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Description of key information

Lina (2014) (90-day oral - diet, rat), K1

Under the conditions of this study the no-observed-adverse-effect level (NOAEL) was placed at the highest level tested, namely 1250 mg/kg diet (≥82 and 91 mg/kg body weight/day for males and females, respectively).

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:
18 April 2013 to 19 July 2013
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
Qualifier:
according to guideline
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
other: Wistar Outbred rats (RccHan™:WIST)
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Age at study initiation: Approximately 6 weeks old
- Weight at study initiation: The body weights at initiation of treatment were within ±20 % of the mean weight for each sex, and ranged from 162 to 201 g (mean 182 g) for males, and from 131 to 158 g (mean 146 g) for females.
- Fasting period before study: No
- Housing: The animals were kept in macrolon cages with wood shavings as bedding material and strips of paper and a wooden block as environmental enrichment. They were housed in groups of five, separated by sex. During FOB testing and motor activity assessment and urine collection, the animals were temporarily kept singly.
- Diet: Feed was provided ad libitum. The rats received a cereal-based rodent diet. The diets were provided as a powder to the rats in stainless steel cans, covered by a perforated stainless steel plate to prevent spillage. Once weekly, the feed in the feeders was replaced by fresh portions from the freezer.
- Water: Drinking water was provided ad libitum. Each cage was supplied with domestic mains tap-water in polypropylene bottles, which were cleaned weekly and filled as needed.
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature: 20 to 24 °C
- Humidity: 45 to 65 %
- Air changes: ca. 10 air changes per hour
- Photoperiod: Lighting was artificial with a sequence of 12 hours of light and 12 hours of dark.

IN-LIFE DATES: From: 04 April 2014 To: 18 July 2014 (males) and 19 July 2014 (females)
Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): Fresh batches of the experimental diets were prepared three times during the study (on 17 April, 14 May and 19 June 2013).
- Mixing: Experimental diets were prepared by mixing in a mechanical blender (the same batch of) diet with the appropriate amounts of test material.
- Storage of food: The experimental diets were stored in closed plastic bags in a freezer (< -18 °C) in portions sufficient for 7 days.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
From all three batches of diets prepared in the study, samples were taken immediately after diet preparation and analysed on the same day (except for the samples prepared on 14 May 2013, which were analysed on 22 May 2013 after storage in a freezer (<-18 °C) pending analysis).
The following analyses were conducted in the study:
- Homogeneity: The homogeneity of the test material in the experimental diets was demonstrated in the first batch prepared.
- Concentration: The concentration of test material at each dietary level was determined in all three batches.
- Stability: The stability of the test material in the feed under experimental conditions was not determined in this study. The stability under similar conditions and at similar dietary levels was confirmed in a previous study conducted at the testing facility. In this study, the test material was stable in the diet at levels of 200, 500 and 1250 mg/kg diet when stored for 7 days, and when stored in the freezer (<-18 °C) for at least 4 weeks.

TEST MATERIAL ANALYSIS IN THE FEED
The concentration of the test material in the diet was determined using the following procedure: after addition of an internal standard to the diet, samples were extracted with acetate buffer (pH 4.5). Derivatisation was performed by adding 20 % sodium tetraethyl borate (STEB) solution in tetrahydrofuran (THF). After addition of hexane, the mixture was shaken and incubated at 60 °C. The hexane extract was cleaned up by adding 2 M HCl and shaking. Samples were analysed with Gas Chromatography – Mass Spectrometry (GC-MS). Quantitation was obtained by comparison of Q values (ratio peak area test material / peak area internal standard) in the sample extracts with those in calibration solutions containing known amounts of the test material.

VALIDATION CRITERIA
Before analysis of study samples, the analytical method was validated by analysing three spiked samples per dose level, to conform to the following criteria:
> Linearity: the correlation coefficient of the calibration curve should be greater than or equal to 0.99;
> Recovery: the mean recovery of the test material from the diet should be between 80 and 120 % at each of the dose levels of the study;
> Repeatability: the relative standard deviation in the percentage recovery, when the recovery test is performed three times at each of the dose levels to be used in the toxicity study, should be less than 15 %.
With respect to specificity: the signal obtained for samples should be corrected for the signal obtained with blank samples in case the signal obtained for blank samples is ≥5 % of the signal obtained for low-dose samples.

- Preparation of validation samples
Validation samples with nominal concentrations of 0, 200, 500 and 1250 mg/kg diet were prepared in triplicate by adding an aliquot of the test material in methanol stock solution to 2 g diet. All validation and blank samples were extracted as described above and analysed as described below.

SAMPLE PREPARATION
Study samples were analysed in duplicate. Test material was extracted from the diet using the following method:
Approximately 2 g diet was accurately weighed and 50 μL internal standard solution (approximately 0.6 mg/mL methanol), 20 mL methanol, 5 mL acetate buffer pH 4.5 (approximately 80 g sodium acetate/L milliQ water, acidified to pH 4.5 using acetic acid), 2.0 mL 20 % STEB in THF and 20.0 mL hexane was added. The mixture was shaken at 200 rpm for 45 minutes and placed in a water bath at 60 °C for 45 minutes. To approximately 3 mL hexane extract, approximately 3 mL 2 M HCl was added and the mixture was shaken at 200 rpm for 30 minutes. Sample hexane extracts were diluted with blank diet hexane extract to obtain expected concentrations within the calibrated concentration range.
The following dilutions were prepared: 200 mg/kg samples were diluted 13.3 times, 500 mg/kg samples were diluted 40 times and 1250 mg/kg samples were diluted 100 times.

CHROMATOGRAPHY
The concentration of test material in the diluted extracts of the validation samples and the study samples was determined using GC-MS. The GC-MS conditions were as follows:
- Injection: 1 μL, splitless time 1.2 min, PTV (programmable temperature vaporisation) 60 °C x 14.5 °C/min; 300 °C (5 min)
- Carrier gas: Helium
- Column: HP 5MS, 30 m, 0.25 mm ID, 0.25 μm film
- Column temperature: 45 °C (3 min) x 5 °C/min x 80 °C (0 min) x 15 °C/min x 260 °C (15 min)
- Detection: Mass Spectrometry (MS). Quantification: internal standard m/z 277; test material m/z 291. Qualifiers: internal standard m/z 179; test material m/z 179.
- Source temperature: 250 °C
- Interface temperature: 200 °C

CALIBRATION
On each day that the concentration of the test material in the diet was analysed, 2 stock solutions of the test material were prepared by accurately weighing approximately 100 mg into 20 mL volumetric flasks and bringing to volume with methanol. The stock solutions were diluted 50 times with methanol. From the 2 diluted stock solutions 6 calibration samples were prepared by adding 50, 100, 200, 300, 400 and 500 μL from the stock solutions to approximately 2 grams of diet, in an alternating fashion. The calibration samples were extracted and analysed as described previously. Calibration graphs were constructed by plotting the Q value (ratio peak area test material/peak area internal standard) against the concentration of the test material.

CALCULATION
The concentration of the test material in the sample extracts was calculated using the calibration graph. The concentration in the samples was calculated using the following formula:
C = A * D * B
Where:
C = concentration of test material in diet samples (mg/kg)
A = concentration of test material determined in diet sample extracts (expressed as mg/kg)
D = dilution factor for hexane extract
B = correction factor (2 g/weight of sample analysed (g))

DETERMINATION OF HOMOGENEITY AND CONTENT OF TEST MATERIAL IN DIET
- Homogeneity: The homogeneity was assessed in the batch of diets prepared on 17 April 2013. Five samples of each test diet (200, 500 and 1250 mg /kg diet), taken at different locations in the feed container, were analysed.
For each concentration level, a one-way analysis of variance (ANOVA) was performed using the sample location (1 to 5) as grouping factor. An associated F-value with probability p<0.01 was considered to be significant (i.e. the mean concentrations differ significantly at the five locations in the feed container). The test material was considered to be homogeneously distributed in the diet if p≥0.01 and/or if the relative standard deviation (RSD) between the mean concentrations at the five locations was less than or equal to 15 %.
- Content: The content of test material was determined in the batches of diets prepared on 17 April, 14 May and 19 June 2013.
The content of the test material in diet was considered to be “close to intended” if the mean measured concentration was between 80 and 120 % of the intended concentration.

RESULTS AND DISCUSSION
VALIDATION OF THE ANALYTICAL METHOD
- Linearity: All calibration graphs had a correlation coefficient of ≥0.99 and were therefore considered to be linear.
In total 4 calibration curves were recorded, which had correlation coefficients of 0.997 (validation series, 09 April 2013), 0.999 (homogeneity/content 1 series, 17 April 2013), 0.999 (content 2 series, 22 May 2013) and 0.998 (content 3 series, 19 June 2013).
- Specificity: The chromatogram of the extract of blank diet did not show a peak at the position of the test material.
- Recovery: The mean recoveries ranged from 115 to 117 %. All recovery values met the validation criteria set for recovery.
- Repeatability: The relative standard deviations (RSD) in the mean recoveries ranged from 1.0 to 5.5 %, which met the criterion set for the RSD in the percentage recovery.

HOMOGENEITY, STABILITY AND CONTENT OF TEST MATERIAL IN DIET AND QUALITY CONTROL
- Homogeneity: The RSD between the mean concentrations at five different locations was <15 % for all dose levels (200, 500 and 1250 mg/kg diet), i.e. the test material was considered to be homogeneously distributed in the diets.
- Content: The concentration of the test material was close to intended (80 to 120 %) for all diets at all dose levels. Therefore the concentration of the test material was considered to be close to intended at all dose levels.

CONCLUSIONS
The method used for the quantitative analysis of the test material in diet met the pre-set validation criteria. The test material was homogeneously distributed in test diets prepared for all dose levels. The content of the test material in the diet was close to the intended concentration at all dose levels.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Continuous in the diet
Dose / conc.:
0 mg/kg diet
Dose / conc.:
200 mg/kg diet
Dose / conc.:
500 mg/kg diet
Dose / conc.:
1 250 mg/kg diet
No. of animals per sex per dose:
10 animals per sex per dose
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: In a reproduction/developmental toxicity screening assay (OECD 421), statistically significant reductions in absolute and relative thymus weight were observed in maternal rats exposed to 500 and 1250 mg/kg diet of the test material for 6 weeks throughout pre-mating, mating and post-mating.
However, a 13-week study in non-pregnant rats lasts considerably longer and comprises many more toxicological endpoints than a developmental study; it was therefore decided to use the same dietary levels.
Observations and examinations performed and frequency:
CAGE SIDE AND DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Each animal was observed daily in the morning hours by cage-side observations and, if necessary, handled to detect signs of toxicity. The observations included, but were not limited to, the following: general, respiration, mouth, abdomen, faeces, behaviour, skin/fur, head, nose, eyes, ears, penis, perineum, extremities (legs), tail, testes, urethra and urine.
On working days, all cages were checked again in the afternoon for dead or moribund animals to minimise loss of animals from the study. On weekends and public holidays only one check per day was carried out. All abnormalities, signs of ill health or reactions to treatment were recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight of each animal was recorded at initiation of treatment (day 0), and once per week thereafter. In addition, the animals were weighed on their scheduled necropsy date after overnight fasting (terminal body weight to calculate the organ to body weight ratios).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
The intake of test material per kg body weight per day was calculated from the nominal dietary concentration of the test material, the feed consumption and the body weight at the end of the pertaining week.
Feed consumption was measured per cage by weighing the feeders. The consumption was measured over successive weekly periods. The results were expressed in g per animal per day.

FOOD EFFICIENCY: No

WATER CONSUMPTION: Yes
- Time schedule for examinations: Water consumption was measured per cage, by daily weighing of the drinking bottles during 5-day periods in weeks 1, 6 and 12. The results were expressed in g per animal per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations and dose groups examined: Ophthalmoscopic observations were made in all rats in the week prior to the start of treatment and in rats of the control group and high-dose group in the last week of the treatment period (day 90). As no treatment-related ocular changes were observed in the high-dose group, eye examinations were not conducted in animals of the intermediate-dose groups. Eye examination was carried out using an ophthalmoscope after induction of mydriasis by a solution of atropine sulphate.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Haematology was conducted at necropsy (on day 91 for males and on day 92 for females). Blood samples were taken from the abdominal aorta of the rats. EDTA was used as anticoagulant.
- Anaesthetic used for blood collection: Yes; whilst under CO₂/O₂ anaesthesia
- Animals fasted: Yes. The rats were fasted overnight before necropsy (water was freely available).
- How many animals: All animals
- In each sample the following determinations and calculations were carried out: haemoglobin (Hb), packed cell volume (PCV), red blood cell count (RBC), reticulocytes, total white blood cell count (WBC), differential white blood cell counts (lymphocytes (lympho), neutrophils (neutro), eosinophils (eosino), basophils (baso) and monocytes (mono)), prothrombin time (Prothrom Time), thrombocyte count, mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH) and mean corpuscular haemoglobin concentration (MCHC).

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At necropsy. Blood samples were taken from the abdominal aorta; blood was collected in heparinised plastic tubes and plasma was prepared by centrifugation.
- Anaesthetic used for blood collection: Yes; whilst under CO₂/O₂ anaesthesia
- Animals fasted: Yes. The rats were fasted overnight before necropsy (water was freely available).
- How many animals: All animals
- The following measurements were made in the plasma: alkaline phosphatase activity (ALP), aspartate aminotransferase activity (ASAT), alanine aminotransferase activity (ALAT), gamma glutamyl transferase activity (GGT), total protein, albumin, ratio albumin to globulin (calculated), urea, creatinine, (fasting) glucose, bilirubin total (Bilirub Total), cholesterol total, triglycerides, phospholipids, calcium (Ca), sodium (Na), potassium (K), chloride (Cl) and inorganic phosphate (PO₄)

URINALYSIS: Yes
- Time schedule for collection of urine: On day 84 to 85
- Animals fasted: Yes. All rats were deprived of water for 24 hours and of food during the last 16 hours of this period.
- Metabolism cages used for collection of urine: Yes. During the last 16 hours of deprivation, the rats were individually kept in stainless-steel metabolism cages and urine was collected.
- The following determinations were carried out in individual samples: volume, density, appearance, pH, glucose, occult blood, ketones, protein, bilirubin and urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes (arena testing, FOB and motor activity)
In addition to the above daily general clinical observations, detailed clinical examinations (in an arena outside the home cage) were performed on all rats prior to the first exposure and then once weekly throughout the study. Behavioural endpoints (Functional Observation Battery and motor activity assessment) were investigated in all rats in week 13.
> Arena testing
Signs noted included but were not limited to: changes in skin and fur, piloerection, changes in the eyes, gait (including posture), and presence of clonic or tonic movements, stereotypies and bizarre behaviour.
> FOB
The measures included according to functional domain are as follows:
- Autonomic: lacrimation, salivation, pupil response to light, palpebral closure, piloerection, defaecation and urination.
- Neuromuscular: gait, mobility, forelimb and hindlimb gripstrength, landing foot splay and righting reflex.
- Sensorimotor: response to tail pinch, click, touch and approach of a visual object.
- Convulsive: clonic and tonic movements.
- Excitability: ease of removal, handling reactivity, arousal and vocalisations.
- Activity: rearing, posture and motor activity.
- Physiological: body temperature.
> Motor activity
Motor activity was assessed following FOB testing. Changes in spontaneous motor activity were assessed using an automated quantitative microprocessor-based video image analysis system. In addition, habituation of activity was evaluated.
Sacrifice and pathology:
NECROPSY
At the end of the treatment period, the animals were killed on two consecutive days (males on day 91 and females on day 92). The rats were killed in such a sequence that the average time of killing was approximately the same for each group. Prior to sacrifice, the rats were fasted overnight but had free access to water. The animals were killed by exsanguination from the abdominal aorta under deep irreversible CO₂/O₂ anaesthesia and then examined macroscopically for pathological changes.

ORGAN WEIGHTS
At scheduled sacrifice, the following organs were weighed (paired organs together) as soon as possible after dissection to avoid drying, and the relative organ weights were calculated from the absolute organ weights and the terminal body weight: adrenals, brain, epididymides, heart, kidneys, liver , ovaries, prostate, seminal vesicles + coagulating glands, spleen, testes, thymus and uterus.

TISSUE PRESERVATION
Samples of the following tissues and organs of all animals were preserved in a neutral aqueous phosphate-buffered 4 per cent solution of formaldehyde:
adrenals, aorta, axillary lymph nodes, bone marrow (sternum), brain (brainstem, cerebrum, cerebellum), caecum, colon, duodenum, epididymides, exorbital lachrymal glands*, eyes, femur with joint*, heart, ileum, jejunum, kidneys, liver, lungs, mammary gland (females), mandibular (cervical) lymph nodes*, mesenteric lymph nodes, nerve-peripheral (sciatic), ovaries, oviducts (fallopian tubes), oesophagus, pancreas, parathyroid, parotid salivary glands, patches of Peyer (gut associated lymphoid tissue), pituitary, prostate, rectum, seminal vesicles + coagulating glands, skeletal muscle (thigh), skin (flank), spinal cord (cervical, mid-thoracic and lumbar), spleen, stomach (forestomach, fundus and pylorus), sublingual salivary glands, submaxillary salivary glands, testes, thymus, thyroid, trachea/bronchi, urinary bladder, uterus (with cervix), vagina and all gross lesions.
* These tissues were preserved but not processed for histopathological examination.
The carcass containing any remaining tissues was retained in formalin until completion of the histopathological examination and then discarded.

MICROSCOPIC EXAMINATION
The tissues to be examined microscopically were embedded in paraffin wax, sectioned at 5 μm and stained with haematoxylin and eosin.
Histopathological examination (by light microscopy) was performed on all tissues and organs listed above (except those marked with an asterisk) of all animals of the control group and the high-dose group. As no treatment-related microscopic changes were observed in the high-dose group, microscopic examination was not extended to animals of the intermediate-dose groups. Gross lesions were examined in rats of all dose groups.
Statistics:
- Post-Treatment Body Weight Data: ‘AnCova & Dunnett’s Test’ with ‘Automatic’ as data transformation method. Day 0 body weight data are used as covariate (unless removed during data pre-processing).
- Pre-Treatment Body Weight Data, Clinical Pathology (Haematology, Clinical Chemistry, Urinary Volume and Density) and Organ Weight Data: ‘Generalised Anova/Ancova Test’ with ‘Automatic’ as data transformation method.
- Feed/ Water Consumption: Dunnett’s multiple comparison test.
- Semi-quantitative urinalysis results: ‘Kruskal-Wallis & Dunnett Test’ with ‘Rank’ as data transformation method.
- Functional Observational Battery: one-way analysis of variance followed by Dunnett’s multiple comparison tests (continuous data), Kruskal-Wallis non-parametric analysis of variance followed by multiple comparison tests (rank order data) or Pearson chi-square analysis (categorical data).
- Motor Activity Data: total distance moved: one-way analysis of variance followed by Dunnett’s multiple comparison tests; habituation of activity: repeated measures analysis of variance on time blocks.
- Incidences of Histopathological Changes: Fisher’s exact probability test.

Tests are performed as two-sided tests with results taken as significant where the probability of the results is <0.05 or <0.01.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no treatment-related clinical signs.
Mortality:
no mortality observed
Description (incidence):
None of the animals died during the treatment period.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were no treatment related differences in body weights.
In males of all treatment groups, mean body weights were somewhat lower than in controls, but the differences were only small (maximally 5 to 6 %) and not statistically significant, apart from an incidental significancy (p<0.05) in the low-dose group on day 21 (mean: 298 g in controls versus 288 g in the low-dose group).
In high-dose females, an incidental increase (mean: 168 g in controls to 171 g in high dose) reached the level of statistical significance by analysis of covariance on day 7 of the study (p<0.01), but mean body weights were comparable in females at all other stages.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
Feed consumption was not affected by the administration of the test material.
Due to the decreased feed intake per kg body weight with increasing age of the rats, the intake of test material per kg body weight gradually decreased in all groups. The overall mean intake of the test material in the low-, mid- and high-dose group was respectively 13, 32 and 82 mg/kg body weight/day for males, and 14, 37 and 91 mg/kg body weight/day for females.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
There were some fluctuations in water intake but there were no treatment-related differences in water consumption.
In males of the mid-dose group, water intake was consistently lower than in controls throughout the study (overall means in week 1, 6 and 12 were 31, 37 and 38 g/rat/day in controls and 26, 29 and 29 g/rat/day in the mid-dose, respectively), the differences being statistically significant at several occasions (p<0.05). In males of the low- and high-dose group, water intake was statistically significantly lower than in controls on day 2 only. As there was no dose-response relationship, the fluctuations in water consumption were not ascribed to the treatment.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
Ophthalmoscopic examination of the rats at the end of the study did not reveal any treatment-related changes.
Haematological findings:
no effects observed
Description (incidence and severity):
There were no statistically significant changes in red blood cell and clotting variables, apart from an incidental decrease in MCH in females of the mid-dose group.
In males of the mid- and high-dose group, total white blood cell counts were statistically significantly (p<0.05) lower than in controls. The values were well within the range of historical control data. Moreover, there was no clear dose-response relationship and similar findings were not noted in females.
Therefore this finding is considered a chance finding. There were no statistically significant changes in differential white blood cell counts in either sex.
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in clinical chemistry variables.
Endocrine findings:
not examined
Urinalysis findings:
no effects observed
Description (incidence and severity):
The renal concentration test did not reveal statistically significant differences in urinary volume and density. There were no treatment-related differences in urinary appearance or in semi-quantitative urinary observations
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
The results of the neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test material in rats.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
There were no statistically significant differences in absolute organ weights or in organ-to-body weight ratios.
Gross pathological findings:
no effects observed
Description (incidence and severity):
Macroscopic observations at necropsy did not reveal treatment-related abnormalities.
All macroscopic observations occurred only incidentally or at comparable incidence between test groups and controls, and represent normal background pathology of rats of this strain and age.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
Microscopic examination revealed no treatment-related abnormalities. The histopathological findings were considered unremarkable and part of the background pathology of rats of this strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Dose descriptor:
NOAEL
Effect level:
82 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: The NOAEL was placed at the highest level tested, 1250 mg/kg diet, which was equivalent to an overall mean intake of 82 mg/kg body weight/day for males.
Dose descriptor:
NOAEL
Effect level:
91 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: The NOAEL was placed at the highest level tested, 1250 mg/kg diet, which was equivalent to an overall mean intake of 91 mg/kg body weight/day for females.
Critical effects observed:
not specified

Table 1: Summary of Mean Body Weight Data

Dose Group (ppm)

Sex

Body Weight (g) on Day

-1

0

7

14

21

28

35

42

49

56

63

70

77

84

90

0

M

175.45

182.36

227.13

267.33

297.68

323.51

342.38

360.46

373.64

385.83

396.23

407.87

413.50

419.60

421.94

F

144.73

146.81

168.16

184.92

200.60¹

208.78

218.40

225.91

233.75

236.09

239.11

242.90

246.72

245.04

246.73

200

M

177.01

184.34

227.73

264.62

288.16

311.75

332.73

347.23

358.27

368.48

378.85

390.81

398.10

404.32

409.82

F

144.92

146.29

170.39

185.47

199.53

207.00

219.31

224.37

232.20

236.39

239.14

243.12

245.27

243.49

245.39

500

M

176.18

182.24

227.54

264.03

289.14

314.84

333.89

350.48

363.74

374.99

384.10

395.63

401.37

406.49

410.65

F

144.57

145.41

167.92

184.86

195.69

205.53

214.28

221.45

230.55

233.88

238.49

241.80

244.71

242.05

245.59

1250

M

175.55

180.44

223.71

259.89

285.03

307.46

326.28

341.18

353.08

364.04

373.49

382.40

391.16

398.47

402.64

F

144.27

144.59

171.36²

183.93

196.45

206.16

217.18

224.50

231.30

233.37

240.17

243.06

243.02

244.37

247.04

¹ Dunnett 2 sided p <0.05

² Dunnet 2 sided p <0.01

 

Table 2: Mean Red Blood Cell and Coagulation Parameters

 Dose Group (ppm)

Sex

RBC (10E12/L)

Hb (mmol/L)

PCV (L/L)

MCV (fL)

MCH (fmol)

MCHC (mmol/L)

Reticulocytes (%)

Thrombocytes (10E9/L)

Prothrom Time (s)

0

M

9.400

10.32

0.5086

54.11

1.098

20.29

2.042

1037.3

41.83

F

8.701

10.07

0.4857

55.81

1.157¹

20.73

2.279

878.5

33.63

200

M

9.510

10.27

0.5102

53.72

1.081

20.13

2.162

1018.6

40.68

F

8.805

10.03

0.4831

54.92

1.141

20.77

2.474

991.3

33.64

500

M

9.491

10.57

0.5158

54.38

1.113

20.48

1.890

941.7

41.05

F

9.163

10.29

0.5002

54.62

1.125²

20.59

2.445

970.7

33.80

1250

M

9.608

10.38

0.5145

53.55

1.080

20.17

1.940

1097.1

41.80

F

8.690

10.04

0.4825

55.51

1.155

20.81

2.580

1031.5

34.38

¹ Anova p <0.05

² Dunnett 2 sided p < 0.05

 

Table 3: Total and Differential White Blood Cell Counts

 Dose Group (ppm)

Sex

WBC (10E9/L)

Lympho Absolute (10E9L)

Neutro Absolute (10E9/L)

Eosino Absolute (10E9/L)

Baso Absolute (10E9/L)

Mono Absolute (10E9/L)

Lymphocytes (%)

Neutrophils (%)

Eosinophils (%)

Basophils (%)

Monocytes (%)

0

M

7.03

5.61

1.16

0.075

0.015

0.173

79.32

17.03

1.07

0.20

2.38

F

3.89

3.20

0.52

0.053

0.007

0.107

81.70

13.99

1.38

0.19

2.73

200

M

6.01

4.71

1.09

0.073

0.016

0.122

78.85

17.71

1.17

0.27

1.99

F

4.08

3.35

0.56

0.051

0.008

0.113

81.95

13.95

1.29

0.18

2.62

500

M

5.13¹

4.03

0.92

0.061

0.008

0.115

79.18

17.36

1.19

0.14

2.14

F

3.57

2.77

0.64

0.042

0.006

0.104

77.77

18.08

1.13

0.16

2.85

1250

M

5.24¹

4.23

0.84

0.047

0.009

0.113

80.99

15.96

0.87

0.16

2.06

F

4.18

3.38

0.63

0.050

0.010

0.117

80.35

15.50

1.17

0.22

2.77

¹ Dunnett 2 sided p < 0.05

Conclusions:
Under the conditions of this study the no-observed-adverse-effect level (NOAEL) was placed at the highest level tested, namely 1250 mg/kg diet (≥82 and 91 mg/kg body weight/day for males and females, respectively).
Executive summary:

A sub-chronic study was conducted in order to investigate the toxicity potential of the test material in the Wistar rat. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 408 and EU Method B.26.

The test material was administered at constant concentrations in the diet at levels of 0 (control), 200, 500 and 1250 ppm for a period of 13 weeks to 10 rats per sex per dose group. These dietary levels provided an overall mean intake of 13, 32 and 82 mg/kg body weight/day for males, and 14, 37 and 91 mg/kg body weight/day for females.

Observations and measurements to detect possible adverse effects included daily clinical observations, neurobehavioural testing, ophthalmoscopic examination, growth, feed and water intake, haematology, clinical chemistry, urinalysis, organ weights and macroscopic and microscopic examination of organs and tissues.

The administration of the test material was well tolerated at all dose levels and did not induce any treatment-related changes in any of the variables investigated.

None of the animals died during the study and there were no treatment-related clinical signs. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test material. Ophthalmoscopic examination did not show any treatment-related ocular changes. There were no relevant changes in body weight, feed intake or water intake. Haematology did not reveal any treatment-related changes in red blood cell variables or clotting potential. There were no effects on differential white blood cell counts (a decrease in total white blood cell counts in males of the mid- and high-dose group was not ascribed to treatment). Clinical chemistry did not reveal any differences in clinical chemistry variables. Urinalysis did not reveal any treatment-related changes in renal concentrating ability, or in in semi-quantitative urinary measurements.

There were no treatment-related differences in absolute and relative organ weights and macroscopic examination at necropsy and microscopic examination of organs and tissues did not reveal treatment-related findings.

Therefore, under the conditions of this study the no-observed-adverse-effect level (NOAEL) was placed at the highest level tested, namely 1250 mg/kg diet (≥82 and 91 mg/kg body weight/day for males and females, respectively).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
82 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The key study is conducted under GLP conditions and in accordance with standardised guidelines. Two of the available supporting studies are also conducted under GLP conditions. The quality of the database is therefore considered to be high.

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

A sub-chronic study was conducted in order to investigate the toxicity potential of the test material in the Wistar rat. The study was conducted under GLP conditions and in accordance with the standardised guidelines OECD 408 and EU Method B.26.

Rationale for dose-level selection:

The rationale for dose-level selection was based on effects obtained with related organotins coumpounds. In particular, as it is mentioned in the study report, the results of the 90 days oral toxicity study (TNO report V 3963, OECD 408) with MOTC (monoctyltin trichloride), a potential gastric hydrolysis product , were taken in account for dose-level selection. In this study, treatment-related changes were observed at 500 ppm in the diet (increased ALP levels, decreased thymus weight, and lymphoid depletion of the thymus). The NOAEL for MOTC was 100 ppm (6.4 and 6.8 MOTC/kg/day). Assuming that MOTE is rapidely converted in the stomach to MOTC and taking into account the molecular weight of MOTE (841.9 comprising 27.5% monooctyltin) and MOTC (338.3 comprising 68.5% monoctyltin), MOTE could be expected to be 2.5 times less toxic compared to MOTC, based on bioavailability. This consideration justified the selection of 1250 ppm MOTE in the diet (2.5 x 500 ppm for MOTC) as the high dose-level in the current study to induce more toxicity. Furthermore we also considered for the selection of the dose-levels previous studies with MOTE (2-dose-range finding studies and an OECD 421) exposed in shorter duration (2 or 6 weeks) where statistically significant changes were observed in the thymus weight of lactating females at 500 and 1250 ppm. Finally, the highest dose-level of 1250 ppm was selected for this 13 weeks study based on data on the hydrolysis compound in a comparable 13 weeks study and supporting data exposed to shorter duration to MOTE (OECD 421).

In conclusion, the dose-levels were selected in full compliance with the test guidelines "taking into account any existing toxicological and toxickinetic data available for the test compound or related materials".

The test material was administered at constant concentrations in the diet at levels of 0 (control), 200, 500 and 1250 ppm for a period of 13 weeks to 10 rats per sex per dose group. These dietary levels provided an overall mean intake of 13, 32 and 82 mg/kg body weight/day for males, and 14, 37 and 91 mg/kg body weight/day for females.

Observations and measurements to detect possible adverse effects included daily clinical observations, neurobehavioural testing, ophthalmoscopic examination, growth, feed and water intake, haematology, clinical chemistry, urinalysis, organ weights and macroscopic and microscopic examination of organs and tissues.

The administration of the test material was well tolerated at all dose levels and did not induce any treatment-related changes in any of the variables investigated.

None of the animals died during the study and there were no treatment-related clinical signs. Neurobehavioural observations and motor activity assessment did not indicate any neurotoxic potential of the test material. Ophthalmoscopic examination did not show any treatment-related ocular changes. There were no relevant changes in body weight, feed intake or water intake. Haematology did not reveal any treatment-related changes in red blood cell variables or clotting potential. There were no effects on differential white blood cell counts (a decrease in total white blood cell counts in males of the mid- and high-dose group was not ascribed to treatment). Clinical chemistry did not reveal any differences in clinical chemistry variables. Urinalysis did not reveal any treatment-related changes in renal concentrating ability, or in in semi-quantitative urinary measurements.

There were no treatment-related differences in absolute and relative organ weights and macroscopic examination at necropsy and microscopic examination of organs and tissues did not reveal treatment-related findings.

Therefore, under the conditions of this study the no-observed-adverse-effect level (NOAEL) was placed at the highest level tested, namely 1250 mg/kg diet (≥82 and 91 mg/kg body weight/day for males and females, respectively).

The NOAEL obtained with MOTE in the 13 weeks repeated dose study was set at 1250 ppm (82-91 mg/kg/day), this means that according to CLP criteria, MOTE is not classified for STOT RE exposure as no observed effects were observed at dose higher than >10 and lower <100 mg/kg/day. Increasing the dose-levels will not add more information for Human Health harmonized classification.

Supporting Information

There are two weeks range-finding rat studies available, performed in order to select the dose-levels of a GLP screening reprotox study (OECD 421). There is also the repeated exposure data obtained in the screening reprotox study (Waalkens and Berendsen, 2009) which has a duration of exposure of 28 days in the males and at least 6 weeks exposure in the females. Groups of 12 rats per sex were dosed via the diet with 0, 200, 500 and 1250 mg/kg diet. This study provides information on parental toxicity, in particular on body weight, food consumption, organ weight and microscopic examination of the organs sampled.

Mortality and clinical signs were checked daily. Body weight and food consumption were recorded at designated intervals at least weekly.

No mortalities and no remarkable clinical signs were observed in the male and female animals. Mean body weights of male and female animals were comparable between the control and the treated groups during the pre-mating and mating period of the males and during the pre-mating, mating period, gestation and lactation periods of the females. Mean food consumption of the male and female animals was comparable between the control and the treated groups during the entire study.

Mean absolute and relative organ weights (testes, epididymides and thymus) of the male animals were comparable in all groups.

In pregnant females, no effect was observed in the 200 mg/kg diet group while a statistically significant decrease of absolute and relative thymus weight was observed in the 500 and 1250 mg/kg diet groups. This was considered not to be of significance on the basis that the values found for the control group were quite high and outside the historical control range, the absolute and relative thymus weights of the 500 and 1250 mg/kg diet groups were not statistically significantly decreased compared to the 200 mg/kg diet group, and at microscopic examination no effects were observed in the thymus of the pregnant female animals of the 1250 mg/kg diet group.

Macroscopic examination at necropsy did not reveal treatment-related findings. Microscopic examination of the sampled organs of the control and high-dose groups at the end of the main study did not reveal treatment-related histopathological changes in the 1250 mg/kg group.

Under the conditions of this study, the oral administration at 200, 500 and 1250 mg/kg diet was well tolerated at all dose-levels.

The no adverse effect level (NOAEL) for parental toxicity was 1250 mg/kg diet [approx. 72 and 96 mg/kg body weight/day for male and female animals, respectively].


Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
The study was conducted to the appropriate guidelines under GLP conditions. It encompasses the most appropriate period of test material administration.

Justification for classification or non-classification

The NOAEL obtained with MOTE in the 13 weeks repeated dose study was set at 1250 ppm (82-91 mg/kg/day), this means that according to CLP criteria, MOTE is not classified for STOT RE exposure as no observed effects were observed at dose higher than >10 and lower <100 mg/kg/day. Increasing the dose-levels will not add more information for Human Health harmonized classification.

In accordance with the criteria for classification as defined in Annex I, Regulation (EC) No. 1272/2008, the substance does not require classification with respect to repeated dose toxicity.

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