Reactive Orange 143

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

07 February 2012 to 06 March 2012

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2012

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Test guideline 2

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, 2000.

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

yes

Test material

Test material information

Constituent 1

Details on test material:

- Name of test material (as cited in study report): FAT 40854/A TE
- Physical state: Solid
- Storage condition of test material: At room temperature in the dark

Specific details on test material used for the study:

Identification: FAT 40854/A TE
Description: Reddish-brown powder (determined at NOTOX)
Batch: TZ 5719 / BOP 02-11
Content: 46.2 % (4 main constituents)
Test substance storage At room temperature in the dark
Stability under storage conditions: Stable
Expiry date: 01 April 2016
Solubility in Water: More than 80 g/L

Test animals

Species:

rat

Strain:

Wistar

Remarks:

Crl:WI(Han)

Details on species / strain selection:

Test system: Rat: Crl:WI(Han) (outbred, SPF-Quality).
Rationale: Recognized by international guidelines as the recommended test system (e.g. EPA, FDA, OECD and EC).

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Charles River Deutschland, Sulzfeld, Germany.
- Age at study initiation: Young adult animals (approx. 6 weeks old)
- Weight at study initiation: Body weight variation was within ±20 % of the sex mean.
- Housing: Group housing of 3 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18.7 – 23.3 ºC
- Humidity (%): 41 - 61 %
- Air changes (per hr): approx. 15
- Photoperiod (hrs dark / hrs light): 12/12

On one day during acclimatization period the lights were turned off for 20 minutes, due to a technical error. Deviation was of a slight and incidental nature, and was therefore considered not to have adversely affected the study outcome.

IN-LIFE DATES: From: 07 February 2012 to 06 March 2012

Administration / exposure

Route of administration:

oral: gavage

Details on route of administration:

Oral gavage, using a plastic feeding tube. Formulations were placed on a magnetic stirrer during dosing.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. No correction was made for the purity of the test substance.

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated according to the latest body weight.

On Day 3 and 11 the use of magnetic stirrer was not recorded in the study daybook. The use of the magnetic stirrer is standard in these kinds of studies. Furthermore, the formulation was considered to be homogeneous to visually acceptable levels by the bio-technicians prior to dosing. Based on the low incidence of this deviation and the accepted homogeneity to visually acceptable levels, not recording the use of magnetic stirrer is considered to have no adverse influence on the study outcome.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analyses were conducted on a single occasion during the pretreatment and/or treatment phase, according to a validated method (NOTOX project 497818). Samples of formulations were analyzed for homogeneity (highest and lowest concentration) and accuracy of preparation (all concentrations). Stability in vehicle over 6 hours at room temperature under protection from light was also determined (highest and lowest concentration). The accuracy of preparation was considered acceptable if the mean measured concentrations were 90-110 % of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation was ≤ 10 %. Formulations were considered stable if the relative difference before and after storage was maximally 10 %. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10 %). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily, 7 d/w.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Remarks:

Control

Doses / concentrations 2

Dose / conc.:

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

Remarks:

Low dose

Doses / concentrations 3

Dose / conc.:

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

Remarks:

Middle dose

Doses / concentrations 4

Dose / conc.:

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

Remarks:

High dose

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Dose levels are based on results of a10-day dose range finding study with FAT 40854/A TE (NOTOX project 497845).

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

Mortality / Viability: At least twice daily.
Clinical signs: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals immediately after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded. Signs were graded for severity and the maximum grade was predefined at 3 or 4. Grades were coded as slight (grade 1), moderate (grade 2), severe (grade 3) and very severe (grade 4). For certain signs, only its presence (grade 1) or absence (grade 0) was scored.
Functional Observations: During week 4 of treatment, the following tests were performed on all animals after dosing (abbreviations mentioned in the respective tables indicated between brackets): - hearing ability (HEARING), pupillary reflex (PUPIL L/R), static righting reflex (STATIC R) and grip strength (GRIP) (Score 0 = normal/present, score 1 = abnormal/absent). - locomotor activity (recording period: 1-hour under normal laboratory light conditions, using a computerized monitoring system, Kinder Scientific LLC, Poway, USA). Total movements and ambulations are
reported. Ambulations represent movements characterized by a relocation of the entire body position like walking, whereas total movements represent all movements made by the animals, including ambulations but also smaller or more fine movements like grooming, weaving or movements of the head.
Body weights: Weekly.
Food consumption: Weekly.
Water consumption: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

Clinical laboratory investigations:
Blood samples were collected under anaesthesia using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) between 7.00 and 10.30 a.m. on the day of necropsy at the end of the treatment. Animals were deprived of food overnight (for a maximum of 20 hours), but water was available. Blood samples were drawn from the retro-orbital sinus of all rats/sex/group and collected into tubes (Greiner Bio-One GmbH, Kremsmünster, Austria) prepared with EDTA for haematological parameters (0.5 mL), with citrate for clotting tests (0.45 mL) and Li-heparin treated tubes for clinical biochemistry parameters (0.5 mL). An additional blood sample (0.25 mL) was collected into untreated tubes for determination of bile acids.

Sacrifice and pathology:

Animals surviving to the end of the observation period and all moribund animals were deeply anaesthetized using isoflurane (Abbott B.V., Hoofddorp, The Netherlands) and subsequently exsanguinated and subjected to a full post mortem examination. All animals assigned to the study were necropsied and descriptions of all macroscopic abnormalities recorded. Samples of the tissues and organs as per guideline were collected from all animals at necropsy and fixed in 10 % buffered formalin. Tissues/organs mentioned in parentheses were not examined by the pathologist, since no signs of toxicity were noted at macroscopic examination.
Organ weights:
The following organ weights and terminal body weight were recorded from the surviving animals on the scheduled day of necropsy: Adrenal glands, Spleen, Brain, Testes, Epididymites, Thymus, Heart, Uterus (including cervix), Kidneys, Prostate, Liver, Seminal vesicles including coagulating glands, Ovaries, Thyroid including parathyroid.

Histotechnology
All organ and tissue samples, as defined under Histopathology (following), were processed,
embedded in paraffin wax (Klinipath, Duiven, The Netherlands), cut at a thickness of 2-4 micrometers and stained with haematoxylin and eosin (Klinipath, Duiven, The Netherlands).
Histopathology:
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all Main group 1 and 4 animals,
- all tissues from all animal of all dose groups which died spontaneously or were terminated in extremis,
- stomach and Peyer’s patches of all animals of groups 2 and 3 (males and females), based on (possible) treatment-related changes in these organs in group 4,
- kidneys of all animals of groups 2 and 3 (males), based on (possible) treatment-related changes in this organ in group 4,
- all gross lesions.
All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
Motor activity data was subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup differences followed by the Wilcoxon test to compare the treated groups to the control group. All tests were two-sided and in all cases p <0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

However, red faeces were noted in the highest dose group of males and females. This is considered to have arisen as a result of treatment (red test substance). Incidental findings in single animals that were noted included salivation, scabs and/or a wound. These findings occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, these were considered signs of no toxicological significance.

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

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:

no effects observed

Description (incidence and severity):

Any statistically significant changes at 100 and 300 were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and/or remained within the range considered normal for rats of this age and strain. These findings included: Slightly lower mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC) in males at 300 mg/kg and higher neutrophils (%) and lower lymphocytes (%) in females at 300 mg/kg and lower basophils (%) in females at 100 mg/kg.
The increases of neutrophil counts with concurrently reduced lymphocyte counts, which were noted in females without a treatment related distribution, could be considered to be a secondary non-specific response to stress and to be of no toxicological significance.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

Lower bilirubin levels were noted in males and females at 1000 mg/kg, which remained within the range considered normal for rats of this age and strain. A lower level bilirubin is considered to be not relevant in toxicological terms.
Furthermore, any statistically significant changes in females at 100 and 300 mg/kg were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These findings included higher level of aspartate aminotransferase (ASAT) and chloride and lower level of inorganic phosphate.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals. A lower total movement count was noted in females at 1000 mg/kg and a higher total movement count was noted in males at 100 mg/kg. These changes occurred in the absence of a clear dose related distribution, supportive clinical signs and similar results in the opposite sex. Therefore the variation in motor activity is considered to be of no toxicological significance.
All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

no effects observed

Description (incidence and severity):

Statistically significant changes were noted in seminal vesicles weights (males 300 mg/kg), relative adrenal weight (males 100 mg/kg) and relative spleen weight (females 300 mg/kg) between treated and control animals. These findings were considered not to be a sign of toxicity as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Reddish discolouration of the glandular mucosa of the stomach was noted in one male at 1000 mg/kg. This finding is considered to be caused by pigment of the test substance, corresponding with the microscopic finding in the stomach.
The incidence of other incidental findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, and did not show a dose-related incidence trend. These necropsy findings were therefore considered to be of no toxicological relevance, and included scab formation, foci in the stomach, discolouration of the thymus and fluid in the uterus.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In the kidney a dose-dependent increased incidence and severity was noted in hyaline droplets (up to moderate) in males at 300 and 1000 mg/kg/day. In this study the occurrence of cortical hyaline droplets was accompanied by a slightly increased incidence and severity of corticomedullary tubular basophilia in males at 300 and 1000 mg/kg/day compared to control. (Group 1: 3/5 grade 1, Group 2: 1/5 grade 1, Group 3: 1/5 grade 1 and 1/5 grade 2, and Group 4: 1/5 grade 1 and 2/5 grade 2).
Red/brown pigment was noted in the mucosa of the stomach (minimal to slight) in half of the animals of Group 4. In addition red/brown pigment was noted in the Peyer’s patches (minimal) in half of the animals of Group 4. The nature and severity of these microscopic findings were not considered to be toxicologically relevant and not adverse.
All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both control and treated rats.

Other effects:

no effects observed

Details on results:

Analysis of dose preparations:
The purpose of this part of the study was to determine the accuracy of preparation, homogeneity and stability of the test substance in formulations. The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 90 % and 110 %). No test substance was detected in the Group 1 formulation. The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤10 %). Formulations at the entire range were stable when stored at room temperature under normal laboratory light conditions for at least 6 hours.

Effect levels

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: Based on study findings

Target system / organ toxicity

Critical effects observed:

not specified

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for FAT 40854/A TE was 1000 mg/kg.

Executive summary:

A GLP-compliant repeated dose 28-day oral toxicity study with FAT 40854/A by daily gavage in the rat was carried out according to EU method B.7 and OECD guideline 407. Based on the results of a 10-day range finding study the dose levels for this 28-day oral gavage study were selected to be 0, 100, 300 and 1000 mg/kg. The test substance formulated in Water was administered daily for 28 days by oral gavage to SPF-bred Wistar rats. One control group and three treated groups were tested, each consisting of 5 males and 5 females. Chemical analyses of formulations were conducted once during the study to assess accuracy, homogeneity and stability over 6 hours. The parameters evaluated were clinical signs daily; functional observation tests in week 4; body weight and food consumption weekly; clinical pathology and macroscopy at termination; organ weights and histopathology on a selection of tissues. Formulation analyses confirmed that formulations of test substance in Water were prepared accurately and homogenously and were stable over at least 6 hours. No toxicologically significant changes in clinical appearance, functional observations, body weight, food consumption, clinical investigations, macroscopic examination and organ weights were noted in this study with FAT 40854/A up to 1000 mg/kg. The red color of the FAT 40854/A may have caused red faeces as noted males and females at 1000 mg/kg. At this dose leveI red/brown pigment was also noted at macroscopic and/or microscopic examination in the mucosa of the stomach and in the Peyer’s patches. In the kidney a dose-dependent increased incidence and severity was noted in hyaline droplets (up to moderate) in males at 300 and 1000 mg/kg. These droplets were considered to represent alpha-2µ globulin, a normal protein in male rats that undergoes reabsorption in the proximal cortical tubules. A range of chemicals is known to increase hyaline droplet formation beyond the physiological capacity of the tubular epithelium, which may result in tubular cell degeneration. The occurrence of cortical hyaline droplets was accompanied by an increased incidence and/or severity of tubular basophilia (compared to control animals). This specific male rat response is not observed in normal female rats and higher species of either sex, including humans. Therefore, this finding is not toxicological relevant for humans. Based on these results a No Observed Adverse Effect Level (NOAEL) for FAT 40854/A of at least 1000 mg/kg was established.