Registration Dossier

Administrative data

Description of key information

A LOAEL of 0.5 mg/kg bw/day was established from a 90-day study in rat by oral gavage, at which level leukocytosis was seen, inflammatory responses in mesenteric lymph nodes and lungs, and effects in the hindlegs and/or tail of few animals that were histopathologically supported by occurrence of arthritis with or without hyperostosis.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August-November 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study has been performed according to OECD and/or EC guidelines and according to GLP principles.
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2008
Deviations:
no
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
Qualifier:
according to
Guideline:
other: Japanese Chemical Substances Control Law 1973, Notification of Mar. 31, 2012 by MHLW (0331 No 7), METI (No 5) and MOE (No 110331009).
Deviations:
no
Qualifier:
according to
Guideline:
EPA OPPTS 870.3100 (90-Day Oral Toxicity in Rodents)
Version / remarks:
1998
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no
Species:
rat
Strain:
other: Crl:WI (Han)
Sex:
male/female
Details on test animals and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: approx. 6 weeks old
- Weight at study initiation: 149-180 g for males and 124-145 g for females.
- Housing: Group housing of 5 animals per sex in labeled Macrolon cages with sterilized sawdust as bedding material and paper as cage-enrichment
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7-21.4
- Humidity (%): 38-70
- Air changes (per hr): at least 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 25 August 2014 to 28 Nov 2014
Route of administration:
oral: gavage
Vehicle:
propylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Formulations (w/w) were prepared daily within 6 hours prior to dosing, and were homogenized to visually acceptable levels. Adjustment was made for specific gravity of the test substance and vehicle. No correction was made for the purity/composition of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): based on trial formulations performed at WIL Research Europe and on information from the sponsor.
- Specific gravity: 1.036

DOSE VOLUME: 5 ml/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
METHOD:
Homogeneity of preparation was analyzed for samples of Group 2 and 4 formulations in Weeks 1 and 6, and for samples of Group 2 and 3 formulations in Week 13. Accuracy of preparation was analyzed for formulations of all groups in Weeks 1, 6 and 13. Group 4 formulations were not analyzed in Week 13 since remaining Group 4 animals were sacrificed on Day 65. Stability in vehicle over 6 hours at room temperature under normal laboratory light conditions was also determined from Group 2 and 4 formulations in Week 1.
The accuracy of preparation was considered acceptable if the mean measured concentrations were 85-115% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation is ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:
90 days
Frequency of treatment:
Once daily, 7 d/w.
Remarks:
Doses / Concentrations:
0, 0.5, 2.2 and 8.8 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels were selected by the sponsor based on the results of a previously conducted 28-day study with the test substance.
Positive control:
No.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals after dosing. Once prior to start of treatment and at weekly intervals, this was also performed outside the home cage in a standard arena. The time of onset, grade and duration of any observed signs were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined weekly and mean daily diet consumption calculated as g food/kg bw/d: Yes

WATER CONSUMPTION
- Time schedule for examinations: subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: at pretest (all animals) and at week 13 (control and 2.2 mg/kg bw/d group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: According to test guidelines (no check at monthly intervals or midway through the test)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to scheduled post mortem examination
- Animals fasted: Yes, overnight
- How many animals: all animals
- Parameters checked: According to test guidelines (no check at monthly intervals or midway through the test)

URINALYSIS: urine was collected for possible future examination, but urine was not investigated as no effects were expected based on the results of the study
- Time schedule for collection of urine: from animals suriving until scheduled necropsy urine was collected overnight before necropsy; from animals sacrificed in extremis urine was collected from the bladder at necropsy, if possible.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes, during overnight sampling period

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during week 12/13
- Dose groups that were examined: first 5 surviving animals/sex/group
- Battery of functions tested: hearing ability, pupillary reflex and static righting reflex; fore- and hind-limb grip strength; motor activity test (total movement and ambulations)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS:
According to test guidelines with additionally prostate.

HISTOPATHOLOGY: Yes
According to test guidelines
Other examinations:
ESTROUS CYCLE:
All females has a daily lavage from day 76 up to and including day 91 to determine the stage of estrous.

RECTAL TEMPERATURE:
once weekly from weeks 9-13, at least 4 hours after dosing
Statistics:
The following statistical methods were used to analyze the data:
If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
The Steel-test (many-to-one rank test) was applied if the data could not be assumed to follow a normal distribution.
The Fisher Exact-test was applied to frequency data.
The Kruskal-Wallis nonparametric ANOVA test was applied to motor activity data to determine intergroup differences.

All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.
Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
effects observed, treatment-related
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
only volume investigated
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
8.8 mg/kg bw/d: 5/10 males dead (one found dead on day 42 and 4 sacrificed in extremis on days 42, 58, 64 and 58, resp.); 7/10 females were sacrificed in extremis at days 54, 54, 36, 42, 64, 64 and 55, resp.
The male found dead was believed to be due to pleuritis (marked inflammation of the pleura, watery-clear contents in the thoracic cavity and moderate infiltrate of the serosa of the thymus).
The animals sacrificed in extremis were diagnosed with swollen, painful, red tarsal and metatarsal area (uni- or bilateral), and/or swollen hindleg, and/or muscle atrophy of hindleg, and/or swollen and/or blue-red discoloured tailbase and hindlegs, and/or abnormal gait/not using hindleg.
The remaining males and females were sacrificed on day 65. Major clinical signs consisted of hunched posture and abnormal gait/hypotonia/swelling of the hindlegs.
2.2 mg/kg bw/d: 1 male on day 64 and 4 females on days 84, 54, 64 and 71 sacrificed in extremis
0.5 mg/kg bw/d: 1 male on day 84 and 1 female on day 79

Clinical signs noted in all dose groups consisted of swelling of the hindlegs/heel, paw and/or tail, general or focal erythema of the hindlegs/heel/paw (and testicles at 8.8 mg/kg bw/d), thickening of the tail and/or hindlegs/heel (and testicles at 8.8 mg/kg bw/d), abnormal gait (general or hindleg/heel) and hypotonia of the hind and/or forelegs/paw. Other clinical signs at 2.2 and 8.8 mg/kg bw/d consisted of lethargy, hunched posture, piloerection and/or lean appearance. These clinical signs generally showed an increasing trend with regard to the number of animals affected and earlier onset of symptoms with increasing doses.
Salivation seen after dosing at 0.5, 2.2 and 8.8 mg/kg bw/d was considered not toxicologically relevant, considering the nature and minor severity of the effect and its time of occurrence (i.e. after dosing). This sign was considered to be a physiological response related to taste of the test substance rather than a sign of systemic toxicity.
No abnormalities were noted during weekly arena observations different to those observed during clinical observations. Any other clinical signs noted during the treatment period occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study and did not show any apparent dose-related trend. At the incidence observed, these were considered to be unrelated to treatment.

BODY WEIGHT AND WEIGHT GAIN
8.8 mg/kg bw/d: males and females showed a lower body weight/body weight gain during the second half of their treatment period, achieving a level of statistical significance on most occasions.
2.2 mg/kg bw/d: males showed a statistically significantly lower body weight/body weight gain from week 78 onwards
0.5 mg/kg bw/d: no effect

FOOD CONSUMPTION
8.8 mg/kg bw/d: food consumption before or after correction for body weight of females appeared lower than controls througout treatment, and for males during the last 4-5 weeks of treatment.
2.2 mg/kg bw/d: absolute food consumption of males appeared lower than controls essentially throughout the treatment phase
0.5 mg/kg bw/d: food consumption before or after correction for body weight appeared similar to control levels.

OPHTHALMOSCOPIC EXAMINATION
No ophthalmology findings were noted that were considered to be related to treatment.

HAEMATOLOGY
The following (statistically significant) changes in haematology parameters distinguished treated from control animals:
 Higher white blood cell counts in males and females at 2.2 mg/kg bw/d, and in males also at 0.5 mg/kg bw/d.
 Higher absolute and relative neutrophil counts in males and females at 0.5 and 2.2 mg/kg bw/d.
 Higher absolute monocyte counts in males and females at 2.2 mg/kg bw/d, and in males also at 0.5 mg/kg bw/d.
 Higher absolute eosinophil counts in females at 2.2 mg/kg bw/d.
 Lower haemoglobin in males and females at 0.5 and 2.2 mg/kg bw/d.
 Lower haematocrit in males and females at 0.5 and 2.2 mg/kg bw/d.
 Lower mean corpuscular volume (MCV) in males and females at 2.2 mg/kg bw/d, and in males also at 0.5
mg/kg bw/d.
 Lower mean corpuscular haemoglobin (MCH) in males and females at 2.2 mg/kg bw/d, and in males also
at 0.5 mg/kg bw/d.
 Lower mean corpuscular haemoglobin concentration (MCHC) in females at 2.2 mg/kg bw/d.
 Higher platelet counts in males and females at 2.2 mg/kg bw/d (not statistically significant for females).

The lower relative lymphocyte counts in males and females at 0.5 and 2.2 mg/kg bw/d were ascribed to the higher white blood cell counts since absolute lymphocyte counts showed no clear dose-related changes. The shorter activated partial thromboplastin time (APTT) of males at 2.2 mg/kg bw/d was considered not toxicologically relevant since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
The statistically significantly lower relative basophil counts of males at 2.2 mg/kg bw/d were similar to control levels when based on absolute counts. Therefore this change was considered to be unrelated to treatment. Any other statistically significant changes in haematology parameters were also considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

CLINICAL CHEMISTRY
The following (statistically significant) changes in clinical biochemistry parameters distinguished treated from control animals:
 Lower total protein in males and females at 2.2 mg/kg bw/d.
 Lower albumin in males and females at 0.5 and 2.2 mg/kg bw/d.
 Lower glucose in males at 0.5 and 2.2 mg/kg bw/d.
 Lower cholesterol in males at 2.2 mg/kg bw/d.
 Higher chloride in males at 2.2 mg/kg bw/d.
 Lower calcium in males and females at 2.2 mg/kg bw/d, and in males also at 0.5 mg/kg bw/d.
The statistically significantly lower alanine aminotransferase activity and total bilirubin of males and females at 2.2 mg/kg bw/d and lower total bilirubin of females at 0.5 mg/kg bw/d was considered not toxicologically relevant given the relatively minor magnitude of change and since the opposite effect (i.e. an increase) would be expected in case of target organ toxicity.
Any other statistically significant changes in clinical biochemistry parameters were considered to be unrelated to treatment as these occurred in the absence of a dose-related trend.

URINE VOLUME
A statistically significantly higher urinary volume was recorded for males and females at 2.2 mg/kg bw/d.

NEUROBEHAVIOUR
Grip strength of both fore-and hindlegs showed an apparent trend towards a reduction across male and female dose groups (without being statistically significant).
Hearing ability and pupillary reflex were normal in all examined animals. No toxicologically relevant changes in static righting reflex were noted. Motor activity was similar between treated and control groups. All groups showed a similar motor activity habituation profile with a decreasing trend in activity over the duration of the test period.
One female at 2.2 mg/kg bw/d and one male at 0.5 mg/kg bw/d showed an apparent impairment in righting reflex. This was considered to be secondary to the observed clinical signs noted for the hindlegs of these animals around the time of conduct of this observation. Also, since other animals of these groups showed a normal static righting reflex, and the incidence of these occurrences did not show a dose-related trend, this was not considered to be of toxicological relevance.

ORGAN WEIGHTS
At 2.2 mg/kg bw/d, a statistically significant higher absolute and relative spleen weight was recorded for females (spleen weights were not recorded at 8.8 mg/kg bw/d).
Other statistically significant changes in organ weights and organ to body weight ratios were considered to be unrelated to treatment, since these changes occurred in the absence of a dose-related trend, were considered secondary to lower terminal body weights and/or occurred in the absence of histopathological correlates.

GROSS PATHOLOGY
The following macroscopic findings were considered to be related to treatment (including unscheduled
deaths):
8.8 mg/kg bw/d:
- Emaciated: 1/10 females.
- Lung: grown together with pleura in 2/10 males.
- Liver: enlarged in 2/10 males and 1/10 females.
- Spleen: enlarged in 3/10 males and 2/10 females.
- Mesenteric lymph node: enlarged in 9/10 males and 9/10 females.
- Popliteal lymph node(s): enlarged in 3/10 males and 5/10 females.
- Iliac lymph node: enlarged in 9/10 males and 9/10 females.
- Renal lymph node: enlarged in 1/10 males and 1/10 females.
- Hindfoot(s): thickened in 6/10 males and 9/10 females.
- Skin (base of) tail: thickened in 1/10 males and 3/10 females.
- Knee region: thickened in 1/10 females.
- Thigh muscle(s): reduced in size in 3/10 males and 6/10 females.
- Thoracic cavity: watery clear fluid in 1/10 males.

0.5 and 2.2 mg/kg bw/d:
- Lung: grown together with pleura/diaphragm/lobe/pericardium/thymus in 4/10 males and 1/10 females at 2.2 mg/kg bw/d.
- Lung: (several) reddish/gray-white foci in 1/10 males at 0.5 mg/kg and in 1/10 males and 1/10 females at 2.2 mg/kg bw/d.
- Mesenteric lymph node: enlarged in 2/10 males and 1/10 females at 0.5 mg/kg bw/d and in 10/10 males and 10/10 females at 2.2 mg/kg bw/d.
- Popliteal lymph node(s): enlarged in 3/10 females at 2.2 mg/kg bw/d.
- Iliac lymph node: enlarged in 1/10 males and 1/10 females at 0.5 mg/kg, and in 1/10 males and 3/10 females at 2.2 mg/kg bw/d.
- Hindfoot(s): thickened in 1/10 males and 2/10 females at 0.5 mg/kg, in 2/10 males and 4/10 females at 2.2 mg/kg bw/d.
- Skin (base of) tail: thickened in 1/10 males and 1/10 females at 0.5 mg/kg and in 1/10 male and 3/10 females at 2.2 mg/kg bw/d.
- Thigh muscle(s): reduced in size in 1/10 males and 4/10 females at 2.2 mg/kg bw/d.

The incidence of other necropsy findings among control and treated animals was within the background range of findings that are encountered among rats of this age and strain, did not show any apparent dose-related incidence trend and/or had no treatment-related histopathological correlates. These necropsy findings were therefore considered to be unrelated to treatment with the test substance.

HISTOPATHOLOGY: NON-NEOPLASTIC
The following microscopic findings noted among intercurrent sacrifices and animals surviving until
scheduled necropsy were considered to be related to treatment with the test substance:
8 mg/kg bw/d (organs with macroscopic findings only):
General observation
- Leucocytosis (mostly detectable in the spleen and liver), in 3/10 males and 3/10 females. The low
incidence of the highest dose group could be underestimated due to a limited amount of liver and
spleen available for histopathologic examination.
Lung
- Inflammation pleura in 2/2 males (1 moderate, 1 marked).
- Inflammation alveolar in 1/2 males (slight).
- Infiltrate eosinophils in 1/2 males (slight).
Thymus
- Serosa infiltrate in 1/1 males (minimal).
Duodenum:
- Granulocytic (necrotizing) inflammation in 1/2 females (minimal).
Liver:
- Granulomas with or without necrosis (slight) in 1/10 females (slight).
Spleen:
- Granulomas with or without necrosis in 2/3 males (1 slight, 1 marked).
- Lymphoid hyperplasia in 1/3 males (minimal) and 2/2 females (minimal).
Mesenteric lymph node:
- Granulomas with or without necrosis in 9/9 males (1 minimal, 1 slight, 1 moderate, 4 marked, 2
massive) and 10/10 females (1 slight, 4 moderate, 5 marked).
- Extranodal inflammation with or without peritonitis in 9/9 males (1 minimal, 3 slight, 5 moderate) and
10/10 females (5 slight, 5 moderate).
- Macrophage foci in 2/9 males (2 slight) and 2/10 females (1 minimal, 1 moderate).
- Increased cellularity of cortex and/or medullary cords in 8/9 males (1 minimal, 5 slight, 2 moderate)
and 10/10 females (5 slight, 5 moderate).
- Medullary sinus ectasia in 2/9 males (1 slight, 1 moderate).
Iliac lymph node
- Increased cellularity of cortex and/or medullary cords was noted in 9/9 males (1 minimal, 6 slight, 2
moderate) and 9/9 females (2 minimal, 7 slight).
- Lymphangectasia in 3/9 females (1 minimal, 2 slight).
Renal lymph node:
- Increased cellularity of cortex and/or medullary cords in 1/1 males (1 moderate) and 1/1 females (1
moderate).
- Lymphangectasia in 1/1 females (1 minimal).
Popliteal lymph node:
- Increased cellularity of cortex and/or medullary cords in 3/3 males (slight) and 5/5 females (4 slight, 1
moderate).
- Lymphangectasia in 1/3 males at 8 mg/kg.
- Medullary sinus ectasia in 1/3 males (minimal) and 1/5 females (minimal).
Tarsal joints:
- Arthritis with or without hyperostosis in 6/6 males (marked) and 8/9 females (4 marked, 4 massive).
Knee joint:
- Arthritis with or without hyperostosis in 1/1 females (moderate).
Tail vertebra:
- Arthritis with or without hyperostosis in 1/1 males (marked) and 3/3 females (1 moderate, 2 marked).
Gastrocnemius muscle(s):
- Myofiber atrophy in 1/3 males (minimal) and 5/6 females (3 minimal, 2 slight).

0.5 and 2 mg/kg bw/d:
General:
Leucocytosis (mostly detectable in the spleen and liver), in 7/10 males and 5/10 females at 0.5 mg/kg bw/d,
in 9/10 males and 10/10 females at 2 mg/kg bw/d.
Thoracic cavity:
Lung:
- Inflammation pleura in 1/10 males (slight) at 0.5 mg/kg and in 6/10 males (2 minimal, 2 slight, 2
moderate) and 4/10 females (2 minimal, 1 slight, 1 moderate) at 2 mg/kg bw/d.
- Inflammation granulomatous in 1/10 males (minimal) at 0.5 mg/kg bw/d and in 1 male (minimal) and 1
female (moderate) at 2 mg/kg bw/d.
- Inflammation alveolar at increased incidence and severity in 5/10 males (3 minimal, 2 slight) and
4/10 females (3 minimal, 1 slight) at 0.5 mg/kg bw/d and in 5/10 males (4 minimal, 1 slight) and 3/10
females ( 2 minimal, 1 slight) at 2 mg/kg bw/d, compared to minimal degree in 3/10 males and 2/10
females of the control group.
- Infiltrate eosinophils at increased incidence and severity in 6/10 males (4 minimal, 2 slight) and 3/10
females (1 minimal, 2 slight) at 0.5 mg/kg bw/d and in 8/10 males (5 minimal, 3 slight) and 9/10 females (5
minimal, 3 slight, 1 moderate) at 2 mg/kg bw/d, compared to 1/10 male (minimal) at 0 mg/kg bw/d.
- Infiltrate lymphocytes in 5/10 males (3 minimal, 2 slight) at 2 mg/kg bw/d.
Aorta:
- Serosa infiltrate in 2/10 females (1 minimal, 1 slight) at 2 mg/kg bw/d.
Thymus
- Serosa infiltrate in 1/10 males (minimal) and 1/10 females (1 minimal) at 2 mg/kg bw/d.

Abdominal cavity:
Jejunum:
- Granulocytic (necrotizing) inflammation in 1/10 males (marked) and 1/10 females (minimal) at 2
mg/kg bw/d.
Ileum:
- Granulocytic (necrotizing) inflammation in 9/10 males (2 minimal, 4 slight, 3 moderate) and 4/10
females (2 minimal, 1 slight, 1 moderate) at 2 mg/kg bw/d.
- (Foamy) macrophages in 9/10 males (6 minimal, 3 slight) and 10/10 females (9 minimal, 1 slight) at 2
mg/kg bw/d.
Liver:
- Increased severity of granulomas (slight) in 2/10 females at 2 mg/kg, compared to minimal degrees
in 1/10 male and 2/10 females at 0.5 mg/kg bw/d.
Spleen:
- Lymphoid hyperplasia in 5/10 females (minimal) at 2 mg/kg bw/d.
Mesenteric lymph node:
- Granulomas with or without necrosis in 5/10 males (4 moderate, 1 marked) and 4/10 females (2
minimal, 1 slight, 1 moderate) at 0.5 mg/kg bw/d and in 9/10 males (4 moderate, 3 marked, 2 massive)
and 10/10 females (1 slight, 4 moderate, 4 marked, 1 massive) at 2 mg/kg bw/d.
- Extranodal inflammation with or without peritonitis in 5/10 males (1 minimal, 4 slight) and 4/10
females (2 slight, 2 moderate) at 0.5 mg/kg bw/d and in 9/10 males (5 slight, 4 moderate) and 10/10
females (7 slight, 3 moderate) at 2 mg/kg bw/d.
- Increased incidence and/or severity of macrophage foci in 9/10 males (2 minimal, 5 slight, 2
moderate) and 7/10 females (1 minimal, 6 slight) at 0.5 mg/kg bw/d compared to 3/10 males (2 minimal, 1
slight) and 2/10 females (2 minimal) at 2 mg/kg and 0/10 males and 2/10 females( 2 minimal) in the
control group. It was considered that incidences and severities did not further increase at 2 mg/kg bw/d,
due to the increase in granulomas.
- Increased cellularity of cortex and/or medullary cords was noted in 2/10 males (minimal) and 3/10
females (1 minimal, 2 slight) at 0.5 mg/kg bw/d and in 8/10 males (4 minimal, 4 slight) and 10/10 (1
minimal, 6 slight, 2 moderate, 1 marked) females at 2 mg/kg bw/d.
- Medullary sinus ectasia in 3/10 females (2 minimal, 1 moderate) at 2 mg/kg bw/d.
Iliac lymph node
- Increased cellularity of cortex and/or medullary cords in 1/1 males (minimal) and 1/1 females (1
slight) at 0.5 mg/kg bw/d and in 1/1 males (1 slight) and 3/3 females (3 slight) at 2 mg/kg bw/d.
- Medullary sinus ectasia in 1/1 males (1 minimal) at 0.5 mg/kg bw/d and in 1/1 males (moderate) at 2
mg/kg bw/d.
Adrenal glands
- Serosa infiltrate inflammatory cells, in 1/10 females (slight) at 2 mg/kg bw/d.
Pancreas
- Acinar atrophy at increased incidence and/or severity in 3/10 males (2 minimal, 1 slight) and 4/10
females (4 minimal) at 2 mg/kg bw/d , compared to minimal severity in 1/10 males at 0 mg/kg and in 1/1
males at 0.5 mg/kg bw/d.

Hindlegs, tail and draining lymph node:
Popliteal lymph node
- Increased cellularity of cortex and/or medullary cords in 3/3 females (1 minimal, 2 slight) at 2 mg/kg bw/d.
- Medullary sinus ectasia in 1/3 females (1 slight) at 2 mg/kg bw/d.
Tarsal joints
- Arthritis with or without hyperostosis in 4/10 males (3 minimal, 1 marked) and 3/10 females (1
minimal,1 slight, 1 marked) at 0.5 mg/kg bw/d and in 8/10 males (5 minimal, 1marked, 2 massive) and
7/10 females (2 minimal, 1 moderate, 1 marked, 3 massive) at 2 mg/kg bw/d.
Tail vertebra
- Arthritis with or without hyperostosis in 1/1 males (moderate) and 1/2 females (minimal) at 0.5 mg/kg bw/d
and in 2/3 females (moderate) at 2 mg/kg bw/d.
Gastrocnemius muscle(s):
- Myofiber atrophy in 1/4 females (minimal) at 2 mg/kg bw/d.
Sciatic nerve:
- Infiltrate surrounding tissue in 2/10 males (1 minimal, 1 slight) and in 1/10 females (minimal) at 2
mg/kg bw/d.

Female reproductive tract:
Uterus:
- Inactive uterus in 4/10 females at 2 mg/kg bw/d.
Cervix:
- Inactive uterine cervix in 1/1 females at 0.5 mg/kg bw/d.
Vagina:
- Epithelial atrophy was present in 1/1 females at 0.5 mg/kg bw/d and in 1/10 females at 2 mg/kg bw/d.
- Epithelial mucification in 4/10 females (3 minimal, 1 slight) at 2 mg/kg bw/d.

Sublingual glands and sternum, including bone marrow:
Sublingual glands:
- Acinar atrophy was in 1/10 males ( minimal) and 2/10 females (1 minimal,1 slight) at 2 mg/kg bw/d.
Sternal bone
- Arthritis with or without hyperostosis at the costo-sternal articulation in 1/10 females (1 slight) at 2
mg/kg bw/d.
(Sternal) bone marrow:
- Myeloid hyperplasia in 9/10 males (minimal) and 6/10 females (minimal) at 0.5 mg/kg bw/d and in 10/10
males (3 minimal, 7 slight) and 10/10 females (4 minimal, 6 slight) at 2 mg/kg bw/d.

OTHER FINDINGS
No toxicologically relevant changes in rectal temperature were recorded.

Estrous cycle length/regularity showed no treatment-related effect.
Dose descriptor:
LOAEL
Effect level:
0.5 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: serious inflammatory effects in small intestine, mesenteric lymph node, liver, lung, spleen and hindlegs
Critical effects observed:
not specified

RESULTS ANALYSIS:

Accuracy of preparation:

The concentrations analysed in the formulations of Group 2, Group 3 and Group 4 were in agreement with target concentrations (i.e. mean accuracies between 85% and 115%).

The slightly higher accuracies may in part have resulted from the higher nominal concentrations, since nominal amounts of test substance and vehicle used for formulation were higher than targeted concentrations. Accuracy was based on target concentrations.

Small responses at the retention time of the test substance were observed in the chromatograms of the Group 1 formulations. It was considered to derive from carry over since similar responses were obtained in the analytical blanks.

Homogeneity:

The formulations of Group 2 and Group 4 were homogeneous (i.e. coefficient of variation ≤ 10%).

Stability:

Analysis of Group 2 and Group 4 formulations after storage yielded a relative difference of ≤ 10%. Based on this, the formulations were found to be stable during storage at room temperature under

normal laboratory light conditions for at least 6 hours.

Conclusions:
In a 90-day oral repeated dose toxicity study with rats, the LOAEL was determined to be 0.5 mg/kg bw/day.
Executive summary:

In light of the corrosive properties of the substance, and the possible local nature of the effects that could affect a clear evaluation of systemic effects, dosing by dietary route was recommended for this 90-day study by oral route. However, after various attempts no adequate analytical method could be developed, and it was decided to proceed with dose by gavage. Besides, in view of the low dose levels selected for the study, interference through local effects that are expected to occur especially in the forestomach (serves as a storage reservoir in rodents), were considered to be minimal.

Wistar rats were administered 0, 0.5, 2.2 or 8.8 mg/kg bw/d of the test substance by gavage daily for 90 days. The control group received the vehicle propylene glycol. Examinations were performed according to OECD 408 with additionally rectal temperature and estrous cycle determination.

A total of 12/20 animals at 8.8 mg/kg bw/d were sacrificed in extremis or were found dead between weeks 6 and 10. Additionally, 2/20 animals at 0.5 mg/kg bw/d and 5/20 animals at 2.2 mg/kg bw/d were sacrificed in extremis in week 12 and between weeks 8 and 12, respectively. All remaining animals at 8.8 mg/kg bw/d were sacrificed in week 10. Morbidity was related to marked arthritis of the tarsal joints and/or granuloma(s), with central necrosis of the mesenteric lymph nodes, and at 8.8 mg/kg bw/d also in one case due to pleuritis. The incidence and onset of mortality showed an apparent dose-related trend.

The hindlegs and/or tail of several rats treated at 0.5 mg/kg bw/d and the majority of rats treated at 2.2 and 8.8 mg/kg bw/d were affected, showing clinical symptoms such as abnormal gait/hypotonia/swelling. Occasionally, testicles showed erythema at 8.8 mg/kg bw/d. These clinical signs generally showed an increasing trend with regard to the number of animals affected and earlier onset of symptoms with increasing doses. These animals also displayed supportive macroscopic findings such as thickened hindlegs/tail and/or enlarged popliteal lymph nodes and/or reduced size of gastrocnemius muscle(s). Histopathologically, these findings were supported by a dose-related occurrence of arthritis with or without hyperostosis in the tarsal joints (up to marked severity at 0.5 mg/kg bw/d and up to massive severity at 2.2 mg/kg bw/d). In general, lower severities were related to acute inflammations with edema of the soft tissues surrounding tarsal bones and joints as hallmark, whereas higher severities were more frequently noted in chronic inflammations including hyperostosis of bones and in some cases with fissures of bones. In several animals a similar histopathology was noted in tail vertebrae (2.2 and 8.8 mg/kg bw/d) and/or knee joint (8.8 mg/kg bw/d) which in a few cases was accompanied by atrophy of the gastrocnemius muscle (2.2 and 8.8 mg/kg bw/d). This atrophy was regarded as a feature of inactivity. All these findings in the hindlegs can be correlated with in-life clinical symptoms, such as abnormal gait /hypotonia/swelling. One female treated at 2.2 mg/kg bw/d showed arthritis of the rib-sternal junction.

The draining popliteal lymph node showed increased cellularity of the common cell types of lymph nodes (without a clear predominance) in the cortex and/or medullary cords, similar as noted in the iliac and renal lymph nodes (supported at necropsy by enlargement of these nodes). Increased cellularity was also noted for the liver at 8 mg/kg and correlated to enlargement of the liver.

Granulocytic inflammation (up to marked) observed in the small intestines (especially in the ileum) at 2.2 mg/kg bw/d was considered to be due to a direct exposure of the test item or its metabolite. Furthermore, there were (foamy) macrophages in the lamina propria up to slight degree noted in most rats at 2.2 mg/kg bw/d. At 0.5 mg/kg bw/d, there were no test item-related findings in the small intestines.

At 0.5 mg/kg bw/d and above, the draining mesenteric lymph node showed up to massive granulomas with or without central necrosis (especially at the outer side of the cortex), most times merging into extranodal inflammation and/or necrosis with or without peritonitis (up to moderate). Incidences and severity showed a dose related response. Enlarged mesenteric lymph nodes recorded at necropsy at 0.5 mg/kg bw/d and above correlated to increased cellularity, granuloma(s), central necrosis and/or medullary sinus ectasia.

It was remarkable that there were rats with granulomas of the mesenteric lymph nodes without a clear relation with histopathology of the small intestines (granulocytic inflammations). This started in males at 0.5 mg/kg bw/d with moderate and marked degrees of granulomas of the mesenteric lymph node without any finding in the small intestines.

Granulomas were also noted at low incidence in the spleen at 8.8 mg/kg bw/d (up to marked, only from rats with a macroscopically enlarged spleen) and liver at 2.2 and 8.8 mg/kg bw/d. Capsules of these abdominal organs were unaffected (one spleen at 8.8 mg/kg bw/d excluded), making it less plausible that the liver and spleen findings were caused by peritonitis. The higher spleen weight recorded for females at 2.2 mg/kg bw/d were attributed to leucocytosis and granulomas in the spleen.

In the thoracic cavity, major findings were present in the lung, consisting of adhesions seen at necropsy at 2.2 and 8.8 mg/kg bw/d correlating to inflammation of the pleura (up to marked), and a clear increase in incidence and severity of perivascular eosinophilic/lymphocytic infiltrates. Furthermore, there was a slight increase in severity and/or incidence of alveolar and/or granulomatous inflammation (correlating to reddish/gray-white foci at necropsy at 0.5 and 2.2 mg/kg bw/d). Secondary changes to the pleural inflammation consisted in a few cases of serosa infiltrates with inflammatory cells of thymus or aorta and in one rat (at 8.8 mg/kg bw/d) of watery clear fluid in the thoracic cavity.

Myeloid hyperplasia of the sternal bone marrow (up to slight) and leucocytosis (mainly detectable in spleen and liver, and correlating with the macroscopic finding enlarged) started at 0.5 mg/kg bw/d and are considered to be a consequence of the inflammatory processes. The neutrophilia recorded for males and females at 0.5 and 2.2 mg/kg bw/d was regarded to be related to several inflammatory processes, most severely present in mesenteric lymph nodes and tarsal joints. Myeloid hyperplasia in the sternal bone marrow could account for this increase in neutrophils.

Inactive uterus (cervix) epithelium and/or atrophy/mucification of the vagina epithelium in 1/1 females at 0.5 mg/kg bw/d and 4/10 females at 2.2 mg/kg bw/d, and increased incidence and/or severity of acinar atrophy in the pancreas and/or sublingual salivary glands at 2.2 mg/kg bw/d were considered to be related to the poor health condition, affecting normal estrous cycling behavior.

Summarizing, it remained unclear whether most findings were caused systemically by direct exposure to the test item or its metabolite (either by vascular or lymphatic circulation) or indirectly (e.g. by an elicited immune-mediated response, such as release of cytokines/chemotaxis from the inflammatory processes in primarily ileum and/or mesenteric lymph node). Overall, it was considered that exposure of intestines lead to (necrotizing) inflammation and granulomas up to massive degrees of draining mesenteric lymph nodes with possible breakthrough of granulomas into the abdominal cavity. However, this possibly induced peritonitis cannot explain the several findings (which are not restricted to surfaces of organs) in many organs at several locations.

At 2.2 and 8.8 mg/kg bw/d, a lower body weight/body weight gain and lower food consumption was noted for males and/or females, essentially during the second half of the treatment period.

Next to changes in (differential) white blood cell counts, haematological changes consisted of lower haemoglobin, haematocrit, mean corpuscular volume and mean corpuscular haemoglobin, and higher platelet counts in males and/or females at 0.5 and/or 2.2 mg/kg bw/d. Changes in clinical biochemistry parameters consisted of lower total protein, albumin, cholesterol, glucose and calcium, and higher chloride in males and/or females at 0.5 and/or 2.2 mg/kg bw/d. Additionally, a higher urinary volume was recorded for both sexes at 2.2 mg/kg bw/d.

The apparent trend towards a reduction in grip strength of both fore-and hindlegs across dose groups of both sexes may be secondary to the observed clinical signs and resulting impairment of movements. Other functional observation parameters remained unaffected.

There were no indications of possible reproductive toxicity based on the parameters determined in this study. Estrous cycle length/regularity appeared unaffected during the period in which estrous cycle length was determined (Day 76-91), and histopathological examination of the male and female reproductive organs did not show lesions that were directly related to treatment.

No toxicologically relevant changes in rectal temperature and ophthalmology findings were noted.

Based on these results no NOAEL could be established and the LOAEL was determined to be 0.5 mg/kg bw/d.

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
LOAEL
0.5 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The study is GLP compliant and has Klimisch score 1

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

Several repeated oral gavage dose studies in rats on Etherdiamine C13i/acetate are available with varying duration, involving14-day treatment (RF to 28-day; OECD 414 full & RF, see under developmental toxicity), a 28-day (OECD 407) study, and a 90-day (OECD 408) study.

 

A 28day study on Etherdiamine C13i/acetate was performed to current OECD/EU protocols carried out according to GLP. Prior to this study dose range finding studies were done.

These studies showed that levels of Etherdiamine C13i/acetate of 100 and 300 mg/kg bw resulted to death or sacrifice by day 4 resp. 8.

A 14-day RF with dose levels 0, 3, 10 or 30 mg/kg bw/day, resulted to clinical signs at 30 mg/kg from treatment day 5 onwards including dyspnea, reduced food consumption and body weight development. Differences in the hematology and clinical biochemistry parameters were largely indicative of non-specific inflammation and stress, coupled with some indication of effects upon the liver and possible renal alterations which are generally associated with glomerular damage. The NOAEL from this study could be considered to be 10 mg/kg, as the small changes seen at this level in WBC (elevated in males), liver weights (males) and spleen weights do not show full dose relation.

 

Dose levels of 3, 9 and 25 mg/kg/day were selected for the main 28-day study with Etherdiamine C13i/acetate. Dose groups consisted of 5 males and 5 females and included 14-day recovery groups for the control and high dose.

Four animals (20%) of the high dose group died and one from the mid dose. Clinical signs were confined to the high dose groups, and were resolved the second day of the recovery period. There was a decreased food intake, with lower BW gain in group 4 with small improvements during recovery period.

Microscopic examinations showed treatment-related lesions in the forestomach of main test animals of the high dose groups, representing a localized stomach reaction to a repeatedly gavaged slightly irritant test material.

 

The findings from this study indicate to disseminated inflammation with increased levels of neutrophils, accompanied by possibly stress related increased adrenal weights and decreased thymus weights. Histopathology also indicates inflammation processes in various organs with increased (foamy) macrophages and granulomas.

Blood enzymes show no significant elevation, which indicates there is no real organ/cytotoxicity occurring.

Most of the parameters improve partial or complete during recovery.

Necrotic and/or inflammatory processes were observed in stomach, small intestine, mesenteric lymph node, liver, thymus, heart and joints. Although the lesions observed in the small intestine and mesenteric lymph node were considered to be possibly due to the route of application, it was considered that based on these histopathological findings present in all treated groups, a NOAEL cannot be established thus resulting to a LOAEL of 3 mg/kg bw/day.

 

In light of the corrosive properties of the substance, and the possible local nature of the effects that could affect a clear evaluation of systemic effects, dosing by dietary route was recommended for the subsequent 90-day study by oral route. However, after various attempts no adequate analytical method could be developed, and it was decided to proceed with dose by gavage. Besides, in view of the low dose levels selected for the study, interference through local effects that are expected to occur especially in the forestomach (serves as a storage reservoir in rodents), were considered to be minimal.

Wistar rats were administered 0, 0.5, 2.2 or 8.8 mg/kg bw/d of the test substance by gavage daily for 90 days. The control group received the vehicle propylene glycol. Examinations were performed according to OECD 408 with additionally rectal temperature and estrous cycle determination.

A total of 12/20 animals at 8.8 mg/kg bw/d were sacrificed in extremis or were found dead between weeks 6 and 10. Additionally, 2/20 animals at 0.5 mg/kg bw/d and 5/20 animals at 2.2 mg/kg bw/d were sacrificed in extremis in week 12 and between weeks 8 and 12, respectively. All remaining animals at 8.8 mg/kg bw/d were sacrificed in week 10. Morbidity was related to marked arthritis of the tarsal joints and/or granuloma(s), with central necrosis of the mesenteric lymph nodes, and at 8.8 mg/kg bw/d also in one case due to pleuritis. The incidence and onset of mortality showed an apparent dose-related trend.

Haematology and biochemistry findings, supporting as associated findings to inflammatory reactions.

At 0.5 mg/kg bw/d and above, the draining mesenteric lymph node showed up to massive granulomas with or without central necrosis (especially at the outer side of the cortex), most times merging into extranodal inflammation and/or necrosis with or without peritonitis (up to moderate). Incidences and severity showed a dose related response. Enlarged mesenteric lymph nodes recorded at necropsy at 0.5 mg/kg bw/d and above correlated to increased cellularity, granuloma(s), central necrosis and/or medullary sinus ectasia.

Overall, it was considered that exposure of intestines lead to (necrotizing) inflammation and granulomas up to massive degrees of draining mesenteric lymph nodes with possible breakthrough of granulomas into the abdominal cavity,and more systemic inflammatory responses indicated by granulocytic (necrotizing) inflammations in other organs and the increase of neutrophils.

Based on these results no NOAEL could be established and the LOAEL was determined to be 0.5 mg/kg bw/d, at which level leukocytosis was seen, inflammatory responses in mesenteric lymph nodes and lungs, and effects in the hindlegs and/or tail of few animals that were histopathologically supported by occurrence of arthritis with or without hyperostosis.

 

 

Small intestinal and mesenteric lymph node lesions with foamy macrophageshave consistently been observed with similar substances fatty amine like substances. A mode of action has not been established but it is possible to suspect the known corrosivity to be at least partially involved. It is indicative that the observed effects are local and they are by some interpreted as phospholipidosis, something commonly observed following treatment with cationic amphiphilic material, including marketed pharmaceuticals, and considered to be non-adverse. When taking into consideration the relatively strong corrosive effects of this substance, and for substances belonging to the same group of chemicals, and the route of administration, it cannot be excluded that the overall toxicity reflects a point-of-first-contact effect.

 

Information on the chronology for pathogenesis by Etherdiamine C13i/acetate can be obtained from comparing the available data from several repeated oral gavage dose studies in with varying duration, involving 14-day treatment (RF to 28-day; OECD 414 full & RF), a 28-day (OECD 407) study, and a 90-day (OECD 408) study, specifically on elements that (could) point at inflammatory responses: reported macroscopic observations, histopathology reporting and the BWC, specifically for the neutrophils.

A distinction can be made between local inflammatory responses in GI-tract (thickened intestinal wall and enlarged mesenteric lymph nodes, foamy macrophages in lamina propria of intestinal wall and in the mesenteric lymph nodes, as well as granulocytic (necrotizing) inflammations in these tissues) and more systemic inflammatory responses indicated by granulocytic (necrotizing) inflammations in other organs and the increase of neutrophils. Actual systemic inflammatory responses extending to organs outside the gastro-intestinal tract do occur after longer duration of dosing of Etherdiamine C13i/acetate at lower dose levels. Form these available studies it seems that the increase of the neutrophils (> 100% compared to control) is possibly a good marker for the development of inflammatory response observed following the dosing of the etherdiamine.

 

Extrapolation from oral to

- Inhalation: Effects are mainly following from effects on local lymph nodes. It is uncertain whether similar effects in lung are to be expected. Based on the available data and progression of effects from mesenteric lymph nodes to lungs, similar sensitivity is assumed.

- Dermal: Effects are mainly on local lymph nodes following absorption. On skin local corrosive/irritant effects and lower absorption are to be expected. However, based on local irritant effects that are possibly inflammatory mediated, same sensitivity is assumed.

 

The likelihood of exposures via inhalation or by dermal route is low considering use and physical properties: The substance is severely corrosive and itsuse is limited to industrial settings under controlled conditions. Alsothe high boiling point (> 300 °C) and low vapour pressure (0.005 Pa at 25°C) indicate that the potential for inhalation is not significant.

 

 

The available studies show that repeated exposures lead to a generalised inflammatory response that is both increasing with dose and duration. Because the current 90-day study did not provide a clear NOAEL and there is uncertainty related to further increasing exposure durations, a chronic study is proposed.


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
Only available study of longest duration (90-days) of high validity.

Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
Likelihood of exposures via inhalation is low considering use and physical properties: The use is limited to industrial settings under controlled conditions. Also the high boiling point (> 300 °C) and low vapour pressure (0.005 Pa at 25°C) indicate that the potential for inhalation is not significant to justify this study.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
Lack of exposure

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
Etherdiamine C13i/acetate is very corrosive to the skin and is not expected to easily pass the skin. The skin is therefore not a preferred route when studying repeated dose systemic toxicity.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: ileum

Justification for classification or non-classification

STOT-RE Cat,1 is required in case of significant toxicity at levels at levels ≤ 10 mg/kgbw/d in 90-day studies or ≤ 30 mg/kgbw/d in case of 28-day studies.

Under DSD, R48 is required with ‘Harmful’ at levels between 5 and 50 mg/kgbw/d in 90-day studies or 15 - 150 mg/kgbw/d in case of 28 -day studies, and with 'Toxic' at lower levels.

 

The 28-day study in rat by oral gavage with Etherdiamine C13i/acetate showed at 25 mg/kg bw/day necrotic and/or inflammatory processes in stomach, small intestine, mesenteric lymph node, liver, thymus, heart and joints and resulted to 20% mortality. The primary target organs for the test item were considered to be the gastrointestinal tract organs, mesenteric lymph node and liver.

The 90-day study resulted to severe toxicity and mortality at 8.8 mg/kg bw/day. Also the 2.2 mg/kg dose level resulted to severe toxicity

 

Consequently classification is required for STOT-RE Cat.1 [Danger] with H372 Causes damage to organs through prolonged or repeated exposure.

For DSD, these results lead to classification T; R48/25 Toxic: danger of serious damage to health by prolonged exposure if swallowed.