4,5-dihydro-5-oxo-4-[[4-[[2-(sulphooxy)ethyl]sulphonyl]phenyl]azo]-1-(4-sulphophenyl)-1H-pyrazole-3-carboxylic acid, sodium salt

Endpoint summary

Description of key information

Dose range finding 14-day repeated dose toxicity

A dose range finding study was performed which preceeded the presented key 28-day repeated dose study. To assess the possible health hazards which could arise from repeated exposure of Reactive Yellow 42 via oral administration to rats, the test item was administered daily to 3 groups of test animals at 300, 600 and 1000 mg/kg bw/day for a treatment period of 14 days.

There are no regulatory guidelines for this type of dose range-finding study, but the study design was based on the principles specified in OECD TG 407, the study was non-GLP.

No NOAEL was determinded for this study as it served as a dose range finder.

28-Day repeated dose toxicity

One key repeated dose toxicity study was performed according to OECD TG 422 and in compliance to GLP to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day.

The NOAEL for the test item under the condition of this study is considered to be > 1000 mg/kg bw/day for general toxicity.

Repeated dose toxicity via other routes

There are no studies available in which the repeated dose toxicity via inhalation or dermal route is assessed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

08.02-23.08.2017

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:

July, 2016

Deviations:

no

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-368

Version / remarks:

July, 2000

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Species: Wistar rat, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 327 - 487 g (mean: 397.89 g); females: 213 - 277 g (mean: 240.33 g)
- Fasting period before study: not reported
- Housing: Animals were housed in groups of 5 per sex in type IV polysulphone cages
- In each cage Altromin saw fibre was used as bedding
- Diet (ad libitum): free access to Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least 5 days.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours artificial light / 12 hours dark

Route of administration:

oral: gavage

Details on route of administration:

Prior to the start of the treatment period: a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration. Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10 females/ group) with regular estorus cycle (4-5 day cycle) were used in the study. The test item and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups. Each animal was marked with its identification number by individual ear tattoo marking.

Vehicle:

water

Details on oral exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females.
Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period:
- formulation analysis for stability and homogeneity of the test item in the selected vehicle.
- pre-start stability analysis for high and low dose group samples, at 0h, 6h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day (-15 to -35 °C) (10 samples).
- pre-start homogeneity investigation for samples from various dose levels (top, middle and bottom) of high dose and low dose groups (6 samples).
Result: the test item was homogenous (after 60 min without stirring) ==> during the study samples were collected for the investigation of homogeneity and samples for substance concentration were only collected in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and the last week of the study (gestation / lactation) from all dose groups (12 samples). Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

Treatment with test item formulation or vehicle: 7 days/week with max. of 63 days, i.e. during 14 days of pre-mating and max. 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to postnatal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days / week

Dose / conc.:

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

Remarks:

control group

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 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

100 animals (40 males and 60 females) were included in the study.

Prior to the start of the treatment:
- detailed clinical observation was made
- no pathological signs observed before the first administration

Before the first administration:
- all animals were weighed & assigned to the experimental groups (homogenous variation in body weight throughout the groups of males and females)(randomisation with IDBS Workbook 10.1.2 software).

Dose selection rationale:
- Based on DRF study 3 dose groups were determined (LD = low dose, MD = medium dose, HD = high dose).

Body weight and food consumption:
- weighed once before the assignment to the experimental groups
- weighed on the first day of dosing
- weighed weekly
- weighed at the end of the study
During pregnancy, females:
- were weighed on gestation days (GD) 0, 7, 14 and 20
- weighed within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13 along with pups.
- all animals were weighed directly before termination.

Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Positive control:

not applicable

Observations and examinations performed and frequency:

Clinical observations:

eneral clinical observations:
- at least once per day (preferably at the same time)
- health condition was recorded
- 2x/day observed for morbidity and mortality (except on weekends and public holidays ==> 1x/day)

Detailed clinical observations:
- 1x before first exposure + min. 1x one week thereafter
- observations: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional observations:
- multiple detailed behavioural observations: 1 week before first treatment, during last week of treatment in 5 randomly selected males, during last week of lactation of the lactation period in 5 randomly selected (lactating) females
- Observations: sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Haematology:
- in 5 randomly selected males and females (only lactating females) from each group at the end of the treatment prior to or as part of the sacrifice of the animals.
- Examined parameters: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc).

Blood coagulation:
- in 5 randomly selected males and females (only lactating females) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
- parameters: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical biochemistry:
- in 5 randomly selected males and females (only lactating females) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
- parameters: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total bilirubin (TBIL), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Urinalysis:
- in 5 randomly selected males and females (only lactating females) prior to or as part of the sacrifice of the animals.
- urine colour/ appearance was recorded
- parameters: specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Sacrifice and pathology:

Males: sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days)
Females: sacrificed on respective PND 13 by using anesthesia (ketamine/xylazine). Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal smear or from the last day of mating period.

All animals: subjected to a detailed gross necropsy including careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ weights at necropsy:
- wet weight of the organs of 5 randomly selected male and female animals (only lactating females) from each group.
- paired organs were weighed together.
- organ weights of animals found dead were not recorded.
- Reproductive organs were weighed from all animals.
- Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/parathyroid glands was measured after fixation.
- The following tissues/organs were examined: testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight),
thyroid/parathyroid glands (from all adult males and females), liver, kidneys (paired weight), heart.
Further tissues/organs from the same selected animals were preserved.

All animals found dead: subjected to a gross necropsy and the organs preserved for a histopathological examination.

Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination.

Histopathology:
- a full histopathology on the preserved organs and tissues of the selected animals of the control and HD group which were sacrificed at the end of the treatment period.
- evaluation of thyroid/parathyroid glands from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males sacrificed on PND 13.
- a full histopathology was carried out on the preserved organs and tissues of all animals which died during the study.

Any gross lesion macroscopically identified was examined microscopically in all animals.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test.
Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests.
These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

- Salivation & moving bedding on few days, observed immediately after the dose administration for few minutes. Considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no adverse toxicological relevance.
- During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, two mortalities were observed:
- Female (Control) was found dead on post natal day (PND) 13. No specific clinical signs were observed in this animal before death or during entire study period
- Male (Low Dose) was found dead on premating day (PMD) 9. Clinical sign observed before death was severely increased salivation on PMD 8

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

- Males and females: no test item treatment related effect for body weight and body weight gain in dose groups (DGs)
- No statistically significant differences for body weight and body weight gain between DGs and control except statistically significantly lower body weight gain in LD males during premating day 1-7 and statistically significantly higher body weight gain during gestation day 14-20 in HD group when compared with the controls. These differences were either marginal, within the biological variation or due to lack of dose dependenc, not considered to be adverse due to treatment with test item.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

- food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group (in correlation with bw and bw gain).
- no test item related or statistically significant effect on food consumption in males and females during the whole study period except statistically significantly higher food consumption in HD group females during PND 0-4 when compared with the controls. This marginal increase in food consumption has no toxicological significance.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

- males sacrificed at the end of treatment period: no test item related adverse effects (treatment vs control)
- females sacrificed at the end of treatment period: no test item related effect or statistically significant effect observed on any of the haematology parameters except statistically lower reticulocytes in MD group when compared with the controls. Due to lack of dose dependency, this effect on reticulocytes in MD group was considered as incidental.

All other group mean and most of the individual values for haematological parameters in male and females were comparable with the controls. No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

- males sacrificed at the end of treatment period: no test item related or statistically significant effect on any clinical biochemistry parameter except statistically significantly lower ASAT group mean value in all treatment groups and ALAT in MD group. These lower liver enzyme values have no toxicological significance. In the light of no histopathological findings in liver and all individual values were within the historical data range (ASAT- 26-175.5 U/L, ALAT- 14.8-126.5 U/L), this statistically significant effect in males was considered as incidental and not related to test item.

- females sacrificed at the end of treatment period: statistically significantly higher ASAT in HD group and lower ASAT, ALAT, Creatinine, urea, cholesterol, glucose and sodium in LD group was observed when compared with the controls. As all individual values were within the historical data range (ASAT- 21.9- 219.8 U/L) and due to lack of dose dependency, this effect on few clinical chemistry parameters in females was considered as incidental and toxicologically irrelevant.
All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls.
There were no biologically relevant differences observed in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

- Males & females: no statistically significant differences in the absolute and relative organ weights except statistically significantly higher relative thyroid/parathyroid and prostate with seminal vesicles weights were observed in LD group males when compared with controls. Due to lack of dose dependency and in the light of fact that no test item related histopathological findings were observed in these organs, this marginal increase in organ weights was not considered to be toxicologically relevant.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.

- predominant macroscopic changes observed: right kidney dilatation (male no. 81 of LD group), cyst on ovary (female no. 46 of control group). Few organs like gastrointestinal tract, lung, kidney and spleen from female no. 49 of control group were autolytic.

Macroscopic findings correlating with histopathological observations were observed in following animals:
- Animal no. 46 the cyst observed at necropsy histologically correlated with an ovarian follicular cyst
- Animal no. 81 the dilatation observed at the right kidney during the necropsy correlated histologically with pelvic dilatation
The above mentioned findings were deemed incidental.
All other observed gross lesions reflected the animal physiology or, in absence of corresponding histopathological findings, were considered of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

-1 control group female (Animal No. 49) & 1 LD group male (Animal No. 85) were found dead on study day 51 and 9 respectively. For both the cause of morbidity was not evident at histopathological evaluation.
All findings observed at necropsy were considered not to be test item related and deemed to be incidental.
The histopathology evaluation did not reveal any test item related morphological evidence of human relevant toxicities in the organs and tissues examined. The accumulation of hyaline droplets in renal tubular epithelial cells from males belonging to the control and high dose group is a male rat specific phenomenon with no toxicological relevance in humans.

The histopathological NOAEL: 1000 mg//kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

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: general toxicity

Key result

Critical effects observed:

no

Conclusions:

The NOAEL for the test item Reactive Yellow 42 in this study is considered to be 1000 mg/kg bw/day for general toxicity.

Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day.

In conclusion:

- there were two mortalities observed in the study (female, control group and male, low dose group). Histopathologically the cause of the death was not evident.

- no adverse effects of the test item were found on male and female rats after clinical observations of systemic toxicity, functional observations, body weight development, food consumption and thyroid hormone analysis in parental males, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

The NOAEL of the test item Reactive Yellow 42 in this study for general toxicity is considered to be 1000 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017-01-10 to 2017-01-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October, 2008

Principles of method if other than guideline:

There are no regulatory guidelines for this type of dose range-finding study, but the design of this study is based on the principles specified in following test guidelines on the toxicity testing of chemicals.
OECD Guideline for the Testing of Chemicals (Section 4 Health Effects), Test No. 407, Repeated Dose 28-Day Oral Toxicity Study in Rodents (October 03, 2008)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
- female animals were non-pregnant and nulliparous
- Age at the start of the treatment period: 7-8 weeks old
- Body weight at the allocation of the animals to the experimental groups:
-- males: 180 – 210 g (mean: 194.86 g, ± 20% = 155.89 – 233.83 g)
-- females: 155 – 187 g (mean: 168.86 g, ± 20% = 135.09 – 202.63 g)
Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were group housed (3 animals/sex/cage) in IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding
- Adequate acclimatization period (at least five days)

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dose formulation analysis have not been conducted for this dose range-finding study.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

7 days per week for a period of 14 days.

Dose / conc.:

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

Remarks:

control (C)

Dose / conc.:

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

Remarks:

low dose (LD)

Dose / conc.:

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

Remarks:

medium dose (MD)

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Dose Level:
The dose level was selected based on a previous acute oral toxicity study in rats where the LD50 was > 2000 mg/kg. The dose levels selected for this study will be 300, 600 and 1000 mg/kg for low, mid and high dose groups, respectively.Control group will be administered with vehicle.

Number and Sex of Animals:
- 24 animals (12 males and 12 females) were used for the study (3 male and 3 female animals per group).
The control group animals (3 males and 3 females) were used together with another study.

Preparation of the Animals:
- Prior to the start of the treatment period a detailed clinical observation outside the home cage was made
- Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively
- Each animal was marked for individual identification with an ear tattoo and/or tail number

Evaluation of Results:
The findings of this study were evaluated in terms of the observed effects.
Parameters like body weight gain were calculated for each animal and food consumption for each cage as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and presented as percentage.

All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).

Deviations:
Inadvertently, the weighed organs were not preserved. This deviation did not influence the quality or integrity of the present study.

Positive control:

not applcable

Observations and examinations performed and frequency:

Body Weight and Food Consumption:
The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the treatment period as well as on the day of necropsy.
Food consumption was measured on study days 1, 8 and 14 for each cage.

Clinical Observations:
All Animals were observed for clinical signs during the entire treatment period of 14 days.
General clinical observations were made at least once a day, approximately at the same time each day and considering the peak period of anticipated effects after dosing.
The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

Sacrifice and pathology:

Haematology:
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
The following haematological parameters were examined: haematocrit value (HCT), haemoglobin content (Hb), red blood cell count (RBC),platelet count (PLT), white blood cells (WBC).

Clinical Biochemistry:
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total cholesterol (Chol), sodium (Na), potassium (K).

Pathology:
On study day 15, all animals of the study were sacrificed using anesthesia (ketamine, xylazin) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight:
The wet weight of the following organs was taken from all sacrificed animals as soon as possible. Paired organs were weighed together.
Organ weights at necropsy: liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix thymus, thyroid/parathyroid glands, spleen, brain, heart.

Other examinations:

not specified

Statistics:

not specified

Clinical signs:

no effects observed

Description (incidence and severity):

No test item related clinical signs were observed in any male and female animals during the entire study period.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study and all animals were sacrificed on day 15.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

In males and females, mean body weight increased with the progress of the study in all treatment groups when compared with the controls and no test item related effect was observed on body weight and body weight gain in treatment groups during the entire study period. However body weight gain was decreased during few study intervals in males and females. Due to lack of dose dependency and consistency, this effect on body weight development was not considered to be toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females, no test item related effect on food consumption was observed during the whole study period. In correlation to the body weight and body weight gain, the food consumption was comparable in treatment groups when compared with the control group.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

In males and females, no test item related effect on haematological parameters was observed in treated groups when compared with the controls. All individual and group mean values were within the biological variation and historical control data range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males and females, no test item related effect on clinical biochemistry was observed in treated groups and all individual and group mean values were comparable with controls and within the historical control data range.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, no test item related organ weight effects were observed in all treatment groups when compared with the controls except statistically significantly higher thymus and kidney relative (to body weight) were observed in LD group males. Due to lack of dose dependency and consistency, this effect on organ weight in LD group male was not considered to be test item related and toxicologically relevant.
There was increase or decrease in few absolute and relative organs weights (testes, spleen, thyroid/parathyroid, adrenal gland, heart, epididymides, liver and prostate with seminal vesicles and coagulating gland in males and brain, spleen, thymus, thyroid/parathyroid, adrenal gland, heart, liver, ovaries, uterus with cervix in females) observed when compared with the controls. However, due to lack of dose dependency, this effect was not considered to be test item related.

Gross pathological findings:

no effects observed

Description (incidence and severity):

At necropsy of males and females, macroscopic examination of the animals revealed no macroscopic findings except in females fluid filled ureters (1/3 in C and MD group) and fluid filled uterus (1/3 in C) were observed. This gross pathological finding in one female was spontaneous in nature and as such not due to a systemic effect due to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Remarks on result:

other: other: Not determinable because of methodological limitations. This RSS relates to the Dose Range Finder study. The dose descriptor is determined in the main test.

Conclusions:

Under the conditions of the present study, the repeated oral application of the test item to male and female Wistar rats at doses of 300, 600 and 1000 mg/kg body weight for 14 days was associated with no major signs of toxicity or mortality.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Reactive Yellow 42 via oral administration to rats over a period of 14 days. There are no regulatory guidelines for this type of dose range-finding study, but the study design is based on the principles specified in OECD TG 407. The study was non-GLP.

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 14 days. Animals of an additional control group were handled identically as the dose groups but received the vehicle only (sterile water). The 4 groups comprised 3 male and 3 female Wistar rats each at doses of 0 (control), 300 (low dose), 600 (mid dose) and 1000 mg/kg bw/day (high dose).

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in sterile water and administered daily during a 14-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurement.

During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken.

No mortality occurred in the control or any of the dose groups during the treatment period of this study andall animals were sacrificed on day 15. No test item related clinical signs were observed in anymale and femaleanimals during the entire study period.

In males and females, mean body weight increased with the progress of the study in all treatment groups when compared with the controls and no test item related effect was observed on body weight and body weight gain in treatment groups during the entire study period. However body weight gain was decreased during few study intervals in males and females.

In males and females, no test item related effect on food consumption was observed during the whole study period. In correlation to the body weight and body weight gain, the food consumption was comparable in treatment groups when compared with the control group.

In males and females, no test item related effect on haematological and clinical biochemistry parameters was observed in treated groups when compared with the controls.

At necropsy of males and females, macroscopic examination of the animals revealed no macroscopic findings except in females fluid filled ureters (1/3 in the control and medium dose group) and fluid filled uterus (1/3 in the control group) were observed. These macroscopic findings were considered to be spontaneous in nature.

In males and females, no test item related organ weight effects were observed in all treatment groups when compared with the controls.

In conclusion, in males and females, no test item related organ weight effects were observed in all treatment groups when compared with the controls except statistically significantly higher thymus and kidney relative (to body weight) were observed in the low dose group males. Due to lack of dose dependency and consistency, this effect on organ weight in the low dose group males was not considered to be test item related and toxicologically relevant.

There was increase or decrease in few absolute and relative organs weights observed when compared with the controls. However, due to lack of dose dependency, this effect was not considered to be test item related.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

No issues with the quality of the study

System:

other: general toxicity

Organ:

kidney

liver

lungs

ovary

parathyroid gland

seminal vesicle

spleen

thyroid gland

ventral prostate gland

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Dose range finding 14-day repeated dose toxicity

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of Reactive Yellow 42 via oral administration to rats over a period of 14 days to 3 male and 3 female Wistar rats per dose at doses of 300, 600 and 1000 mg/kg bw/day. (non-GLP study with design based on the principles specified in OECD TG 407)

No mortality occurred in the control or any of the dose groups during the treatment period of this study and all animals were sacrificed on day 15. No test item related clinical signs were observed in males or femals. No differences in haematolgical or clinical biochemistry parameters were observed in males or females compared to controls. At necropsy of males and females, macroscopic examination revealed no macroscopic findings except in females where fluid filled ureters (1/3 in the control and medium dose group) and fluid filled uterus (1/3 in the control group) were observed. These macroscopic findings were considered to be spontaneous in nature.

In males and females, no test item related organ weight effects were observed in all treatment groups when compared with the controls except statistically significantly higher thymus and kidney relative (to body weight) were observed in the low dose group males. Due to lack of dose dependency and consistency, this effect on organ weight in the low dose group males was not considered to be test item related and toxicologically relevant. There was increase or decrease in few absolute and relative organs weights observed when compared with the controls. However, due to lack of dose dependency, this effect was not considered to be test item related.

28-Day repeated dose toxicity

The aim of this study, performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day.

There were two mortalities observed in the study (female, control group and male, low dose group). Histopathologically the cause of the death was not evident. No adverse effects of the test item were found on male and female rats after clinical observations of systemic toxicity, functional observations, body weight development, food consumption and thyroid hormone analysis in parental males, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

Justification for classification or non-classification

28-Day repeated dose toxicity

The key study, performed according to OECD TG 422 and in compliance to GLP, assessed the possible adverse effects of Reactive Yellow 42 on male and female Wistar rats after repeated dose administration with dose levels of 100, 300, and 1000 mg/kg body weight/day.

The NOAEL of the test item Reactive Yellow 42 in this study for general toxicity is considered to be 1000 mg/kg bw/day. The substance did not induce any adverse effects after 28 days of repeated dosing.

According to the criteria set out in the CLP Regulation No 1272/2008 in section 3.9, the substance does not meet the criteria for classification according to CLP regulation. The substance is therefore found to be not toxic after repeated dosing.