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Diss Factsheets

Administrative data

Description of key information

OECD 408 - NOAEL >= 1000 mg/kg bw/d (no adverse effects observed)


OECD 422 - NOAEL >= 1000 mg/kg bw/d (no adverse effects observed)

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:
July 2021 - June 2022
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Reason / purpose for cross-reference:
reference to other study
Remarks:
dose-range finding study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
25 June 2018
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Details on species / strain selection:
The Sprague Dawley SD rat was the species and strain of choice because it is accepted by many regulatory authorities and there are ample experience and background data on this species and strain. The experimental laboratory has sufficient historical control data for the strain used.
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: ordered from Envigo RMS srl, San Pietro al Natisone (UD), Italy and supplied by Envigo RMS B.V., Kreuzelweg 53, 5961 NM Horst, Netherlands
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 27-29 days old
- Weight at study initiation: 95-121 g for males and 91-113 g for females
- Fasting period before study: no
- Housing: up to 5 of one sex in a cage (in clear polysulfone solid bottomed cages)
- Diet (e.g. ad libitum): 4 RF 21,Mucedola S.r.l., Via G. Galilei 4, 20019 SettimoMilanese (MI), Italy) was offered ad libitum
- Water (e.g. ad libitum): drinking water ad libitum
- Acclimation period: 12 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22°C ± 2°C
- Humidity (%): 55% ± 15%
- Air changes (per hr): approx. 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 01-July-2021 (Arrival of animals) To: 13--Oct-2021 (Last day of necropsy)
Route of administration:
oral: gavage
Details on route of administration:
Oral administration is adequate since the substance is not handled in inhalable form and dermal penetration is not expected based on the data available. The oral route has been selected as it has been specifically requested by the Regulatory Authorities.
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
0.5 % aqueous carboxymethylcellulose (0.5% CMC)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The required amount of test item was suspended in the vehicle.
The preparations were made daily/weekly (concentrations of 10, 30 and 100 mg/mL).
Concentrations were calculated and expressed in terms of test item as supplied.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analysis was performed in a separate study in order to validate the analytical method and the preparation procedure and to verify the stability of the preparations.

The analytical method was validated in the range from 10 to 100 mg/mL. Linearity, accuracy and precision were within the limits stated in the validation protocol (r > 0.995; accuracy 85-115%; precision CV < 10%).
A 27 hour stability at room temperature and an 8 day stabiliy at 2-8°C were verified in the range from 10 to 100 mg/mL. Suspensions are considered to be stable if concentration and homogeneity are still acceptable (85%-115% for concentration and CV < 10% for homogeneity) after the defined period of storage.
The proposed preparation procedure for the test item was checked in the range from 10 to 100 mg/mL by chemical analysis (concentration and homogeneity) to confirm that the method was suitable. Final results for all levels were within the acceptability limits for concentration (85-115%) and homogeneity (CV < 10%).
Samples of the suspensions prepared on Week 1 (Day 1) and Last week (Day 86) were analysed to check their homogeneity and concentration. Results of the analyses were within the acceptability limits for suspensions (85-115% for concentration and CV < 10% for homogeneity) and are available.
The validated software used for this activity was Chromeleon.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: Dose levels have been selected based on the results obtained in a previous oral toxicity study in rats, performed according to OECD Guideline 422 - Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test. In this study, no adverse effects were noted at all dose levels (100, 300 and 1000 mg/kg bw/day) and the dose level ≥ 1000 mg/kg bw/day was considered the NOAEL in this study. Therefore, the doses were selected to include the limit dose of 1000 mg/kg bw/d and dose spacing of ca. 3 was used as standard dose spacing.

- Dose range finding studies: please refer to cross-referenced study according to OECD Guideline 422

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Animals were checked for mortality twice per day on weekdays and once per day on weekends and public holidays. Observations for clinical signs were conducted once per day after dosing.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Once before commencement of treatment and at least once per week during the study. Each animal was observed in an open arena. The test included observation and record of changes in gait and posture, reactivity to handling, presence of clonic or tonic movements, stereotypies or bizarre behaviour and effects on the autonomic nervous system (e.g. lachrymation, piloerection, unusual respiratory pattern). Changes in fur, skin, eyes, mucous membranes, occurrences of secretions and excretions was also recorded.

BODY WEIGHT: Yes
- Time schedule for examinations: day of allocation to treatment group, start of treatment, weekly thereafter and just prior to necropsy

FOOD CONSUMPTION:
- TIme schedule: recorded at weekly intervals starting from treatment (the group mean daily intake per rat was calculated). Measurements were performed by cage.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: all animals were examined prior to the commencement of treatment, eyes of all animals from high dose and control groups were re-examined during Week 13 of treatment

SERUM HORMONES: Yes
- Time of blood sample collection: last week of treatment
- Animals fasted: Yes
- How many animals: all
- Hormone parameters: T3, T4, TSH (determined by RadioImmuneAssay (RIA))

HAEMATOLOGY: Yes
- Time schedule for collection of blood: prior to necropsy
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Coagulation (Prothrombin time, APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: prior to necropsy
- Animals fasted: Yes
- How many animals: all
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Blood urea nitrogen, Creatinine, Glucose, Triglycerides, Inorganic phosphorus, Total bilirubin, Total cholesterol, HDL, LDL, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

OESTROUS CYCLE: YES
- Time schedule: at the end of the treatment and just prior to necropsy, vaginal smears were taken from all animals

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Once during weeks 13 of treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (e.g. auditory, visual and proprioceptive stimuli) / grip strength / motor activity (at 10 minute intervals over 60 minutes)

URINALYSIS: NO

IMMUNOLOGY: NO
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
Samples of all the tissues listed below were fixed and preserved in 10% neutral buffered formalin (except eyes, optic nerves, Harderian glands, testes and epididymides which were fixed inModified Davidson’s fluid and preserved in 70% ethyl alcohol).

TISSUES PROCESSED:
see "any other information on methods including tables"
Statistics:
Standard deviations were calculated as considered appropriate. For continuous variables the significance of the differences amongst groups was assessed by analysis of variance.
Differences between each treated group and the control group were assessed by Dunnett’s test using a pooled error variance. The homogeneity of the data was verified by Bartlett’s test before Dunnett’s test. If the data were found to be inhomogeneous aModified t test (Cochran and Cox) was applied.
The mean values, standard deviations and statistical analysis were calculated from the actual values in the computer without rounding off.
Clinical signs:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related clinical signs were observed.

100 mg/kg bw/d, 1 male animal: Ocular transparent discharge and coloured staining around both eyelid in some occasions during the study
--> considered unrelated to treatment with the test item (absence of a dose-relation and other clinical signs)


No treatment-related changes were observed at the weekly clinical examination, which included an evaluation of neurotoxicity

100 mg/kg bw and 300 mg/kg bw, males and females: slight fluctuations in the number of rearing events during dosing period in a single occiasion
1000 mg/kg bw, males and females: occasional decrease in the number of rearing evens
--> not considered to be treatment-related (absence of a clear dose-relation and episodical occurence)
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Description (incidence and severity):
When compared to control animals, no changes were noted in mean body weights and
body weight gain in both genders during the study.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No treatment-related changes were observed in food consumption in male and female
animals, during the study.
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No findings were detected in both eyes of all animals, from high dose and control groups,
following examination performed duringWeek 13 of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
100 mg/kg bw, males: higher monocytes (+78%)
--> considered to be incidental (within the range of historical data)

1000 mg/kg bw males: lower erythrocytes (-5%)
--> considered to be non-adverse (5/10 animals slightly below the historical data and slight severity of the effect; even though the relation with the test item cannot be definitively excluded)

1000 mg/kg bw, females: higher leucocytes (+41%)
--> considered to be incidental (within the range of historical data)
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related changes were recorded
300 and 1000 mg/kg bw, males: higher inorganic phosphorous (14% and 15%, respectively; 3/10 and 4/10 animals outside of historical date, respectively)
--> considered to be incidental (slight severity, absence of other related findings)
1000 mg/kg bw, females: decreased chloride (-2%)
--> considered to be incidental (within the range of historical data)
Endocrine findings:
no effects observed
Description (incidence and severity):
Thyroid hormone determination: no changes were recorded.
Urinalysis findings:
not examined
Behaviour (functional findings):
effects observed, non-treatment-related
Description (incidence and severity):
No differences that could be considered treatment-related were observed at functional tests (sensory reaction to stimuli, landing footsplay, grip strength, motor activity) performed at the end of the treatment period.
300 and 1000 mg/kg bw, males and females: lower values in males and higher values in females of grip strength measurement (first, second trials and mean values)
--> considered to be incidental (absence of a clear dose-relation and to the inconsistency of the variations (higher or lower)

No treatment-related changes were observed at the weekly clinical examination, which included an evaluation of neurotoxicity
100 mg/kg bw and 300 mg/kg bw, males and females: slight fluctuations in the number of rearing events during dosing period in a single occiasion
1000 mg/kg bw, males and females: occasional decrease in the number of rearing events
--> not considered to be treatment-related (absence of a clear dose-relation and episodical occurence)
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
All differences between the test item-treated groups and controls were small, never reached
statistical significance and were interpreted to reflect individual variations.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All macroscopic observations were spontaneous in nature and bore no relationship to
treatment.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
1000 mg/kg bw, males and females: aggregates of large eosinophilic macrophages in the sinuses of mesenteric lymph nodes with a score up to moderate
300 mg/kg bw, 1 female: aggregates of large eosinophilic macrophages in the sinuses of mesenteric lymph nodes with a minimal severity
--> possibly reflects accumulation of the test item; considered non adverse (due to absence of other changes in the node architecture and of associated clinical pathology changes)

1000 mg/kg bw, females: higher incidence and severity of squamous metaplasia of uterine glands
--> spontaneous squamous metaplasia is rare (indeed a higher incidence than usual is noted in controls and at intermediate dose levels in the present study); relationship to treatment cannot be completely excluded but increased incidence is likely a chance event given the high background in this study and the absence of differences in ovarian cycle distribution between control and high dose groups
Histopathological findings: neoplastic:
no effects observed
Key result
Dose descriptor:
NOAEL
Effect level:
>= 1 000 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Critical effects observed:
no

Table 1: Clinical Signs - Group Incidence














































































































































































































MalesFemales
Dosing Dosing 
 Dose group [mg/kg bw/d]01003001000  Dose group [mg/kg bw/d]01003001000
 Number of Animals10101010 Number of Animals10101010
 Number Examines10101010 Number Examines10101010
CategoryObservationababababCategoryObservationabababab
No significant signsNo significant signs1092.51088.51092.51092.5No significant signsNo significant signs1092.51092.51092.51092.5
APPEARANCEStaining, Both eyelids, Right, Moderate, Brown0012.00000          
Staining, Both eyelids, Right, Slight, Brown00118.00000          
Staining, Both eyelids, Right, Slight, Red0011.00000          
Staining, Eye, Slight, Brown0011.00000          
EYE - EAR - MOUTH-NOSEOcular discharge, Right, Transparent00119.00000          

a - number of animals affected


b - number of days with clinical sign/animal


 


Table 2: Body Weight - Group mean data





















































































































































































































































































































































































































































































































Males
  QuarantineDosing
Dose [mg/kg bw/d]Body Weight (g)Day 8Day 1Day 8Day 15Day 22Day 29Day 36Day 43Day 50Day 57Day 64Day 71Day 78Day 85Day 92
0(n)101010101010101010101010101010
Mean150.68186.47236.80281.32312.78339.15357.60374.00388.44396.51408.26417.40431.78435.04432.00
SD8.8279.99610.98810.76711.48613.27415.54715.20613.86412.82514.22014.13915.68115.02821.392
100(n)101010101010101010101010101010
Mean150.30186.64239.17287.20318.98345.68369.36388.32402.51410.19422.80429.83442.59445.40448.40
SD8.24812.74613.75417.39017.69321.58322.03523.35521.65422.08224.32422.78121.30922.94830.329
300(n)101010101010101010101010101010
Mean150.10184.05235.98280.84312.24338.58361.19379.70392.97403.37413.48423.71436.85439.08440.44
SD7.4496.5537.8359.18812.88017.79619.34722.13119.48424.61125.79027.48530.49827.67133.514
1000(n)101010101010101010101010101010
Mean150.52184.17234.38279.46309.39328.27350.81368.01383.58390.55404.03410.94424.02423.31422.51
SD8.7549.97611.71114.01614.06517.23018.04721.31122.23921.75722.80824.09023.69926.42828.406
Females
  QuarantineDosing
Dose group [mg/kg bw/d]Body Weight (g)Day 8Day 1Day 8Day 15Day 22Day 29Day 36Day 43Day 50Day 57Day 64Day 71Day 78Day 85Day 92
0(n)101010101010101010101010101010
Mean135.00150.48170.47191.19199.22209.94220.58226.76234.38233.67240.22241.98254.34246.63245.33
SD5.7126.9157.8337.5428.3508.6479.92410.20310.8869.71913.34712.84010.66713.6818.958
100(n)101010101010101010101010101010
Mean135.90150.82169.36187.70196.72209.38216.37222.46228.59234.15233.75237.49241.86241.94240.66
SD6.3835.3827.83110.6439.5619.56411.39613.52313.56115.11614.16017.00315.78818.13917.121
300(n)101010101010101010101010101010
Mean134.63151.08171.75185.76199.71211.50221.59227.10232.45237.24240.17243.19246.98247.21244.47
SD5.4765.5569.24811.48513.68215.30517.60017.87715.45818.12117.19717.70318.09018.83815.847
1000(n)101010101010101010101010101010
Mean133.95149.97170.25188.09199.05212.24217.10222.82230.33232.76236.21239.07244.73245.27242.53
SD6.1306.2838.50211.83410.44610.00810.73312.91311.81115.02114.08712.31615.30813.89016.520

 


Table 3: Haematology - Group mean data


































































































































































































































































































































































































































































































































































































































































































Dosing Day 92
Males
Dose group [mg/kg bw/d] RBC
(x10^6/μL)		HGB
(g/dL)		HCT
(%)		MCV
(fL)		MCH
(pg)		MCHC
(g/dL)		RET
(x10^9/L)		RETR
(%)		WBC
(x10^3/μL)		NEU
(x10^3/μL)		LYM
(x10^3/μL)		MON
(x10^3/μL)		EOS
(x10^3/μL)		BAS
(x10^3/μL)		LUC
(x10^3/μL)		NEUR
(%)		LYMR
(%)		MONR
(%)		EOSR
(%)		BASR
(%)		LUCR
(%)		PLT
(x10^3/μL)
0(n)10101010101010101010101010101010101010101010
Mean8.07914.5844.6255.2418.0532.72159.461.9735.5340.9964.2120.1440.1340.0230.02417.6976.402.602.460.430.40921.4
SD0.34140.4832.1441.4530.4450.54910.9230.094514.5950.781112.3980.07140.05910.01160.013510.66910.3241.0240.9120.1250.15692.55
100(n)8888888888888888888888
Mean8.26614.6944.6554.0817.7932.91157.091.9055.6611.2733.9440.256+D0.1310.0260.02521.3170.854.60+D2.280.460.49974.9
SD0.30730.2530.8541.6490.6940.46111.3260.156513.0500.78060.56190.08830.03360.00740.00767.3866.9771.2190.3280.0520.18950.05
300(n)10101010101010101010101010101010101010101010
Mean7.98414.5344.6355.9218.2132.56155.921.9534.8240.8963.6240.1570.1110.0200.01819.0274.523.292.360.410.40903.4
SD0.31260.3021.4801.1920.4360.57616.0100.17930.77990.11230.83020.04990.04230.00670.01143.9935.5681.0431.1120.1370.24986.77
1000(n)10101010101010101010101010101010101010101010
Mean7.683*D14.2242.9055.8218.5033.14148.731.9335.5191.0174.1590.2070.0910.0210.02319.4674.473.691.590.370.40935.7
SD0.42981.0113.0511.6190.6220.47419.9130.228214.5430.1484Feb 350.08030.03840.01100.01064.8734.1940.6790.4380.0950.09473.36
Females
Dose group [mg/kg bw/d] RBC
(x10^6/μL)		HGB
(g/dL)		HCT
(%)		MCV
(fL)		MCH
(pg)		MCHC
(g/dL)		RET
(x10^9/L)		RETR
(%)		WBC
(x10^3/μL)		NEU
(x10^3/μL)		LYM
(x10^3/μL)		MON
(x10^3/μL)		EOS
(x10^3/μL)		BAS
(x10^3/μL)		LUC
(x10^3/μL)		NEUR
(%)		LYMR
(%)		MONR
(%)		EOSR
(%)		BASR
(%)		LUCR
(%)		PLT
(x10^3/μL)
0(n)10101010101010101010101010101010101010101010
Mean6.87513.0538.1355.4618.9834.25138.652.0183.4920.5082.7780.1170.0610.0110.01915.0979.003.271.800.310.53917.5
SD0.40070.7782.4731.4520.5920.53620.8380.29850.99540.13060.86270.05460.01850.00570.00743.7244.0340.8650.4940.1290.16493.98
100(n)10101010101010101010101010101010101010101010
Mean6.70112.8137.6956.2619.1133.96151.882.2704.1380.7003.2140.1350.0550.0110.02116.5478.153.181.35*C0.290.49907.9
SD0.26010.5491.4480.8440.4890.45518.5820.304112.9850.42930.93940.05800.02550.00880.01295.3295.7240.9530.3500.1200.20855.16
300(n)10101010101010101010101010101010101010101010
Mean6.70212.7337.2555.6019.0434.23140.632.1024.1940.6243.3050.1470.0800.0150.02415.3178.323.561.920.300.60916.1
SD0.25280.4001.3631.3490.4250.30619.6890.30590.82390.11980.77700.04670.03060.00530.00974.1564.5760.8920.7180.0820.19473.22
1000(n)10101010101010101010101010101010101010101010
Mean6.81313.1238.1155.9519.2434.40153.142.2554.931*D0.7543.8730.1860.0650.0170.03415.4778.483.661.34*C0.360.68905.3
SD0.30960.4731.5581.1660.5100.40332.9090.517510.5230.22490.86820.08750.01780.00670.01964.0994.7061.2650.2950.1070.336101.97

HCT: HAEMATOCRIT [%]


RBC: RED BLOOD CELL COUNT [10^6/μL]


HGB: HAEMOGLOBIN [g/dL]


MCV: MEAN RED BLOOD CELL VOLUME [fL]


MCH: MEAN CORPUSCULAR HAEMOGLOBIN [pg]


MCHC: MEAN CORPUSCULAR HAEMOGLOBIN CONCENTRATION [g/dL]


PLT: PLATELETS [10^3/uL]


WBC: WHITE BLOOD CELL COUNT [10^3/uL]


NEUR/NEU: NEUTROPHILS [% or 10^3/μL]


LYMR/LYM. LYMPHOCYTES [% or 10^3/μL]


MONR/MON: MONOCYTES [% or 10^3/μL]


EOSR/EOS: EOSINOPHILS [% or 10^3/μL]


BASR/BAS: BASOPHILS [% or 10^3/μL]


LUCR/LUC: LARGE UNSTAINED CELLS [% or 10^3/μL]


RETR/RET: RETICULOCYTES [% or 10^3/μL]


*D Dunnett LSD Test Significant at the 0.05 level


+D Dunnett LSD Test Significant at the 0.01 level


*C Cochran and Cox Test Significant at the 0.05 level


 


Table 4: Clinical Chemistry - Group mean data
































































































































Dosing Day 92
MalesFemales
Dose group [mg/kg bw/d] Cl [nmol/L]IP [mg/dL]Group Cl [nmol/L]IP [mg/dL]
0(n)1010Control(n)1010
Mean103.175.823Mean104.806.112
SD0.8010.7791SD1.2090.8488
100(n)10102(n)1010
Mean102.876.028Mean105.386.255
SD0.7470.6315SD1.9360.7440
300(n)10103(n)1010
Mean103.646.637*DMean104.706.687
SD0.6700.5913SD1.0220.7779
 (n)10104(n)1010
1000Mean103.156.671*DMean103.06*D6.447
 SD1.0840.6880SD1.6320.6767

Cl: Chloride


IP: inorganic phosphorus


*D Dunnett LSD Test Significant at the 0.05 evel


 


Table 5: Clinical Chemistry - Historical Control Data




















































































































Historical Contral Data
MalesFemales
 Age: 9-16 weeksAge: > 16 weeks Age: 9-16 weeksAge: > 16 weeks
 Cl [nmol/L]IP [mg/dL]Cl [nmol/L]IP [mg/dL] Cl [nmol/L]IP [mg/dL]Cl [nmol/L]IP [mg/dL]
no25686369199no22475385214
Mean102.77.53103.45.81Mean103.76.10103.75.14
SD2.20.662.61.30SD2.51.292.50.99
Min obs93.65.2796.14.15Min obs94.64.0497.32.76
Max obs109.28.92111.017.14Max obs111.111.67110.88.16
Range min (perc. 5%)99.66.4099.44.61Range min (perc. 5%)100.04.4899.73.67
Range max (perc. 95%)105.78.54107.96.93Range max (perc. 95%)107.27.83107.96.89

 


Table 6: Thyroid hormones determination - Group mean data
























































































































































Dosing Day 88
MalesFemales
Dose group [mg/kg bw/d] T3 [nmol/L]T4 [nmol/L]TSH [ng/mL]Dose group [mg/kg bw/d] T3 [nmol/L]T4 [nmol/L]TSH [ng/mL]
0(n)1010100(n)101010
Mean0.95347.112.68Mean1.27139.910.31
SD0.15943.905.648SD0.14536.574.047
100(n)101010100(n)101010
Mean0.96748.911.47Mean1.22439.47.16
SD0.17614.864.181SD0.15015.681.260
300(n)101010300(n)101010
Mean1.03152.39.97Mean1.27138.78.68
SD0.12636.733.345SD0.14978.151.734
1000(n)1010101000(n)101010
Mean1.04252.311.69Mean1.34642.58.71
SD0.223606.574.888SD0.15313.272.822

 


Table 7: Estrous Cycle 



























































































































GroupStageDays SeenGroupStageDays SeenGroupStageDays SeenGroupStageDays Seen
ControlProestrus92100 mg/kg bw/dOestrus92300 mg/kg bw/dDioestrus921000 mg/kg bw/dProestrous92
Oestrus92Oestrus92Oestrus92Oestrus92
Oestrus92Oestrus92Dioestrus92Oestrus92
Oestrus92Oestrus92Oestrus92Oestrus92
Oestrus92Oestrus92Dioestrus92Proestrous92
Dioestrus93Oestrus93Oestrus93Oestrus93
Dioestrus93Metaoestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93
Oestrus93Oestrus93Oestrus93Oestrus93

 


Table 8: Open Field Observation of Rearing - Group Mean Data



























































































































































































































































































































































































































































































Males
  AllocationDosing
Dose group [mg/kg bw/d] Day 2Day 3Day 10Day 17Day 24Day 31Day 38Day 45Day 52Day 59Day 66Day 73Day 80Day 89
0(n)1010101010101010101010101010
Mean10.46.78.212.912.88.79.16.97.06.97.811.25.86.1
SD0.701.251.622.181.401.161.663.981.254.251.231.553.851.37
100(n)1010101010101010101010101010
Mean10.67.28.513.88.4+D8.08.64.98.96.27.811.53.96.4
SD0.521.321.051.552.121.701.311.522.334.831.402.221.791.43
300(n)1010101010101010101010101010
Mean11.78.28.114.47.6+D8.38.77.18.95.7.769.24.66.1
SD28.1261.691.521.261.071.421.063.382.424.992.071.231.901.60
1000(n)1010101010101010101010101010
Mean12.7+C6.87.613.08.4+D8.38.46.47.85.15.87.3+D5.37.7
SD44.7141.320.841.491.171.571.512.411.621.912.972.832.581.77
Females
  AllocationDosing
Dose group [mg/kg bw/d] Day 5Day 4Day 11Day 18Day 25Day 32Day 39Day 46Day 53Day 60Day 67Day 74Day 81Day 90
0(n)1010101010101010101010101010
Mean15.116.517.213.914.214.913.912.212.912.515.517.612.112.0
SD24.1081.581.231.521.815.973.413.362.691.961.081.172.382.45
100(n)1010101010101010101010101010
Mean15.516.417.514.412.916.112.413.113.013.115.916.1*D11.611.8
SD16.1321.171.960.842.778.053.603.932.982.281.200.742.222.04
300(n)1010101010101010101010101010
Mean13.616.717.214.613.314.614.412.813.513.215.616.612.212.3
SD27.7912.001.621.172.985.582.722.572.272.101.071.432.101.49
1000(n)1010101010101010101010101010
Mean16.115.614.6+D14.513.714.611.213.512.613.816.015.5+D12.512.2
SD23.4081.841.511.182.835.254.292.802.272.572.110.971.652.30

*D Dunnett LSD Test Significant at the 0.05 level


+C Cochran and Cox Test Significant at the 0.01 level


+D Dunnett LSD Test Significant at the 0.01 level


 


Table 9: Sensory Reactivity / Grip Strength - Group Mean Data
























































































































































Dosing Day 85
MalesFemales
Dose group [mg/kg bw/d] GS1 (N)GS2 (N)GSMEDose group [mg/kg bw/d] GS1 (N)GS2 (N)GSME
0(n)1010100(n)101010
Mean17.2317.7517.490Mean46.63113.3949.315
SD2.2222.69322.704SD1.1431.3420.8377
100(n)101010100(n)101010
Mean18.3817.2017.790Mean22.92028.00410.190*D
SD2.4072.71020.556SD1.5581.0810.3658
300(n)101010300(n)101010
Mean15.18*C14.52+C14.850+CMean11.46+D11.43+D11.445+D
SD0.9851.34710.376SD1.0781.4610.7798
1000(n)1010101000(n)101010
Mean15.51*C15.43*C15.470*CMean11.63+D10.95*D11.290+D
SD0.8250.5250.4911SD1.2371.3130.5962

GS1 (N): Grip strength 1 (N) - First reading of the animal grips on horizontal bar with forelimbs.


GS2 (N): Grip strength 2 (N) - Second reading of the animal grips on horizontal bar with forelimbs.


GSME (N): Grip strength mean (N) - Averaged reading of the animal grips on horizontal bar with forelimbs.


*C Cochran and Cox Test Significant at the 0.05 level


+C Cochran and Cox Test Significant at the 0.01 level


*D Dunnett LSD Test Significant at the 0.05 level


+D Dunnett LSD Test Significant at the 0.01 level


 


Table 10: Organ Weights (absolute) - Group Mean Data





















































































































































































































































































































































































































































Males
Dose group [mg/kg bw/d] Terminal Body Weight
(g)		Adrenal glands
(g)		Brain
(g)		Epididymides
(g)		Heart
(g)		Kidneys
(g)		Liver
(g)		Pituitary gland
(g)		Seminal vesicales
(g)		Spleen
(g)		Testes
(g)		Thymus
(g)		Thyroid gland
(g)
0(n)10101010101010101010101010
Mean418.760.05721.8851.48841.4592.84211.3450.010229.3670.85163.98800.38780.0250
SD15.1480.010700.10620.115890.09940.22560.93400.001480.255470.123090.202270.088630.00279
100(n)10101010101010101010101010
Mean430.830.05471.8201.42041.4652.95211.1600.010129.1490.81683.86980.38230.0254
SD24.4560.006990.11940.167000.10520.21850.69290.002180.452060.126340.266080.049320.00334
300(n)10101010101010101010101010
Mean423.030.05501.8431.45011.4362.90011.1660.010129.5470.82984.07720.37490.0250
SD27.2230.006000.11240.158320.10170.141911.3510.001660.317160.089520.194360.058750.00397
1000(n)10101010101010101010101010
Mean404.630.05501.7941.41481.3982.77210.7910.010028.7220.78343.93530.40100.0245
SD28.8620.008840.12490.137080.13390.29060.89770.002160.382760.090600.245110.085680.00375
Females
Dose group [mg/kg bw/d] Terminal Body Weight
(g)		Adrenal glands
(g)		Brain
(g)		Heart
(g)		Kidneys
(g)		Liver
(g)		Ovaries
(g)		Pituitary gland
(g)		Spleen
(g)		Thymus
(g)		Thyroid gland
(g)		Uterus
(g)		-
0(n)101010101010101010101010 
Mean235.090.06211.6240.8871.5975.7960.10240.01230.52860.26710.02170.9126 
SD11.2520.005880.08840.08550.12370.50740.019350.002750.042180.021620.004600.36216 
100(n)101010101010101010101010 
Mean227.460.06171.6060.8861.4615.4700.10050.01200.50950.24090.021111.748 
SD17.2370.011850.12800.07140.11950.48640.017570.002110.077150.054430.004910.75968 
300(n)101010101010101010101010 
Mean233.980.06571.6520.8881.5695.7380.11430.01330.54510.23110.02260.9112 
SD17.0120.009500.09650.04720.13230.59010.020860.002450.043200.031270.005170.47996 
1000(n)101010101010101010101010 
Mean229.270.06341.6280.9331.5945.7590.10030.01320.54610.25750.02551.0257 
SD12.8130.009960.10030.09700.16930.48180.018240.004290.083100.048520.003570.84612 

 


Table 11: Microscopic observations


































































































































































Incidence and severity of selected microscopic findings (End of Treatment)



Gender



Males



Females



Group



1



2



3



4



1



2



3



4



Dose (mg/kg bw/d)



0



100



300



1000



0



100



300



1000



Mesenteric lymph node


 

Aggregates, macrophages


 

   Examined



10



10



10



10



10



10



10



10



   Absent



10



10



10



2



10



10



9



1



   Minimal



0



0



0



1



0



0



1



6



   Slight



0



0



0



4



0



0



0



3



   Moderate



0



0



0



3



0



0



0



0



Uterus


 

Squamous metaplasia


 

Examined



NA



NA



NA



NA



10



10



10



10



   Absent



-



-



-



-



7



6



7



3



   Minimal



-



-



-



-



1



2



2



2



   Slight



-



-



-



-



1



1



1



2



   Moderate


    

1



1



0



3



NA: not applicable

Conclusions:
On the basis of the above results and under the conditions tested, no adverse effects were observed at all dose levels investigated following treatment with the test substance when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg/day, when compared to controls. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg/day.
Executive summary:

The test substance was tested in a GLP-conform OECD TG 408 study:


The toxicity of the test substance was investigated in Sprague Dawley SD rats after daily oral administration for 13 weeks.


Three groups, each of 10 male and 10 female rats, received the test item by gavage at dosages of 100, 300 and 1000 mg/kg/day for 13 consecutive weeks. A fourth similarly constituted group received the vehicle alone (0.5% aqueous carboxymethylcellulose) and acted as a control. All doses were administered at a constant volume of 10 mL/kg body weight.


 


The following investigations were performed: mortality and morbidity check, daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, oestrus cycle evaluation, ophthalmoscopy, clinical pathology investigations (including haematology and coagulation, clinical chemistry and thyroid hormones determination), terminal body weight, organ weights, macroscopic observations and microscopic examination, which included the identification of the stages of the spermatogenic cycle and the evaluation of the oestrus cycle.


 


No mortality occurred and no treatment-related clinical signs were observed during the study.


No relevant changes in detailed clinical observation, that can be considered related to treatment, were observed in animals at removal from cage and in an open arena. No treatment-related changes were observed in treated animals at functional tests (sensory reaction to stumuli, landing foot splay, grip strength). No treatment-related differences were noted in the motor activity measurements.


Body weight, food consumption and oestrus cycle were not affected by treatment.


No treatment-related findings in opthalmoscopy were recorded.


No changes in haematology and coagulation which were considered to be adverse were observed. The statistically significant differences of monocytes in males and leucocytes in females were of slight severity and incidence, therefore they were considered to be incidental. The decrease of erythrocytes recorded in males dosed at 1000 mg/kg/day was considered to be non-adverse, even though the relation with the test item cannot be definitively excluded.


No treatment-related changes were recorded in coagulation and clinical chemistry.


No treatment-related changes were recorded in thyroid hormone determination.


No changes in organ weights or macroscopic effects were noted.


In microscopic observations, aggregates of large macrophages were observed in both sexes in mesenteric lymph nodes at the dose of 1000 mg/kg/day, and in a single female at the dose of 300 mg/kg/day. This change was considered non-adverse. A high incidence of squamous metaplasia of the uterus was observed at 1000 mg/kg/day, in a context of an unusually high prevalence of this finding in the study, including in control animals. It was therefore considered a chance event, unrelated to the test item.


On the basis of the above results and under the conditions tested, no adverse effects were observed at all dose levels investigated following treatment with the test substance when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg/day, when compared to controls. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg/day.

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

Repeated dose - 13 weeks (OECD408):


No adverse effects were observed in a GLP compliant OECD 408 study in rats at dose levels of 100, 300, 1000 mg/kg bw with an oral administration of 13 consecutive weeks in male and female animals. The test material was dosed by gavage as a suspension in aqueous carboxymethyl cellulose (0.5%). The OECD 422 study (described below) served as a dose range finding study.


The following investigations were performed: mortality and morbidity check, daily clinical signs, weekly detailed clinical signs (removal from cage and open field observations), evaluation of sensory reactivity to stimuli and motor activity, body weight, food consumption, oestrus cycle evaluation, ophthalmoscopy, clinical pathology investigations (including haematology and coagulation, clinical chemistry and thyroid hormones determination), terminal body weight, organ weights, macroscopic observations and microscopic examination, which included the identification of the stages of the spermatogenic cycle and the evaluation of the staining of the oestrus cycle.


Neither mortality nor treatment-related clinical signs were observed throughout the course of the study. Furthermore, neurotoxicity assessment including open field measurements and motor activity assessment did not reveal any treatment-related changes. Upon necropsy, body weights, organ weights, ophthalmoscopic and macroscopic examination did not show any treatment-related findings. Coagulation and thyroid hormone measurements were without treatment-related findings. Changes observed in hematological and clinical chemistry examinations were within the range of the laboratory's historical control data. In high dose males, a decreased in erythrocytes was observed of -5%, which was in 5/10 animals below the historical control values for this parameter. However, due to the slight severity of this finding, it was considered not adverse even though a relation with treatment could not be definitively excluded. 


Microscopic examination of tissues showed aggregates of large macrophages in the mesenteric lymph nodes of both sexes at the dose of 1000 mg/kg/day, and in a single female at the dose of 300 mg/kg/day. In the absence of other changes in the node architecture and of associated clinical pathology changes, this finding was considered non-adverse. A higher incidence of squamous metaplasia of the uterus was observed at 1000 mg/kg/day, in a context of an unusually high prevalence of the change in the study, including in control animals. Additionally, no differences in ovarian cycle distribution were observed between control and high dose animals. The increased squamous metaplasia of the uterine glands was therefore considered a chance event, unrelated to the test item.


On the basis of these results, no adverse effects were observed at all dose levels investigated following treatment with the test substance when administered by oral gavage for 13 consecutive weeks at the dosages of 100, 300 and 1000 mg/kg/day, when compared to controls. Therefore, it can be concluded that the No Observed Adverse Effect Level (NOAEL) for this study is 1000 mg/kg/day.


 


Repeated dose - Reprotox Screening (OECD422):


No adverse effects were observed in the GLP compliant OECD 422 study in rats at the nominal doses of 100, 300 and 1000 mg/kg bw, including the 14-day postobservation groups for the high dose and control group animals. The test material was dosed by gavage as a suspension in aqueous carboxymethyl cellulose (0.5%).


The study was performed according to the OECD testing guideline in its version of 2016. This involves a longer treatment of females to account for the longer observation of pups.


No treatment-related finding was observed for any parameter.


The test material was found to be stable in formulations for 7 days and to be homogenously distributed. Aliquots of dosing samples taken throughout the study were frozen and analysed later versus a standard of the solid test material. The mean recovery was 84% indicating that the acutal dosed volume might have been only up to 840 mg/kg bw. As no spiked storage control samples were included, loss of the absorbing test material during storage might explain the lower recovery. Considering the absence of adverse effects, this does not give rise of an undetected hazard.


In the 14-day dose-range finding study in rats, no adverse clinical signs were noted during treatment with 300 or 1000 mg/kg bw. Body weight gain was not affected. Weights of assessed organs (liver, spleen, kidney, adrenal gland) were within the normal range. The substance was applied as a suspension in aqueous carboxymethyl cellulose.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008


The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. No adverse effects were observed at doses up to 1000 mg/kg bw in subacute and subchronic oral toxicity studies in rats. Since there were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats the substance does not need to be classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fourteenth time in Regulation (EC) No. 2020/217.