, pink discoloration was observed in numerous organs/tissues including the stomach, small and large intestine, kidneys, pancreas, testes, seminal vesicles, prostate, epididymides, vagina, uterus and cervix, mandibular salivary gland, mandibular and mesenteric lymph nodes, thymus, tongue, trachea, urinary bladder, eye, lacrimal gland, skin, oral, nasal and conjunctival mucosa, fatty (subcutaneous abdominal and thoracic) and connective tissues (subcutaneous abdominal, thoracic, intramuscular) at necropsy.

At 250 mg/kg bw/day, pink discoloration of the stomach, kidneys or trachea was also seen with low incidence in single animals.

These changes were considered due to the expected colouring effect of the test item (dye). All other macroscopic changes were regarded as incidental.

Microscopic Findings: Test item-related findings microscopically observed in the kidneys and stomach and were in correlation with the observations noted at necropsy. The changes were characterized as minimal to mild degeneration/regeneration of the cortical tubules in the kidneys of most animals at 1000 mg/kg bw/day and minimal tubular degeneration in single males at 250 mg/kg bw/day. These effects are considered to be caused by adaptive changes in order to absorb, catabolise and eliminate the dye substance and not cytotoxic effects on kidney cells. Furthermore, minimal to mild degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach were seen in animals dosed 1000 mg/kg bw/day and to a lesser degree in single animals at 250 mg/kg bw/day. These effects are considered to be caused by a high salt load administered directly to the stomach mucosa by gavage via a stomach tube and reflects a local irritation effect of and is hence without relevance for humans.

All effects seen, showed to be reversible in the recovery groups, as the effects seen in main animals in kidneys and stomach were found with a much lower incidence and severity in recovery animals and led to an almost complete regression of the lesions in the stomach.

There was no evidence of treatment-related histopathological findings in reproductive organs. Histopathological evaluation of the male gonads as well as testicular interstitial cell structure, the spermatogenic cells representing different phases of the development and differentiation of the spermatozoons were similar in Control and High Dose males. The follicular, luteal and interstitial compartments of the ovary as well as epithelial capsule and stroma were similar histological structure in both Control and High Dose females.

F1 Generation:

Mortality and clinical observations: The test substance administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level.

At 1000 mg/kg bw/day, the mean number of viable pups was minimally lower than control on PND0 and PND4, only attaining statistical significance when evaluated for the total number of pups. Hence, survival index was minimally lower on PND0 and PND4, although without attaining statistical significance.

The pups found dead and cannibalised were counted and sex determined if possible, but not further examined macroscopically.  No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. Findings such as cold to touch, occasional cannibalization or lack of suckling were noted with low incidence, within the physiological range expected in the population of Wistar rats and considered without toxicological significance, or to reflect a test item or adverse effect.

In the Control group (131 pups examined), 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), and another pup was found dead on PND2 without displaying any clinical signs prior to their death.

In the 62.5 mg/kg bw/day Low dose group with 155 pups examined, 3 pups were found dead on PND0, with negative pulmonary floating test (possibly stillborn), 4 were not suckled on PND0, then survived with no clinical signs until the scheduled necropsy on PND4, and another pup was found dead on PND3 without displaying any clinical signs prior to their death. 

In the 250 mg/kg bw/day Mid dose group, with 141 pups examined, 4 pups were found dead on PND0, with negative floating test (possibly stillborn), one was found dead and had positive pulmonary floating test, 5 were cannibalized and 2 cold or not suckled.

In the 1000 mg/kg bw/day High dose group with 113 pups examined, 7 pups were found dead on PND0, with negative floating test (possibly stillborn), 2 were found dead on PND0, with positive pulmonary floating test, 1 was found dead on PND1, and 6 were cold, not suckled and/or cannibalized.

There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.

Body weight and body weight gain: There were no effects considered adverse on the offspring weight or weight gain at 62.5 and 250 mg/kg bw/day. When evaluated per litter basis or for all pups, the mean litter weights on PND 0 and 4, pups body weight and/or body weight gain evaluated on PND 0 and 4 showed no differences compared to controls in the F1 generation that could be considered toxicologically relevant or related to test item administration.

At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control on both PND0 and PND4. When evaluated for all pups, the mean body weights were statistically significant, minimally lower than control on PND0 and on PND4. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained.

Pathology evaluation

FOUND DEAD

F1 Generation: Twenty three pups were found dead between Days 0-3 post partum. Necropsy was performed on 4, 4, 5 and 10 pups from the Control, Low, Mid and High Dose groups, respectively.

Negative floating test was observed in 3/4, 3/4, 4/5 and 7/10 animals from the Control, Low, Mid and High Dose groups.  Positive floating test was observed in 1/5 and 2/10 Mid and High dose of the pups found dead on PND 0 (no floating test was required on the remaining pups which died between PND1-3).  White liquid/creamy material in the stomach was present in 1/4, 1/4 and 1/10 pups from Control, Low and High Dose. The stomach was empty in 1/5 and 2/10 offsprings from the Mid and High Dose groups. A cause of death of these pups could not be determined.

TERMINAL (MAIN)

Macroscopic Findings:

No macroscopic changes were seen in F1 offspring generation euthanized and examined externally at scheduled termination on PND 4.

In conclusion, under the conditions of this study, the no observed adverse effect level (NOAEL) for parental/adult systemic toxicity is considered to be 250 mg/kg bw/day. For reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250  mg/kg bw/day for F1 offspring, based on a possible test substance-related slight effect on pups survival and weight at 1000 mg/kg bw/day. As the effects seen are considered to be secondary effects caused by the inflammation of the stomach mucosa due to gavage effects, they were not considered to be relevant to humans. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.  The NOAEL is considered to be >= 1000 mg/kg bw/day.

Reference 2

Endpoint:

one-generation reproductive toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 Jul 2001 to 12 Dec 2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 415 [One-Generation Reproduction Toxicity Study (before 9 October 2017)]

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.34 (One-Generation Reproduction Toxicity Test)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Winkelmann GmbH, Gartenstrasse 27, 33178 Borchen, Germany
- Age at study initiation: approximately 6 weeks
- Housing: single
- Diet (ad libitum): sniff R/M-Z (V1324)
- Water (ad libitum): tap
- Acclimation period: at least five days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 to 25
- Humidity (%): 30 to 70
- Air changes (per hr): 16-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 31 July 2001 To: 12 December 2001

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
The test item was dissolved daily in deionized water in concentrations of 12.5 mg/mL, 50 mg/mL and 200 mg/mL.

VEHICLE: deionized water
- Concentration in vehicle: 12.5 mg/mL, 50 mg/mL and 200 mg/mL
- Amount of vehicle: 5 mL/kg body weight

Details on mating procedure:

- M/F ratio per cage: 1:1 (1:2-mating was performed in three high dose females because of mortality in males)
- Length of cohabitation: three weeks
- Proof of pregnancy: vaginal plug or sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was individually caged

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

-

Duration of treatment / exposure:

Males : 10 weeks pre-mating, treatment continued during mating (ca. 3 weeks)
Females : 4 weeks pre-mating, treatment continued during mating (ca. 3 weeks) and during lactation until day 21 post partum

Frequency of treatment:

daily

Details on study schedule:

NA

Remarks:

Doses / Concentrations:
12.5 mg/mL, 50 mg/mL and 200 mg/mL
Basis:
nominal conc.

Remarks:

Doses / Concentrations:
62.5, 250 and 1000 mg/kg body weight per day
Basis:

No. of animals per sex per dose:

28

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose rationale was based on a subacute 28-day ora toxicity study with the test compound in rats, which did not show any adverse findings up to and including the limit dose of 1000 mg/kg body weight. Accordingly, dose levels of 0, 62.5, 250 and 1000 mg/kg body weight per day were selected for the present study.

Positive control:

NA

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: No data

BODY WEIGHT: Yes
- Time schedule for examinations: once weekly in both sexes during the pre-mating period
in females on day 0, 7, 14 and 21 during gestation and on day 0, 4, 7, 14 and 21 of lactation period.

FOOD CONSUMPTION: Food consumption was recorded together with the body weights (except the mating period for both genders, and except on day 4 of lactation for the females).

OTHER:
- Clinical Chemistry: 10 male and 10 female animals per group at scheduled sacrifice

Oestrous cyclicity (parental animals):

daily during mating period

Sperm parameters (parental animals):

Parameters examined in all P male parental generations: testis weight, epididymis weight, prostate weight, seminal vesicles weight
histopathology of testis, epididymis, prostate, seminal vesicles

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 8 pups/litter (4/sex/litter as nearly as possible); excess pups were killed and discarded.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring: number and sex of pups, stillbirths, live births, postnatal mortality, presence of gross anomalies, weight gain, viability, physical or behavioural abnormalities

GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was determined for pups born or found dead.

Postmortem examinations (parental animals):

SACRIFICE
- Male animals: All surviving animals were killed in the third week of the mating period
- Maternal animals: All surviving animals were killed on day 22 (or until day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday

GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations. All abnormal findings with special attention paid to the organs of the reproductive system were recorded

HISTOPATHOLOGY / ORGAN WEIGHTS
The following tissues or organs (or pieces of them) were preserved in Bouin's solution (testes) and formaldehyde solution and processed for histopathological investigations: Epididymides, Kidneys, Liver, Ovaries with oviducts, Pituitary, Prostate, Seminal vesicle, Testes, Uterus, Vagina, all other gross lesions.
Histopathological examinations were carried out of the control and high dose animals on these organs, as well as on on heart, spleen, lung, pancreas and gastro-intestinal tract from those animals with macroscopically visible changes, i.e., blueish colored pigmentation storage of the test compound.
The following organs were weighed: Epidymides, Kidneys, Liver, Ovaries, Pituitary, Prostate, Seminal vesicle, Testes, Uterus

OTHER: In order to investigate the cause of the dental findings in the late treatment period of the high dose animals, in total five affected incisors of the high dose males and five incisors of the control animals were analyzed for calcium and phosphorous content (two high dose and two control animals, data not presented, filed in the raw data). Secondly they were extended to fluoride, calcium and phosphorous content on the remaining 3 high dose incisors and control incisors.

Postmortem examinations (offspring):

SACRIFICE
- The F1 offspring not selected as parental animals were sacrificed at 4 days of age.
- Dead or moribund pups and pups killed at day 4 were examinated for defects.

- All surviving F1-animals were killed on day 22 (or until day 24, after weekends), after birth. Animals with necropsy date on weekend were killed the next weekday

Statistics:

All Parameters: The assumption of a monotonic dose-response relationship for all parameters justifies the restriction of the significance level to 5 percent (per parameter and sex), using the method of: HOTHORN L, LEHMACHER W.: A Simple Testing Procedure "Control versus k Treatments" for One-sided Ordered Alternatives, with Application in Toxicology, Biom. J. 33, 179-189, Akademie Verlag
Bodyweights: The changes of parameter values compared to the treatment-free baseline values are analyzed with the t-Test:
HARTUNG J., ELPERT B., KLÖSENER K. H., Lehr- und Handbuch der angewandten
Statistik (1989), R. Oldenbourg Verlag, München
Clinical Pathology Data: Wilcoxon's Test: HOLLANDER M., WOLFE, D. A:, Nonparametric statistical methods
Organ weights (absolute): t-Test
Organ weights (relative to bodyweight): Wilcoxon's Test

Reproductive indices:

Copulatory index (%): Number of sperm positive females x 100 / Number of mated females
Fertility index - Males (%): Number of fertile males x 100 / Number of mated males
Fertility index - Females (%): Number of pregnant females x 100 / Number of mated females
Gestation index (%): Number of females with viable pups x 100 / Number of pregnant females
Sex ratio: (Number of pups examined - Number of males (females)) x 100 / Number of pups examined

Offspring viability indices:

Intra uterine mortality: (Number of implantations - Number of newborns) x 100 / Number of implantations
Total mortality: (Number of implantations - Number of viable pups) x 100 / Number of newborns
Viability index (%): Number of viable pups on day 4 (7, 14, 21) x 100 / Number of viable pups on day 0 (4, 7, 14)
Lactation index (%): Number of viable pups on day 21 x 100 / Number of viable pups on day 0 of lactation
Weaning index (%)

Clinical signs:

effects observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not examined

Reproductive performance:

effects observed, treatment-related

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
There were no intercurrent deaths in the control-, low- and mid-dose group animals. In the high dose group (1000 mg/kg body weight), 1 male and 1 female animal was found dead early with unknown pathogenesis. In addition, further 6/28 males and 4/27 females were found dead or had to be killed on human grounds from study week 6-7 onwards. Animal No. 128 was killed by mistake on day 51.

Behavior and health status was not affected in low- and mid-dose group animals with the exception of 4 males exhibiting broken off incisors from week 6 onwards. Several high-dose animals had broken off- and white-discolored incisors, generally starting to occur from study week 6 onwards. Some of those animals developped general clinical signs (stilted gait, hypoactivity, coat bristling, irregular respiration, respiratory sounds diarrhea, snout encrusted blood colored or swollen etc.) and some of those ended up in a general poor condition.

Blue discolored feces were observed in all P-generation male and female animals of the 250 and 1000 mg/kg body weight groups.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
Body weight gain was significantly decreased for high dose animals that had dental problems.
Those high dose animals that were found dead from week 6 onwards or were killed on human grounds did not take up food a few days before death. Mean absolute food consumption in all remaining animals of the high dose group (1000 mg/kg) was slightly to moderately decrerased. This was in line with the lower body weight gains recorded for this group. Hence, relative food consumption was generally comparable in all groups throughout the study, except for high dose females, who exhibited a significant decrease of relative food consumption during the lactation period

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
There were no test item related differences in the estrous cycle.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
Due to the lower food consumption resulting from broken-off incisors the pregnancy index was lower in high-dose females
The mean number of implantations counted, mean live pups/litter, birth index were comparable in all groups. In addition, supernumerary
implantation sites, percentage of implantations, were not influenced by administration of the test compound.
Mean gestation length was comparable in all groups.

ORGAN WEIGHTS
In high dose males, liver, kidney, testes, epididymides, prostate and seminal vesicles weight were slightly lower, with statistical significance, which was due to the reduction of terminal body weight and hence, not related to target organ toxicity.
The same applied for high dose females, where liver, kidney and uterus weight was slightly lower, with statistical significance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Males and females from the mid-dose group exhibited kidneys with dark brown discolorations. In addition, the kidneys of one male in this groups was bluish discolored.
The main relevant findings were discolorations in several organs animals of the high dose group. Further major alterations were white discolored or broken incisors in nearly all animals of this group.

HISTOPATHOLOGY (PARENTAL ANIMALS)
Histopathological findings in parental animals of the high-dose group at terminal killing revealed intratubular pigment in kidneys in 10 male and 5 female animals. Single animals exhibited degenerations or necrosis of tubular cells. Increased number of necrotic/apoptotic cells were found in the liver. Mixed cellular infiltrations in the submucosal area of the stomach were found particular in males.

Dose descriptor:

NOAEL

Remarks:

General health

Effect level:

62.5 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: broken-off incisors (fluorose) from 0.3% fluor impurity

Dose descriptor:

NOAEL

Remarks:

Reproductive performance

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: All effects observed were due to broken-off incisors resulting in lower food consumption and a lower pregnancy index. This effect was due to fluorosis of the rats' teeth caused by the 0.3% fluor impurity

Clinical signs:

no effects observed

Mortality / viability:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Sexual maturation:

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

no effects observed

Histopathological findings:

not examined

VIABILITY (OFFSPRING)
no effects

CLINICAL SIGNS (OFFSPRING)
no effects

BODY WEIGHT (OFFSPRING)
Mean body weight of live pups during lactation was significantly decreased in the high dose offspring (1000 mg/kg bw.) from day 14, post partum onwards. Mean body weight was not affected in any other group

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

1 000 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: effects observed on body weight of high-dose pups were due to broken-off incisors in dams resulting in lower food consumption. This effect was due to fluorosis of the rats' teeth caused by the 0.3% fluor impurity

Reproductive effects observed:

not specified

This study is read across to the structural analogue 02, which is considered to be structurally equivalent to the substance to be registered. The read-across justification can be found in section 13.

Conclusions:

Daily oral administration of the test substance to rats during the premating, mating, gestation and lactation period at dose levels of 62.5 or 250 mg/kg body weight did not affect food consumption, body weight development, male or female mating/reproductive performance, fertility, gestation length as well as development of their progenity.
Daily oral administrations of 1000 mg/kg body weight (high-dose group) were well tolerated in rats within the first 5 weeks of treatment, but thereafter, from week 6 onwards, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). This finding was time-dependent, with a threshold dose of 250 mg/kg body weight for males, and could be related to the fluoride impurity (0.3%) of this batch tested.
Although there was marked pigment storage of the test compound in several organs, there was no clear functional or histopathological correlate that could be related to compound-induced systemic toxicity and/or specific reproductive toxicity. Impairment of reproduction and fertility at high dose parental animals was primarily the result of severe dental problems.
In the presence of severe dental problems at 1000 mg/kg bw and threshold dose of 250 mg/kg bw for this finding, there was no evidence of selective reproductive toxicity in rats.

Executive summary:

The present study was conducted in order to determine the effects of the test substance on reproduction when administered orally by gavage to male and female Sprague Dawley rats during pre-mating, mating, gestation and lactation.

Groups of 28 male and 28 (27 in the high-dose group) female Spraque Dawley rats received the test substance orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight for a period of 10 weeks (males) and 4 weeks (females), prior to mating. Dosing of males was continued during the whole mating period until sacrifice (approx. week 11 - 13 of the study). Treatment of mated females was continued until day 21 after littering. The dosing volume was 5 mL/kg, corresponding to concentrations of 0, 12.5, 50 and 200 mg/mL. At start of the study, the animals were 5-9 weeks of age with mean body weights of 240 g for males, and 206 g for females.

Behavior and state of health were observed daily in all groups. Body weight development and food consumption were recorded throughout the study in females, and during pre-mating period in males. After the mating period the males were killed and necropsied. The dams were allowed to litter and rear their progeny to the stage of weaning. Growth, development and behavior of the

progeny were assessed during lactation. The dams as well as surviving pups were killed on day 22-24 post partum. Animals scheduled for necropsy on weekend were killed the next weekday.

At the time of sacrifice or death during the study the animals of the P generation were examined for macroscopically visible abnormalities. The main organs were weighed and the organ to body weight ratios calculated. Special attention was paid to the organs of the reproductive system. Histopathology of listed organs was performed in case of macroscopic visible changes. Moreover,

dental mineral analyses (fluoride, calcium and phosphorus) were performed externally. In addition, clinical chemistry investigations, in particular for serum electrolytes, were performed in 10 animals per sex and group as amended to the protocol.

Body weights, food consumption, clinical chemistry data, absolute and relative organ weights and litter parameters were analyzed with the aid of a statistical program to show differences compared to the controls.

RESULTS

High-dose group (1000 mg/kg body weight): There were 7 males and five females that were found dead or killed on humane grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. In addition, one female was killed with dead pups at birth, another one with live pups was killed on lactation day 6 due to

inability to suckle them properly. Teeth trimming were carried out to insure food uptake during mating procedures for as many animals concerned as possible. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length, (ca. 23.0 days), was not affected. Because of these

unscheduled deaths the number of pregnancies was markedly reduced (12 cf./22 of control). The absolute number of females at term with live pups was reduced (11 cf./21 of control), with lower absolute number of implantations. One dam had dead pups only. However, relative numbers of live pups, the mean number of implatations and birth index, was not adversely affected when

related to the number of females at term with live pups. During early lactation, 4/11 females had to be killed on humane grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offpring up to the end of the lactation period, however, mean pup body weight gains were significantly decreased from day 14 post partum up to the end of the study. Mean viability index, weaning index, survival rate at day 21 was not affected. There was 1 unreared litter recorded for this group. The pups did not show any macroscopically visible abnormalities.

Apart from significantly decreased total bilirubin levels, clinical pathology was unobstrusive, also with regard to serum electrolytes. Anatomic pathology revealed severe dental lesions (broken off, deformed and white discolored incisors), which were confirmed to contain a 3-fold concentration of fluoride. Fluoride (0.3%) was identified as an impurity of the test compound batch, tested in

this study. Massive bluish discolorations of the whole carcasse and in several inner organs were also detected at necropsy. Microscopy confirmed intratubular pigment storage in the kidneys, increased number of necrotic/apoptotic cells in the liver as a histopathological correlate of clinical starvation, and mixed cellular infiltrations in the submucosal area of the stomach, probably as a

result of irritating effects of the test compound. There were no selective changes in sexual organs that could be related to selective reproductive toxicity in these dose group animals, nor were there any correlates of target organ toxicity.

Mid-dose group (250 mg/kg body weight): There were no premature deaths. No compound related clinical findings were recorded for the females. Four males had broken-off incisors during the late treatment period (weeks 6 -12). However, food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their

offspring remained unaffected by administration of the test compound. Clinical chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were generally unobstrusive, apart from pigment storage (dark brownish/or bluish discolorations) in the kidneys.

Low-dose group (62.5 mg/kg body weight): There were no premature deaths. No compound-related clinical signs were recorded in the P-generation male and female animals. Food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their offspring remained unaffected by administration of the test compound. Clinical Chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were unobstrusive.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A modified one-generation study was conducted in order to determine the effects of the test substance on reproduction when administered orally by gavage to male and female Sprague Dawley rats during pre-mating, mating, gestation and lactation. Groups of 28 male and 28 (27 in the high-dose group) female Sprague Dawley rats received the test substance orally once daily at dose levels of 0, 62.5, 250 or 1000 mg/kg body weight for a period of 10 weeks (males) and 4 weeks (females), prior to mating. Dosing of males was continued during the whole mating period until sacrifice (approx. week 11 - 13 of the study). Treatment of mated females was continued until day 21 after littering. The dosing volume was 5 mL/kg, corresponding to concentrations of 0, 12.5, 50 and 200 mg/mL. At start of the study, the animals were 5-9 weeks of age with mean body weights of 240 g for males, and 206 g for females.

Behaviour and state of health were observed daily in all groups. Body weight development and food consumption were recorded throughout the study in females, and during pre-mating period in males. After the mating period the males were killed and necropsied. The dams were allowed to litter and rear their progeny to the stage of weaning. Growth, development and behaviour of the progeny were assessed during lactation. The dams as well as surviving pups were killed on day 22-24 post partum. Animals scheduled for necropsy on weekend were killed the next weekday.

At the time of sacrifice or death during the study the animals of the P generation were examined for macroscopically visible abnormalities. The main organs were weighed and the organ to body weight ratios calculated. Special attention was paid to the organs of the reproductive system. Histopathology of listed organs was performed in case of macroscopic visible changes. Moreover, dental mineral analyses (fluoride, calcium and phosphorus) were performed externally. In addition, clinical chemistry investigations, in particular for serum electrolytes, were performed in 10 animals per sex and group as amended to the protocol.

Body weights, food consumption, clinical chemistry data, absolute and relative organ weights and litter parameters were analysed with the aid of a statistical program to show differences compared to the controls.

High-dose group (1000 mg/kg body weight): There were 7 males and five females that were found dead or killed on humane grounds due to starvation and bad general health condition as a cause of broken off incisors and subsequent disability of food uptake. In addition, one female was killed with dead pups at birth, another one with live pups was killed on lactation day 6 due to inability to suckle them properly. Teeth trimming were carried out to ensure food uptake during mating procedures for as many animals concerned as possible. Mean food consumption and body weight development was decreased during pre-mating (males) and during the lactation period (surviving females). Mean gestation length, (ca. 23.0 days), was not affected. Because of these unscheduled deaths the number of pregnancies was markedly reduced (12 cf./22 of control). The absolute number of females at term with live pups was reduced (11 cf./21 of control), with lower absolute number of implantations. One dam had dead pups only. However, relative numbers of live pups, the mean number of implantations and birth index, was not adversely affected when related to the number of females at term with live pups. During early lactation, 4/11 females had to be killed on humane grounds, as they were not able to rear their healthy offspring due to starvation. The remaining 7 females reared their healthy offspring up to the end of the lactation period, however, mean pup body weight gains were significantly decreased from day 14 post partum up to the end of the study. Mean viability index, weaning index, survival rate at day 21 was not affected. There was 1 non-reared litter recorded for this group. The pups did not show any macroscopically visible abnormalities.

Apart from significantly decreased total bilirubin levels, clinical pathology was unobtrusive, also with regard to serum electrolytes. Anatomic pathology revealed severe dental lesions (broken off, deformed and white discoloured incisors), which were confirmed to contain a 3-fold concentration of fluoride. Fluoride (0.3%) was identified as an impurity of the test compound batch, tested in this study. Massive bluish discolorations of the whole carcass and in several inner organs were also detected at necropsy. Microscopy confirmed intratubular pigment storage in the kidneys, increased number of necrotic/apoptotic cells in the liver as a histopathological correlate of clinical starvation, and mixed cellular infiltrations in the submucosal area of the stomach, probably as a result of irritating effects of the high salt content of the test compound. There were no selective changes in sexual organs that could be related to selective reproductive toxicity in these dose group animals, nor were there any correlates of target organ toxicity.

Mid-dose group (250 mg/kg body weight): There were no premature deaths. No compound related clinical findings were recorded for the females. Four males had broken-off incisors during the late treatment period (weeks 6 -12). However, food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their offspring remained unaffected by administration of the test compound. Clinical chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were generally unobtrusive, apart from pigment storage (dark brownish/or bluish discolorations) in the kidneys.

Low-dose group (62.5 mg/kg body weight): There were no premature deaths. No compound-related clinical signs were recorded in the P-generation male and female animals. Food consumption, body weight development, mating and reproductive performance, fertility, mean gestation length, rearing and development of their offspring remained unaffected by administration of the test compound. Clinical Chemistry, as well as anatomic pathology (necropsy, organ weights, histopathology) in particular of the sexual organs were unobtrusive.

In contrary to Reactive Red 198, the structural analogue contains a mono-chloro, di-fluoro-pyrimidine group. This led to a higher content (0.3%) of fluoride in the test substance resulting in fragility of the incisors and a very low food uptake in high-dose dams. Consequently, the survival rate in the high-dose group was rather low. Daily oral administrations of 1000 mg/kg body weight were well tolerated in rats within the first 5 weeks of treatment, but thereafter, from week 6 onwards, caused mortality due to dental lesions with subsequent disability of food uptake and starvation (clinical picture of dental fluorosis). Consequently, due to the fact that no fluoride is contained in Reactive Red 198, a NOAEL of 1000 mg/kg body weight is expected for the test substance for parental reproductive performance and their offspring.

 

A study has been performed in accordance with the OECD Guideline for Testing of Chemicals No. 422 (22nd March 1996) in the Rat, to obtain information on the possible toxic effects of the test item following repeated daily administration by oral gavage to Wistar rats. Reversibility of any treatment-related changes was evaluated following a 14-day Recovery period. The study also comprised a reproductive/developmental toxicity screening test, intended to provide initial information on possible effects on male and female reproductive performance such as gonadal function, mating behaviour, conception, pregnancy, parturition and also on the development of the F1 offspring from conception to Day 4 post-partum. In the Main Groups, male and female Wistar rats were treated for 2 weeks pre-mating, then during the mating/postmating period, males for 28 days and females throughout gestation period and up to and including postpartum/lactation Day PPD5. Additional 5 male and 5 female rats from the Control and High dose Recovery Groups 1 and 4 scheduled for follow-up observations were not mated, but treated up to the first scheduled euthanasia of the Main dams (Day 41), then kept at least for 14 days without treatment to detect delayed occurrence, or persistence of, or recovery from toxic effects, and subjected to necropsy with macroscopic examination on Day 56.

Parameters measured during the study included signs of morbidity and mortality, detailed observation of clinical signs, neurological and ophthalmoscopic assessment, body weight and food consumption, and clinical pathology evaluation, including haematology, coagulation, clinical chemistry and urinalysis. In addition, the reproductive performance and indices, pregnancy, parturition and postpartum/lactation period were monitored in the adult Main animals, and viability, clinical signs and development were evaluated in their F1 offspring until PND4. At termination, necropsy with macroscopic examination was performed. Weights of selected organs were recorded and representative tissues/organs were sampled and preserved in appropriate fixatives from the adult animals. For the adult animals, detailed histological examination was performed on the selected list of retained organs in the Control and High dose groups (Main and Recovery), all macroscopic findings (abnormalities) from all animals and kidney and stomach samples from the Mid and Low dose Main groups.

No mortality or systemic adverse effects occurred during the study. During the treatment period, slight to moderate dark pink discoloration of the skin was noted in the High dose animals after Day 12 of treatment, until the completion of the study up to Day 55 during the Recovery period. Furthermore, dark red discoloration of the faeces was noted at 1000 mg/kg bw/day from Day 1, and of the urine, from Day 2 onwards. The discoloration of the faeces persisted in the 1000 mg/kg bw/day Recovery animals for up to 3 days after the last dose administration (up to Day 44) and of the urine, until completion of the recovery phase and termination. These changes were ascribed to elimination of the test item or its metabolites through faeces or urine and an expected staining effect. There were no test item-related differences to Control in the mean daily food consumption.

No test item related effects, or changes considered toxicologically significant were noted in the haematology parameters at up to and including 250 mg/kg bw/day dose levels. At 1000 mg/kg bw/day, a slight increase in WBC was noted.

A slight increase in bile acids and a lower albumin/globulin ratio and ALT levels were recorded in the High dose animals. At urinalysis, pink urine was noted animals evaluated of the 250 mg/kg bw/day group and red urine, in the 1000 mg/kg bw/day animals. These changes were ascribed to elimination of the test item or its metabolites through urine (urine collection in metabolic cages) and an expected staining effect.

There were no statistically significant differences between the Control and test item-treated groups with regard to reproductive ability or in the mating or gestation indices, or effects considered adverse or toxicologically significant in correlation with the test item administration. Test item administration was considered to have no impact on the duration of the mating period or  on the duration of pregnancy. No abnormalities in the gestation outcome ascribed to the treatment were observed. All the parturitions were normal. The number of corpora lutea and implantation sites were comparable in control and treated groups.

There were no statistically significant differences or effects that could be ascribed to treatment on the pre/post-implantation, post-natal or total mortality values (%) at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, the intrauterine mortality, post-natal mortality and subsequently the total mortality were higher than control. However, only the post-natal mortality attained statistical significance. These effects were considered to be possibly related to the test item administration at the High dose level, although a role of the individual variability cannot be excluded. The test item administered to parental generation under the conditions of this study did not lead to any adverse effects considered related to treatment or toxicologically significant in the F1 generation at up to and including 250 mg/kg bw/day dose level. At 1000 mg/kg bw/day, the mean number of viable pups was slightly lower than in the control, attaining statistical significance when evaluated for the total number of pups. These results correlated with the higher post-natal and total mortality observed in the High dose dams. Survival index slightly lower than control in the High dose group, although without attaining statistical significance.

No external abnormalities ascribed to treatment were detected at the clinical or external macroscopic examinations of the pups. There were no adverse effects, or effects considered related to test item administration on the sex ratios, and no statistical or toxicological significant differences to control were noted.

There were no effects considered adverse on the offspring weight or weight gain at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, when evaluated per litter basis, there were no statistically significant differences to control, however, the mean litter weights were lower than control. As the differences were minor and the dose response was not clear, a test item related adverse effect on the offspring development following administration of 1000 mg/kg bw/day to the parental generation in the conditions of this study could not be ascertained.

At pathology evaluation, pink/red discoloration of numerous organs/tissues was observed at necropsy in the High dose group. In the Mid dose group, discoloration was only observed in trachea, stomach and kidneys in single animals. Histopathologically, treatment-related changes were observed in the kidney and stomach of High and Mid dose rats. The severity and/or incidence of these changes indicated a relationship to dose. Degeneration/regeneration of the cortical tubules in the kidneys were present in High dose rats and Mid dose males. Degeneration/regeneration of epithelial cells of the glandular mucosa in the stomach accompanied by a minimal to mild inflammatory influx in the submucosa, were recorded in rats at dose levels of 250 and 1000 mg/kg bw/day. There was clear dose responded decrease in the severity when compared High Dose (1000 mg/kg bw/day) and Mid Dose (250 mg/kg bw/day) animals. A minimal epithelial degeneration/regeneration without influx of inflammatory cells was observed in two Low Dose males.

After a 14 day of recovery period, a residual effect of treatment was microscopically seen in the kidneys and stomach in the High Dose groups. In the kidneys, decreased severity of the test item-related microscopic alterations from mild to minimal was more evident in the males compared to the females. Cessation of treatment for 14 days resulted in almost completed regression of the test item-related lesions in the stomach. The decrease of the severity from mild to minimal was observed in all High Dose animals. Also, mitotic index has been decreased and inflammatory influx as well as formation of the submucosal lymphoid follicles, were no more observed. Pups were found dead, intact and were subjected to necropsy between Days 0-3 post partum. A cause of death of these pups could not be determined. In the Main animals, there were no toxicologically relevant changes in the organ weight values, or effects considered adverse or test item related at up to and including 250 mg/kg bw/day. At 1000 mg/kg bw/day, the absolute kidneys weight was minimally higher than control in the High dose males, without attaining statistical significance. No similar effects were observed in the High dose females, however, kidneys weights were higher than control in both male and female Recovery animals. In view of the microscopic findings recorded, these kidneys weight variations were considered to reflect a possible test item re-absorption at 1000 mg/kg bw/day.

In conclusion, under the conditions of this study, for reproductive toxicity, the no observed effect level (NOEL) for parental/adult effects is considered to be 1000 mg/kg bw/day for the males and 250 mg/kg bw/day for the females, due to the higher intrauterine, postnatal and total foetal mortality at 1000 mg/kg bw/day, correlated with a NOEL of 250  mg/kg bw/day for F1 offspring, based on a possible test substance-related slight effect on pups survival and weight at 1000 mg/kg bw/day. As the effects seen are considered to be secondary effects caused by the inflammation of the stomach mucosa due to gavage effects, they were not considered to be relevant to humans. No structural adverse effects were observed in the surviving pups at up to and including 1000 mg/kg bw/day, under the conditions of this study.  The NOAEL is considered to be >= 1000 mg/kg bw/day.

Effects on developmental toxicity

Description of key information

No teratogenic effect was observed.

Link to relevant study records

Reference

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 September to 25 November 1993

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 414 (Prenatal Developmental Toxicity Study)

GLP compliance:

yes

Limit test:

yes

Species:

rat

Strain:

Wistar

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Hoechst AG
- Age at study initiation: 65 to 75 days
- Weight at study initiation: 193.1 +/- 14.2
- Fasting period before study: NA
- Housing: single
- Diet: Altromin 1310 ad libitum
- Water: tap water ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 22°C
- Humidity (%): 48 to 61%
- Air changes (per hr): 16 to 20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14. Sep To: 25. Nov 1993

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS:
- Frequency of preparation: daily
- Administration: within 3 hours after preparation

VEHICLE
- Concentration in vehicle: 200 mg/kg nominal
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

not specified

Details on analytical verification of doses or concentrations:

-

Details on mating procedure:

- Impregnation procedure: cohoused
- M/F ratio per cage: 1:1
- Length of cohabitation: over night (3:30 pm to 7:30 am next day)
- Verification of same strain and source of both sexes: yes - own breeding facility
- Proof of pregnancy: sperm in vaginal smear, referred to as day 1 of pregnancy

Duration of treatment / exposure:

7. - 16. day of pregnancy

Frequency of treatment:

daily

Duration of test:

cesarean section on Day 21 of pregnancy

No. of animals per sex per dose:

20 to 24 mated females

Control animals:

yes

Details on study design:

- Dose selection rationale: test item was tolerated in the acute and subacute studies without adverse effects

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE : Yes
- Time schedule for examinations: weekly
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/100 g body weight/day: Yes

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 21
- Organs examined: all organs examined macroscopically
uterus - live and dead fetuses, resorption sites, placentas
ovaries - corpora lutea

OTHER:
- diameter of conceptuses undergoing resorption
- placenta weights
- presence of iron in uterus walls with ammonium sulphide to detect invisible implantation sites

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Placenta weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Body weight: Yes: all per litter
- Crown-rump length: Yes: all per litter
- Soft tissue examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: No data

Statistics:

comparison to actual control group and historical controls
MANOVA: body weight development, fetal weight, placental weight
PURI&SEN rank order test: food intake
Mantel-Haenszel's chi-squared test: live fetuses, intrauterine fetal death, number of implants, number of corpora lutea
multivariate analysis of variance: litter means of fetal weights, crown-rump length, placental weights
Fisher test: autopsy findings, body cross-sections, skelettal examination

Dams which had no live fetuses were excluded from the calculation of mean values and statistical evaluation

Indices:

No data

Historical control data:

Yes

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food efficiency:

no effects observed

Gross pathological findings:

no effects observed

Details on results:

Clinical examinations
The dams of the compound group and the control dams showed no impairment to behaviour and General condition and survived until the scheduled study end. All dams in the compound group excreted black-discoloured faeces on at least eight days during and after the treatment period, mostly, however, in the period from day 8 - 19 of pregnancy. Blue-discoloured urine was voided by three dams from days 8 - 10 or days 8 - 11 of pregnancy. One dam from the compound group displayed pallor from day 8 - 11 of pregnancy. Compound solution escaped from the mouths of two dams after the 3rd and/or 4th administration, presumably due to the dosing method. The amounts, however, were small and without relevance for assessing the test compound. Local abdominal hair loss was noted in another dam from the compound group on days 20 and 21 of pregnancy.
The administration of C. I. Reactive Black 5 had no influence on the feed consumption of the dams. The dams consumed comparable amounts of feed to those of the controls both during and after the treatment period.
The body weight gain of the dams treated with C. I. Reactive Black 5 was likewise unaffected. The dams treated with the test compound gained in weight to the same extent as the controls.
Autopsy of the dams
The concluding autopsy revealed a marked dilatation of one renal pelvis in one dam each from the compound and control groups. No further alterations to the internal organs could be seen in the macroscopic examination.

Number of abortions:

no effects observed

Pre- and post-implantation loss:

no effects observed

Total litter losses by resorption:

no effects observed

Early or late resorptions:

no effects observed

Dead fetuses:

no effects observed

Changes in pregnancy duration:

no effects observed

Changes in number of pregnant:

no effects observed

Details on maternal toxic effects:

Examinations after caesarean section
All 20 dams in the compound group and 19 dams in the control group carried live conceptuses to term and were delivered by caesarean section. One control dam had no live foetuses, but merely 8 empty implantation sites in the uterus, indicating early embryonic death shortly after implantation. In this animal, the corpora lutea were quite small and could not therefore be determined exactly. The number of corpora lutea, which provides information on the number of ovulations prior to start of treatment, and the number of implants and live foetuses in the remaining dams of the compound group did not differ markedly from those of the control animals and lay in the range of our previous control values.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Fetal body weight changes:

no effects observed

Reduction in number of live offspring:

no effects observed

Changes in sex ratio:

no effects observed

Changes in litter size and weights:

no effects observed

External malformations:

no effects observed

Skeletal malformations:

no effects observed

Visceral malformations:

no effects observed

Details on embryotoxic / teratogenic effects:

Examination of foetuses
The live foetuses delivered in the compound group were normally developed. Their body weights and lengths did not differ from those of the control foetuses and lay in the range of our previous control values. The number of live foetuses weighing less than 3.0 g and thus qualifying according to our definition as slightly retarded or retarded was even lower than that of the control group. The sex ratio was relatively balanced in both groups: the males predominated slightly in the control group and the females predominated slightly in the compound group.
Dead embryofoetal primordia were found in both groups. They consisted exclusively of conceptuses undergoing resorption. These had died at a relatively early stage and exhibited diameters of up to 6.3 mm (compound group) or 6.6 mm (control group). Dead foetuses were not found in either of the two groups. The number of dead embryofoetal primordia in the dams of the compound group was low and did not differ from that of the control dams.
The placentas of the foetuses in the compound group were unremarkable macroscopically and in respect of their weights, which corresponded to those of the control group.
The findings observed at the morphological examination of the foetuses show that no malformations appeared in any foetus in the compound group. One control foetus exhibited a right-sided microphthalmia.
The autopsy and body cross-section examinations revealed blood in the pericardium or abdominal cavity, completely fused or bipartite pulmonary lobes or a haematoma in a liver lobe as well as a distention of one or both renal pelves or one ureter in individual or some foetuses. Furthermore, one foetus in the compound group displayed blood in the vicinity of the right kidney. Findings confined to individual foetuses in the control group were: blood in the thoracic cavity or in the right kidney and a stomach which was relatively large and tautly filled with fluid.
The skeletons of the foetuses in the compound group were at roughly the same stage of development as those of the control foetuses. Their stage of ossification corresponded to the 21st day of pregnancy. The number of foetuses in the substance-treated group displaying a poor or missing ossification of sternebrae, 5th metacarpal bone and the ossification of fewer than two caudal vertebral centres did not differ markedly from that of the control foetuses. The number of foetuses in which individual cranial bones were not-yet or only poorly ossified was, significantly increased compared to the simultaneous control group but still lay within the range of previous control values (maximum frequency 61.5 %).
Apart from these findings, individual or several foetuses in both groups exhibited longitudinally displaced, dislocated, fragmented sternebrae or waved and/or thickened ribs while numerous foetuses displayed an additional short rib anlage, uni- or bilaterally, at the 1st lumbar vertebra. In individual foetuses of the compound group, a dysplastic 10th or 11th and 12th thoracic vertebral centre or a 14th thoracic vertebra with a short, analogous rib pair were observed. Three foetuses in the control group showed an additional short rib, left- or both-sided, at the 7th cervical vertebra.

Key result

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Remarks on result:

not determinable due to absence of adverse toxic effects

Key result

Abnormalities:

no effects observed

Key result

Developmental effects observed:

no

Conclusions:

No adverse effects were observed after daily administration of 1000 mg/kg/day in dams or their fetuses.
Maternal NOEL: 1000 mg/kg bw/day
Fetal NOEL: 1000 mg/kg bw/day

Executive summary:

In this limit test, the test substance, dissolved in distilled water, was administered orally by stomach tube in a single daily dose of 1000 mg/kg body weight to a group of 20 pregnant female Wistar rats from the 7th - 16th day of pregnancy. A simultaneous control group of the same size received the vehicle without test compound. On the 21st day of pregnancy, the dams were killed and delivered by caesarean section. The foetuses delivered by caesarean section were then examined morphologically for developmental disorders.

The study showed that the repeated oral administration of the test substance, at a dose of 1000 mg/kg body weight in the sensitive phase of organogenesis for the conceptuses, did not lead to any impairment of the general physical condition of the dams or impaired intrauterine development of conceptuses.

The morphological examination of the foetuses with regard to stage of development, outwardly detectable anomalies as well as anomalies of the internal organs and the skeleton showed no indication of an embryotoxic or teratogenic effect of the compound. The findings observed are to be regarded as spontaneous in origin.

On the basis of the results of this limit test, the “no observed adverse effect level” in rats following oral administration lies at 1000 mg/kg body weight/day or above with regard to maternal and embryofoetal toxicity and teratogenicity.

No teratogenic effect was observed.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a supporting study with a structural analogue, the test substance, dissolved in distilled water, was administered orally by stomach tube in a single daily dose of 1000 mg/kg body weight to a group of 20 pregnant female Wistar rats from the 7th - 16th day of pregnancy. A simultaneous control group of the same size received the vehicle without test compound. On the 21st day of pregnancy, the dams were killed and delivered by caesarean section. The foetuses delivered by caesarean section were then examined morphologically for developmental disorders. The study showed that the repeated oral administration of the test substance, at a dose of 1000 mg/kg body weight in the sensitive phase of organogenesis for the conceptuses, did not lead to any impairment of the general physical condition of the dams or impaired intrauterine development of conceptuses.

The morphological examination of the foetuses with regard to stage of development, outwardly detectable anomalies as well as anomalies of the internal organs and the skeleton showed no indication of an embryotoxic or teratogenic effect of the compound. The findings observed are to be regarded as spontaneous in origin.

On the basis of the results of this limit test, the “no observed adverse effect level” in rats following oral administration lies at >= 1000 mg/kg body weight with regard to maternal and embryofoetal toxicity and teratogenicity.

Justification for classification or non-classification

No classification necessary