3-C12-18-(even numbered)-alkylamido-N,N-dimethylpropan-1-amino oxide

Endpoint summary

Description of key information

Horne (2014) performed a 90-day oral repeated dose toxicity study in rats. The test was performed according to OECD guideline 408 and in compliance with GLP requirements. The No Observed Adverse Effect Level (NOAEL) was considered to be 50 mg/kg bw/day. The study is scored as reliable (K1) and selected as key study.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2013-03-04 - 2014-03-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-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:

- Name of test material (as cited in study report): Amides, C12-C18 (even numbered), N-[3-dimethylamino) propyl], N’-oxides or Rewominox B 204
- Physical state: clear liquid
- Analytical purity: 35.6%
- Purity test date: 18 January 2013
- Lot/batch No.: ST03102288
- Expiration date of the lot/batch: 31 January 2015
- Stability under test conditions: 14 days at 2 to 8°C
- Storage condition of test material: 2-8°C in the dark

Species:

rat

Strain:

other: CR:CD (Sprague-Dawley)

Details on species / strain selection:

The rat was selected because it is a rodent species known to be accepted by various regulatory authorities and because of the availability of background data within these laboratories.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Margate, UK.
- Age at study initiation: 7-8 weeks
- Weight at study initiation: Males: between 202.1 and 263.3g. Females: between 150.8 to 205.1
- Fasting period before study: None (but one fasting night during week 13 for blood sampling)
- Housing: The animals were housed in a single, exclusive room, and in groups up to 4 rats of the same sex and dose level par cage.
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 17 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 to 24°C
- Humidity (%): 45 to 65 %
- Air changes (per hr): 15-20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12 hours nominal

IN-LIFE DATES: From: 22 March 2013 to 24 June 2013

Route of administration:

oral: gavage

Details on route of administration:

The oral (gavage) route of administration was chosen because it is a possible human exposure route.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test article was formulated as a solution in purified water following dispensary standard operation procedures and a formulation method develpoped in the laboratory for this study. Formulations were prepared weekly and stored refrigerated (2 to 8°C) in a sealed container, protected
from the light. The formulations were stirred on arrival at the animal room and were stirred continuously before and throughout dosing.

The dose levels of 50, 150 and 500 mg/kg/day were expressed as the active ingredient. The test article was supplied as a 356 mg/mL solution and final concentrations of 5, 15 and 50 mg/mL were formulated by serial dilution.

OTHER:
After the dose formulation was drawn into the syringe, the catheter was washed in potable water, wiped clean and then washed again before dosing the animal. he test article was administered orally by gavage, as it is possible human exposure route. A dose volume of 10 mL/kg was used. Individual dose volumes were based on individual body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

- SAMPLE COLLECTION:
Samples (3 x 1 mL aliquots from test formulations; 2 x 1 mL aliquots from control formulations) prepared for use on Week 1 and Week 13 were taken for analysis of achieved concentration. Samples were stored at ambient until and during analysis that occurred within 2 hours of the formulations being prepared.

- SAMPLE ANALYSIS:
A set of standard solutions over the approximate concentration range 100 to 600 μg/mL was analysed by HPLC. A six point calibration graph was constructed for the standard solutions. Injections of the lowest calibration standard diluted by factors of 100 and 50 were used to determine a limit of detection (LOD). Formulations were diluted to within the range of the calibration line (approximately100.0 to 600.0 μg/mL) using water.

Duration of treatment / exposure:

The animals were dosed once for at least 13 weeks excluding the day of necropsy.

Frequency of treatment:

The test material was administered daily.

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

Dose / conc.:

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

Remarks:

active ingredient, actual ingested

No. of animals per sex per dose:

10 animals per sex per dose
5 spare males and females were available to replace any animals due to extremes of body weight, ophthalmoscopy changes or ill health.

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale:
- High dose: The high dose of 500 mg/kg/day was chosen as, based on the background information (28 day toxicity study), it was not expected to reveal overt toxicity and should set a No-Observed-Adverse-Effect Level (NOAEL).
- Intermediate dose: The intermediate dose of 150 mg/kg/day was chosen to set an NOAEL if effects were seen at 500 mg/kg/day.
- Low dose: The low dose of 50 mg/kg/day was chosen to set a No-Observed-Effect Level (NOEL).
- Background information:
In a previous 28 day toxicity study conducted by the Sponsor (Study number 625/051) a dose of 1000 mg/kg/day resulted in the death of a single female on Day 12. Noisy respiration, increased salivation, red/brown staining of the fur and wet fur, and hunched posture were recorded in animals at this level with diuresis, tiptoe gait, gasping respiration, pallor of the extremities and staining around the ano-genital region also recorded sporadically. No overt signs were recorded in animals at 150 or 15 mg/kg/day. Body weight gain and food consumption were initially affected but improved as the study progressed. Haemolytic anaemia was noted in animals at 1000 or 150 mg/kg/day. Histopathology identified target organs as liver, spleen, kidneys, urinary bladder and stomach with adverse findings recorded at 150 or 1000 mg/kg/day. Findings included centrilobular hepatocyte enlargement in the liver, splenic extramedullary haemopoiesis in the liver, splenic extramedullary haemopoiesis, haemosiderin pigment deposition/accumulation in the liver, spleen and kidneys, hyperplasia in the urinary bladder and acanthosis, hyperkeratosis occasionally associated with subepithelial inflammatory cell infiltrates in the (fore)stomach.

Positive control:

No positive control.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at the beginning and the end of the working day.
- Cage side observations : animals were observed for signs of ill health or overt toxicity.

DETAILED CLINICAL OBSERVATIONS: Yes
Each animal was given a detailed physical examination at daily intervals, one hour after dosing only. An individual record was maintained of the clinical condition of each animal. Post dose observations were also performed in relation to the time of dosing to coincide with clinical examinations one hour post dose daily from Day 1 to termination.

BODY WEIGHT: Yes
Individual body weights were recorded twice weekly, before dose, from Dosing Day 1 to Week 4, and at weekly intervals from Week 5 to Week 13 and before necropsy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of food consumed by each cage of animals was determined twice weekly.

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
The amount of water consumed by each cage of animals was determined daily for a three day period (Friday to Monday) in Week 1, 6 and 12.

OPHTHALMOSCOPIC EXAMINATION: Yes
Investigations were performed on all animals pre-treatment and on Groups 1 and 4 in Week 12.

HAEMATOLOGY and CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Once at Day 90.
- Animals fasted: Yes, samples were collected after an overnight period without food.
- How many animals: Investigations were performed on all surviving animals from each test and control group at the end of the study (Day 90)
- Parameters examined: Haematology Tests: haemoglobin, red blood cell count, packed cell volume, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, reticulocyte count, red cell distribution width, haemoglobin distribution width, total and differential white cell count, platelet count (includes platelet clump assessment - clump count below 100 considered as none detected; clump count over 100 considered as plateletclumps present and confirmed by review of Advia cytogram or blood film examination), platelet crit, mean platelet volume, platelet distribution width. Coagulation Tests: prothrombin time activated partial thromboplastin time. Clinical Chemistry Tests: aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, total cholesterol, total bilirubin, total protein, albumin, globulin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphate, creatinine, urea, glucose.

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: before initiation of treatment and at once weekly intervals thereafter. In Week 13, an detailed assessment was done.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity, grip strength, motor activity and some open filed test were performed weekly. Functional observationanl battery, open field examinations and locomotor activity were assessed in Week 13.

Sacrifice and pathology:

NECROPSY AND GROSS PATHOLOGY: All animals including the decedent animal were subjected to necropsy. The scheduled necropsies were performed in Week 14 (Day 95), after an overnight period without food. Where possible, they were carried out in replicate order. Each animal was given an overdose of isoflurane anaesthesia. Once a suitable deep plane of anaesthesia was established, major blood vessels were severed to exsanguinate the animal. A full macroscopic examination was performed under the general supervision of a pathologist and all lesions were recorded.

ORGAN WEIGHTS: Animals were weighed immediately before being killed for necropsy. The organs denoted by ‘W’ in the tissue list were dissected free from fat and other contiguous tissue and weighed before fixation (see table below). Left and right organs were weighed together. For 2F 54, the heart was weighed with adhered pericardium.

HISTOPATHOLOGY: The tissues from each animal presented in the following table were preserved in 10% neutral-buffered formalin unless otherwise indicated.
The following tissues were embedded in paraffin wax BP (block stage), sectioned at anominal 5 μm and stained with haematoxylin and eosin:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

MICROSCOPIC OBSERVATIONS
The following tissues were examined microscopically by the Principal Investigator for Histopathology:
Group 1 and Group 4: All tissues denoted by ‘E’ in the tissue list above
Group 2 and Group 3: Lungs, liver, spleen, kidney, urinary bladder, (fore)stomach and gross lesions
Decedent: All tissues denoted by ‘E’ in the tissue listed below

Other: Bone Marrow Smear Evaluation
Bone marrow smears were prepared at necropsy. Tissues were taken into bovine serum albumin. The smears were prepared, air dried and then were fixed in methanol, but not examined.

Statistics:

Data from treated animals were compared with control data. Statistical analyses were performed where appropriate.
The control group (Group 1) was taken as the baseline group with which the treated groups (Groups 2, 3, 4) were compared.
Body weight gains, terminal body weights, functional observational battery, motor activity, haematology and clinical chemistry variables were analysed using one-way analysis of variance (ANOVA), separately for each sex. Levene's test for equality of variances among the groups was performed. Where this showed no evidence of heterogeneity (P>0.05), and the group effect from the ANOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.
Where Levene’s test was significant (P≤0.05), a rank-transformation was applied to the data prior to analysis. No analysis was performed for variables with values above or below the limit of the assay. Some variables were not analysed due to there being too few values for meaningful analysis.
Organ weights were analysed using Analysis of Covariance (ANCOVA), for each sex separately, using the terminal body weight as the covariate. This analysis depends on the assumption that the relationship between the organ weights and the covariate is the same for all groups and the validity of this assumption was tested. Where there was no evidence of heterogeneity among the slopes (P>0.01), and the group effect from the ANCOVA was significant (P≤0.05), pairwise comparisons with the control group were made using Dunnett's test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Post dosing observations of salivation and staining around the mouth are indicative of the treated animals not liking the taste of the test article and/or being dosed with an irritant material and are considered a secondary action of the test article. Noisy breathing was generally recorded for animals given 500 mg/kg/day but also in a single male at 150 mg/kg/day and a single female at 50 mg/kg/day; this finding being commonly associated with oral administration of irritant materials.

Mortality:

mortality observed, treatment-related

Description (incidence):

A single male dosed at 500 mg/kg/day was found dead during Week 12; although a cause of death could not be established, as the macroscopic findings (large caecum and liver, dark lungs and thymus, a thickened stomach and red fluid in the thoracic cavity) along with the microscopic findings (congestion of the liver, aggregates of foamy alveolar macrophages within the lung (graded as slight, multifocal), squamous cell hyperplasia with hyperkeratosis (both graded as moderate), together with erosion/ulcer (slight, focal) in the (fore)stomach, and minimal transitional cell hyperplasia in the urinary bladder) match the findings in the high dose animals, it is considered that this death was related to the treatment of the test substance.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

When compared with control values, body weight gains for males during the first 8 weeks of treatment did not reveal an effect of treatment with the test substance. Over the second half of the study gains were low for males given 150 or 500 mg/kg/day, when compared with control, resulting in an overall slightly low gain in males at 500 mg/kg/day. There was no effect on gains for males given 50 mg/kg/day. Body weight gains for female at 500 mg/kg/day were variable, but high, over the first 5 weeks of treatment resulting in slightly high overall gains. There was no effect on body weight gains for females at 50 or 150 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

There was no effect on food consumption in any test substance-treated male group or for females given 50 or 150 mg/kg/day. Food consumption values were high for females given 500 mg/kg/day.

Food efficiency:

not specified

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption values were high in males and females given 500 mg/kg/day. There was no effect seen in groups given 50 or 150 mg/kg/day. The increase in the water consumed could be associated with the oral dosing of an irritant material resulting in the behavioural response to drink. There was no histopathological evidence of any effect on the kidney although transitional cell hyperplasia in the urinary bladder was recorded, suggesting urinary excretion of an irritant material.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no ocular effects after treatment with the test substance in any group and sex.

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Statistically significant haematological changes (decreased haemoglobin, red blood cell counts, mean cell haemoglobin concentration, haemoglobin distribution width and red cell distribution width and increased reticulocyte counts and percentage, mean cell volume and mean cell haemoglobin) consistent with a decreased red blood cell mass associated with regeneration were seen in all treated males and female groups with the severity of the finding being dose dependant. The haemopoietic response in the spleen is considered to account for the increased weight of this organ.
For haemoglobin levels and red blood cell numbers, females given 500 mg/kg/day showed a change of between 7 and 17% from control values while males showed a change of between10 and 19% meaning the findings are on the border of non adverse and adverse. Males given 150 mg/kg/day showed a change of between 7 and 11% compared to controls with females showing a change between 1 and 5% with these findings being considered non-adverse due to the smaller magnitude of change. The low dose (50 mg/kg/day) percentages were between 0 and 2% for females and 4 and 6% for males and also not considered adverse. A reduced red blood cell mass of more than 30% is considered adverse and a change of less than 10% is considered non-adverse for rodents (Principles of Methods of Toxicology 5th Ed. (A Wallace Hays) 2003; In Chapter 26 (Robert L Hall and Nancy E Everds) p1331-1332.) This finding was not considered to be anaemia as it wasn’t associated with any additional findings such as an increase plasma and urinary bilirubin.
Compound-related changes in the haematology parameters consisted of generally dose-dependent and statistically significant decreases in a number of erythroid parameters (erythrocyte count and haemoglobin concentrations) in compound-treated rats of both genders. These changes were accompanied by evidence for a robust regenerative response, strongly suggesting an appropriate response by the bone marrow. This regenerative response was indicated by one or more of the following in the compound-treated groups in both genders: 1) robust and dose-dependent reticulocyte response; 2) generally dose-dependent increase in the mean corpuscular volume and decreases in the mean corpuscular haemoglobin concentration which is consistent with increased number of reticulocytes. With this convincing evidence of appropriate bone marrow regeneration (increased reticulocyte response) an investigation of the bone marrow smears was not required (References: Provencher Bollinger, A. Cytologic evaluation of bone marrow in rats: Indications, methods, and normal morphology. Veterinary Clinical Pathology 33(2):58-67, 2004; Reagan, W.J., Irizarry-Rovira, A., Poitout-Belissent, F., Provencher Bollinger, A., Ramaiah, S.K., Travlos, G., Walker, D., Bounous, D., Walter, G. (Bone Marrow Working Groups of the ASVCP/STP). Best practice for evaluation of bone marrow in nonclinical toxicity studies. Toxicologic Pathology 39: 435-448, 2011).

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical chemistry changes revealed low cholesterol and high urea levels in males and female at 500 mg/k/g/day and high inorganic phosphate in males at 500 mg/kg/day. However, the reason for these findings are not clear and in the absence of any histopathological correlate in the liver and kidney, they were considered slight and not adverse they were not taken into consideration when setting the NOAEL.

Endocrine findings:

not specified

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

There was no effect on functional observational battery tests or locomotor activity.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Treatment related organ weight changes included a statistical decrease in organ weight was seen in the thymus of males at 500 mg/kg/day. Liver weights were high in females at 500 mg/kg/day, with spleen weights high in females at 150 mg/kg/day and males and females at 500 mg/kg/day. Adrenal gland weights were high in males given 500 mg/kg/day compared to concurrent controls. Increased adrenal and decreased thymus weights are considered to be an effect of the low terminal body weight recorded in the males and the high liver weight likely to be a consequence of the high body weight gain seen in the females and as there is no histopathological correlate, these changes are considered not to be of toxicological significance.
There was no effect on organ weights for animals given 50 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

On macroscopic examination, findings related to treatment with the test substance were present in the stomach and caecum of animals treated with 500 mg/kg/day: the (fore)stomach of 5/10 males and 2/10 females of the 500 mg/kg/day treated group showed macroscopic abnormalities consisting of thickening and in some of those 7 animals also a change in colour (recorded as abnormal and pale), and a distention was noted in the caecum of 5/10 males treated with this dose level. These findings comprised: thickening and/or an abnormal/pale colour of the (fore)stomach, and a large or distended caecum.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

On microscopic examination, findings related to treatment with the test substance were observed in the (fore)stomach, lung, spleen, liver and urinary bladder.
In the stomach, changes observed comprised squamous cell hyperplasia, erosions/ulcers within the affected epithelium, some acute inflammatory changes and submucosal oedema. These changes were noted in a single male treated with 150 mg/kg/day and in males and females treated with 500 mg/kg/day.
In the spleen an increase in severity of (brownish) pigment was observed in males and females from the 50 mg/kg/day treated group onwards: The pigment is believed to be haemosiderin. The incidence of the pigment in the spleen at 50 mg/kg bw/day was 7/10 (slight) and 3/10 (moderate) in males and 1/10 (slight), 7/10 (moderate) and 2/10 (severe) in males. At 150 mg/kg bw/day, 1/10 (minimal), 6/10 (slight) and 3/10 (moderate) in males and, in females, 8/10 (moderate) and 2/10 (severe). At 500 mg/kg bw/day, 5/10 (slight) and 5/10 (moderate) in males and, in females, 9/10 (moderate) and 1/10 (severe). This pigment appeared even in the controls at a moderate grade in females (7/10) and slight in males (8/10).
The changes observed in the urinary bladder were all mild and comprised diffuse transitional cell hyperplasia and/or increased epithelial folding. The transitional epithelium of the urinary bladder showed an increased epithelial folding in a 2/10 males treated with 150 mg/kg/day and in 5/10 males and 7/10 females of the 500 mg/kg/day treated group, while a low grade diffuse transitional cell hyperplasia was observed in few males and one female treated with 500 mg/kg/day. These findings are considered to be due to the irritating properties of the test substance. The epithelium of the urinary bladder is closely related to the squamous epithelium in the (fore)stomach and it is considered that the changes in both organs are related.
An increase in the presence of aggregates of (slight foamy) alveolar macrophages was noted in the lung of the 500 mg/kg/day treated animals, with the significance of these findings unclear.
A treatment-related increase in the incidence of pigmented Kupffer’s cells was observed in the liver of females of the 500 mg/kg/day group only. No microscopic findings were observed in the liver and in the caecum to support the liver weight changes observed in females dosed at 500 mg/kg/day, and the gross changes observed in the caecum of animals dosed at 500 mg/kg/day.

The kidney was not considered target tissue in this 13-week study.

No treatment-related histopathological changes were noted in any of the reproductive organs investigated in this study.

Histopathological findings: neoplastic:

not specified

Other effects:

no effects observed

Description (incidence and severity):

The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. Test article was
not detected in the control samples.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

other: Toxicologically significant changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg bw/day and in the (fore)stomach at 150 mg/kg bw/day.

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: gastrointestinal, urinary, and hematopoietic system

Organ:

bladder

blood

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

Daily oral administration of the test substance to Sprague-Dawley rats for 13 weeks (test performed according to OECD 408) at dose levels of 50, 150 and 500 mg/kg/day, resulted in toxicologically significant effects at dose levels of 500 mg/kg/day and 150 mg/kg/day. The changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg/day and the changes in the (fore)stomach at 150 mg/kg/day were considered adverse and toxicologically significant. No adverse effect was observed at 50 mg/kg/day. Therefore the No Observed Adverse Effect Level is set at 50 mg/kg/day. With the LOAEL set at 150 mg/kg/day, the substance is not to be classified as STOT RE toxicant according to CLP Regulation.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

1999-09-17 - 2000-06-30

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)

Version / remarks:

adopted 27 July 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

- Name: NINOX HCDO
- Purity: approximately 81%
- Lot/batch No.: 876 TK, sample IX
- Physical state: off white paste
- Storage condition of test material: room temperature, in the dark

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River (UK)
- Age at study initiation: approximately five to seven weeks old.
- Weight at study initiation: the males weighed 139 to 173 g, and the females weighed 127 to 155 g
- Housing: The animals were housed in groups of five by sex in polypropylene grid-floor cages suspended over trays lined with absorbent paper
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: nine days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2 ºC
- Humidity (%): 55 ± 15%
- Air changes (per hr): approximately fifteen changes per hour
- Photoperiod (hrs dark / hrs light): twelve hours continuous light and twelve hours darkness

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The test substance was prepared as a solution in distilled water. The analytical results indicate that the prepared formulations were within ±10 % of the nominal concentration. The volume of test and control material administered to each animal was based on the most recent bodyweight and was adjusted at weekly intervals.

VEHICLE
- Concentration in vehicle: 3.68, 36.8, and 245 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentration of the test substance was determined by gas chromatography (GC) using an external standard technique. Samples were taken from the top, middle and bottom of the container. Sampling was performed in triplicate. The test material formulations were sampled and analysed initially and then after storage at approximately +4ºC in the dark for fourteen days.

Duration of treatment / exposure:

Twenty-eight consecutive days.

Frequency of treatment:

The test material was administered daily.

Dose / conc.:

18.4 CFU/kg bw/day (actual dose received)

Remarks:

15 mg/kg bw/day of active ingredient, actual ingested

Dose / conc.:

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

Remarks:

150 mg/kg bw/day of active ingredient, actual ingested

Dose / conc.:

1 227 mg/kg bw/day (actual dose received)

Remarks:

1000 mg/kg bw/day of active ingredient, actual ingested

No. of animals per sex per dose:

5 females and 5 males per dose.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
A range-finding toxicity study in Sprague-Dawley rats was performed. Six animals, 3 males and 3 females, were included in the study. The test material was administered daily, for fourteen consecutive days, by gavage. Control animals were treated in an identical manner with 5 ml/kg/day of distilled water. The studied dose was 1250 mg/kg/day. The level doses were defined based on the results of necropsy data, bodyweights and clinical observations.

Observations and examinations performed and frequency:

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: before dosing and one and five hours after dosing during the working week. Animals were observed immediately before dosing and one hour after dosing at weekends.

BODY WEIGHT: Yes
- Time schedule for examinations: Individual bodyweights were recorded on Day 1 and on Days 7, 14, 21 and 28. Bodyweights were also recorded at terminal kill.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Time schedule: Food consumption was recorded for each cage group at weekly intervals throughout the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was observed daily, for each cage group, by visual inspection of the water bottles for any overt changes.

HAEMATOLOGY AND CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study
- Animals fasted: No
- How many animals: investigations were performed on all surviving animals from each test and control group at the end of the study (Day 28)
- Parameters checked: see tables below.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Battery of functions tested: sensory activity, grip strength and motor activity.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes. All animals were subjected to a full external and internal examination, and any macroscopic abnormalities were recorded.

ORGAN WEIGHTS: Yes. The following organs removed from animals that were killed at the end of the study, were dissected free from fat and weighed before fixation: adrenals, kidneys, testes, brain, liver, thymus, epididymides, ovaries, heart, spleen.

HISTOPATHOLOGY: Yes. Samples of the following tissues were removed from all animals and preserved in buffered 10% formalin: adrenals, aorta (thoracic), bone and bone marrow (femur including stifle joint), bone and bone marrow ( sternum), (including cerebrum, cerebelum and pons), caecum, colon, duodenum, epididymides, eyes, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), muscle (skeletal), oesophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands (submaxillary), sciatic nerve, seminal vesicles, skin (hind limb), spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.

From all control animals and all the animals at the 1000 mg/kg/day dose group, the following tissues were preapred as paraffin blocks, sectioned and stained with haematoxylin and eosin for subsequent microscopic examination: adrenals, bone & bone marrow (sternum), brain (including cerebrum, cerebelum and pons), caecum, colon, duodenum, epididymides, gross lesions, heart, ileum, jejunum, kidneys, liver, lungs (with bronchi), lymph nodes (cervical and mesenteric), ovaries, prostate, rectum, sciatic nerve, seminal vesicles, spinal cord (cervical), spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder and uterus.
The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Statistics:

Data were assessed for dose response relationships by linear regression analysis followed by one way analysis of variance (ANOVA) incorporating Levene's test for homogeneity of variance. Where variances were shown to be homogenous pairwise comparisons were conducted using Dunnetts's test. Where Levene's test showed unequal variances the data were analysed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney "U" test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Animals treated with 1000 mg/kg/day began to show clinically observable signs of toxicity from Day 3. Symptoms gradually worsened and became more prevalent during weeks 1 and 2 before stabilising during the second half the study. Signs seen in most animals included fur loss, noisy respiration, increased salivation, red/brown staining of the fur, wet fur and hunched posture. The only observations seen in animals from the 150 or 15 mg/kg/day dose groups were incidents of fur loss, which is a common finding in group housed rats and in isolation is not toxicologically significant and a single incident of increased salivation entirely associated with the dosing procedure and not indicative of toxicity.

Mortality:

mortality observed, treatment-related

Description (incidence):

One female treated with 1000 mg/kg/day was found dead on Day 12 of the study.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed a substantially reduced bodyweight gain during week 1 of the study only. In subsequent weeks bodyweight gain in these animals was similar to controls but a full recovery was not apparent in males with terminal bodyweight remaining notably lower than controls. No adverse effect on bodyweight development was detected for animals of either sex from the 150 or 15 mg/kg/day dose groups.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed reduced food consumption and reduced food efficiency during the first week of the study only. No adverse effects were detected in animals of either sex treated with 150 or 15 mg/kg/day.

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

Water consumption measurement, initiated on Day 15, revealed a substantial increase for animals of either sex treated with 1000 mg/kg/day, which persisted until the end of the study. There was no convincing effect on water consumption in animals from the 150 or 15 mg/kg/day dose groups.

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Haematological changes consistent with haemolytic anaemia were detected in animals of either sex treated with 1000 or 150 mg/kg/day. The condition was dose related in severity and involved statistically significant reductions in erythrocyte count, haemoglobin, haematocrit and mean corpuscular haemoglobin and increases in mean corpuscular volume, mean corpuscular haemoglobin and reticulocyte count. The majority of 1000 mg/kg/day reticulocyte values were outside the normally expected range for rats of the strain and age used. Males and females from the 1000 mg/kg/day dose group also showed a statistically significant increase in neutrophil numbers, but not in the total leucocyte count, together with a slightly elevated prothrombin time. Animals of either sex treated with 15 mg/kg/day showed no toxicologically or statistically significant haematological changes.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed statistically significant elevations in plasma aspartate aminotransferase (ASAT) and alanine aminotransferase (ALAT) including individual values outside the normally expected range for rats of the strain and age used, particularly for ALAT. The group mean ALAT concentration for males from the 150 mg/kg/day dose group was also significantly elevated and there was a slight, but not statistically significant increase in this and ASAT levels for animals of either sex at this dose level. Plasma bilirubin levels were increased for animals of either sex from the 1000 mg/kg/day dose group but statistical significance was confined to the males.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Weekly behavioural assessments supported the clinical observations recorded during the study with incidents of tiptoe gait, hunched posture and noisy respiration seen at each observation time for animals treated with 1000 mg/kg/day. There were no treatment-related changes in the functional performance parameters measured. Males treated with 1000 mg/kg/day showed an enhanced starle reflex compared with controls. The percentage peak response was statistically significantly elevated and the percentage average response and RMS response were also higher than controls, although the differences did not achieve statistical significance.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Males and females treated with 1000 mg/kg/day showed a statistically significant increase in spleen weight, both absolute and relative to bodyweight, compared with controls. Many of the individual values were outside the normally expected range for rats of the strain and age used and the effect extended to either sex from the 150 mg/kg/day dose group, although statistical significance was not always achieved at this dose level. A statistically significantly higher relative testes weight for 1000 mg/kg/day males was considered to result from the lower terminal bodyweight for these animals and was not indicative of a target organ effect. Reductions in absolute liver and kidney weight for 1000 mg/kg/day males were not reflected in the relative weights and were considered to be of no toxicological importance. There were no toxicologically significant effects on organ weight for animals of either sex treated with 15 mg/kg/day.

Gross pathological findings:

effects observed, treatment-related

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following treatment-related changes were observed:
- Liver: centrilobular hepatocyte enlargement was observed for three males and scattered deposits of pigment were seen in a few animals of either sex dosed at 1000 mg/kg/day. One female dosed at 150 mg/kg/day similarly demonstrated hepatic pigment accumulation. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Spleen: increased severities of extramedullary haemopoiesis and pigment accumulation were observed in relation to treatment for rats of either sex dosed at 1000 or 150 mg/kg/day. Female rats receiving 15 mg/kg/day demonstrated increased severities of splenic pigment accumulation but no alteration in extramedullary haemopoiesis compared with control animals. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Kidneys: an increased severity of pigment accumulation was observed in the renal tubular epithelium of animals of either sex dosed at 1000 mg/kg/day. The accumulated pigment was Perl´s positive and therefore probably haemosiderin.
- Urinary bladder: hyperplasia of the transitional cell epithelial lining of the urinary bladder was seen for males and females dosed at 1000 mg/kg/day.
- Stomach: acanthosis and hyperkeratosis, occasionally with associated subepithelial inflammatory cell infiltrates, were observed in relation to treatment in the forestomach of animals of either sex receiving 1000 mg/kg/day of the test material.
- No treatment-related changes were reported in the ovaries, protate, seminal vesicles, testes and uterus.

Histopathological findings: neoplastic:

no effects observed

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

act. ingr.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

water consumption and compound intake

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

other: hematopoietic, urinary, gastrointestinal and hepatobiliary system

Organ:

bladder

kidney

liver

spleen

stomach

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Conclusions:

The "No Observed Effect Level" (NOEL) was considered to be 15 mg/kg/day.

Executive summary:

The Repeated dose 28-Day Oral toxicity Study in Rodents for the test substance was performed in Sprague-Dawley rats.The test material was administered by gavage to three groups, each of five male and five female, for up to twenty-eight consecutive days, at dose levels of 15, 150 and 1000 mg/kg/day of active ingredient (incorporating a correction factor for test substance purity). A control group of five males and five females was dosed with vehicle alone (distilled water). Clinical signs, functional observations, bodyweight gain and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination. Histopathological evaluation of selected tissues was performed in all control animals (0 mg/kg/day) and animals at the highest dose group (1000 mg/kg/day). The liver and spleen from all 15 and 150 mg/kg/day dose group animals were also processed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg/day.

In the blood chemistry, elevations in plasma aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted at the dose of 1000 mg/kg/day; ASAT and ALAT were also elevated for 150 mg/kg/day animals. Haematological changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time were observed at doses of 1000 and 150 mg/kg/day.

A statistically significant increase in spleen weight was observed in males and females treated with 1000 mg/kg/day. The effect extended to the 150 mg/kg/day dose group although statistical significance was not always achieved.

Treatment-related changes in liver and spleen were detected at 1000 mg/kg/day and 150 mg/kg/day. Histopathological changes were also observed in kidneys, urinary bladder and stomach of animals treated with 1000 mg/kg/day. No treatment-related changes were reported in the ovaries, prostate, seminal vesicles, testes and uterus of control animals or animals at 1000 mg/kg/day.

Oral administration of the test substance to rats for a period of up to twenty-eight consecutive days at dose levels of up to 1000 mg/kg/day resulted in toxicologically significant effects at dose levels of 1000 and 150 mg/kg/day. No such effects were observed in animals of either sex treated with 15 mg/kg/day. The "No Observed Effect Level" (NOEL) was, therefore, considered to be 15 mg/kg/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

50 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: dermal

Data waiving:

study scientifically not necessary / other information available

Justification for data waiving:

a short-term toxicity study does not need to be conducted because a reliable sub-chronic (90 days) or chronic toxicity study is available, conducted with an appropriate species, dosage, solvent and route of administration

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

Repeated dose toxicity: oral

Two reliable studies are available for this endpoint: a 28-day oral repeated dose toxicity study and a 90-day oral repeated dose toxicity study.

Horne (2014) performed a 90-day repeated oral dose toxicity study in rats according to OECD guideline 408 and in compliance with GLP. In this study Sprague-Dawley rats (10 females and 10 males per dose) were exposed daily by oral gavage with the active ingredient at 50 mg/kg bw/day, 150 mg/kg bw/day and 500 mg/kg bw/day of active ingredient). These doses were selected based on the results of the 28 -day repeated oral dose toxicity study described here above. A control group was dosed with the vehicle only (distilled water).

Clinical signs (health monitoring, clinical examinations and post-dose observations), bodyweight, food and water consumption were monitored during the study. All animals were subjected to a functional observational battery weekly and at the Week 13 before sacrifice. Blood samples were collected after an overnight period without food from all the animals at the end of the study and haematology, coagulation and clinical chemistry parameters were evaluated for all animals. All animals were subjected to a gross necropsy examination. Selected organs were weighed. Histopathological evaluation of selected tissues was performed (including reproductive organs) in all control animals (0 mg/kg bw/day) and animals at the highest dose group (500 mg/kg bw/day). The lungs, liver, kidney, urinary bladder, (fore)stomach, spleen and other gross lesions observed at the macroscopic evaluation were also processed for animals of the 50 and 150 mg/kg bw/day dose group.

Only one animal dosed at 500 mg/kg bw/day was found dead during Week 12 and this death was considered to be treatment-related.

Findings for animals at 500 mg/kg bw/day included salivation, staining around the mouth and noisy breathing. This is indicative of irritant test materials with the animals not liking the taste of the formulation. Body weight gains were low and, as there was no effect on food consumption; this was considered non-treatment related toxicity. Water consumption values were high for males and females which again was associated with the oral dosing of an irritant material. The changes in the haematology parameters were consistent with a decrease in red cell mass showing regeneration and the magnitude of change (between 7% and 19%) was considered borderline adverse as a reduced red blood cell mass of more than 30% is considered adverse and a change of less than 10% is considered non-adverse for rodents. This finding was not considered to be anaemia as it was not associated with any additional findings such an increase of plasma bilirubin.

Microscopic examination revealed adverse findings in the (fore)stomach (squamous cell hyperplasia) and urinary bladder (transitional cell hyperplasia) which was considered a result of the irritating properties of the test article. The epithelium of the urinary bladder is closely related to the squamous epithelium in the (fore)stomach and it is considered that the changes in both organs are related.

Findings for animals at 150 mg/kg bw/day included low body weight gains and as there was no effect on food consumption, this was considered non-specific toxicity. The changes in the red blood cell mass were not considered adverse at this dose level as the magnitude of change was less than 11%. Forestomach squamous cell hyperplasia was noted in a single male at 150 mg/kg bw/day and as this finding matched those recorded at 500 mg/kg bw/day this was considered adverse. The only treatment-related change seen in males at 50 mg/kg bw/day were statistically relevant minor changes in the red blood cell parameters, indicating that the change in red blood cell mass was affecting these animals but as the magnitude of change was much lower than animals treated at 500 mg/kg bw/day, these findings were considered treatment-related but not adverse in nature.

In conclusion, daily oral administration of the test substance, to rats for 13 weeks at dose levels of up to 500 mg/kg bw/day, resulted in toxicologically significant effects at dose levels of 500 mg/kg bw/day and 150 mg/kg bw/day. The changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg bw/day and the changes in the (fore)stomach at 150 mg/kg bw/day were considered adverse and toxicologically significant. No adverse effect was observed at 50 mg/kg bw/day. Therefore, the NOAEL is set at 50 mg/kg bw/day.

No treatment-related histopathological changes were noted in any of the reproductive organs investigated in this study.

Jones et al. (2000) performed a 28-day repeated dose toxicity study according to OECD 407 and EU method B.7, and in compliance with GLP. In this supporting study, Sprague-Dawley rats (5 females and 5 males per dose) were exposed daily by oral gavage with the test substance at 18.4 mg/kg bw/day (15 mg/kg bw/day of active ingredient), 184 mg/kg bw/day (150 mg/kg bw/day of active ingredient), 1227 mg/kg bw/day (1000 mg/kg bw/day of active ingredient). A control group was dosed with the vehicle only (distilled water).

Clinical signs, functional observations, bodyweight gain and food and water consumption were monitored during the study. Haematology and blood chemistry were evaluated for all animals at the end of the study. All animals were subjected to a gross necropsy examination. Histopathological evaluation of selected tissues was performed in all control animals (0 mg/kg bw/day) and animals at the highest dose group (1000 mg/kg bw/day). The liver and spleen from all 15 and 150 mg/kg bw/day dose group animals were also processed.

Clinical signs of toxicity, functional incidents as hunched posture, sensory reactivity, reduced food consumption, increase of water consumption and reduced bodyweight gain were observed at dose of 1000 mg/kg bw/day.

In the blood chemistry, elevations in plasma aspartate aminotransferase (ASAT), alanine aminotransferase (ALAT) and bilirubin was noted at the dose of 1000 mg/kg bw/day; ASAT and ALAT were also elevated for 150 mg/kg bw/day animals. Haematological changes as haemolytic anaemia, significant elevation in neutrophil numbers, and slightly elevated prothrombin time were observed at doses of 1000 and 150 mg/kg bw/day.

A statistically significant increase in spleen weight was observed in males and females treated with 1000 mg/kg bw/day. The effect extended to the 150 mg/kg bw/day dose group although statistical significance was not always achieved.

Treatment-related changes in liver and spleen were detected at 1000 mg/kg bw/day and 150 mg/kg bw/day. Histopathological changes were also observed in kidneys, urinary bladder and stomach of animals treated with 1000 mg/kg bw/day. No treatment-related changes were reported in the ovaries, prostate, seminal vesicles, testes and uterus of control animals or animals at 1000 mg/kg bw/day.

Based on the above-mentioned results, the No Observed Effect Level (NOEL) was considered to be 15 mg/kg bw/day.

Although the 28-day repeated oral dose toxicity study yields to the lowest NOEL/NOAEL value, this study is considered less predictive compared to the 90-day repeated oral dose toxicity study as the duration of exposure is shorter and less animals are used per group. In addition, the spacing between doses in the 28-day study of 10-fold (compared to 2-3 fold in the 90-day study) also increases the uncertainty about the derived NO(A)EL. Therefore, the oral 90-day NOAEL value is considered more reliable than the 28-day NOEL value and thus the 90-day repeated oral dose toxicity study is selected as key study for this endpoint. The study is scored as reliable (K1).

 Repeated dose toxicity: dermal

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the dermal route of exposure. 

Repeated dose toxicity: inhalation

A key study is available for the oral route of exposure. According to the REACH Regulation, only one route of exposure should be tested for repeated dose toxicity (column 2, annex VIII, section 8.6.1). Therefore, it is not necessary to perform a repeated dose toxicity study via the inhalation route of exposure.

Justification for classification or non-classification

The administration of the test material to rats in a 90-day repeated oral (gavage) dose toxicity study performed according to OECD 408 guideline resulted in toxicologically significant effects at dose levels of 500 and 150 mg/kg bw/day (changes in the (fore)stomach, urinary bladder and red blood cell parameters at 500 mg/kg bw/day and changes in the (fore)stomach at 150 mg/kg bw/day). No adverse effects were observed at 50 mg/kg bw/day and this dose was considered to be the NOAEL value.

According to the CLP Regulation (Regulation (EC) No 1272/2008, a substance is classified (or not) based on the dose level at which significant/serious adverse effects are observed. This level should be compared to the corresponding Guidance Values (GVs) in the EU CLP guideline. The lowest dose at which significant/severe effects are observed in the 90-day repeated dose study is 150 mg/kg bw/day but the NOAEL is 50 mg/kg bw/day, that means below the GV for a 90-day study (100 mg/kg bw/day; Table 3.9.3 in the Regulation). Therefore, the first dose at which significant/serious adverse effects are observed (Effective Dose, ED) should be derived by interpolation, as indicated in the ECHA CLP guidance document on classification, labelling and packaging (CLP) of substances and mixtures (Version 4.0 November 2013). Based on the observed results and the interpolation, the ED is estimated to be 126,66 mg/kg bw/day (> GV for a 90-day study 100; Table 3.9.3 in the Regulation). Thus, and according to the criteria of the CLP Regulation, the substance does not need to be classified for STOT RE. In addition, the changes observed in the stomach can be considered as an adaptive response to the repeated oral gavage with a corrosive substance.