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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 June 2013 to 21 October 2013
Reliability:
1 (reliable without restriction)

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2014
Report Date:
2014

Materials and methods

Test guideline
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
no
GLP compliance:
yes (incl. certificate)

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
solid: particulate/powder
Remarks:
migrated information: powder
Details on test material:
Trade name : Bruggolite® E02
Chemical names: Sodium hydroxymethanesulfinate dihydrate, Hydroxymethane
sulfinic acid, monosodium salt
Label name . Bruggolite E02 Pulver
Batch number : 13042401
Date of expiry : April 2014
Appearance : white hygroscopic powder
Storage conditions: room temperature, protected from humidity
CAS Number : 149-44-0
EC Number : 205-739-4
Molecular formula: CH3O3SNa

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
A total of 120 Wistar Hannover (Hsd Brl Han: WIST) rats (60 males and 60 females), 27-29 days old and with body weight of approximately 60-99
g, were ordered from and supplied by Harlan Italy s.r.l., San Pietro al Natisone (UD), Italy.
After arrival the weight range for each sex was determined and the animals were temporarily identified within the cage by means of a coloured mark
on the tail. A health check was then performed by a veterinarian.
An acclimatisation period of 18 days was allowed before the start of treatment, during which time the health status of the animals was assessed by
thorough observations.
The animals were housed in a limited access rodent facility. Animal room controls will be set to maintain temperature and relative humidity at 22°C
± 2°C and 55% ± 15% respectively; actual conditions were monitored, recorded and the records retained. There were approximately 15 to 20 air
changes per hour and the rooms were lit by artificial light for 12 hours each day.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
The test item was administered orally by gavage at a dose volume of 10 mL/kg body weight. Control animals received the vehicle alone at the same
dose volume.
The required amount of Sodium Hydroxymethansulfinate was dissolved/suspended in the vehicle. The formulation were prepared daily
(concentrations of 10, 25 and 60 mg/mL). Concentrations will be calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Prior to commencement of treatment, analysis was performed to confirm that the proposed formulation procedure was acceptable and that the
stability of the formulation was satisfactory in the range from 10 to 100 mg/mL. The stability was found to be 24 hours at room temperature and 8
days at +4°C.
Samples of the formulations prepared in weeks 1 and 13 of the study were also analysed to check the concentration. Results of analyses were
within
the limits of acceptance.
Duration of treatment / exposure:
All animals were dosed once a day, 7 days a week, for a minimum of 13 consecutive weeks, followed by a recovery period of 4 weeks for 5 males
and 5 females from groups 1 and 4. Animals in the main phase were dosed up until the day before necropsy.
Frequency of treatment:
Once daily
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
100 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
250 mg/kg/day
Basis:
nominal in water
Remarks:
Doses / Concentrations:
600 mg/kg/day
Basis:
nominal in water
No. of animals per sex per dose:
Each main group comprised 10 male and 10 female rats. Control and high dose groups included 5 additional animals per sex, sacrificed after 4
weeks of recovery.
Control animals:
yes
Details on study design:
Dose levels of 100, 250 and 600 mg/kg/day were selected in consultation with the Sponsor based on information from preliminary studies.

Examinations

Observations and examinations performed and frequency:
Mortality

Throughout the study, all animals were checked early in each working day and again in the afternoon. At weekends and Public Holidays a similar
procedure was followed except that the final check was carried out at approximately mid-day.

Clinical signs and neurotoxicity assessment

All clinical signs were recorded for individual animals.
Once before commencement of treatment and once daily during the study, each animal was observed and any clinical signs were recorded.
Observations were performed at the same time interval each day.
Once before commencement of treatment and at least once per week from the start of treatment, each animal was given a detailed clinical
examination. Each animal was observed in an open arena. The test included observation of changes in gait and posture, reactivity to handling,
presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation,
piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions were also
recorded.
Once during weeks 12 or 13 of treatment and once during week 4 of recovery an evaluation of sensory reactivity to stimuli of different modalities
(e.g. auditory, visual and proprioceptive stimuli) and an assessment of grip strength were also performed.

Motor activity assessment (MA)

The motor activity (MA) of all animals was measured over a period of 5 minutes once during week 12 or 13 of treatment and once during week 4 of
recovery by an automated activity recording. Measurements were performed using a computer generated random order.

Body weight

Each animal was weighed on the day of allocation to treatment group, on the day that treatment commenced, weekly thereafter and just prior to
necropsy.

Food consumption

The weight of food consumed by each cage of rats was recorded at weekly intervals following allocation. The group mean daily intake per rat was
calculated.

Ophthalmoscopy

Both eyes of all animals assigned to the study were examined just prior to the commencement of treatment by means of an ophthalmoscope, and
by a slit-lamp microscope, after the instillation of 0.5% Tropicamide (Visumidriatic®, Visufarma, Rome, Italy). Two male and 2 female animals with
nonresolving
lesions were replaced with spare animals showing no ocular abnormality, from the batch initially ordered for the study. The eyes of all
animals from high-dose and control groups were re-examined during week 13 of treatment.

Clinical pathology investigations

At the end pf week 13 of treatment, just prior to necropsy, samples of blood were withdrawn under isofluorane anaesthesia from the abdominal
vena cava of the first 10 surviving male and the first 10 surviving female animals from each group, under conditions of food deprivation. At the
same time interval, individual overnight urine samples were also collected from the same animals under the same conditions. Before starting urine
collection water bottles were removed from each cage and each animal received approximately 10 mL/kg of drinking water by gavage, in order to obtain urine samples suitable for analysis.
During week 4 of the recovery period blood and urine samples were also taken (after consultation with the Sponsor) from all surviving animals
under identical conditions in order to re-evaluate clinical chemistry and urine parameters.

The blood samples collected were divided into tubes as follows:
EDTA anticoagulant for haematological investigations
Heparin anticoagulant for biochemical tests
Citrate anticoagulant for coagulation tests

The measurements performed on blood and urine samples are listed below:

Haematology
Haematocrit
Haemoglobin
Red blood cell count
Reticulocyte count
Mean red blood cell volume
Mean corpuscular haemoglobin
Mean corpuscular haemoglobin concentration
White blood cell count
Differential leucocyte count - Neutrophils
- Lymphocytes
- Eosinophils
- Basophils
- Monocytes
- Large unstained cells
Platelets
Prothrombin time
Clinical chemistry
Alkaline phosphatase
Alanine aminotransferase
Aspartate aminotransferase
Gamma-glutamyltransferase
Urea
Creatinine
Glucose
Triglycerides
Phosphorus
Total bilirubin
Total cholesterol
Total protein
Albumin
Globulin
A/G Ratio
Sodium
Potassium
Calcium
Chloride

Urinalysis

Appearance
Volume
Specific gravity
pH
Protein
Glucose
Ketones
Bilirubin
Urobilinogen
Blood

The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, were examined microscopically for:

Epithelial cells
Leucocytes
Erythrocytes
Crystals
Spermatozoa and precursors
Other abnormal components
Sacrifice and pathology:
Euthanasia

Animals were killed by exsanguination under isofluorane anaesthesia. All animals, including those found dead, were subjected to necropsy,
supervised by a pathologist.
The clinical history of the animal was studied and a detailed post mortem examination was conducted (including examination of the external
surface and orifices).
Changes were noted, the requisite organs weighed and the required tissue samples preserved in fixative and processed for histopathological
examination.

Organ weights

From all animals completing the scheduled test period, the organs indicated in Annex 1 were dissected free of fat and weighed.
The ratios of organ weight to body weight were calculated for each animal.

Tissues fixed and preserved

Samples of all the tissues listed in Annex 1 were fixed and preserved in 10% neutral buffered formalin (except eyes, testes and epididymides which
were fixed in Modified Davidson's fluid and preserved in 70% ethyl alcohol).

Histopathological examination

The tissues required for histopathological examination are listed in Annex 1. After dehydration and embedding in paraffin wax, sections of the
tissues were cut at 5 micrometre thickness and stained with haematoxylin and eosin.

The examination was as detailed below:

a)Tissues specified in Annex 1 from all animals in the control and high dose groups dying during the treatment period or killed at the end of the 13
weeks of treatment.
b) All abnormalities in all main phase groups

Organs / Tissues Weight Fixation Preservation Microscopic Examination

Abnormalities x x
Adrenal glands x x x
Aorta x x
Bone marrow (from sternum) x x
Brain (cerebrum, cerebellum,
medulla/pons) x x x
Caecum x x
Colon x x
Duodenum x x
Epididymides x x x
Eyes x
Femur with joint x
Heart x x x
Ileum x x
Jejunum (including Peyer’s patches) x x
Kidneys x x x
Liver x x x
Lungs
(including mainstem bronchi) x x
Lymph nodes - cervical x x
Lymph nodes - mesenteric x x
Mammary area x x
Oesophagus x x
Ovaries x x x
Oviducts a x x
Pancreas x x
Parathyroid glandsb x x
Pituitary gland x x
Prostate gland x x
Rectum x x
Salivary glands x x
Sciatic nerve x x
Seminal vesicles x
Skeletal muscle x
Skin x x
Spinal column x
Spinal cord (cervical, mid-thoracic, lumbar) x x
Spleen x x x
Stomach x x
Testes x x x
Thymus (where present) x x x
Thyroid x x
Trachea x x
Urinary bladder x x
Uterus – cervix x x x
Vagina x x

a: weighed and preserved with ovaries;
b: preserved with thyroid gland
Statistics:
Statistics
For continuous variables the significance of the differences amongst group means were assessed by Dunnett’s test or a modified t test, depending
on the homogeneity of data.
Statistical analysis of histopathological findings was carried out by means of the non parametric Kolmogorov Smirnov test.

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
(Daily clinical signs in surviving animals were limited to slight skin/fur staining of the ventral region, seen in the majority of the animals dosed at 600 mg/kg/day during the last five days of treatment period and during the duration of recovery period.)
Mortality:
mortality observed, treatment-related
Description (incidence):
(Daily clinical signs in surviving animals were limited to slight skin/fur staining of the ventral region, seen in the majority of the animals dosed at 600 mg/kg/day during the last five days of treatment period and during the duration of recovery period.)
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
(Statistically significant body weight reductions were observed in the high dose females on Days 1 and 8 of recovery. Body weight changes were occasionally slightly reduced in the high dose females during treatment and recovery periods. )
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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
(Increases of urea and calcium, decrease of creatinine in males from the low and/or high dose groups, decreases of glucose and increase of phosphorus in females from the low and/or high dose groups)
Urinalysis findings:
no effects observed
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
(yellowing staining observed in the ventral abdomen of the animals dosed at 600 mg/kg/day)
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
600 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'
Dose descriptor:
NOEL
Effect level:
100 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see "Remark"

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Two females receiving 600 mg/kg/day were found dead on Day 50 of the treatment period of the

study. The changes observed at macroscopic and microscopic pathology did not give any

indication of the cause of death of these animals.

No significant signs of toxic or neurotoxic effects were seen during the “in-life” phase of the study.

No lesions were recorded at ophthalmological examination.

No changes of toxicological relevance were observed in haematological, coagulation and urine

parameters.

Minor fluctuations of clinical chemistry parameters were recorded in animals dosed with 250

and/or 600 mg/kg/day. The majority of the above changes were reversible at the end of the

recovery period.

No treatment-related findings were reported at post mortem observations and at histopatological

examination.

Applicant's summary and conclusion

Conclusions:
It can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study was 600 mg/kg/day and the No Observed Effect Level
(NOEL) was 100 mg/kg/day.
Executive summary:

Study design

The toxicity of Sodium Hydroxymethansulfinate was investigated in Wistar Hannover rats after

daily oral administration for 13 weeks and recovery from any treatment-related effects during a

recovery period of 4 weeks.

Three groups, each of 10 male and 10 female Sprague Dawley rats, received the test item by

gavage at dosages of 100, 250 and 600 mg/kg/day for 13 consecutive weeks. A fourth similarly

constituted group received the vehicle alone (purified water) and acted as a control. Five

additional animals for each sex were included in the high dose and control groups for recovery

assessment.

The following investigations were performed: daily clinical signs, weekly detailed (open field

observations) clinical signs, evaluation of sensory reactivity to stimuli and motor activity, body

weight, food consumption, ophthalmoscopy, clinical pathology investigations, terminal body

weight, organ weights, macroscopic observations and histopathological examination.

Mortality and daily clinical signs

Two females receiving 600 mg/kg/day were found dead on Day 50 of the treatment period of the

study (Nos. 95750085 and 95750089). No clinical signs or other signs of toxicity were observed in

these animals up to death.

The changes observed at macroscopic and microscopic pathology did not give any indication of

the cause of death of these animals.

Daily clinical signs in surviving animals were limited to slight skin/fur staining of the ventral region,

seen in the majority of the animals dosed at 600 mg/kg/day during the last five days of treatment

period and during the duration of recovery period.

Weekly detailed clinical signs (open field measurements)

No changes of note were found at the weekly clinical examination which included an evaluation of

neurotoxicity.

Sensory reactivity to stimuli and motor activity

No differences between treated animals and controls which could be considered of toxicological

relevance were observed at evaluations of sensory reaction and motor activity measurements

performed at the end of treatment and recovery periods.

Body weight and body weight changes

No significant differences in body weights were noted between treated animals and controls during

treatment period.

Statistically significant body weight reductions were observed in the high dose females on Days 1

and 8 of recovery.

Body weight changes were occasionally slightly reduced in the high dose females during

treatment and recovery periods.

Food consumption

Food consumption was not affected by treatment.

Ophthalmoscopy

No treatment-related findings were seen at the ophthalmic examination.

Haematology

No changes of toxicological relevance were observed during the treatment and recovery periods

of the study.

Coagulation

No changes of toxicological relevance were observed during the treatment and recovery periods

of the study.

Clinical chemistry

Dosing phase

Some statistically significant fluctuations of biochemical parameters (increases of urea and

calcium, decrease of creatinine in males from the low and/or high dose groups, decreases of

glucose and increase of phosphorus in females from the low and/or high dose groups) were

observed at the end of treatment.

No other treatment-related changes were observed.

Recovery phase

The changes recorded during the dosing phase showed an almost complete reversibility.

Urinalysis

No changes of toxicological relevance were recorded at the end of treatment and recovery

periods.

Terminal body weight and organ weights

No changes which could be considered of toxicological relevance were observed at the end of

treatment and recovery periods.

Macroscopic observations

Final and recovery sacrifice

Treatment-related changes were noted in the ventral abdomen of the animals dosed at 600

mg/kg/day, consisting of yellowing staining.

Microscopic observations

Final sacrifice

No apparent treatment-related changes were noted.

Conclusion

On the basis of the above results, only minor signs of possible treatment-related effects of the test

item, Sodium Hydroxymethansulfinate, were observed in male and female rats at the dose levels

of 250 and 600 mg/kg/day, when administered by oral gavage for 13 consecutive weeks at the

dosages of 100, 250 and 600 mg/kg/day.

These effects included minor clinical signs, slight reductions in body weight gain (high dose

females) and some fluctuations in clinical pathology parameters, mainly observed in the animals

dosed at 600 mg/kg/day. These changes were generally fully reversible and, as such, they were

not considered to be adverse.

No changes were observed in the animals dosed at 100 mg/kg/day.

Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study

was 600 mg/kg/day and the No Observed Effect Level (NOEL) was 100 mg/kg/day.