Registration Dossier
Registration Dossier
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EC number: 226-971-2 | CAS number: 5580-58-5
Endpoint summary
Administrative data
Key value for chemical safety assessment
Effects on fertility
Description of key information
'Yellwo disazo condensation pigments' are too large and insoluble for significant systemic uptake. Therefore, no hazard for toxicity to reproduction is possible. No adverse effects were observed in the screening study at the limit dose (OECD 421/422) for Pigment Yellos 93 (CAS 5580-57-4), Pigment Yellow 128 (CAS 79953-85-8), Pigment Yellow 155 (CAS 68516-73-4). A read-across with these Pigments were performed to fill the data gap for PY94 (CAS 5580-58-5).
Link to relevant study records
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- READ ACROSS ANALOGUE APPROACH
The read-across hypothesis is fundamentally based on the same core structure of five ‘yellow disazo condensation pigments’ which optionally can serve as target or as source substances. None of the pigments are sufficiently soluble, either in water or in octanol for systemic uptake or metabolism. The molecular weight ranges from 716.6 g/mol (Pigment Yellow 155) to 1229.2 g/mol (Pigment Yellow 128). Therefore, the molecular weights of all ‘yellow disazo condensation pigments’ are well above the threshold of 500 g/mol, which is generally considered for low dermal and oral uptake [ECHA Guidance R. 7c, 2017]. Furthermore, for each of the substances, the critical body burden (CBB) is above the octanol solubility, which generally indicates a low uptake in biota and makes toxicity unlikely [ECHA Guidance R. 11, 2017].
An evidence of the low bioavailability for all five pigments is the absence of any systemic toxicity in the repeated-dose toxicity study with Pigment Yellow 128 (highest molecular weight), Pigment Yellow 93 (intermediate molecular weight) and Pigment Yellow 155 (lowest molecular weight). From theoretical considerations, uptake and metabolism should result in the release of aromatic amines; such compounds have a characteristic toxicity profile. As this was not observed, all five substances are not considered to be taken up by the body. This assumption is supported by results of several other toxicological and ecotoxicological studies with the pigments, in which no hazards were identified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: fertility
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: systemic toxicity
- Key result
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Reproductive effects observed:
- no
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- From Nov 2021 to Oct 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- TEST MATERIAL
Batch no. 0004079400
CAS no. 79953-85-8
Purity 99.4 wt%
Expiry/Retest date 27 August 2022
Appearance Yellow powder
Storage conditions Room temperature - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover rat
- Details on species / strain selection:
- The Wistar Hannover rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain.
The oral route was selected as it is a possible route of exposure of the test item in man and has been specifically requested by the Regulatory Authorities. - Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Italia S.p.A., Calco (Lecco), Italy
- Females: nulliparous and non-pregnant
- Age at study initiation: 9 - 11 weeks old
- Weight at acquisition: 212-230 g for males and 182-193 g for females
- Housing arrival - mating: animals were housed up to 5 of one sex to a cage, in polysulfone solid bottomed cages measuring 59.5×38×20 cm, nesting material was provided inside suitable bedding bags and changed at least twice a week
- Housing during mating: animals were housed one male to one female in clear polysulfone cages measuring 42.5×26.6×18.5 cm with a stainless steel mesh lid and floor, each cage tray held absorbent material which was inspected and changed daily
- Housing after mating: the males were re-caged as they were before mating, females were transferred to individual solid bottomed cages for the gestation period, birth and lactation, nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum, via water bottles
- Acclimation period: approx. 2 weeks
DETAILS OF FOOD AND WATER QUALITY:
There was no information available to indicate that any non-nutrient substance likely to influence the effect of the test item was present in the drinking water or the diet.
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-60
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 21 Dec 2021 (allocation) To: 17 Feb 2022 (last day of necropsy) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5% CMC in water, softened by reverse osmosis
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Dose volume: 10 mL/kg body weight, dose volumes for males and females were adjusted once per week for each animal according to the last recorded body weight, dose volumes for females during the gestation and lactation periods were calculated according to the last recorded body weight
- Dosing preparation: preparations were made daily, preparations were kept under magnetic stirring for at least 16 hours at room temperature and up to completion of dosing - Details on mating procedure:
- - M/F ratio per cage: monogamous (one male to one female)
- Length of cohabitation: female was paired with the same male until positive identification of mating
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray, day of successful mating was referred to as day 0 post coitum
- Further matings after two unsuccessful attempts: no
- After successful mating each pregnant female was caged: females were transferred to individual solid bottomed cages for the gestation period, birth and lactation (measuring 42.5×26.6×18.5 cm), nesting material was provided inside suitable bedding bags, additional suitable nesting material was provided as necessary, nesting material was changed at least twice a week - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in a separate study in the range from 10 to 100 mg/mL. In the same study, a 28 hour stability at room temperature and a 8-day stability at 2- 8°C were verified in the range from 10 to 100 mg/mL. Samples of the formulations prepared during the current study (the first and the last week of treatment where possible) were analysed to check the concentration and homogeneity. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity).
- Duration of treatment / exposure:
- Males were treated for 2 consecutive weeks prior to pairing and during pairing with females until the day before necropsy, for a total of 39 days.
Females were treated for 2 consecutive weeks prior to pairing, during pairing and throughout the gestation and lactation periods until day 13 post partum, for a period comprising from 51 to 55 days. - Frequency of treatment:
- daily
- Details on study schedule:
- - Age at mating of the mated animals (P0 generation) in the study: approx. 13-14 weeks (approx. 11-12 weeks old at start of administration, followed by 2 weeks pre-mating phase)
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Group 1
Control - Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Group 2
Low-level dose - Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Group 3
Medium-level dose - Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- Group 4
High-level dose - No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Details on study design:
- - Dose selection rationale: dose levels of 100, 300 and 1000 mg/kg/day were selected based on information from a preliminary non-GLP compliant study
- Rationale for animal assignment: rats were allocated to the groups by computerised stratified randomisation to give approximately equal initial group mean body weights., individuals were uniquely identified within the study by sex, tattoo on the hind feet and ear notch and housed 5 of one sex per cage
- Fasting period before blood sampling for clinical biochemistry: yes - Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Mortality: checked twice daily
- Clinical signs: once before commencement of treatment and at least once daily during the study
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of treatment and at least once a week thereafter
- Observation of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, pupil size, unusual respiratory pattern), changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions (see table No. 1 for observed parameters and evaluation scale)
- Animals were examined in an open arena for a minimum of three minutes
BODY WEIGHT: Yes
- Time schedule for examinations: males were weighed weekly from allocation to termination, females were weighed weekly from allocation to positive identification of mating and on days 0, 7, 14 and 20 post coitum, dams were also weighed on days 1, 4, 7, 13 post partum and just before to necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- the weight of food consumed by each cage of males and females were recorded weekly (whenever possible) during the pre-mating period starting from day 1 of dosing up to mating
- Individual food consumption for mated females were measured on gestation days 7, 14 and 20 post coitum starting from day 0 post coitum and on day 7 and 13 post partum starting from day 1 post partum
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes (except for coagulation test in males)
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 2 were examined
- Coagulation parameters investigated: prothrombin time and activated partial thromboplastin time
CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of treatment period
- Animals fasted: Yes
- How many animals: 5 males and 5 females (with viable litters if possible), randomly selected
- Parameters checked in table No. 3 were examined
SERUM HORMONES: Yes
- Time of blood sample collection: at termination, timing of the blood collection for thyroid hormone determination was as close as possible between animals and at the same time of the day in case of sampling on different days
- Animals fasted: Yes
- How many animals: all parental animals, each litter (1 sample for males and 1 sample for females, when possible), blood samples from different pups was pooled to obtain the required volume
- Hormones analyzed: serum levels of total thyroxine (total T4) and thyroid stimulating hormone (TSH)
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule: once during study towards end of treatment, for males on day 39 and for females (with viable litters) on day 12 post partum
- Number of animals: 5 males and 5 females randomly selected from each dose group
- Studies conducted: grip strength and sensory reactivity to stimuli, motor activity assessment
IMMUNOLOGY: No - Oestrous cyclicity (parental animals):
- - Vaginal smears were taken in the morning from day 1 of dosing up to positive identification of mating
- The vaginal smear data was examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
- Before despatch to necropsy: vaginal smears were also taken from all females and the oestrous cycle phase recorded - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, maximum of 4 pups per sex per litter (partial adjustment was possible); excess pups were killed, and at least one culled male and one culled female per litter were selected for hormone determination
- No pups were eliminated when the litter size dropped below the culling target (8 pups per litter in total)
- If there was only one pup available above the culling target, only one pup was eliminated and used for blood collection for possible hormone determination
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
(i) Number and sex of pups
(ii) Stillbirths, live births
(iii) Body weight measured on days 1, 4, 7 and 13 post partum
(iv) Anogenital distance measured on day 1 post partum, AGD was normalized to the cube root of body weight collected on day 1 post partum
(v) Nipple count check examined in male pups on day 13 post partum
(vi) Necropsy
GROSS EXAMINATION OF DEAD PUPS: Yes
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination
- All pups with abnormalities were retained in a 10% neutral buffered formalin
ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY: No
ASSESSMENT OF DEVELOPMENTAL IMMUNOTOXICITY: No - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: sacrifice after the mating of all females on day 40 of the study
- Maternal animals: females with live pups were killed on day 14 post partum, females showing no evidence of copulation were killed 25 days after the last day of the mating session
GROSS PATHOLOGY: Yes
- Necropsy: the clinical history of adult animals was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices), changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination
- All females were examined also for the following: number of visible implantation sites (pregnant animals), number of corpora lutea (pregnant animals)
ORGAN WEIGHTS:
- Parental animals: from all animals completing the scheduled test period, the organs indicated in table No. 4 were dissected free of fat and weighed, the ratios of organ weight to body weight was calculated for each animal
HISTOPATHOLOGY: Yes
- Samples of all tissues (parental animals) required for histopathological examination are listed in table No. 4
- Samples of the tissue were fixed and preserved in 10% neutral buffered formalin (except testes and epididymides which were fixed in Modified Davidson’s fluid and preserved in 70% ethyl alcohol)
- After dehydration and embedding in paraffin wax, sections of the tissues were cut at 5 micrometer thickness and stained with haematoxylin and eosin, in addition, the testes and epididymides were cut at 2-3 micrometer thickness and stained with Periodic Acid Schiff (PAS), the morphological evaluation of the seminiferous epithelium (staging of spermatogenic cycle) was performed
The examination was restricted as detailed below:
(i) Sexual organs (cervix, clitoral gland, ovaries, uterus and vagina) and thyroid glands from all parental females
(ii) Sexual organs (coagulation gland, epididymides, preputial gland, prostate gland, seminal vesicles and testes) and thyroid glands from all parental males
(iii) Tissues specified in section 4.5.7 from 5 males and 5 females randomly selected (animals evaluated for clinical pathology) in the control and high dose group killed at term
(iv) Morphological evaluation of the seminiferous epithelium (staging of the spermatogenic cycle) from all males in control and high dose groups
(v) All abnormalities in all groups - Postmortem examinations (offspring):
- SACRIFICE
- Pups (F1 offspring) that had completed the scheduled test period (days 4 or 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal
- Pups selected for blood collection for hormone determination were killed on the day of blood sampling
GROSS PATHOLOGY
- All pups found dead in the cage were examined for external and internal abnormalities
- All culled pups sacrificed at day 4 post partum were subjected to an external examination
- All live pups sacrificed on day 14 post partum were examined for external abnormalities
- Gonads were inspected from all pups in order to confirm the sex previously determined by external examination
ORGAN WEIGHTS
- Pups at day 14 post partum: thyroid was weighed from one male and one female pup selected for blood collection and preserved in 10% neutral buffered formalin, the thyroid weight was determined after fixation - Statistics:
- Standard deviations were calculated as appropriate.
For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t-test, depending on the homogeneity of data. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the nonparametric version of the Williams test. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off. - Reproductive indices:
- MALES:
Copulation index (%) = (no. of males with confirmed mating/no. of males cohabitated) X 100
Fertility index (%) = (no. of males which induced pregnancy/ no. of males cohabitated) X 100
MALES AND FEMALES:
Pre coital Interval = the number of nights paired prior to detection of mating
FEMALES:
Copulation index (%) = (no. of females with confirmed mating/no. of females cohabitated) X 100
Fertility index (%) = (no. of pregnant females/ no. of females cohabitated) X 100
Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations - live litter size at birth/no. of visible implantations) X 100
Pre-natal loss at day 0 post partum was calculated as a percentage from the formula:
(total litter size - live litter size/total litter size) X 100
Post-natal loss at day 4 post partum (before culling) was calculated as a percentage from the formula:
(live litter size at birth - live litter size at day 4 (before culling)/live litter size at birth) X 100
Post-natal loss at day 13 post partum (after culling) was calculated as a percentage from the formula:
(live litter size on day 4 (before culling) - live litter size at day 13/live litter size on day 4 (before culling)) X 100 - Offspring viability indices:
- Sex ratios was calculated at birth, on day 4 and on day 14 post partum and was presented as the percentage of males per litter.
- Clinical signs:
- no effects observed
- Mortality:
- mortality observed, non-treatment-related
- Description (incidence):
- One control female was sacrificed for humane reason on Day 22 of gestation phase due to difficulties in parturition.
- Body weight and weight changes:
- no effects observed
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- Food consumption was unaffected by treatment in both genders during the study.
- Food efficiency:
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- no effects observed
- Clinical biochemistry findings:
- no effects observed
- Endocrine findings:
- no effects observed
- Description (incidence and severity):
- The determination of T4 and TSH was performed on
- Samples from all parental males from all groups
- Samples from pups on Day 14 post partum.
No treatment-related changes were recorded. Thyroid stimulating hormone was statistically si
gnificantly lower than controls in parental males (31%). This is due to 2 control animals, which sh
owed values above the range of historical data, therefore the differences observed has no biological/
toxicological significance.
The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of
14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated
animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences. - Urinalysis findings:
- not examined
- Behaviour (functional findings):
- no effects observed
- Description (incidence and severity):
- Observation of treated animals at removal from the cage and in an open arena did not reveal significant changes when compared to controls.
No alterations in motor activity, grip strength, landing footsplay and sensory reactivity to stimuli were observed in any treatment group at the examination performed at the end of treatment. - Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Description (incidence and severity):
- There were no treatment-related microscopic observations at the end of the treatment period. There were no test item-related microscopic observations in the testis (stage were evaluation on PAS-stained slides). Any microscopic observations had a comparable incidence in control and treated groups and/or are characteristically seen in untreated rats of the same age and were considered incidental and unrelated to treatment.
- Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- The oestrous cyclicity of the treated females monitored during the pre-mating period, for a total of
14 days, was not affected by treatment. The mean number of oestrous cycles observed in treated
animals and control group was comparable.
No differences were recorded in pre-coital interval and copulation plugs between treated and control
groups. Copulatory index and fertility index did not show any treatment-related differences. - Reproductive performance:
- no effects observed
- Description (incidence and severity):
- Implantation, pre implantation loss, pre-natal loss data and gestation length of females: Mean gestation period was comparable between control and treated animals, being of 22/23 days. Corpora lutea, number of implantations sites and live litter size did not show dose-related or treatment-related differences, compared to controls. Pre-natal loss (percentage) was similar between control and treated groups.
- Dose descriptor:
- NOAEL
- Remarks:
- reproduction toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Dose descriptor:
- NOAEL
- Remarks:
- systemic toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Description (incidence and severity):
- No treatment-related clinical signs were observed in pups during the 14-day observation period.
- Body weight and weight changes:
- no effects observed
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- no effects observed
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Nipple retention in male pups:
- no effects observed
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Description (incidence and severity):
- No differences were observed in the weight of thyroid in treated pups, when compared to controls.
- Histopathological findings:
- not examined
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Dose descriptor:
- NOAEL
- Remarks:
- developmental toxicity
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Critical effects observed:
- no
- Reproductive effects observed:
- no
- Conclusions:
- The oral administration of the test substance by gavage to male and female Wistar rats revealed no signs of reproduction and developmental toxicity up to the highest tested dose level. Therefore, a NOAEL for general systemic toxicity, reproductive performance as well as for developmental toxicity was set to 1000 mg/kg bw/day.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25. Nov 2021 - 31. Mar 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- Batch no.: 0004460012
Purity: 99.3 wt%
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: Room temperature
The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to a quality system. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Germany, D-97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8-10 weeks
- Weight at study initiation: (P) Males: 208-227 g; Females: 198 -217 g
- Housing: 5 of one sex to a cage until pairing; 1:1 (m/f) during pairing; after pairing: females individually, males as before mating
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2°C
- Humidity: 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 25 Nov 2021 To: Feb 2022 (females), Jan 2022 (males) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5 %
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The preparations were made weekly and every three days at concentration of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray
- After successful mating each pregnant female was caged: individually - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
A 28 hour stability at room temperature and an 8 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in internal SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the formulations prepared on the first and the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in internal SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. - Duration of treatment / exposure:
- Males: 34/35 d
Females: 66 d - Frequency of treatment:
- once daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Vehicle control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose group
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Medium dose group
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- High dose group
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), at least once daily (clinical signs)
- Cage side observations: mortality, clinical signs
BODY WEIGHT: Yes
- Time schedule for examinations: weekly, additionally females on days 0, 7, 14 and 20 p.c.; Dams on days 1, 4, 7 and 13 p.p. and prior necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food consumed by each cage of males and females was recordedweekly during the pre-mating period starting from the beginning of treatment up to mating. Individual food consumption for mated females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.
PARTUITION CHECK:
- three times a day during the working day and twice daily during the weekends and Public Holidays
- from Day 20 to Day 25 post coitum
BLOOD COLLECTION:
- Males: Blood samples (approximately 0.8 mL) were collected on the day of sacrifice under isoflurane anaesthesia from the sublingual vein.
- Females: As part of the necropsy procedure, blood samples (approximately 0.8 mL) were withdrawn under isoflurane anaesthesia from the abdominal vena cava.
IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH) - Oestrous cyclicity (parental animals):
- The assessment of oestrous cycles, by vaginal smears, was performed once daily in the morning in stock females and in allocated females for 2 weeks before the start of dosing.
From Day 1 of treatment, vaginal smears were taken in the morning from all females throughout the mating period until a positive identification of copulation was detected.
The vaginal smear data were examined to determine the following:
1. anomalies of the oestrous cycle
2. the pre-coital interval (i.e., the number of nights paired prior to the detection of mating)
Vaginal smears were also taken from all females, before despatch to necropsy and the oestrous cycle phase recorded. - Litter observations:
- STANDARTISATION OF LITTER:
- on Day 4 post partum,
- litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter
- Partial adjustment (for example, 5 males and 3 females) was acceptable
- no pups were eliminated when litter size dropped below the culling target (8 pups/litter).
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths, live births
- weight gain (weighing on day 1, 4, 7, 13)
- postnatal mortality
- presence of gross anomalies
- anogenital distance (AGD)
- pup weight on the day of AGD
- presence of nipples/areolae in male pups (Day 13/14)
GROSS EXAMINATION OF DEAD PUPS
BLOOD COLLECTION:
- On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 mL (per sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).
In order to obtain the required volume, blood samples were pooled from two or three pups on Day 4 post partum.
IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH) - Postmortem examinations (parental animals):
- SACRIFICE
- Male animals: All surviving animals, on Day 21/22 of the mating phase, after 34/35 days of treatment
- Maternal animals: The dams with live pups were killed on Day 14 post partum. One female showing no evidence of copulation was sacrificed 28 days after the last day of the mating session (Day 43 of the mating phase). The five females which did not give birth 25 days after positive identification of mating were killed shortly after. Two control females dnd one mid-dose female were sacrificed on Day 28 of the gestation phase. One mid-dose female and one high dose female were sacrificed on Day 27 post coitum.
NECROPSY
- The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
- All females were also examined for the number of visible implantation sites (pregnant animals). Uteri from females with no visible implantations, and uterus from one female with implantations only on the right horn, were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
HISTOPATHOLOGY / ORGAN WEIGHTS
The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. - Postmortem examinations (offspring):
- SACRIFICE
Pups that had completed the scheduled test period (Day 4 or Day 14 post partum) were euthanised by intraperitoneal injection of Sodium Thiopenthal. Pups selected for blood collection for hormone determination were killed on the day of blood sampling
GROSS NECROPSY
- The pup from one dam (Group 3) found dead in the cage was examined for external and internal abnormalities.
- All culled pups sacrificed at Day 4 post partum were subjected to an external examination. Sex was determined by internal gonads inspection.
- All live pups sacrificed on Day 14 post partum were examined for external abnormalities and sex confirmation by gonadal inspection.
HISTOPATHOLOGY / ORGAN WEIGTHS
- Thyroid - Statistics:
- Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
- Reproductive indices:
- Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day when the parturition is defined complete (Day 0 post partum).
Males:
Copulatory Index (%) = no. of males with confirmed mating/no. of males cohabitated ×100
Fertility Index (%) = no. of males which induced pregnancy/no. of males cohabitated ×100
Females:
Copulatory Index (%) = no. of females with confirmed mating/no. of females cohabitated ×100
Fertility Index (%) = no. of pregnant females/no. of females cohabitated ×100
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
(Total litter size−Live litter size)/Total litter size ×100
Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations−Live litter size at birth)/no. of visible implantations ×100
Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from
the formula:
(Live litter size at birth−live litter size at Day 4 (before culling))/Live litter size at birth ×100
Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from
the formula:
(Live litter size onDay 4 (after culling)−Live litter size onDay 13)/Live litter size onDay 4 (after culling) ×100
Males and females:
Pre coital Interval = The number of nights paired prior to the detection of mating - Offspring viability indices:
- Sex ratios was calculated at birth, on Day 4 and on Day 14 post partum and was presented as the percentage of males per litter.
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No differences in body weight were recorded between treated and control groups throughout the entire study.
A degree of variability in body weight gain was observed in both males and females over the course of the study and among all groups, but no relationship with the test item treatment was observed. Notably, there was a -58% decrease in body weight gain in high dose females at Day 7 post partum, compared to the control, but without statistical significance.
Being isolated event and without a dose-related trend, this case was not considered to be treatment related. - Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Reproductive function: oestrous cycle:
- no effects observed
- Description (incidence and severity):
- The oestrous cyclicity and the number of copulatory plug (mean value) of the treated females monitored during the pre-mating period, for a total of 14 days, was not affected by treatment.
The mean number of oestrous cycles observed in treated animals and controls was comparable and was of 3/4 cycles. The mean number of copulatory plugs found in the cage was 3 in control, mid and high dose groups, and 4 in low dose group.
Pre-coital interval, copulatory and fertility indices were considered to be unaffected by administration of the test item. The copulatory indices were 90% for controls and 100% in all treated groups of both sexes. The fertility indices in males and females were 70% for controls, 100% in low dose animals and 90% in mid- and high dose animals. - Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- effects observed, non-treatment-related
- Description (incidence and severity):
- The number of implantations, live litter size and the percentage of prenatal loss did not
show treatment-related differences. One female in the mid-dose group had total
resorptions.
Mean gestation period was comparable between control and treated animals.
All dams give birth between Days 22 and 23 post coitum.
The slight differences noted in mean values of total litter size, live litter size at birth and on Days 1, 4 and 13 post partum and litter weight were without a dose-related trend nor statistical significance, therefore considered not related to treatment. Moreover, these data remained almost unchanged within each group throughout the post partum phase.
Post natal loss was observed in low and mid- dose groups: one low dose female and two mid-dose females which lost their pups (missing or found dead) respectively on Day 7 and on Day 4 post partum. On statistical analysis, the mean results of the low and mid-dose groups were found to be significant on these days, but being single cases and in the absence of a dose-related trend, they were not considered to be caused by test item treatment.
The mean pup weight at all stages was always comparable between groups or even higher for pups from treated females respect to the controls.
Sex ratios at birth, on Days 4 and 14, expressed as percentage of males, did not show significant differences between groups. - Key result
- Dose descriptor:
- NOAEL
- Remarks:
- general systemic, reproductive and developental toxicity
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Clinical signs:
- no effects observed
- Mortality / viability:
- mortality observed, non-treatment-related
- Description (incidence and severity):
- There were some missing pups from low (one pup) and mid-dose group (three pups), and one pup found dead from one mid-dose female.
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- one thin pup from one control dam seen only at Day 13 post partum.
- Food consumption and compound intake (if feeding study):
- not examined
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Urinalysis findings:
- not examined
- Sexual maturation:
- not examined
- Anogenital distance (AGD):
- no effects observed
- Nipple retention in male pups:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No nipples in males were noted.
- Organ weight findings including organ / body weight ratios:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistical significance decrease in mean thyroid weight was found in male pups of mid-dose group respect to than from control dams. This decrease (-21%) was slight and not dose related, therefore considered incidental. No other statistically significant changes were noted.
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- no effects observed
- Behaviour (functional findings):
- not examined
- Developmental immunotoxicity:
- not examined
- Key result
- Dose descriptor:
- NOAEL
- Generation:
- F1
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Critical effects observed:
- no
- Key result
- Reproductive effects observed:
- no
- Conclusions:
- The NOAEL for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
- Executive summary:
This screening study for reproductive and developmental toxicity was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats.
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.
On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
- Endpoint:
- screening for reproductive / developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to other study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- Deviations:
- no
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
- Limit test:
- no
- Specific details on test material used for the study:
- - Lot/batch No.: DEB2 146870
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Storage condition of test material: room temperature - Species:
- rat
- Strain:
- Wistar
- Sex:
- male/female
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Pigment Yellow 155 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.
VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100mL, respectively - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: maximum of two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: indiviually - Analytical verification of doses or concentrations:
- yes
- Duration of treatment / exposure:
- males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation) - Frequency of treatment:
- daily
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Parental animals: Observations and examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily
- Cage side observations checked: any signs of morbidity, pertinent behavioral changes and signs of overt toxicity, littering and lactation behavior of the dams, parturition behavior of the dams
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: prior to the administration period and thereafter at weekly intervals
BODY WEIGHT: Yes
- Time schedule for examinations: Before the start of the administration period. During the administration period body weight was determined on study day 0 (start of the administration period) and thereafter once a week at the same time of the day (in the morning).
FOOD CONSUMPTION:
Food consumption was determined once a week for male and female parental animals, except during mating period, during gestation.
WATER CONSUMPTION AND COMPOUND INTAKE: No
OTHER: Functional observational battery was performed in the first five parental males and females (with litter) per group at the end of the administration period. The motor activity assessment (MA) was carried out in the first five parental males and females (with litter) per group at the end of the administration period. - Litter observations:
- STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: no
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, physical or behavioural abnormalities
GROSS EXAMINATION OF DEAD PUPS:
yes, for external and internal abnormalities; possible cause of death was not determined for pups born or found dead. - Postmortem examinations (parental animals):
- SACRIFICE
- Maternal animals: All surviving animals: Males approximately one week post-mating and females after 4 days of lactation and two weeks thereafter.
GROSS NECROPSY
- Gross necropsy consisted of external and internal examinations including the cervical, thoracic, and abdominal viscera.
HISTOPATHOLOGY / ORGAN WEIGHTS
Histopathology: All gross lesions, Adrenal glands, Bone marrow (femur), Brain, Cecum, Cervix, Coagulating glands, Colon, Duodenum, Epididymides, Heart, Ileum, Jejunum, Kidneys, Liver, Lung, Lymph nodes (mesenteric and axillary lymph nodes), Ovaries, Oviducts, Peyer’s patches, Prostate, Rectum, Sciatic nerve, Seminal vesicles, Spinal cord (cervical, thoracic and lumbar cords), Spleen, Stomach (forestomach and glandular stomach), Testes, Thymus, Thyroid glands, Trachea, Urinary bladder, Uterus, Vagina
Organn weights: Epididymides, Testes, Adrenal glands, Brain, Heart, Kidneys, Liver, Spleen, Thymus - Postmortem examinations (offspring):
- SACRIFICE
- The F1 offspring at 4 days of age.
- These animals were subjected to postmortem examinations (macroscopic examination) as follows: All pups without notable findings or abnormalities were discarded after their macroscopic evaluation. Animals with notable findings or abnormalities were evaluated on a case-by-case basis, depending on the type of finding noted.
GROSS NECROPSY
- All pups were examined externally and eviscerated; their organs were assessed macroscopically. - Statistics:
- Food consumption (parental animals), body weight and body weight change (parental animals and pups; for the pup weights, the litter means were used), number of mating days, duration of gestation, number of implantation sites, postimplantation loss and % postimplantation loss, number of pups delivered per litter where analysed by simultaneous com-parison of all dose groups with the control group using the DUNNETT-test (two-sided) for the hypothesis of equal means.
Male and female mating indices, male and female fertility indices, gestation index, females with liveborn pups, females with stillborn pups, females with all stillborn pups, live birth index, pups stillborn, pups died, pups cannibalized, pups sacrificed moribund, viability index, number of litters with affected pups at necropsy where analysed by pairwise comparison of each dose group with the control group using FISHER'S EXACT test for the hypothesis of equal proportions.
Proportions of affected pups per litter with necropsy observations where analysed by pairwise comparison of each dose group with the control group using the WILCOXON-test (one-sided) for the hypothesis of equal medians.
Feces, rearing, grip strength of forelimbs and hindlimbs, landing foot-splay test, motor activity where analysed by non-parametric one-way analysis using KRUSKAL-WALLIS test (two-sided). If the resulting p-value was equal or less than 0.05, a pairwise comparison of each dose group with the control group was performed using WILCOXON-test (two-sided) for the equal medians. - Reproductive indices:
- Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, post implantation loss
- Offspring viability indices:
- Viability index
- Clinical signs:
- effects observed, treatment-related
- Description (incidence and severity):
- yellow discolored feces, no mortality
- Body weight and weight changes:
- no effects observed
- Description (incidence and severity):
- except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)
- Food consumption and compound intake (if feeding study):
- no effects observed
- Description (incidence and severity):
- except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%)
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Histopathological findings: non-neoplastic:
- no effects observed
- Other effects:
- no effects observed
- Reproductive function: oestrous cycle:
- not examined
- Reproductive function: sperm measures:
- not examined
- Reproductive performance:
- no effects observed
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: fertility
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Basis for effect level:
- other: systemic toxicity
- Clinical signs:
- no effects observed
- Mortality / viability:
- no mortality observed
- Body weight and weight changes:
- no effects observed
- Sexual maturation:
- not examined
- Organ weight findings including organ / body weight ratios:
- not examined
- Gross pathological findings:
- no effects observed
- Histopathological findings:
- not examined
- Key result
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Reproductive effects observed:
- no
Referenceopen allclose all
Tab. 5: Summary of female reproduction data (group data)
| control | 100 mg/kg bw | 300 mg/kg bw | 1000 mg/kg bw | |
| paired females | 10 | 10 | 10 | 10 |
| not pregnant | 0 | 2 | 0 | 2 |
| not mated | 0 | 2 | 0 | 0 |
| humane kill | 1 | 0 | 0 | 0 |
| Conceiving 1-5 days | 10 | 6 | 9 | 8 |
| Conceiving 6-14 days | 0 | 0 | 1 | 0 |
| Total litter loss | 0 | 0 | 0 | 0 |
| Unilateral implantation | 0 | 0 | 0 | 0 |
| No of females with live pups on day 14 post partum | 9 | 6 | 10 | 8 |
Table 2: Reproduction data
Groups | 1 | 2 | 3 | 4 |
Paired No. | 10 | 10 | 10 | 10 |
Positive identification of mating No. | 9 | 10 | 10 | 10 |
Not pregnant | 3 | 0 | 1 | 1 |
Conceiving number | 7 | 10 | 9 | 9 |
Total resorptions | 0 | 0 | 1 | 0 |
Total litter loss | 0 | 0 | 0 | 0 |
Positive identification of mating 1 - 5 days | 9 | 9 | 8 | 7 |
Positive identification of mating 6 - 14 days | 0 | 1 | 2 | 3 |
No. of females with live pups on Day 14 post partum | 7 | 10 | 8 | 9 |
Table 3: Reproduction data and litter data - group means
Summary of Reproduction Data | ||||
| Dosage levels mg/kg/day | |||
Observations | 0 | 100 | 300 | 1000 |
No. Dams Inseminated | 9 | 10 | 10 | 10 |
Oestrus cycle (mean number) | 4 | 3 | 4 | 4 |
No. Dams Pregnant | 7 | 10 | 9 | 9 |
Percent Dams Pregnant | 70.0 | 100.0 | 90.0 | 90.0 |
Copulatory interval (1-5 days) | 9 | 9 | 8 | 7 |
Copulatory interval (6-14 days) | 0 | 1 | 2 | 3 |
No. Dams Died During Study | 0 | 0 | 0 | 0 |
No. Dams with Resorptions only | 0 | 0 | 1 | 0 |
No. of Litters | 7 | 10 | 8 | 9 |
Total No. Implantation | 83 | 101 | 88 | 104 |
Mean No. Implants/Pregnancy | 11.9 | 10.1 | 9.8 | 11.6 |
Pre natal Loss (%) | 4.3 | 10.0 | 5.6 | 9.5 |
Post natal Loss (%) | 0.0 | 1.3 | 0.0 | 0.0 |
|
|
|
|
|
Summary of Litter data | ||||
| Dosage levels mg/kg/day | |||
| 0 | 100 | 300 | 1000 |
Pairs started (no.) | 10 | 10 | 10 | 10 |
Dams with live pup at birth (no.) | 7 | 10 | 8 | 9 |
Dams with live pups at Day 4 pp (no.) | 7 | 10 | 8 | 9 |
Live litter size at birth (mean) | 11.3 | 9.00 | 10.1 | 10.7 |
Live litter size at Day 4 (mean) | 11.3 | 9.00 | 9.6 | 10.7 |
Sex ratio (%) at birth (mean) | 47.9 | 54.5 | 52.4 | 48.9 |
Sex ratio (%) at Day 4 (mean) | 47.9 | 54.5 | 52.8 | 48.9 |
Litter weight at Day 1 (mean) | 78.7 | 64.3 | 69.3 | 73.8 |
Litter weight at Day 4 (mean) | 124.1 | 100.9 | 110.3 | 113.6 |
Pup weight at Day 1 (mean) | 7.0 | 7.2 | 7.4 | 7.0 |
Pup weight at Day 4 (mean)
| 11.1 | 11.4 | 11.9 | 10.9 |
Pup weight at the Day of AGD (Day 1pp) |
|
|
|
|
Mean males | 7.30 | 7.30 | 7.30 | 7.30 |
Mean females | 6.81 | 6.81 | 6.81 | 6.81 |
Pup AGD normalized (Day 1pp) |
|
|
|
|
Mean males | 2.23 | 2.23 | 2.23 | 2.23 |
Mean females | 1.23 | 1.23 | 1.23 | 1.23 |
Male pup nipple present at Day 13 (no.) | 0 | 0 | 0 | 0 |
Pup weight at Day 13 (mean) | 31.8 | 32.6 | 33.5 | 31.2 |
Pups with major abnormalities at necopsy |
|
|
|
|
Dams with 0 | 7 | 10 | 8 | 9 |
Dams with 1 | 0 | 0 | 0 | 0 |
Dams with > 2 | 0 | 0 | 0 | 0 |
Pre-natal loss (implantations minus live births)(no.) |
|
|
|
|
Females with 0 | 4 | 6 | 5 | 4 |
Females with 1 | 2 | 1 | 2 | 3 |
Females with 2 | 1 | 1 | 1 | 1 |
Females with > 3 | 0 | 2 | 1 | 1 |
Post-natal (live births minus live at Day 1) (no.) |
|
|
|
|
Females with 0 | 7 | 10 | 6 | 9 |
Females with 1 | 0 | 0 | 1 | 0 |
Females with 2 | 0 | 0 | 1 | 0 |
Females with > 3 | 0 | 0 | 0 | 0 |
Post-natal (live births at Day 4 after culling minus live Day 14) (no.) |
|
|
|
|
Females with 0 | 7 | 9 | 8 | 9 |
Females with 1 | 0 | 1 | 0 | 0 |
Females with 2 | 0 | 0 | 0 | 0 |
Females with > 3 | 0 | 0 | 0 | 0 |
BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS): Mean body weights and mean body weight gain of the F0 males in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire study period. Except the body weight in male animals was significantly decreased in test group 1 (100 mg/kg bw/d) on post-mating day 0 (-4.4%). Mean body weight and mean body weight gain of the F0 females in test groups 1-3 (100, 300 and 1000 mg/kg bw/d) were comparable to the concurrent control throughout the entire premating, gestation and lactation periods.
TEST SUBSTANCE INTAKE (PARENTAL ANIMALS): Food consumption of the male and female F0 generation parental animals in all test substance-treated groups (100, 300 and 1000 mg/kg bw/d) was comparable to the concurrent control group during the entire study period, covering premating, gestation and lactation.
REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS): For all F0 parental males, which were placed with females to generate F1 pups, copulation was confirmed. Thus, the male mating index was 100% in all groups including the controls. Fertility was proven for most of the F0 parental males within the scheduled mating interval to produce F1 litter. Two males of test group 1 (Nos. 12, 20 mated with females Nos. 112, 120) did not generate F1 pups.
The male fertility index ranged between 80% and 100% (see Tab. 1).
The apparently infertile male animals Nos. 12 and 20 (test group 1; 100 mg/kg bw/d) were not examined histopathologically.
These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
The female mating index calculated after the mating period for F1 litter was 100% in all test groups. The mean duration until sperm was detected (GD 0) was 3.2, 3.8, 2.9 and 4.2 days in test groups 0-3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data. All sperm positive rats delivered pups with the exception of females Nos. 112 and 120 (test group 1), which were mated with male Nos. 12 and 20 did not become pregnant. The female fertility index varied between 80% and 100% (Table 1). Female animals Nos. 112 and 120, which delivered no pups, showed no implantation sites. The mean duration of gestation was similar in all test groups (i.e. between 22.0 and 22.3 days). The gestation index was 100% in all test groups.
The rate of liveborn pups was also not affected by the test substance, as indicated by live birth indices of 99.0% in test group 1, 99.1% in test group 3 and 99.2% in control and test group 2. Moreover, the number of stillborn pups was comparable between the groups. The postimplantation loss was 2.3% in test group 0, 0.98% in test group 1, 5.6% in test group 2 and 6.5% in test group 3. These findings reflected the normal range of biological variation inherent in the strain of rats used for this study as all respective values were within the range of the historical control data.
ORGAN WEIGHTS (PARENTAL ANIMALS): The weight decrease in absolute epididymis and liver weight in males of test group 2 (300 mg/kg bw/day) was regarded to be incidental due to a missing dose response relationship and missing histopathologic findings in test group 3 (1000 mg/kg bw). The decrease in thymus weights of females in test group 3 (1000 mg/kg bw/day) was also regarded to be incidental as no histopathologic correlate could explain the weight decrease.
All other mean weight parameters did not show significant differences when compared to the control groups.
GROSS PATHOLOGY (PARENTAL ANIMALS):Several animals of test group 3 (1000 mg/kg bw) revealed a yellow discoloration of the contents of the glandular stomach, small and large intestines. These findings are regarded to be treatment related.
Each one male and female of test groups 1 and 2 (100 and 300 mg/kg bw/day) (animals No. 12, 21, 111, 125) revealed either some or all of the following findings: deposition or foci on the lung, deposition on the diaphragm, discoloration and enlargement of the mediastinal lymph nodes, deposition on the thymus, and deposition on the sternum. All these findings were regarded to be treatment related by gavage errors.
All other gross lesions noted were single observations and they were regarded to have developed spontaneously and unrelated to compound and treatment.
HISTOPATHOLOGY (PARENTAL ANIMALS): The discoloration of the content in the digestive tract was regarded to be a consequence to the oral intake of the test substance which is of yellow color. Therefore, the gross findings in the remaining animals were not investigated in addition.
The discoloration and depositions described for animals No. 12, 21, 111, 125 in several organs of the thoracic cavity were multifocal granulomas with intrahistiocytically located yellow particles, interpreted as test substance. The granulomas were foreign body reactions interpreted as a consequence to a gavage error. The discoloration of the mediastinal lymph nodes was regarded to be the physiologic clearing route of the lung. Particles that were located intraalveolar were transported via macrophages to the regional lymph nodes and caused there the activation of the lymph nodes and the discoloration. Therefore these depositions and discolorations were caused by the test substance due to a gavage error and were not regarded to be an adverse finding.
All other findings noted were either single observations or they were biologically equally distributed between control and treatment group. All of them were considered to be incidental or spontaneous in origin and without any relation to treatment.
CLINICAL SIGNS (OFFSPRING): The surviving F1 pups of any test group did not show adverse clinical signs up to scheduled sacrifice on PND 4.
BODY WEIGHT (OFFSPRING): Mean pup body weights/pup body weight changes of all pups in all test groups were comparable to the control group.
One female runt was seen in test group 0. One male and six female runts were seen in test group 1 (100 mg/kg bw/d); 1 female runt was seen in test group 3 (1000 mg/kg bw/d).
GROSS PATHOLOGY (OFFSPRING): Respectively one male F1 pup of test group 2 (300 mg/kg bw/d) and test group 3 (1000 mg/kg bw/d) showed post mortem autolysis at gross necropsy. Two female pups of test group 1 (100 mg/kg bw/d) were cannibalized. This finding was assessed as being spontaneous in nature and without biological relevance.
OTHER FINDINGS (OFFSPRING): The sex distribution and sex ratios of live F1 pups on the day of birth and PND 4 did not show substantial differences between the control and the test substance-treated groups; slight differences were regarded to be spontaneous in nature (for details see table 2).
Table 10: Fertility index
|
Test group (mg/kg bw/day) |
|||
Test groups (mg/kg bw/day |
0 (0) |
1 (100) |
2 (300) |
3 (1000) |
Male fertility index [%] |
100 |
80 |
100 |
100 |
Female fertility index [%] |
100 |
80 |
100 |
100 |
Table 11: Sex ratio of live F1 pups
PND 0 |
Test group 0 |
Test group 1 |
Test group 2 |
Test group 3 |
Live males [%] |
40.3 |
50.0 |
50.4 |
57.4 |
Live females [%] |
59.7 |
50.0 |
49.6 |
42.6 |
|
|
|
|
|
PND 4 |
|
|
|
|
Live males [%] |
40.3 |
51.0 |
50.4 |
57.4 |
Live females [%] |
59.7 |
49.0 |
49.6 |
42.6 |
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
As discussed in the section of toxicokinetics, 'yellow disazo condensation pigments' are too large and insoluble for significant systemic uptake. Therefore, no hazard for toxicity to reproduction is expected. This was shown for the three members of 'yellow disazo condensation pigments' including the one with the lowest molecular weight (PY155, CAS 68516-73-4):
A GLP-compliant investigation of the toxicological effects resulting from repeated oral-gavage administration to rats was performed following OECD guideline 422 without deviations (BASF, 2012). Pigment Yellow 155 (CAS 68516-73-4) was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Pigment Yellow 155 was administered to male rats for 41 days and to female rats for 52 days including time prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Treatment with the test item up and including 1000 mg/kg bw/day did not reveal any clinical signs or histological findings and did not affect reproduction.
All dose-treated males and females had dose-related yellow discolored feces during the treatment period. This finding is considered to be a typical effect resulting from oral administration of a yellow dyestuff and not adverse.
Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day.
The NOEL (No Observed Effect Level) for reproduction toxicity was considered to be 1000 mg/kg body weight/day.
The effects of the test item Pigment Yellow 128 (CAS 79953-85-89), on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, when administered daily by oral gavage to Wistar Hannover rat. Males were treated for 14 days prior to pairing and during pairing with females until the day before necropsy, up to a total of 35/36 days. Females were treated for 14 days prior to pairing, during pairing and throughout the gestation and lactation periods until Day 13 post partum, up to at least 51 days. No treatment-related mortalities occurred during the study. One control female was sacrificed for humane reasons on Day 22 post coitum due to difficulties in parturition. No treatment-related clinical signs were recorded. No treatment-related signs of neurotoxicity and no treatment-related differences in body weight, body weight gain and food consumption were observed in treated animals, compared to the controls. No treatment-related changes were observed in haematological and clinical chemistry parameters. No changes considered treatment-related were observed in thyroid hormone levels in parental animals or in pups at Days 4 and 14 post partum determinations. No treatment-related anomalies were noted in the oestrous cycle, copulatory and fertility indices of the treated females, when compared to controls. Parturition, lactation, implantation, litter data and sex ratio did not show any changes which could be considered adverse. No significant differences in the anogenital distance was recorded in pups on Day 1 post partum and no nipples were seen between control and treated groups of male pups. Necropsy findings and thyroid weight in pups did not reveal any treatment-related effect. No treatment-related changes were detected at post mortem examination in treated animals, when compared with controls. There were no test item-related microscopic observations in the testis (stage aware evaluation on PAS-stained slides). On the basis of these results the dosage of 1000 mg/kg/day is considered the NOAEL (No Observed Adverse Effect Level) for systemic toxicity, fertility and reproduction parameters for F0 males, F0 females and F1 pups.
This screening study for reproductive and developmental toxicity of Pigment Yellow 93 (CAS 5580-57-4) was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats.
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).
The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.
On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
Effects on developmental toxicity
Description of key information
'Yellwo disazo condensation pigments' are too large and insoluble for significant systemic uptake. Therefore, no hazard for toxicity to reproduction is possible. No adverse effects were observed in the screening study at the limit dose (OECD 421/422) for Pigment Yellos 93 (CAS 5580-57-4) and Pigment Yellow 155 (CAS 68516-73-4). A read-across with these Pigments were performed to fill the data gap for PY94 (CAS 5580-58-5).
Link to relevant study records
- Endpoint:
- developmental toxicity
- Type of information:
- read-across from supporting substance (structural analogue or surrogate)
- Adequacy of study:
- key study
- Justification for type of information:
- READ ACROSS ANALOGUE APPROACH
The read-across hypothesis is fundamentally based on the same core structure of five ‘yellow disazo condensation pigments’ which optionally can serve as target or as source substances. None of the pigments are sufficiently soluble, either in water or in octanol for systemic uptake or metabolism. The molecular weight ranges from 716.6 g/mol (Pigment Yellow 155) to 1229.2 g/mol (Pigment Yellow 128). Therefore, the molecular weights of all ‘yellow disazo condensation pigments’ are well above the threshold of 500 g/mol, which is generally considered for low dermal and oral uptake [ECHA Guidance R. 7c, 2017]. Furthermore, for each of the substances, the critical body burden (CBB) is above the octanol solubility, which generally indicates a low uptake in biota and makes toxicity unlikely [ECHA Guidance R. 11, 2017].
An evidence of the low bioavailability for all five pigments is the absence of any systemic toxicity in the repeated-dose toxicity study with Pigment Yellow 128 (highest molecular weight), Pigment Yellow 93 (intermediate molecular weight) and Pigment Yellow 155 (lowest molecular weight). From theoretical considerations, uptake and metabolism should result in the release of aromatic amines; such compounds have a characteristic toxicity profile. As this was not observed, all five substances are not considered to be taken up by the body. This assumption is supported by results of several other toxicological and ecotoxicological studies with the pigments, in which no hazards were identified. - Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across source
- Reason / purpose for cross-reference:
- read-across: supporting information
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Key result
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Developmental effects observed:
- no
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Study period:
- 25. Nov 2021 - 31. Mar 2022
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 421 (Reproduction / Developmental Toxicity Screening Test)
- Version / remarks:
- 29 July 2016
- GLP compliance:
- yes (incl. QA statement)
- Limit test:
- no
- Specific details on test material used for the study:
- Batch no.: 0004460012
Purity: 99.3 wt%
Expiry/Retest date: 18 Jan 2031
Appearance: Yellow powder
Storage conditions: Room temperature
The determination of the identity, strength, purity, composition and stability of the test item was the responsibility of the Sponsor. The Sponsor declared that the characterisation of the test item was carried out according to a quality system. - Species:
- rat
- Strain:
- Wistar
- Remarks:
- Wistar Hannover
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Germany, D-97633 Sulzfeld
- Females nulliparous and non-pregnant: yes
- Age at study initiation: (P) 8-10 weeks
- Weight at study initiation: (P) Males: 208-227 g; Females: 198 -217 g
- Housing: 5 of one sex to a cage until pairing; 1:1 (m/f) during pairing; after pairing: females individually, males as before mating
- Diet: ad libitum, laboratory rodent diet (4 RF 21,Mucedola S.r.l., Via G. Galilei, 4, 20019 SettimoMilanese (MI), Italy)
- Water: ad libitum
- Acclimation period: 2 weeks
ENVIRONMENTAL CONDITIONS
- Temperature: 22°C ± 2°C
- Humidity: 55% ± 15%
- Air changes (per hr): 15 to 20
- Photoperiod (hrs dark / hrs light): 12/12
IN-LIFE DATES: From: 25 Nov 2021 To: Feb 2022 (females), Jan 2022 (males) - Route of administration:
- oral: gavage
- Vehicle:
- CMC (carboxymethyl cellulose)
- Remarks:
- 0.5 %
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle. The preparations were made weekly and every three days at concentration of 10, 30 and 100 mg/mL. Concentrations were calculated and expressed in terms of test item as supplied.
VEHICLE
- Amount of vehicle (if gavage): 10 mL/kg bw - Analytical verification of doses or concentrations:
- yes
- Details on analytical verification of doses or concentrations:
- The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.99; accuracy 85-115%; precision CV < 10%).
A 28 hour stability at room temperature and an 8 day stability at 2-8°C were verified in the range from 10 to 100 mg/mL.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirmthat the method was suitable. Final results for all levels were within the acceptability limits stated in internal SOPs for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the formulations prepared on the first and the last week were analysed to check the homogeneity and concentration. Results of the analyses were within the acceptability limits stated in internal SOPs for suspensions (85-115% for concentration and CV < 10% for homogeneity).
Chemical analysis was carried out by the Analytical Chemistry Department at ERBC. - Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: 14 d
- Proof of pregnancy: spermidentification, vaginal plug in situ or copulation plugs found in the cage tray
- After successful mating each pregnant female was caged: individually - Duration of treatment / exposure:
- Males: 34/35 d
Females: 66 d - Frequency of treatment:
- once daily
- Dose / conc.:
- 0 mg/kg bw/day (actual dose received)
- Remarks:
- Vehicle control
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Remarks:
- Low dose group
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Remarks:
- Mid dose group
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- Remarks:
- High dose group
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Maternal examinations:
- CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (mortality), at least once daily (clinical signs)
- Cage side observations: mortality, clinical signs
BODY WEIGHT: Yes
- Time schedule for examinations: weekly before mating, on days 0, 7, 14 and 20 p.c.; on days 1, 4, 7 and 13 p.p. and prior necropsy
FOOD CONSUMPTION AND COMPOUND INTAKE:
The weight of food consumed by each cage and females was recorded weekly during the pre-mating period starting from the beginning of treatment up to mating. Individual food consumption for mated females were measured on Days 7, 14 and 20 post coitum starting from Day 0 post coitum and on Days 7 and 13 post partum starting from Day 1 post partum.
PARTUITION CHECK:
- three times a day during the working day and twice daily during the weekends and Public Holidays
- from Day 20 to Day 25 post coitum
OESTROUS CYCLE:
The assessment of oestrous cycles, by vaginal smears, was performed once daily in the morning in stock females and in allocated females for 2 weeks before the start of dosing.
IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH)
POSTMORTEM EXAMINATIONS:
- The clinical history of the males and females of the parental generation was studied and a detailed post mortem examination was conducted (including examination of the external surface and orifices). Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological examination.
- All females were also examined for the number of visible implantation sites (pregnant animals). Uteri from females with no visible implantations, and uterus from one female with implantations only on the right horn, were immersed in a 20% solution of ammonium sulphide to reveal evidence of implantation.
- The tissues indicated in Table 1 were prepared for microscopic examination and weighed, respectively. - Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: No
- Blood sampling:
- As part of the necropsy procedure, blood samples (approximately 0.8 mL) were withdrawn under isoflurane anaesthesia from the abdominal vena cava.
- Fetal examinations:
- STANDARTISATION OF LITTER:
- on Day 4 post partum,
- litter size was adjusted by eliminating extra pups by random selection to yield, as nearly as possible, four pups per sex per litter
- Partial adjustment (for example, 5 males and 3 females) was acceptable
- no pups were eliminated when litter size dropped below the culling target (8 pups/litter).
PARAMETERS EXAMINED
The following parameters were examined in F1 offspring:
- number and sex of pups
- stillbirths, live births
- weight gain (weighing on day 1, 4, 7, 13)
- postnatal mortality
- presence of gross anomalies
- anogenital distance (AGD)
- pup weight on the day of AGD
- presence of nipples/areolae in male pups (Day 13/14)
GROSS EXAMINATION OF DEAD PUPS
BLOOD COLLECTION:
- On Days 4 and 14 post partum, as part of the necropsy procedure, blood samples of approximately 0.5 mL (per sex) were taken from each litter (1 sample for males and 1 sample for females, when possible). Blood samples were withdrawn under light ether anaesthesia from the heart (intracardiac puncture).
In order to obtain the required volume, blood samples were pooled from two or three pups on Day 4 post partum.
IMMUNOANALYSIS:
- Thyroid hormone determination (T4 and TSH) - Statistics:
- Standard deviations were calculated as appropriate. For continuous variables, the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov Smirnov test. The non-parametric Kruskal-Wallis analysis of variance (non-continuous variables) was used for the other parameters. Intergroup differences between the control and treated groups were assessed by the non-parametric version of theWilliams test. The criterion for statistical significance was p < 0.05. The mean values, standard deviations and statistical analysis were calculated from actual values in the computer without rounding off.
- Indices:
- Gestation length was calculated as the time between the day of successful mating (Day 0 post coitum) and the day when the parturition is defined complete (Day 0 post partum).
Males:
Copulatory Index (%) = no. of males with confirmed mating/no. of males cohabitated ×100
Fertility Index (%) = no. of males which induced pregnancy/no. of males cohabitated ×100
Females:
Copulatory Index (%) = no. of females with confirmed mating/no. of females cohabitated ×100
Fertility Index (%) = no. of pregnant females/no. of females cohabitated ×100
Pup loss at Day 0 post partum was calculated as a percentage from the formula:
(Total litter size−Live litter size)/Total litter size ×100
Pre-natal loss was calculated as a percentage from the formula:
(no. of visible implantations−Live litter size at birth)/no. of visible implantations ×100
Post-natal loss at Day 4 post partum (before culling) was calculated as a percentage from
the formula:
(Live litter size at birth−live litter size at Day 4 (before culling))/Live litter size at birth ×100
Post-natal loss at Day 13 post partum (after culling) was calculated as a percentage from
the formula:
(Live litter size onDay 4 (after culling)−Live litter size onDay 13)/Live litter size onDay 4 (after culling) ×100
Males and females:
Pre coital Interval = The number of nights paired prior to the detection of mating - Historical control data:
- T4 and TSH historical data for Wistar Hannover rat in serum were available
- Clinical signs:
- no effects observed
- Mortality:
- no mortality observed
- Body weight and weight changes:
- effects observed, non-treatment-related
- Description (incidence and severity):
- No differences in body weight were recorded between treated and control groups throughout the entire study.
A degree of variability in body weight gain was observed in both males and females over the course of the study and among all groups, but no relationship with the test item treatment was observed. Notably, there was a -58% decrease in body weight gain in high dose females at Day 7 post partum, compared to the control, but without statistical significance.
Being isolated event and without a dose-related trend, this case was not considered to be treatment related. - Food consumption and compound intake (if feeding study):
- no effects observed
- Food efficiency:
- not examined
- Water consumption and compound intake (if drinking water study):
- not examined
- Ophthalmological findings:
- not examined
- Haematological findings:
- not examined
- Clinical biochemistry findings:
- not examined
- Endocrine findings:
- no effects observed
- Urinalysis findings:
- not examined
- Behaviour (functional findings):
- not examined
- Immunological findings:
- not examined
- Organ weight findings including organ / body weight ratios:
- no effects observed
- Gross pathological findings:
- no effects observed
- Neuropathological findings:
- not examined
- Histopathological findings: non-neoplastic:
- no effects observed
- Histopathological findings: neoplastic:
- no effects observed
- Number of abortions:
- no effects observed
- Pre- and post-implantation loss:
- not examined
- Total litter losses by resorption:
- not examined
- Early or late resorptions:
- not examined
- Dead fetuses:
- effects observed, non-treatment-related
- Changes in pregnancy duration:
- no effects observed
- Changes in number of pregnant:
- no effects observed
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- 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
- Anogenital distance of all rodent fetuses:
- no effects observed
- Changes in postnatal survival:
- no effects observed
- External malformations:
- no effects observed
- Skeletal malformations:
- not examined
- Visceral malformations:
- not examined
- Other effects:
- effects observed, non-treatment-related
- Description (incidence and severity):
- A statistical significance decrease in mean thyroid weight was found in male pups of mid-dose group respect to than from control dams. This decrease (-21%) was slight and not dose related, therefore considered incidental. No other statistically significant changes were noted.
- Key result
- Dose descriptor:
- NOAEL
- Effect level:
- >= 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Sex:
- male/female
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
- Conclusions:
- The NOAEL for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
- Executive summary:
This screening study for reproductive and developmental toxicity was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats.
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.
On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
- Endpoint:
- developmental toxicity
- Type of information:
- experimental study
- Adequacy of study:
- key study
- Reliability:
- 1 (reliable without restriction)
- Rationale for reliability incl. deficiencies:
- guideline study
- Reason / purpose for cross-reference:
- reference to same study
- Qualifier:
- according to guideline
- Guideline:
- other: OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
- GLP compliance:
- yes (incl. QA statement)
- Remarks:
- BASF SE Experimental Toxicology and Ecology, 67056 Ludwigshafen, Germany
- Limit test:
- no
- Specific details on test material used for the study:
- - Lot/batch No.: DEB2 146870
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the manufacturer, and the manufacturer holds this responsibility.
- Storage condition of test material: room temperature - Species:
- rat
- Strain:
- Wistar
- Details on test animals or test system and environmental conditions:
- TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH
- Age at study initiation: (P) 10-11 wks
- Fasting period before study: no
- Housing: individually
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 6 days
ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 30-70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12 - Route of administration:
- oral: gavage
- Vehicle:
- water
- Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
Pigment Yellow 155 was applied as a suspension. To prepare this suspension, the appropriate amount of test substance was weighed out depending on the desired concentration. Then, drinking water was filled up to the desired volume, subsequently released with a high speed homogenizer. During administration of the test substance, preparations were kept homogeneous by stirring with a magnetic stirrer. The test substance preparations were produced at least once a week.
VEHICLE
- Concentration in vehicle: 1, 3, or 10 g/100mL, respectively - Analytical verification of doses or concentrations:
- yes
- Details on mating procedure:
- - M/F ratio per cage: 1:1
- Length of cohabitation: maximum of two weeks
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
- After successful mating each pregnant female was caged: indiviually - Duration of treatment / exposure:
- males treated 41 days (pre-mating, post-mating)
females treated 52 days (pre-mating, gestation, 4 days lactation) - Frequency of treatment:
- daily
- Duration of test:
- 52 days
- Dose / conc.:
- 100 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 300 mg/kg bw/day (actual dose received)
- Dose / conc.:
- 1 000 mg/kg bw/day (actual dose received)
- No. of animals per sex per dose:
- 10
- Control animals:
- yes, concurrent vehicle
- Ovaries and uterine content:
- The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: No
- Number of corpora lutea: No data
- Number of implantations: No data
- Number of early resorptions: No
- Number of late resorptions: No
- Other: Histopathology of ovaries and uterus - Fetal examinations:
- - External examinations: Yes: [all per litter]
- Soft tissue examinations: Yes: [all per litter]
- Skeletal examinations: No
- Head examinations: No - Indices:
- Male mating index, male fertility index, female mating index, female fertility index, gestation index, live birth index, post implantation loss
offspring: Viability index - Details on maternal toxic effects:
- Maternal toxic effects:no effects
Details on maternal toxic effects:
Regarding clinical examinations, signs of general systemic toxicity were not observed in male or female parental animals of test groups 1-3 (100, 300 and 1000 mg/kg bw/d) during the entire study period. All animals of test group 2 and 3 showed yellow discolored feces. This finding was substance-related due to the yellow color of the dye stuff.
Regarding clinical pathology no treatment-related, adverse effects were observed up to a dose of the compound of 1000 mg/kg bw/d.
Regarding pathology, macroscopically yellow discoloration of the content of the digestive tract in numerous animals was observed. In several treated animals and several organs of the digestive tract these yellow pigments could also be observed histopathologically. Beside the discoloration no signs of toxicity in the respective tissues were noted. This finding is regarded to be a consequence to the oral intake of the yellow test substance and therefore treatment related but not adverse in nature.
Regarding fertility and reproductive performance, no signs of toxicity were observed in male or female parental animals of all test groups (100, 300 and 1000 mg/kg bw/d) during the entire study. - Key result
- Dose descriptor:
- NOAEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: maternal toxicity
- Key result
- Dose descriptor:
- NOEL
- Effect level:
- 1 000 mg/kg bw/day (actual dose received)
- Based on:
- test mat.
- Basis for effect level:
- other: developmental toxicity
- Details on embryotoxic / teratogenic effects:
- Embryotoxic / teratogenic effects: no effects
Details on embryotoxic / teratogenic effects:
Regarding developmental toxicity, no signs of toxicity were observed in male or female pups of all test groups (100, 300 and 1000 mg/kg bw/d). - Key result
- Remarks on result:
- not determinable due to absence of adverse toxic effects
- Key result
- Abnormalities:
- no effects observed
- Key result
- Developmental effects observed:
- no
Referenceopen allclose all
Table 2: Reproduction data
Groups | 1 | 2 | 3 | 4 |
Paired No. | 10 | 10 | 10 | 10 |
Positive identification of mating No. | 9 | 10 | 10 | 10 |
Not pregnant | 3 | 0 | 1 | 1 |
Conceiving number | 7 | 10 | 9 | 9 |
Total resorptions | 0 | 0 | 1 | 0 |
Total litter loss | 0 | 0 | 0 | 0 |
Positive identification of mating 1 - 5 days | 9 | 9 | 8 | 7 |
Positive identification of mating 6 - 14 days | 0 | 1 | 2 | 3 |
No. of females with live pups on Day 14 post partum | 7 | 10 | 8 | 9 |
Table 3: Reproduction data and litter data - group means
Summary of Reproduction Data | ||||
| Dosage levels mg/kg/day | |||
Observations | 0 | 100 | 300 | 1000 |
No. Dams Inseminated | 9 | 10 | 10 | 10 |
Oestrus cycle (mean number) | 4 | 3 | 4 | 4 |
No. Dams Pregnant | 7 | 10 | 9 | 9 |
Percent Dams Pregnant | 70.0 | 100.0 | 90.0 | 90.0 |
Copulatory interval (1-5 days) | 9 | 9 | 8 | 7 |
Copulatory interval (6-14 days) | 0 | 1 | 2 | 3 |
No. Dams Died During Study | 0 | 0 | 0 | 0 |
No. Dams with Resorptions only | 0 | 0 | 1 | 0 |
No. of Litters | 7 | 10 | 8 | 9 |
Total No. Implantation | 83 | 101 | 88 | 104 |
Mean No. Implants/Pregnancy | 11.9 | 10.1 | 9.8 | 11.6 |
Pre natal Loss (%) | 4.3 | 10.0 | 5.6 | 9.5 |
Post natal Loss (%) | 0.0 | 1.3 | 0.0 | 0.0 |
|
|
|
|
|
Summary of Litter data | ||||
| Dosage levels mg/kg/day | |||
| 0 | 100 | 300 | 1000 |
Pairs started (no.) | 10 | 10 | 10 | 10 |
Dams with live pup at birth (no.) | 7 | 10 | 8 | 9 |
Dams with live pups at Day 4 pp (no.) | 7 | 10 | 8 | 9 |
Live litter size at birth (mean) | 11.3 | 9.00 | 10.1 | 10.7 |
Live litter size at Day 4 (mean) | 11.3 | 9.00 | 9.6 | 10.7 |
Sex ratio (%) at birth (mean) | 47.9 | 54.5 | 52.4 | 48.9 |
Sex ratio (%) at Day 4 (mean) | 47.9 | 54.5 | 52.8 | 48.9 |
Litter weight at Day 1 (mean) | 78.7 | 64.3 | 69.3 | 73.8 |
Litter weight at Day 4 (mean) | 124.1 | 100.9 | 110.3 | 113.6 |
Pup weight at Day 1 (mean) | 7.0 | 7.2 | 7.4 | 7.0 |
Pup weight at Day 4 (mean)
| 11.1 | 11.4 | 11.9 | 10.9 |
Pup weight at the Day of AGD (Day 1pp) |
|
|
|
|
Mean males | 7.30 | 7.30 | 7.30 | 7.30 |
Mean females | 6.81 | 6.81 | 6.81 | 6.81 |
Pup AGD normalized (Day 1pp) |
|
|
|
|
Mean males | 2.23 | 2.23 | 2.23 | 2.23 |
Mean females | 1.23 | 1.23 | 1.23 | 1.23 |
Male pup nipple present at Day 13 (no.) | 0 | 0 | 0 | 0 |
Pup weight at Day 13 (mean) | 31.8 | 32.6 | 33.5 | 31.2 |
Pups with major abnormalities at necopsy |
|
|
|
|
Dams with 0 | 7 | 10 | 8 | 9 |
Dams with 1 | 0 | 0 | 0 | 0 |
Dams with > 2 | 0 | 0 | 0 | 0 |
Pre-natal loss (implantations minus live births)(no.) |
|
|
|
|
Females with 0 | 4 | 6 | 5 | 4 |
Females with 1 | 2 | 1 | 2 | 3 |
Females with 2 | 1 | 1 | 1 | 1 |
Females with > 3 | 0 | 2 | 1 | 1 |
Post-natal (live births minus live at Day 1) (no.) |
|
|
|
|
Females with 0 | 7 | 10 | 6 | 9 |
Females with 1 | 0 | 0 | 1 | 0 |
Females with 2 | 0 | 0 | 1 | 0 |
Females with > 3 | 0 | 0 | 0 | 0 |
Post-natal (live births at Day 4 after culling minus live Day 14) (no.) |
|
|
|
|
Females with 0 | 7 | 9 | 8 | 9 |
Females with 1 | 0 | 1 | 0 | 0 |
Females with 2 | 0 | 0 | 0 | 0 |
Females with > 3 | 0 | 0 | 0 | 0 |
Effect on developmental toxicity: via oral route
- Endpoint conclusion:
- no adverse effect observed
- Dose descriptor:
- NOAEL
- 1 000 mg/kg bw/day
- Study duration:
- subacute
- Species:
- rat
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
As discussed in the section of toxicokinetics, 'yellow disazo condensation pigments' are too large and insoluble for significant systemic uptake. Therefore, no hazard for toxicity to reproduction is expected. This was shown for the three members of 'yellow disazo condensation pigments' including the one with the lowest molecular weight (PY155, CAS 68516-73-4):
A GLP-compliant investigation of the toxicological effects resulting from repeated oral-gavage administration to rats was performed following OECD guideline 422 without deviations (BASF, 2013). Pigment Yellow 155 (CAS 68516-73-4) was administered in water as vehicle at dosages of 100, 300, and 1000 mg/kg body weight/day, and controls received the vehicle only. Pigment Yellow 155 was administered to male rats for 41 days and to female rats for 52 days including time prior to pairing, through the pairing and gestation periods until the F1 generation reached day 4 post partum. Treatment with the test item up and including 1000 mg/kg bw/day did not reveal any clinical signs or histological findings and did not affect development of F1 generation.
All dose-treated males and females had dose-related yellowish discolored feces during the treatment period. This finding is considered to be a typical effect resulting from oral administration of a yellow dyestuff and not adverse.
Based on these results a general NOAEL (No Observed Adverse Effect Level) was considered to be 1000 mg/kg body weight/day.
The NOEL (No Observed Effect Level) for developmental toxicity was considered to be 1000 mg/kg body weight/day.
This screening study for reproductive and developmental toxicity of Pigment Yellow 93 (CAS 5580-57-4) was conducted according to OECD Guideline 421 and GLP. The effects of the test item on male and female reproductive performance, such as gonadal function, mating behaviour, conception, development of conceptuses, parturition and lactation of the offspring were investigated, upon daily administration of the test item by oral gavage toWistar Hannover rats.
Males were treated 14 days before pairing, trough the pairing period and up to one day before sacrifice for a total of 34/35 days. Femaleswere treated 14 days before pairing, trough pairing and gestation periods, and during post partum phase until Day 13 or the day before sacrifice (for a maximum of 66 days of treatment).
The doses used were: 0, 100, 300 and 1000 mg/kg/day and were administered at a dose volume of 10 mL/kg body weight. The control group received the vehicle alone (0.5% carboxymethyl cellulose).
The following investigations were performed on parental animals: mortality, clinical signs, body weight, body weight gain, food consumption, oestrous cycle, mating performance, litter data, sex ratios, thyroid hormones determination (parental males), macroscopic observations and organ weights. Histopathological evaluation was performed on all males and females from control and high dose groups and on the abnormalities detected during post mortem examination. The identification of the stages of the spermatogenic cycle was also performed from all males in the control and high dose groups. Clinical signs, measurement of anogenital distance, post mortem external and/or internal examination were recorded for pups. Thyroid hormone levels were also determined in 1 pup/sex/group randomly selected at Day 14 post partum.
During the study, no mortality or other adverse effects concerning systemic toxicity, reproductive performance or development could be observed.
On the basis of the results obtained, no changes that could be clearly attributable to the treatment with the test item were observed at any dose level investigated. Therefore, the NOAEL (No Observed Adverse Effect Level) for general toxicity, reproductive and developmental toxicity was considered to be 1000 mg/kg/day, for male and female parental animals and pups.
Justification for classification or non-classification
Classification, Labelling, and Packaging Regulation (EC) No 1272/2008
The available experimental test data are reliable and suitable for classification purposes under Regulation (EC) No 1272/2008. Based on available data on toxicity to reproduction, the test item is not classified according to Regulation (EC) No 1272/2008 (CLP), as amended for the tenth time in Regulation (EU) No 2017/776.
Additional information
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