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Diss Factsheets

Administrative data

Description of key information

Oral NOAEL: 1000 mg/kg bw/day (subacute; rat)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From September 06th to December 28th, 2005
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
Study conducted according to internationally accepted testing guidelines and performed in compliance with Good Laboratory Practice. Justification for read across approach is given in the endpoint summary and in the read across justification report attached to the Section 13 of this dossier.
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
July 27, 1995
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Version / remarks:
September 30, 1996
Qualifier:
according to guideline
Guideline:
other: Japanese Guidelines for Screening, Toxicity Testing of Chemicals: Testing Methods for new Substances, enacted July 13, 1974, amended December 5, 1986
GLP compliance:
yes (incl. QA statement)
Species:
rat
Strain:
other: WIST (SPF)
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: RCC Ltd, Laboratory Animal Services CH-4414 Filllinsdorfl Switzerland.
- Age at study initiation: 6 weeks.
- Weight at study initiation: males mean value 138.2 g (130.0 - 145.5); females mean value 1213 g (19.4 - 130.9 g).
- Assigned to test groups randomly: yes.
- Housing: in groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding ('LignoceI' Schill AG, CH-4132 MuttenzISwitzerIand).
- Diet: pelleted standard Provimi Kliba 3433 rat maintenance diet (Provimi Kliba AG, CH-4303 Kaiseraugsti Switzerland) was available ad Iibitum.
- Water: community tap-water from ltingen was available ad libitum in water bottles.
- Acclimation period: 7 days, under test conditions after health examination. Only animals without any visible signs of illness were used for the study.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C.
- Humidity: 30 - 70 %.
- Air changes: 10-15 air changes per hour.
- Photoperiod: art12-hour fluorescent light/12-hour dark cycle.
- Other: music during the light period.
Route of administration:
oral: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet: fresh batches of the feed pellets for the study were prepared weekly.
- Preparation: the test item was weighed into a tared glass beaker on a suitable precision balance and mixed with microgranulated feed. Water (ca 1:10 volume/weight/ratio) was added to aid pelleting. The pellets were dried with air for ca. 48 hours before storage.
- Storage temperature of food:feed was stored at room temperature (ca. 20 °C) in disposable paper bags.
- Control: control feed for the animals of group 1 was prepared similarly without the test item.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Concentration, homogeneity and stability (after 10 days) of the test item in the feed were determined in samples taken before initial feeding. Analyses were performed by the Principal Investigator of the analytical phase, according to a HPLC analytical method supplied by the Sponsor and previously adapted at the testing laboratory. The feed formulations were delivered under ambient conditions.
Duration of treatment / exposure:
28 days
Remarks:
Doses / Concentrations:
0, 50, 200 and 1000 mg/kg/day
Basis:
nominal in diet
No. of animals per sex per dose:
Groups 1 and 4: 10 males; 10 females
Groups 2 and 3: 5 males; 5 females
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: based upon the results of a non-GLP 7-day dose-range-finding and palatability study, in which the test item was administered by feeding to 3 rats per group and sex.
- Post-exposure recovery period: 14 days.
Observations and examinations performed and frequency:
MORTALITY/VIABILITY
Observations for mortality/viability were recorded twice daily.

GENERAL CAGESIDE OBSERVATIONS (WEEKLY)
The animals were observed for clinical signs once daily before commencement of administration; twice daily on days 1-3; as well as once daily on days 4-28, and once daily during days 29-42 (recovery).

DETAILED CLINICAL OBSERVATIONS
The animals were observed in their home cages, outside their home cages in a standard arena and in the hand. These observations were performed in random sequence once before commencement of administration and once weekly (weeks 1-3) thereafter.

FOOD CONSUMPTION
The food consumption was recorded once during the acclimatization period and weekly thereafter, using an on-line electronic recording system consisting of a Mettler balance connected to the testing laboratory computer.

BODY WEIGHTS
Body weights were recorded weekly during acclimatization, treatment and recovery and before necropsy, using an on—Iine electronic recording system consisting of a Mettler balance connected to the testing laboratory computer.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, relevant parameters (presented in Appendix l) from a modified Iwvin screen test were evaluated in all animals.
The results of the Functional Observational Battery are presented in the summary and individual tables of the Detailed Clinical Observations (Weekly) under week 4. This method of data presentation permits a clear evaluation and assessment of weekly clinical signs observed during the study.

GRIP STRENGTH
Forelimb and hind limb grip strength measurements were performed using a push-pull strain gauge (Mecmesin, AFG 25N). The animals were placed with the forepaws inside a triangular grasping ring and with the hind paws outside a triangular grasping ring. Using one hand, the animals were held towards the base of the tail and steadily pulled away or towards the ring until the grip was broken. Each measurement was repeated three times, the means were calculated and recorded.

LOCOMOTOR ACTIVITY
Locomotor (decreased or increased) activity was measured quantitatively with AMS Fohr Medical Instruments GmbH (FMI) and DeMeTec GmbH Activity Monitor System. Animals were monitored during the fourth treatment week for a 60-minute period and the total activity of this time period was recorded.
Low beams count was reported in 10-minute intervals as well as the total activity of the measuring period.

HAEMATOLOGY
Blood sampling: after 4 weeks 17 October 2005 (Allocation A and B); after 6 weeks 31 October 2005 (Allocation B)
Blood samples for hematology were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
The following hematology parameters were determined: Erythrocyte count, Hemoglobin, Hematocrit, Mean corpuscular volume, Red cell volume distribution width, Mean corpuscular hemoglobin, Mean corpuscular hemoglobin concentration, Hemoglobin concentration distribution width, Platelet (thrombocyte) count, Reticulocyte count, Reticulocyte maturity index, Methemoglobin, Total leukocyte count, Differential leukocyte count, Coagulation, Thromboplastin time, Activated partial thromboplastin time.

CLINICAL CHEMISTRY
Blood sampling: after 4 weeks 17 October 2005 (Allocation A and B); after 6 weeks 31 October 2005 (Allocation B)
Blood samples for clinical biochemistry were collected from all animals under light isoflurane anesthesia. The animals were fasted in metabolism cages for approximately 18 hours before blood sampling but allowed access to water ad libitum. Blood samples were collected early in the working day to reduce biological variation caused by circadian rhythms. Blood samples were drawn from the retro-orbital plexus using a micro-hematocrit glass capillary tube.
Urine was collected during the 18-hour fasting period into a specimen vial.
The following clinical biochemistry parameters were determined: Glucose, Urea, Creatinine, Bilirubin total, Cholesterol total, Triglycerides, Phospholipids, Aspartate aminotransferase, Alanine aminotransferase, Lactate dehydrogenase, Glutamate dehydrogenase, Creatine kinase, Alkaline phosphatase,
Gamma-glutamyl-transferase, Sodium, Potassium, Chloride, Calcium, Phosphorus inorganic, Protein total, Albumin, Globulin, Albumin/Globulin ratio.

URINALYSIS
Urine sampling: after 4 weeks 17 October 2005 (Allocation A and B); after 6 weeks 31 October 2005 (Allocation B)
Urine was collected during the 18-hour fasting period into a specimen vial.
The following urinalysis parameters were determined: Volume (18 hours), Specihc gravity (relative density), Colour, Appearance, pH, Nitrite, Protein, Glucose, Ketones, Urobilinogen, Bilirubin, Erythrocytes, Leukocytes.

Sacrifice and pathology:
NECROPSY
Sacrifice: after 4 weeks 17 October 2005 (Allocation A); after 6 weeks 31 October 2005 (Allocation B).
All animals were weighed and neoropsied. Descriptions of all macroscopic abnormalities were recorded. All animals surviving to scheduled necropsy and any moribund animal were anesthetized by intraperitoneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and organs were collected from all animals at necropsy and fixed in neutral phosphate buffered 4 % formaldehyde solution (unless otherwise indicated): Adrenal glands, Aorta, Bone (sternum, femur including joint), Bone marrow (femur), Brain (4 levels), Cecum, Colon, Duodenum, Epididymides (fixed in Bouin's solution), Esophagus, Eyes with optic nerve (fixed in Davidson's solution), Harderian gland (fixed in Davidson's solution), Heart, lleum, with Peyer's patches, Jejunum with Peyer's patches, Kidneys, Larynx, Lacrimal gland (exorbital), Liver, Lungs (infused with formalin at necropsy), Lymph nodes (mesenteric, mandibular), Mammary gland area, Nasal cavity, Ovaries, Pancreas, Pituitary gland, Prostate gland (incl coagulating gland), Rectum, Salivary glands (mandibular, sublingual), Sciatic nerve, Seminal vesicles, Skeletal muscle, Skin, Spinal cord (cervical, midthoracic, lumbar), Spleen, Stomach, Testes (fixed in Bouin's solution), Thymus, Thyroid (incl. parathyroid gland), Tongue, Trachea, Urinary bladder (infused with formalin at necropsy), Uterus, Vagina, Gross lesions.

ABSOLUTE AND RELATIVE GROSS WEIGHT
The organ to terminal body weight ratios as well as organ to brain weight ratios were determined,
The determination of the terminal body weight was performed immediately prior to necropsy.
The following organ weights were recorded on the scheduled dates of necropsy: Brain, Head, Liver, Thymus, Kidneys, Adrenals, Spleen, Testes, Epididymides, Ovaries.

HISTOTECHNIQUE
All organ and tissue samples, as defined under Histopathology (following), were processed, embedded and cut at an approximate thickness of 2 to 4 micrometers, and stained with hematoxylin and eosin.

HISTOPATHOLOGY
Slides of all organs and tissues listed in boldface type (see Necropsy, above) that were collected at scheduled sacrifice from the animals of control, middle and high-dose groups were examined by a pathologist.
Organ and tissue samples taken from the animal which was killed in extremis were evaluated similarly to those organs taken from animals ofthe high~dose group.


Statistics:
The following statistical methods were used to analyze the grip strength, Iocomotor activity, body weight, clinical laboratory data, organ weights and ratios, as well as:
- The Dunnett-test (many to one t-test) based on a pooled variance estimate were applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-one rank test) were applied instead of the Dunnett-test when the data can not be assumed to follow a normal distribution.
- Fisher's exact-test were applied to the macroscopic findings.

References :
C.W. Dunnett: A Multiple Comparison Procedure for Comparing Several Treatments with a Control, J. Amer. Stat. Assoc. 50, 1096-1121 (1955).
S.C. Gad and C.S. Weil: Statistics and Experimental Design for Toxicologists. The Telford Press,Caldwell, New Jersey, 43-45 (1986).
Clinical signs:
no effects observed
Description (incidence and severity):
not significantly affected
Mortality:
no mortality observed
Description (incidence):
not significantly affected
Body weight and weight changes:
no effects observed
Description (incidence and severity):
no test item·related differences of toxicological relevance were noted at any dose level
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
differences with control not considered to be of toxicological relevance
Haematological findings:
no effects observed
Description (incidence and severity):
differences within the ranges of the historical control data
Clinical biochemistry findings:
no effects observed
Description (incidence and severity):
differences within the ranges of the historical control data
Urinalysis findings:
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Description (incidence and severity):
no significant differences with control
Gross pathological findings:
no effects observed
Description (incidence and severity):
no test item-related gross lesions were observed
Histopathological findings: non-neoplastic:
no effects observed
Description (incidence and severity):
no toxicological significance
Details on results:
VIABILITY/MORTALITY
In female n. 55 (1000 mg/kg/day), hunched posture, ruffled fur, pale skin colour and an axillary wound were noted on treatment day 4. It was decided to remove this animal for ethical reasons on day 4 of treatment. It was not considered to be test item related. All other rats survived until scheduled necrcpsy.

GENERAL CAGESIDE OBSERVATIONS (DAILY)
Slight ruffled fur was noted in male nos. 18 and 20 (200 mg/kg/day) and in males 26-30 (1000 mg/kg/day) on treatment day 8. Further clinical signs noted in animal 29 included lethargic behavior, hunched posture and ruffled fur on treatment day 18 (ruffled fur was also seen on treatment clays 19-23 and 25).
In female no 55 (1000 mg/kg/day), hunched posture, ruffled fur, pale skin color and an axillary wound was noted on treatment day 4.

DETAILED CLINICAL OBSERVATIONS (WEEKLY)
During week 1, piloerection, hunched posture, pale discoloration and a wound were noted in a single female treated with 1000 mg/kg/day. During week 3, piloerection, hunched posture and somnolence were noted in a single male treated with 1000 mg/kg/day.
All other animals were without clinical findings.

FUNCTIONAL OBSERVATIONAL BATTERY
During week 4, piloerection was noted in a single male treated with 1000 mg/kg/day. This finding was considered to be incidental.
All other animals were without clinical findings.

GRIP STRENGTH
The mean hind limb grip strength of the females treated with 200 mg/kg/day was significantly increased (p<0.05) when compared with the controls. As no difference was noted in females treated with 1000 mg/kg/day, these differences were considered to be incidental.

LOCOMOTOR ACTIVITY
At 1000 mg/kg/day, elevated mean Iocomotor activity was noted in males from 0-10 minutes (p<0.05) and 10-20 minutes (p<0.05), and in females at 0-10 minutes (p<0.01), 10-20 minutes (p<0.01), 20-30 minutes (p<0.01) and 0-50 minutes (overall, p<0.01).
At 200 mg/kg/day, elevated mean locomotor activity was noted in males from 20-30 minutes (p<0.05) only. At 50 mg/kg/day, the Iocomotor activity was reduced in males from 0-10 minutes (p<0.05) and elevated in females from 10-20 minutes (p<0.01), 20-30 minutes (p<0.01), 30-40 minutes (p<0.01) and 0-60 minutes (overall, p<0.01). These differences were not clearly dose-related and therefore considered to be incidental.

FOOD CONSUMPTION
The mean daily food consumption of the male and female rats treated with 1000 mg/kg/day exceeded that of the respective controls throughout the treatment period, varying from +1 % to +10 % higher in males and +8 % to +18 % in females. These differences were not considered to be of toxicological relevance.
During the recovery period, the mean daily food consumption of the rats previously treated with 1000 mg/kg/day was considered to be similar to those of the controls. The relative food consumption values generally followed the same pattern.

BODY WEIGHTS
No test item·related differences of toxicological relevance were noted at any dose level.
During the treatment period, marginally lower mean body weights were noted in males treated with the test item. The differences were not dose-related and were not considered to be sufficient to represent an effect of the test item administration. The mean body weights of the females were unaffected at all dose levels.
The mean body weight gain of the males treated with 1000 mg/kg/day was slightly lower than that of the control males, whereas males treated with 50 mg/kg/day or 200 mg/kg/day gained approximately the same weight as the control males. The mean body weight gain values of the females were unaffected at all dose levels.
During the recovery period, the mean body weights of the males previously treated with 1000 mg/kg/day remained below those of the control males, whereas the mean body weights of the females previously treated with 1000 mg/kg/day exceeded those of the control females.
The mean body weight gain of both sexes previously treated with 1000 mg/kg/day compared favorably with those of the respective controls.

HEMATOLOGY
After 4 Weeks
In males, no differences of toxicological relevance were noted in the hematology findings at any dose level, although marginal (but statistically significant) reductions were noted in 1000 mg/kg/day (p<0.05). These differences remained within the tolerance limits of the historical control data. In females, the reticulocyte maturity indices were shifted towards the high fluorescence reticulocytes from low fluorescence reticulocytes (p<0.05 for all parameters), although the absolute and relative reticulocyte counts were largely unaffected. The absolute monocyte count (p<0.05) and the mean relative "large unstained cells" (p<0.05) of females treated with 1000 mg/kg/day were elevated when compared with controls. All these differences remained within the ranges of the historical control data.
Only the mean absolute "Iarge unstained ceIl" count noted in females treated with 1000 mg/kg/day (p<0.01) exceeded that of the control females and was identical with the upper range ofthe historical control data.

After 6 Weeks
Although a small number of significant differences persisted in the parameters of males after the recovery period (reduced mean corpuscular volume, p<0.01, increased low fluorescence reticulocytes and decreased high fluorescence reticulocytes (both p<0.05), reduced white blood cells (p<0.05), reduced absolute mean lymphocytes (p<0.05), reduced monocytes (p<0.01), and reduced "large unstained cells" (p<0.05)), all were within the ranges of the historical control data and considered to be test item unrelated.

CLINICAL BIOCHEMISTRY
After 4 Weeks
In males, reduced glucose levels (p<0.05) and elevated sodium levels (p<0.01) were noted at 1000 mg/kg/day. At 200 mg/kg/day, elevated chloride (p<0.05) and decreased albumin (p<0.05) were recorded and at 50 mg/kg/day, males showed elevated potassium (p<0.05) and reduced albumin (p<0.01) when compared with controls. All differences remained within the historical control data.
ln females, reduced cholesterol and phospholipid levels (both p<0.05), increased mean glutamate dehydrogenase (p<0.05) were noted at 1000 mg/kg/day, as well as increased sodium (p<0.01), potassium (p<0.05), calcium (p<0.05) and phosphorus (p<0.01) levels. At 200 mg/kg/day, only increased sodium (p<0.01) and phosphorus (p<0.05) were noted, whereas 50 mg/kg/day showed only elevated sodium (p<0.01) when compared with the controls. All differences remained within the historical control data.

After 6 Weeks
ln males, no differences were ascertained after the recovery period. In females, increased glucose levels (p<0.05) were noted, as were reduced lactate dehydrogenase (p<0.05) and decreased creatine kinase (p<0.05), when compared with the control values. All differences remained within the historical control data.

URINALYSIS
After 4 Weeks
ln males and females, no differences of toxicological relevance were noted at any dose level.

After 6 Weeks
ln males, no differences of toxicological relevance were noted at any dose level.
In females treated previously with 1000 mg/kg/day, the mean 18-hour urine production was significantly decreased (p<0.05) and the mean relative density was significantly increased (p<0.05). These differences were considered to be the result of water bottle leakage in the control group resulting in artificially high output and reduced relative density.

ORGAN WEIGHTS
After 4 Weeks
In males treated with 1000 mg/kg/day, marginally elevated mean absolute kidney weights were noted when compared with the controls. The mean kidney-to-body weight ratios were significantly elevated (p<0.05) in these males, but the mean kidney-to-brain weight ratio did not attain statistical significance. Females treated with 1000 mg/kg/day had marginally elevated mean absolute adrenal weights when compared with controls. ln the absence of microscopical changes in these organs, the findings were considered to be incidental.
The mean absolute and relative organ weights of the rats treated with 50 mg/kg/day or 200 mg/kg/day were considered to be unaffected.

After 6 Weeks
The mean absolute liver weights of the males previously treated with 1000 mg/kg/day were significantly lower (p<0.05) than those of the control males, and the brain-to-body weight ratio was increased (p<0.05), the testes-to-body weight rate ratio was increased (p<0.01) and the epididymide-to-body weight ratio was increased (p<0.05) when compared with the controls. The liver-to-brain weight ratio was significantly reduced in males previously treated with 1000 mg/kg/day (p<0.05) when compared with the controls.
All other organ weights were both similar to those of the control group and/or without microscopical changes, and therefore considered to be unaffected.

MACROSCOPIC FINDINGS
At the end of the treatment and following recovery period, no test item-related gross lesions were observed. The few macroscopic findings such as dark red or reddish foci in the fundic mucosa of the stomach, reddish foci in the seminal vesicles, dark red foci in the thymus, pelvic dilatation of the kidneys seen in few animals and distributed among control and treated groups were considered to be within the range of normal background lesions, which may be observed in rats of this strain and age in 4-week studies and were considered incidental, reflecting the usual individual variability.

MICROSCOPIC FINDINGS
At the end of the treatment and following recovery period, no test item-related microscopic lesions were observed.
The moderate centrilobular necrosis seen in the liver of one female (no. 55) treated with 1000 mg/kg/day was most likely secondary to the hypoxia that probably affected this animal. Marked extramedullary hematopoiesis was present in the liver and in the spleen of this animal (killed in extremis on day 4 of the treatment period). Based on this assumption, it is unlikely that the liver necrosis could correlate with the treatment with the test item.
The remaining few microscopic findings, regularly distributed among the groups, were within the range and severity of spontaneous background lesions which may be observed in rats of this strain and age in this laboratory and considered to be of no toxicological significance.
Dose descriptor:
NOEL
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified

TEST ITEM INTAKE

Allocation and target dose level Group 1
0 mg/kg/day
Group 2
50 mg/kg/day
Group 3
500 mg/kg/day
Group 4
1000 mg/kg/day
Males
Week 1 0 mg/kg/day 43 mg/kg/day 172 mg/kg/day 899 mg/kg/day
Week 2 0 mg/kg/day 52 mg/kg/day 215 mg/kg/day 1156 mg/kg/day
Week 3 0 mg/kg/day 59 mg/kg/day 236 mg/kg/day 1182 mg/kg/day
Week 4 0 mg/kg/day 53 mg/kg/day 213 mg/kg/day 1046 mg/kg/day
Mean  0 mg/kg/day 51 mglkglday 209 mg/kg/day 1071 mg/kg/day
Deviation (%) - +2.0 % +4.5 % +7.1 %
Females
Week 1 0 mg/kg/day 49 mg/kg/day 186 mg/kg/day 994 mg/kg/day
Week 2 0 mg/kg/day 48 mg/kg/day 183 mg/kg/day 1036 mg/kg/day
Week 3 0 mg/kg/day 51 mg/kglday 207 mg/kg/day 1148 mg/kg/day
Week 4 0 mg/kg/day 47 mg/kg/day 206 mg/kg/day 1037 mg/kg/day
Mean  0 mg/kg/day 49 mglkg/day 195 mg/kg/day 1054 mg/kg/day
Deviation (%) - -2.0 % -2.5 % +5.4 %

ln males treated with target levels of 50, 200 or 1000 mg/kg/day, the mean test item intake values were 51, 209 and 1071 mg/kg/day, respectively, whereas in females, the mean test item intake values were 49, 195 and 1054 mg/kg/day, respectively.

Conclusions:
Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL).
Executive summary:

ln this subacute toxicity study, the test item was administered in the diet to rats at target dose levels of 50, 200 and 1000 mg/kg/day for a period of 28 days. A control group received similar feed without the test item. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg/day. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during acclimatization, treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 4. At the end of the dosing and the treatment—free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control, middle and high dose animals, and all gross lesions from all animals.

One female (no 55, 1000 mg/kg/day) was killed for ethical reasons on day 4 of treatment. All other rats survived until scheduled necropsy. A small number of clinical signs were noted in two males at 200 mg/kg/day on day 8 of treatment and in five males at 1000 mg/kg/day on day 8. Further clinical signs noted in one male at 1000 mg/kg/day included lethargic behavior, hunched posture and ruffled fur on treatment day 18 (the latter finding was also seen on treatment days 19-23 and 25). During weekly observations (weeks 1-3), piloerection, hunched posture, pale discoloration and a wound were noted during week 1 in a single female at 1000 mg/kg/day. Piloerection, hunched posture and somnolence were noted during week 3 in a single male at 1000 mg/kg/day. ln view of the transient nature and limited number of affected animals, these findings were considered to be unrelated to the treatment with the test item. All other animals were without clinical findings. During the functional observational battery at week 4, no findings of toxicological relevance were noted at any dose level. Grip Strength No differences of toxicological relevance were noted in mean fore- or hindlimb grip strength at any dose level. Locomotor Activity Minor differences in the mean Iocomotor activity were not considered to be of toxicological relevance. The mean daily food consumption and the mean relative food consumption were considered to be unaffected by the treatment with the test item. The target dose levels were largely attained. No test item-related differences of toxicological relevance were noted at any dose level.

All minor differences noted in the hematology parameters of test item-treated rats remained within the historical control values and were therefore considered to be incidental.

All minor differences noted in the clinical biochemistry parameters of test item-treated rats remained within the historical control values and were therefore considered to be incidental. In males and females, no differences of toxicological relevance were noted after 4 weeks' treatment or 2 weeks’ recovery at any dose level. After four weeks treatment and two weeks’ recovew, none of the minor differences in the mean absolute or relative organ weights were found to be related to microscopical changes and therefore considered to be incidental. At the end of the treatment and following recovery periods, no test item-related macroscopic or microscopic findings were recorded.

Conclusion

Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL).

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

There are no experimental data available on the Leucophor 1111X, thus a read across approach with the structural analogous has been proposed.

A subacute toxicity study is available on the analogous substance 01. The test item was administered in the diet to rats at target dose levels of 50, 200 and 1000 mg/kg/day for a period of 28 days. A control group received similar feed without the test item. The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg/day. These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed. One female was killed for ethical reasons on day 4 of treatment. All other rats survived until scheduled necropsy. A small number of clinical signs were noted in two males at 200 mg/kg/day and in five males at 1000 mg/kg/day. Further clinical signs noted in one male at 1000 mg/kg/day included lethargic behavior, hunched posture and ruffled fur. During weekly observations, piloerection, hunched posture were noted in a single female and in a single male at 1000 mg/kg/day; in view of the transient nature and limited number of affected animals, these findings were considered to be unrelated to the treatment with the test item. All other animals were without clinical findings. During the functional observational battery, no findings of toxicological relevance were noted at any dose level. Locomotor Activity Minor differences in the mean Iocomotor activity were not considered to be of toxicological relevance. All minor differences noted in the hematology parameters of test item-treated rats remained within the historical control values and were therefore considered to be incidental. All minor differences noted in the clinical biochemistry parameters of test item-treated rats remained within the historical control values and were therefore considered to be incidental. In males and females, no differences of toxicological relevance were noted after 4 weeks' treatment or 2 weeks’ recovery at any dose level. After four weeks treatment and two weeks’ recovew, none of the minor differences in the mean absolute or relative organ weights were found to be related to microscopical changes and therefore considered to be incidental. At the end of the treatment and following recovery periods, no test item-related macroscopic or microscopic findings were recorded. Based on the results of this study, 1000 mg/kg body weight/day of the test item was established as the no-observed-effect-level (NOEL) (Braun, 2006).

No further investigations have been proposed because a 24 -months chronic toxicity investigation is available; the experiment involved two generations of hamsters. Furthermore, another 24 -months study was conducted on rats. Both the experiments were conducted on the analogous substance 07. The studies are reported in the 7.7 carcinogenicity IUCLID section.

Both analogous substance 01 and analogous substance 07 are Stilbene derivatives Fluorescent Whitening Agents salts. They display similar structural and physicochemical properties; all of them exhibit high degree of dissociation in water and very low octanol/water partition coefficients because to a higher affinity with water phase than the octanol one. The water solubility is due to the sulphonated groups in the molecules. The differences occurring in the structure formulas (i.e. substituents) are expected to not significantly impact the toxicological characterisation.

Further details about the justification for read across approach are given in the report attached to the Section 13 of this dossier.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgement, on the basis of the weight of all evidence available, including the use of recommended guidance values, which take into account the duration of exposure and the dose/concentration, which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

In order to help reach a decision about whether a substance shall be classified or not, and to what degree it shall be classified (Category 1 or Category 2), dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. Nevertheless, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for 28-day study the guidance values are increased by a factor of three.

The No Observed Adverse Effect Level was established at 1000 mg/kg bw/day, on the basis of the results from the subacute study of 28 days, on rats.

In conclusion, the available experimental data are adequate for classification and labelling and the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC 1272/2008).