Tetralithium 5,5'-[vinylenebis[(3-sulphonato-4,1-phenylene)azo]]bis[3-methylsalicylate]

Endpoint summary

Description of key information

A dietary concentration of 1000 ppm is a clear NOAEL for the rat. Only the presence of hyaline droplets in male kidneys prevents an adjusted dietary concentration of 2500 ppm (highest dose tested) being classified as being a NOAEL and, given the non-relevance of this finding to man, this dietary level may be regarded as a NOAEL when assessing the hazard of the Test Item to man.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Experimental Starting Date: 14 December 2016, Experimental Completion Date: 17 April 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

Physical State/Appearance: Red/brown powder
Storage Conditions: Stored at ambient temperature in darkness over silica gel. Used/formulated in light at ambient humidity.
Expiry Date: 27/02/2017

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected for this study as it is a readily available rodent species historically used in safety evaluation studies and is acceptable to appropriate regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animal Information
A sufficient number of male and female Wistar Han™:RccHan™:WIST strain rats were obtained from Envigo RMS (UK) Limited, Oxon, UK. On receipt the animals were examined for signs of ill-health or injury. The animals were acclimatized for six days during which time their health status was assessed. A total of forty animals (twenty males and twenty females) were accepted into the study. At the start of treatment the males weighed 166 to 188g, the females weighed 142 to 164g, and were approximately six to eight weeks old.

Animal Care and husbandry
The animals were housed in groups of five by sex in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK). The animals were allowed free access to food and water. A ground diet (Rat and Mouse SQC Ground Diet No. 1, Special Diet Services Dietex International Ltd, Witham, Essex UK) was used. Mains drinking water was supplied from polycarbonate bottles attached to the cage. Environmental enrichment was provided in the form of wooden chew blocks and cardboard fun tunnels (Datesand Ltd., Cheshire, UK). The diet, drinking water, bedding and environmental enrichment were considered not to contain any contaminant at a level that might have affected the purpose or integrity of the study.
The animals were housed in a single air-conditioned room within the Envigo Research Limited, Shardlow, UK Barrier Maintained Rodent Facility. The rate of air exchange was at least fifteen air changes per hour and the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness. Environmental conditions were continuously monitored by a computerized system, and print-outs of hourly temperatures and humidities are included in the study records. The Study Plan target ranges for temperature and relative humidity were 22 ± 3 °C and 50 ± 20% respectively; there were no deviations from these targets.
The animals were randomly allocated to treatment groups using a stratified body weight randomization procedure and the group mean body weights were then determined to ensure similarity between the treatment groups. The cage distribution within the holding rack was also randomized. The animals were uniquely identified within the study by an ear punching system routinely used in these laboratories.

Route of administration:

oral: feed

Details on route of administration:

The oral route was selected as the most appropriate route of exposure, based on the physical properties of the test item, and the results of the study are believed to be of value in predicting the likely toxicity of the test item to man.

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

The dietary concentrations were chosen based on the results of previous toxicity work including a fourteen day range-finding study in the rat.
The test item was incorporated into the diet at concentrations of 500, 1000 and 2500 ppm as follows:
A known amount of test item was mixed with a small amount of basal laboratory diet until homogeneous in a Robot Coupe Blixer 4 set at a constant speed. This pre-mix was then added to a larger amount of basal laboratory diet and mixed for a further thirty minutes at a constant speed, setting 1 in a Hobart H800 mixer.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Investigation of the actual concentration of the dietary formulations at nominal concentrations of 500 and 1000 ppm revealed lower than expected results during the study. Subsequent investigations showed that at these lower concentrations the diet matrix was interfering with the analytical procedure resulting in the analytical response being no longer linear (see Envigo Research Limited study NR32HS). As such, the apparent measured concentration of test item in the diet at these dietary concentrations was considered to be lower than the actual concentration in the diet. It is considered that the calculation of achieved dosages using the nominal concentrations represents the most accurate estimation of the dosage received by the animals.
Achieved concentration were similar for both sexes within each dietary concentration and the achieved intakes reflected the selected intervals between the dietary concentration (i.e. the two and five fold increase from the lowest dietary level).

Duration of treatment / exposure:

28 days

Frequency of treatment:

continuous

Dose / conc.:

500 ppm

Dose / conc.:

1 000 ppm

Dose / conc.:

2 500 ppm

No. of animals per sex per dose:

5 males and 5 females at each dose.

Control animals:

yes, plain diet

Observations and examinations performed and frequency:

General Observations/Measurements
Clinical Observations
All animals were examined for overt signs of toxicity, ill-health or behavioral change daily from the start of treatment. All observations were recorded.
Body Weight
Individual body weights were recorded on Day 1 (prior to the start of treatment) and at weekly intervals thereafter. Body weights were also performed prior to terminal kill.
Food Consumption
Food consumption was recorded for each cage group at weekly intervals throughout the study. Food conversion efficiency and mean achieved dosage were calculated retrospectively.
Water Consumption
Water intake was measured and recorded daily for each cage group.

Special Evaluations
Functional Observations
Prior to the start of treatment and on Days 7, 14, 21 and 25, all animals were observed for signs of functional/behavioral toxicity. Functional performance tests were also performed on all animals during Week 4, together with an assessment of sensory reactivity to different stimuli.
Behavioral Assessments
Detailed individual clinical observations were performed for each animal using a purpose built arena. The following parameters were observed:
Gait
Hyper/Hypothermia
Tremors
Skin color
Twitches
Respiration
Convulsions
Palpebral closure
Bizarre/Abnormal/Stereotypic behavior
Urination
Salivation
Defecation
Pilo-erection
Transfer arousal
Exophthalmia
Tail elevation
Lachrymation
This test was developed from the methods used by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.

Functional Performance Tests
Motor Activity. Twenty purpose built 44 infra-red beam automated activity monitors were used to assess motor activity. Animals of one sex were tested at each occasion and were randomly allocated to the activity monitors. The tests were performed at approximately the same time each occasion (at least two hours after dosing), under similar laboratory conditions. The evaluation period was one hour for each animal. The time in seconds each animal was active and mobile was recorded for the overall one hour period and also during the final 20% of the period (considered to be the asymptotic period, Reiter and Macphail 1979).
Forelimb/Hindlimb Grip Strength. An automated grip strength meter was used. Each animal was allowed to grip the proximal metal bar of the meter with its forepaws. The animal was pulled by the base of the tail until its grip was broken. The animal was drawn along the trough of the meter by the tail until its hind paws gripped the distal metal bar. A record of the force required to break the grip for each animal was made. Three consecutive trials were performed for each animal. The assessment was developed from the method employed by Meyer et al (1979).

Sensory Reactivity
Each animal was individually assessed for sensory reactivity to auditory, visual and proprioceptive stimuli. This assessment was developed from the methods employed by Irwin (1968) and Moser et al (1988). The scoring system used is outlined in The Key to Scoring System and Explanation for Behavioral Assessments and Sensory Reactivity Tests.
The following parameters were observed:
Grasp response
Touch escape
Vocalization
Pupil reflex
Toe pinch
Blink reflex
Tail pinch
Startle reflex
Finger approach

In-Life Sampling and Analysis
Hematological and blood chemical investigations were performed on all animals from each test and control group at the end of the study (Day 28). Blood samples were obtained from the lateral tail vein. Where necessary repeat samples were obtained by cardiac puncture prior to necropsy on Day 29. Animals were not fasted prior to sampling.

Hematology
Hemoglobin (Hb)
Erythrocyte count (RBC)
Hematocrit (Hct)
Erythrocyte indices
- mean corpuscular hemoglobin (MCH)
- mean corpuscular volume (MCV)
- mean corpuscular hemoglobin concentration (MCHC)
Total leukocyte count (WBC)
Differential leukocyte count
- neutrophils (Neut)
- lymphocytes (Lymph)
- monocytes (Mono)
- eosinophils (Eos)
- basophils (Bas)
Platelet count (PLT)
Reticulocyte count (Retic)
Prothrombin time (CT) was assessed by ‘Innovin’ and Activated partial thromboplastin time (APTT) was assessed by ‘Actin FS’ using samples collected into sodium citrate solution (0.11 mol/L).

Blood Chemistry
The following parameters were measured on plasma from blood collected into tubes containing lithium heparin anti-coagulant:
Urea
Inorganic phosphorus (P)
Glucose
Aspartate aminotransferase (ASAT)
Total protein (Tot.Prot.)
Alanine aminotransferase (ALAT)
Albumin
Alkaline phosphatase (AP)
Albumin/Globulin (A/G) ratio (by calculation)
Creatinine (Creat)
Sodium (Na+)
Triglycerides (Trigs/Tri)
Potassium (K+)
Total cholesterol (Chol)
Chloride (Cl-)
Total bilirubin (Bili)
Calcium (Ca++)
Bile acids

Sacrifice and pathology:

Necropsy
On completion of the dosing period all surviving animals were killed by intravenous overdose of sodium pentobarbitone followed by exsanguination.
All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

Thyroid Hormone Assessment
At termination, blood samples were taken from the exsanguination procedure and the serum from each animal was stored frozen at below -60 °C. No treatment-related effects on the pituitary-thyroid axis were identified, therefore these samples were discarded.

Organ Weights
The following organs, removed from animals that were killed either at the end of the dosing period or at the end of the treatment-free period, were dissected free from fat and weighed before fixation:
Adrenals
Liver
Brain
Ovaries
Epididymides
Spleen
Heart
Testes
Kidneys
Thymus
Pituitary (weighed after partial fixation)
Thyroid/Parathyroid (weighed after partial fixation)
Prostate and Seminal Vesicles (with coagulating glands and fluids)
Uterus with Cervix (and oviducts)

Histopathology
Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin, except where stated:
Muscle (skeletal)~
Adrenals~
Ovaries~
Aorta (thoracic)
Pancreas
Bone & bone marrow (femur including stifle joint)
Pituitary~
Bone & bone marrow (sternum)~
Prostate~
Brain (including cerebrum, cerebellum and pons)~
Rectum~
Salivary glands (submaxillary)
Caecum~
Sciatic nerve~
Colon~
Seminal vesicles (with coagulating glands and fluids)~
Duodenum~
Epididymides ♦~
Skin
Esophagus
Spinal cord (cervical, mid thoracic and lumbar)~
Eyes *~
Gross lesions~
Spleen~
Heart~
Stomach~
Ileum~
Testes ♦~
Jejunum~
Thymus~
Kidneys~
Thyroid/Parathyroid~
Liver~
Trachea~
Lungs (with bronchi)#~
Urinary bladder~
Lymph nodes (mandibular and mesenteric)~
Uterus & Cervix~
Mammary gland~
Vagina~
♦ Preserved in modified Davidson’s fluid
* Eyes fixed in Davidson’s fluid
♦ Preserved in modified Davidson’s fluid
# Lungs were inflated to approximately normal inspiratory volume with buffered 10% formalin before
immersion in fixative
All tissues were dispatched to the histology processing Test Site (Envigo CRS Limited, Eye, Suffolk, IP23 7PX) for processing (Principal Investigator: D Roberts). The tissues shown marked with a ~ from all control and 2500 ppm dose group animals were prepared as paraffin blocks, sectioned at a nominal thickness of 5 µm and stained with Hematoxylin and Eosin for subsequent microscopic examination. Any macroscopically observed lesions were also processed. In addition, sections of testes from all Control and 2500 ppm males were stained with Periodic Acid-Schiff (PAS) stain and examined.
Since there were indications of treatment-related changes, examination was subsequently extended to include similarly prepared sections of kidneys from males in the low and intermediate groups.

Pathology
Microscopic examination was conducted by the Study Pathologist (J Stewart at Envigo CRS Limited, Woolley Road, Alconbury, Huntingdon, Cambridgeshire, PE28 4HS under the supervision of the Principal Investigator D Roberts). A peer review of the histopathology results for the study was conducted by the Test Facility. A histology and histopathology examination phase report is presented in Annex 1 and represents the consensus view of both pathologists.

Statistics:

Please see any other information on materials and methods section

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs apparent during the study.

Mortality:

no mortality observed

Description (incidence):

There were no unscheduled deaths on the study.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

For males at an adjusted dietary concentration of 500 ppm, body weight gain was statistically significantly lower than control during the first week of the study. Although body weight gain remained lower than control during Weeks 2 and 3, there was no statistical significance and body weight gain was higher than control during Week 4. The observed differences in body weight gain resulted in lower overall body weight gain but there was no statistical significance.
There was no effect of dietary exposure on body weight gain for either sex at adjusted dietary concentration of 500 or 1000 ppm or for females at an adjusted dietary concentration of 2500 ppm throughout the study.
For males at an adjusted dietary concentration of 2500 ppm, lower body weight gain during the final week of treatment attained statistical significance when compared to control. However, the water bottle for this cage of animals leaked overnight and it believed that the final recorded body weight was adversely affected by this event. The lower body weight gain observed was therefore considered to be unrelated to dietary exposure to the Test Item.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

For males at an adjusted dietary concentration of 2500 ppm, food consumption was lower than control throughout the study. This lower intake may reflect poor palatability of the diet at this dietary concentration.
There was no obvious effect of dietary exposure on food consumption for either sex at adjusted dietary concentrations of 500 or 1000 ppm or for females at an adjusted dietary concentration of 2500 ppm throughout the study.

Food efficiency:

no effects observed

Description (incidence and severity):

For males at an adjusted dietary concentration of 2500 ppm, food conversion efficiency appeared lower than control during the first week of dietary exposure. Thereafter, intergroup differences in food conversion efficiency did not indicate any effect of dietary exposure.
Intergroup differences in food conversion efficiency did not indicate any obvious effect of dietary exposure for either sex at adjusted dietary concentration of 500 or 1000 ppm or for females at an adjusted dietary concentration of 2500 ppm throughout the study.

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

effects observed, treatment-related

Description (incidence and severity):

For both sexes at an adjusted dietary concentration of 2500 ppm, water consumption appeared higher than control throughout the study.
For males at adjusted dietary concentrations of 500 or 1000 ppm, although water consumption was often slightly higher than control, there was generally no dosage-relationship and, in view of this, the observed differences in water intake were considered to reflect normal biological variation.
For females at an adjusted dietary concentration of 500 ppm, water consumption was generally higher than control throughout the treatment period, with values often close to or occasionally higher, than observed at 2500 ppm. However, water intake at an adjusted dietary concentration of 1000 ppm tended to be only slightly higher than control and values were often clearly lower than observed at the lowest dietary inclusion level.

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Intergroup differences observed for hematology parameters for either sex did not indicate any effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item.
For both sexes at all adjusted dietary concentrations (500, 1000 and 2500 ppm), higher mean reticulocyte counts attained statistical significance when compared to control, although only male values showed a consistent dosage relationship. All values for treated animals were within the respective historical control ranges, while one male control value was below this historical range. In the absence of any supporting differences from control in erythrocyte parameters for these treated animals or any evidence of histopathological change, this finding was considered to be incidental and of no toxicological significance.
For males at an adjusted dietary concentration of 2500 ppm, higher mean numbers of eosinophils attained statistical significance when compared with control. However, only one value for these treated animals exceeded the historical control range and, the distribution of the data indicated that it was generally unsuitable for statistical analysis. In isolation this finding was considered incidental and unrelated to treatment.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Intergroup differences observed for blood chemistry parameters for either sex did not indicate any effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item.
For males at adjusted dietary concentrations of 1000 or 2500 ppm, higher mean inorganic phosphorus levels attained statistical significance when compared with control. However, all values for these treated animals were within the historical range and, in isolation, and in the absence of any supporting histopathological change, this finding was considered incidental and unrelated to treatment.
For females at adjusted dietary concentrations of 1000 or 2500 ppm, lower mean triglycerides levels attained statistical significance when compared with control. However, these differences appeared to be principally attributable to one atypically high control value which exceeded the historical control range and all values for these treated animals were within the historical range. This finding, in the absence of any supporting histopathological change, was considered incidental and unrelated to treatment.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

Behavioral Assessments
Weekly assessment of the animals in a standard arena did not reveal any effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item for either sex.

Functional Performance Tests
Assessment of functional performance using grip strength and motor activity did not indicate any effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item for either sex.
For males receiving 2500 ppm and females receiving 1000 ppm, fore limb grip strength at the second trial was statistically significantly higher than control but, in the absence of any similar increase in the other trials of grip strength for these animals, this finding was considered to be incidental and unrelated to treatment.

Sensory Reactivity Assessments
Intergroup differences observed in the scores for sensory reactivity did not indicate any obvious effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item for either sex.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

For males at an adjusted dietary concentrations of 2500 ppm, lower absolute and body weight relative kidney weights attained statistical significance when compared with control. For this organ, body weight relative values are normally considered to be the more accurate indicator of toxicological effect and all individual body weight relative values for these treated males were within the historical control range while one control value exceed this historical range. Although, males at this dietary level did show evidence of histopathological change (increased hyaline droplets), this finding, in the absence of necrosis, would not necessarily lead to a decrease in organ weight, and an association with the lower kidneys weights and exposure to the test item was considered equivocal.
For males at all adjusted dietary concentrations, lower absolute and body weight relative testes weights attained statistical significance when compared with control, although body weight relative values showed no dosage relationship. All individual absolute and body weight relative values were within the historical control ranges while one body weight relative control values exceeded the historical control range. For this organ, body weight relative values are normally considered to be the more accurate indicator of toxicological effect and all individual body weight relative values for these treated males were within the historical control range while one control value exceed this historical range. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.
For females at adjusted dietary concentrations of 1000 or 2500 ppm, higher absolute and body weight relative thyroid weights attained statistical significance when compared with control, although values showed no dosage relationship. At both 1000 and 2500 ppm, two individual absolute and two individual relative values exceeded the historical control ranges compared to only one absolute control value. In the absence of any supporting histopathological change this finding was considered incidental and unrelated to treatment.
For females at an adjusted dietary concentration of 2500 ppm, higher absolute and body weight relative brain and pituitary weights attained statistical significance when compared with control. All individual values for these treated females were within the historical control ranges and, in the absence of any supporting histopathological change, this finding was considered incidental and unrelated to treatment.
For females at an adjusted dietary concentration of 500 ppm, higher absolute and body weight relative uterine weights attained statistical significance when compared with control. One individual absolute value and two individual relative values exceeded the historical control ranges for these treated females, however in the absence of any statistically significant increase in mean values at higher dietary concentrations this finding was considered incidental and unrelated to treatment.

Gross pathological findings:

no effects observed

Description (incidence and severity):

Macroscopic findings at terminal necropsy did not indicate any obvious adverse effect of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item for either sex.
Yellow or orange coloured contents were observed in the stomach of 3/5 males and 1/5 females at an adjusted dietary concentration of 500 ppm, 3/5 males and 2/5 females at an adjusted dietary concentration of 1000 ppm and 4/5 males and 4/5 females at an adjusted dietary concentration of 2500 ppm. This finding was considered to reflected the colored nature of the Test Item, however no similar colored staining of the internal tissues was apparent during macroscopic necropsy.
One male receiving an adjusted dietary concentration of 500 ppm showed mottled kidneys. Additionally the kidneys for one male receiving an adjusted dietary concentration of 1000 ppm and the left kidney for one female receiving an adjusted dietary concentration of 2500 ppm showed increased pelvic space. The nature and distribution of these kidney findings did not indicate any association with treatment.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Hyaline droplets were seen in the kidneys of males at an adjusted dietary concentration of 2500 ppm.
The type, incidence and distribution of other microscopic findings observed at histopathological examination did not indicate any effects of exposure to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm of the test item for either sex.

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOAEL

Remarks:

rat

Effect level:

1 000 ppm

Based on:

test mat.

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

when assessing the hazard of the Test Item to man

Effect level:

2 500 other: ppm (approx. 212 mg/kg bw)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: The finding of hyaline droplets in male kidneys of the rats is not relevant to man.

Critical effects observed:

no

Exposure to dietary (adjusted for purity) concentrations of 500, 1000 or 2500 ppm of the test item was well tolerated by both sexes, with no clinical signs being apparent at any dietary level throughout the study. 

For males at 2500 ppm, body weight gain was statistically significantly lower than control during the first week of the study, perhaps reflecting an initial reluctance to eat the diet at this concentration. Male food consumption was lower than control throughout the study but this lower food intake did not result in any further statistically significant differences in body weight gain from control. Although, food conversion efficiency was lower during the first week of dietary exposure, subsequent values for the remainder of the study did not indicate any underlying effect on the efficiency of food utilization.

There was no obvious effect of dietary exposure on body weight gain, food consumption or food conversion efficiency for either sex at adjusted dietary concentration of 500 or 1000 ppm or for females at an adjusted dietary concentration of 2500 ppm throughout the study.

For both sexes at an adjusted dietary concentration of 2500 ppm, water consumption appeared higher than control throughout the study, which may reflect palatability of the diet. At lower dietary concentrations, intergroup differences in water intake showed no consistent dietary concentration dependency that indicated an effect of treatment.

Although differences from control for a number of hematology and blood chemistry parameters for treated animals attained statistical significance, these were considered, in the absence of supporting histopathological change, to be of no toxicological significance. 

At necropsy, yellow or orange colored contents were observed in the stomach of both sexes at adjusted dietary concentration of 500, 1000 and 2500 ppm, however this finding was consistent with the colored nature of the Test Item and in the absence of any obvious effect on the tissues, was considered to be of no toxicological significance. Occasional macroscopic kidneys findings were also apparent for isolated treated animals, but there appeared to be no correlation with histopathological changes observed for this organ and these findings were considered incidental and unrelated to treatment.

Males at all adjusted dietary concentrations showed statistically significant lower absolute and body weight relative testes weights compared with control, although relative values showed no dosage relationship. As all individual values for treated animals were within the historical control ranges and, as there was no supporting histopathological change, this finding was considered incidental and unrelated to treatment. This conclusion is consistent with the findings for an Oral (Dietary) Reproduction/Developmental Toxicity Screening Test in the Rat (OECD 421) (Envigo Study Number DT26XP) with this Test item, where no effect on fertility or on male reproductive organ weights or histopathology was apparent.

At an adjusted dietary concentration of 2500 ppm, statistically significantly lower absolute and body weight relative male kidney weights were apparent compared to control. For this organ, body weight relative values are normally considered to be the more accurate indicator of toxicological effect and all individual relative values for these treated males were within the historical control range. Microscopic examination of the male kidneys showed increased hyaline droplets, however, this finding, in the absence of necrosis, would probably be expected to lead to an increase rather than a decrease in organ weight, and an association with the lower kidneys weights and exposure to the test item was considered equivocal.

Differences from control for the following absolute and body weight relative organ weights all attained statistical significance when compared to control; higher thyroid weights for females at adjusted dietary concentrations of 1000 or 2500 ppm, higher brain and pituitary weights for females at an adjusted dietary concentrations of 2500 ppm and higher uterine weights for females at an adjusted dietary concentrations of 500 ppm. All of the majority of individual values for these indicated parameters and animals were within the respective historical control ranges and, in the absence of any supporting histopathological change, these findings were considered incidental and unrelated to treatment.

Treatment related findings observed at histopathological examination of tissues were restricted to hyaline droplets in the kidneys of males at an adjusted dietary concentration of 2500 ppm. These droplets represent accumulation of protein in the lysosomes and this is often seen in male rats due to accumulation of alpha 2u-globulin, the major urinary protein in rats, when it binds to test item and clearance is retarded. This type of hyaline droplet nephropathy is regarded as being unique to the male rat and of little relevance to man.  Therefore, while it represents an adverse effect for the male rat, it is not predictive of an adverse effect in humans. 

Overall, the male body weight and food consumption effects observed during this study at an adjusted dietary concentration of 2500 ppm (equivalent to an average dosage of 212 mg/kg bw/day) were considered insufficient to preclude this dietary level from being a No Observed Adverse Effect Level (NOAEL). With the exception of hyaline droplets in the kidneys of males, there were no other findings apparent in the study that prevented this dietary level as being classified as a NOAEL. While the presence of hyaline droplets in the male kidneys means that 2500 ppm is an adverse effect level for the rat, the non-relevance of this finding to man means that this can be regarded as a NOAEL when assessing the hazard of the Test Item to man. A dietary concentration of 1000 ppm is a clear NOAEL for the rat.

Conclusions:

A dietary concentration of 1000 ppm is a clear NOAEL for the rat. Only the presence of hyaline droplets in male kidneys prevents an adjusted dietary concentration of 2500 ppm (highest dose tested) being classified as being a NOAEL and, given the non-relevance of this finding to man, this dietary level may be regarded as a NOAEL when assessing the hazard of the Test Item to man.

Executive summary:

This study was designed to investigate the systemic toxicity of the test item. It is compatible with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

This study was also designed to be compatible with Commission Regulation (EC) No 440/2008 of 30 May 2008, laying down test methods pursuant to Regulation (EC) No 1907/2006 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH).

The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 500, 1000 and 2500 ppm (adjusted for purity), equivalent to an estimated mean achieved dosage of approximately 45, 88 and 212 mg/kg bw/day (adjusted for purity) for males and 45, 88 and 213 mg/kg bw/day (adjusted for purity) for females respectively. A control group of five males and five females were treated with basal laboratory diet over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Following results from the initial histopathology examinations, histology processing was extended to include the kidneys from males of the low and intermediate dosage groups.

Results

Achieved Dosages

Investigation of the actual concentration of the dietary formulations at normal concentrations of 500 and 1000 ppm revealed lower than expected results during the study. Subsequent investigations showed that at these lower concentrations the diet matrix was interfering with the analytical procedure resulting in the analytical response being no longer linear. As such, the apparent measured concentration of test item in the diet at these dietary concentrations was considered to be lower than the actual concentration in the diet and achieved dosages were calculated using the nominal concentrations. 

Achieved concentrations were similar for both sexes within each dietary concentration and the achieved intakes reflected the selected intervals between the dietary concentration (i.e. the two and five fold increase from the lowest dietary level).

Mortality

There were no unscheduled deaths on the study.

Clinical Observations

There were no clinical signs apparent during the study.

Behavioral Assessment

Results from behavioural assessments did not indicate any effect of exposure for either sex to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm.

Functional Performance Tests

Results from functional performance tests did not indicate any effect of exposure for either sex to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm.

Sensory Reactivity Assessments

Results from sensory reactivity assessments did not indicate any effect of exposure for either sex to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm.

Body Weight

For males at an adjusted dietary concentration of 500 ppm, body weight gain was statistically significantly lower than control during the first week of the study. Although body weight gain remained lower than control during Weeks 2 and 3, there was no statistical significance and body weight gain was higher than control during Week 4. The observed differences in body weight gain resulted in lower overall body weight gain but there was no statistical significance. 

There was no effect of dietary exposure on body weight gain for either sex at adjusted dietary concentration of 500 or 1000 ppm or for females at an adjusted dietary concentration of 2500 ppm throughout the study.

For males at an adjusted dietary concentration of 2500 ppm, lower body weight gain during the final week of treatment attained statistical significance when compared to control. However, the water bottle for this cage of animals leaked overnight and it believed that the final recorded body weight was adversely affected by this event. The lower body weight gain observed was therefore considered to be unrelated to dietary exposure to the Test Item. 

Food Consumption

For males at an adjusted dietary concentration of 2500 ppm, food consumption was lower than control throughout the study. There was no effect on food intake for females at this dietary concentration. 

There was no effect of dietary exposure on food consumption for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study.

There was no obvious effect of dietary exposure on food conversion efficiency for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study.

Water Consumption

For both sexes at an adjusted dietary concentration of 2500 ppm, water consumption was higher than control throughout the study. 

There was no obvious effect of dietary exposure on water consumption for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study. 

Hematology

There was no effect of dietary exposure on for hematology parameters for either sex at adjusted dietary concentrations of 500, 1000 and 2500 ppm.

Blood Chemistry

There was no effect of dietary exposure on for blood chemistry parameters for either sex at adjusted dietary concentrations of 500, 1000 and 2500 ppm.

Necropsy

Yellow or orange coloured contents, consistent with the colored nature of the test item, were observed in the stomach of some animals (both sexes) at adjusted dietary concentration of 500, 1000 or 2500 ppm, but no staining of the internal tissues was apparent.

Organ Weights

For males at an adjusted dietary concentration of 2500 ppm, lower absolute and body weight relative kidney weights attained statistical significance when compared with control. 

There was no effect of dietary exposure on the organ weights measured for females at an adjusted dietary concentration of 2500 ppm or for either sex at adjusted dietary concentrations of 500 and 2500 ppm.

Histopathology

Hyaline droplets were seen in the kidneys of males at an adjusted dietary concentration of 2500 ppm.

Conclusion

A dietary concentration of 1000 ppm is a clear NOAEL for the rat. Only the presence of hyaline droplets in male kidneys prevents an adjusted dietary concentration of 2500 ppm being classified as being a NOAEL and, given the non-relevance of this finding to man, this dietary level may be regarded as a NOAEL when assessing the hazard of the Test Item to man.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

212 mg/kg bw/day

Study duration:

subacute

Species:

other: highest dose tested; human-relevant NOAEL from subacute rat study (OECD TG 407)

Quality of whole database:

In this repeated dose (28-day) feed study a NOAEL was found at 2500 ppm for female rats (eq. to 213 mg/kg bw) and a LOAEL at this concentration for male rats (eq. to 212 mg/kg bw). However, the finding observed in males at the LOAEL was an effect often seen in male rats due to accumulation of alpha 2µ-globulin, a major urinary protein in rats, when it binds to test item and clearance is retarded. This effect is not relevant for humans, since the protein alpha 2µ-globulin does not exist in humans. Therefore, the human relevant NOAEL from the study is 212 mg/kg.
It is important to note that 2500 ppm was the highest dietary concentration that was practical to test, as higher levels were precluded due to the decreased food consumption considered to reflect the palatability of the dietary formulations (documented in the 14-day range finder study NR32HS)

System:

other: non human relevant alpha 2µ globulin kidney effects in male rats observed at highest dose tested (212 mg/kg bw)

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

This study was designed to investigate the systemic toxicity of the test item.  It is compatible with the requirements for notification of a new chemical substance in the EC and follows the testing method described in Commission Directive 96/54/EC (Method B7) and OECD Guidelines for Testing of Chemicals No. 407 "Repeated Dose 28 Day Oral Toxicity Study in Rodents" (adopted 03 October 2008).

The test item was administered by continuous dietary admixture to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dietary concentrations of 500, 1000 and 2500 ppm (adjusted for purity), equivalent to an estimated mean achieved dosage of approximately 45, 88 and 212 mg/kg bw/day (adjusted for purity) for males and 45, 88 and 213 mg/kg bw/day (adjusted for purity) for females respectively.  A control group of five males and five females were treated with basal laboratory diet over the same treatment period.

Clinical signs, functional observations, body weight change, dietary intake and water consumption were monitored during the study. Hematology and blood chemistry were evaluated for all animals at the end of the study.

All animals were subjected to gross necropsy examination and histopathological evaluation of selected tissues from high dose and control animals was performed. Following results from the initial histopathology examinations, histology processing was extended to include the kidneys from males of the low and intermediate dosage groups.

Results

It is important to note that 2500 ppm was the highest dietary concentration that was practical to test, as higher levels were precluded due to the decreased food consumption considered to reflect the palatability of the dietary formulations (documented in the 14-day range finder study NR32HS).

There were no unscheduled deaths on the study. There were no clinical signs apparent during the study. Results from behavioural assessments, functional performance tests, and sensory reactivity assessments did not indicate any effect of exposure for either sex to dietary (adjusted for purity) concentrations of 500, 1000 and 2500 ppm.

For males at an adjusted dietary concentration of 2500 ppm, body weight gain was statistically significantly lower than control during the first week of the study.  Body weight gain during Weeks 2 and 3 and overall body weight gain was also lower than control but was not statistically significant.  There was no effect on body weight gain for females at this dietary concentration. There was no effect of dietary exposure on body weight gain for either sex at adjusted dietary concentration of 500 or 1000 ppm throughout the study.

For males at an adjusted dietary concentration of 2500 ppm, food consumption was lower than control throughout the study. There was no effect on food intake for females at this dietary concentration. There was no effect of dietary exposure on food consumption for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study. For males at an adjusted dietary concentration of 2500 ppm, food conversion efficiency appeared lower than control during the first week of dietary exposure. There was no effect on food conversion efficiency for females at this dietary concentration. There was no effect of dietary exposure on food conversion efficiency for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study.

For both sexes at an adjusted dietary concentration of 2500 ppm, water consumption was higher than control throughout the study. There was no obvious effect of dietary exposure on water consumption for either sex at adjusted dietary concentrations of 500 or 1000 ppm throughout the study.  

There was no effect of dietary exposure on for hematology parameters or blood chemistry parameter for either sex at adjusted dietary concentrations of 500, 1000 and 2500 ppm.

Yellow or orange coloured contents, consistent with the colored nature of the test item, were observed in the stomach of some animals (both sexes) at adjusted dietary concentration of 500, 1000 or 2500 ppm, but no staining of the internal tissues was apparent.

For males at an adjusted dietary concentration of 2500 ppm, lower absolute and body weight relative kidney weights attained statistical significance when compared with control. There was no effect of dietary exposure on the organ weights measured for females at an adjusted dietary concentration of 2500 ppm or for either sex at adjusted dietary concentrations of 500 and 2500 ppm.

Hyaline droplets were seen in the kidneys of males at an adjusted dietary concentration of 2500 ppm.

Conclusion

A dietary concentration of 1000 ppm is a clear NOAEL for the rat. Only the presence of hyaline droplets in male kidneys prevents an adjusted dietary concentration of 2500 ppm (highest concentration tested) being classified as being a NOAEL and, given the non-relevance of this finding to man, this dietary level may be regarded as a NOAEL when assessing the hazard of the Test Item to man.

Justification for classification or non-classification

No classification concluded for Repeated Dose Toxicity according to Regulation (EC) No 1272/2008, Annex I.