2-piperidinoethanol

Endpoint summary

Description of key information

OECD 422: NOAEL for systemic effects = 250 mg/kg bw/d (highest tested dose) in males and 75 mg/kg bw/d in females

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Sep 2018 - Dec 2018

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

29 Jul 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

certified by Landesamt für Umwelt Rheinland-Pfalz

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Lot/batch No. of test material: B1056-070518
- Purity: 99.8 area % (GC, RTX-5); 99.9 area-% (GC, DB-Wax UI)
- Purity test date: 30 May 2018
- Expiry date of lot: 07 May 2020

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: room temperature
- Stability under test conditions: The stability of the test substance under storage conditions over the test period was guaranteed by the sponsor, and the sponsor holds this responsibility. The stability of test substance in deionized water was demonstrated for a period of 7 days at room temperature.

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: The test substance was weighed in a graduated flask depending on the dose group, topped up with deionized water and intensely mixed with a homogenizer. During administration, the preparations were kept homogeneous with a magnetic stirrer.

FORM AS APPLIED IN THE TEST (if different from that of starting material): diluted in deionized water

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The test guideline requires the rat to be used as the animal species. This rat strain was selected since extensive historical control data are available for Wistar rats.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: about 13 - 15 weeks
- Weight at study initiation: males: 371.9 g (mean); females: 207.4 g (mean)
- Housing: individually (exceptions: During overnight matings, male and female mating partners were housed together. Pregnant animals and their litters were housed together.)
- Diet: ad libitum; ground Kliba maintenance diet mouse-rat “GLP” (supplied by Garanovit AG, Kaiseraugst, Switzerland)
- Water: ad libitum
- Acclimation period: 21 days

DETAILS OF FOOD AND WATER QUALITY: The drinking water was regularly assayed for chemical contaminants as well as for the presence of (pathogenic) microorganisms. On the basis of the analytical findings the drinking water was found to be suitable. The food used in the study was assayed for chemical and for microbiological contaminants. On the basis of duration of use and the analytical findings with respect to chemical and microbiological contaminants, the diet was found to be suitable.

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

IN-LIFE DATES: From: 18 Sep 2018 To: 18 Oct and 08 Nov 2018 (F0 males); 04 Dec 2018 (F0 females); 27 Nov - 01 Dec 2018 (litters)

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

deionized water

Details on oral exposure:

VEHICLE
- Concentration in vehicle: 0.25, 0.75 and 2.5 g/100 mL
- Amount of vehicle (if gavage): 10 mL/kg bw/d

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical verifications of the stability of the test substance in deionized water for a period of 7 days at room temperature had been initiated prior to the start of the study in the same batch. At the beginning (during premating), twice during gestation and once during lactation of the study each 1 sample was taken from the low, mid and high concentration for a concentration control analysis. The samples collected at the beginning of the administration period and during lactation were analyzed via capillary electrophoresis (CE) with indirect UV/VIS detection.
All measured values for the test substance were in the expected range of the target concentrations (90 - 110%) demonstrating the correctness of the preparations.

Duration of treatment / exposure:

males: 30 days; females: 56 days
The duration of treatment covered a 2-weeks premating and mating period in both sexes, 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.

Frequency of treatment:

once daily at approximately the same time in the morning (females in labor were not treated)

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

low dose group

Dose / conc.:

75 mg/kg bw/day (nominal)

Remarks:

mid dose group

Dose / conc.:

250 mg/kg bw/day (nominal)

Remarks:

high dose group

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In an acute oral toxicity study in rats, the calculated LD50-value for males and females was 1100 mg/kg bw/d. Severe clinical signs (e.g. dyspnoe, apathy, staggering, trembling, poor general state) and mortality in both sexes were observed at 1000 mg/kg bw/d and above. At the lowest tested dose of 464 mg/kg bw/d, one female died within the first 24 hours.
In a repeated dose test study, the test substance was orally (gavage) applied at concentrations of 0, 150 and 500 mg/kg bw/d for 14 days to four rats per test group and sex. At 500 mg/kg bw/d, one male animal was found dead on study day 10 and one further male was sacrificed in moribund state due to severe clinical findings (gasping, labored respiration, respiration sounds) on study day 10. Furthermore, significantly reduced food consumption, decreased body weights, and hematological as well as gross pathological findings were observed at this dose level. At 150 mg/kg bw/d, no adverse findings were observed.
Based on the above-mentioned effects, the following dose levels were selected: 25, 75 and 250 mg/kg bw/d.

- Fasting period before blood sampling for clinical biochemistry: 16-20 hours

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes (for any signs of morbidity, pertinent behavioral changes and/or signs of overt toxicity; parturition and lactation behavior of the dams)
- Time schedule: at least once daily
- A check for moribund or dead animals was made twice daily on working days or once daily (Saturday, Sunday or on public holidays).

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once prior to the first administration (day 0) and at weekly intervals during the administration period
- Examined parameters: abnormal behavior in handling, fur, skin, posture, salivation, respiration, activity/arousal level, tremors, convulsions, abnormal movements, gait abnormalities, lacrimation, palpebral closure, exophthalmos (protruding eyeball), assessment of the feces excreted during the examination (appearance/consistency), assessment of the urine excreted during the examination, pupil size

BODY WEIGHT: Yes
- Time schedule for examinations: once a week at the same time of the day (in the morning) until sacrifice
• During the mating period, the females were weighed on the day of positive evidence of sperm (GD 0) and on GD 7, 14 and 20.
• Females with litter were weighed on the day of parturition (PND 0), PNDs 4, 7, 10 and 13.
• Females without positive evidence of sperm, without litter and females after weaning (PND 13) were weighed weekly.

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Time schedule for examinations: once a week with the following exceptions:
• Food consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (male and female parental animals).
• Food consumption of the females with evidence of sperm was determined on GD 0 - 7, 7 - 4 and 14 - 20.
• Food consumption of the females which gave birth to a litter was determined on PND 1 - 4, 4 - 7, 7 - 10 and 10 - 13.
• Food consumption was not determined in females without positive evidence of sperm during the mating and the gestation period and in females without litter during the lactation period.

WATER CONSUMPTION: Yes
- Time schedule for examinations: once a week as representative value over a period of 3 days for the male and female parental animals, with the following exceptions:
• Water consumption was not determined after the 2nd premating week (male parental animals) and during the mating period (female parental animals)
• Water consumption of the females with evidence of sperm was determined on gestation days (GD) 0-1, 6-7, 13-14 and 19-20.
• Water consumption of the females, which gave birth to a litter was determined for PND 1-2, 3-4, 6-7 and 12-13.
• Water consumption was not determined in the females without positive evidence of sperm during mating and gestation periods and in the females without litter during lactation period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Leukocyte count (WBC), Erythrocyte count (RBC), Hemoglobin (HGB), Hematocrit (HCT), Mean corpuscular volume (MCV), Mean corpuscular hemoglobin (MCH), Mean corpuscular hemoglobin concentration (MCHC), Platelet count (PLT), Differential blood count, Reticulocytes (RETA), Prothrombin time (Hepato Quick’s test) (HQT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at termination (males); at PND 14 (females)
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: the first 5 surviving parental males and the first 5 females with litters (in order of delivery) per group
- Parameters examined: Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Alkaline phosphatase (ALP), γ-Glutamyltransferase (GGT), Sodium (NA), Potassium (K), Chloride (CL), Inorganic phosphate (INP), Calcium (CA), Urea (UREA), Creatinine (CREA), Glucose (GLUC), Total bilirubin (TBIL), Total protein (TPROT), Albumin (ALB), Globulins (GLOB), Triglycerides (TRIG), Cholesterol (CHOL), Bile acids (TBA)

NEUROBEHAVIOURAL EXAMINATION: Yes; functional observational battery and motor activity assessment (see "OTHER")

OTHER:
Functional observational battery (FOB):
- A functional observational battery was performed in the first five parental male animals per test group and the first five surviving females with litter (in order of delivery) of all test groups at the end of the administration period starting at about 10.00 h on study day 28 (males) and 55 (females).
- Examined parameters:
• Home cage observations: posture, tremors, convulsions, abnormal movements, gait, other findings
• Open field observations: behavior on removal from the cage, fur, skin, salivation, nasal discharge, lacrimation, eyes/ pupil size, posture, palpebral closure, respiration, tremors, convulsions, abnormal movements/ stereotypes, gait, activity/ arousal level, feces excreted within 2 minutes (appearance/ consistency), urine excreted within 2 minutes (amount/ color), rearing within 2 minutes, other findings
• Sensory motor tests/ reflexes: reaction to an object being moved towards the face (approach response), touch sensitivity (touch response), vision (visual placing response), pupillary reflex, pinna reflex, audition (startle response), coordination of movements (righting response), behavior during handling, vocalization, pain perception (tail pinch), grip strength of forelimbs, grip strength of hindlimbs, landing foot-splay test, other findings

Motor activity measurement:
- measured from 14:00 h onwards on the same day as the FOB was performed in the first five parental males and the first five surviving females with litter (in order of delivery) per group

Thyroid hormones (males only)
- Time schedule for collection of blood: at termination
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (about 16 to 20 hours)
- How many animals: all surviving males at termination
- Parameters examined: Total thyroxine (T4), Thyroid stimulating hormone (TSH)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 1)

HISTOPATHOLOGY: Yes (see table 1)

Other examinations:

Organ weights:
determined in all animals sacrificed on schedule: Epididymides, Ovaries, Prostate (ventral and dorsolateral part together, fixed), Seminal vesicles with coagulating glands (fixed), Testes, Thyroid glands (with parathyroid glands) (fixed), Uterus with cervix

determined in 5 animals per sex/test group sacrificed on schedule (females with litters only, same animals as used for hematological and clinical chemistry examinations): Adrenal glands (fixed), Brain, Heart, Kidneys, Liver, Spleen, Thymus (fixed)

Statistics:

see table 2

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- salivation immediately after dosing (up to 2 hours post dosing) during the whole treatment period in several male and all female animals (not considered to be adverse)
- piloerection in 3/10 females on PND 6 (assessed as treatment-related but not adverse, since this minor finding was occasionally seen in three females during one study phase)

Mortality:

no mortality observed

Description (incidence):

- One male animal of the control showed respiration sounds between premating days 3 - 5 and 7 - 9 and piloerection during premating days 4 - 9 and was sacrificed moribund on premating day 9.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- statistically significantly reduced mean body weight changes in females during lactation (PND 0 – 13: 19.4 g vs. 32.9 g in control) showing a body weight loss in the beginning of the lactation phase (PND 0 – 4: -1.5 g vs. 11.9 g in control) (assessed as treatment-related and adverse)

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- statistically significantly reduced food consumption in females during the entire premating period (up to 10 %), during gestation (GD 0 – 7: 9 %) and during several parts of the lactation period (up to 23 % below control) (assessed as treatment-related and adverse)

Food efficiency:

not examined

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

effects observed, treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- statistically significantly increased mean water consumption in males during premating days 7 - 10 (about 24 %) (not assessed as treatment-related and adverse, since this minor finding occurred only within a short treatment phase of four days and no further parameter in males was altered)
- reduced water consumption in females during premating (up to 18 % below control, without statistical significance), beginning of gestation (GD 0-1: 21 % below control, without statistical significance) and lactation (up to 23 % below control, with statistical significance) (assessed as treatment-related and adverse, since the reduction in water consumption was present during almost the whole treatment period)

Mid-dose group (75 mg/kg bw/d):
- statistically significantly reduced water consumption in females during PND 6 – 7 (about 17 % below control) (not assessed as treatment-related and adverse, since all other treatment phases were not affected)

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Low-dose group (25 mg/kg bw/d):
- significantly lower absolute reticulocyte counts in males at the end of the administration period (regarded as incidental and not treatment-related, since this alteration was not dose-dependent)

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- significantly increased glucose levels in females at the end of the administration period (regarded as incidental and not treatment-related, since the mean was within the historical control range (females, glucose 5.58 - 6.98 mmol/L))

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Low-dose group (25 mg/kg bw/d):
- weakly significantly increased absolute and relative spleen weights of males (regarded as incidental, since these changes did not show dose dependency)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

All gross findings occurred individually. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

All findings occurred either individually or were biologically equally distributed over control and treatment groups. They were considered to be incidental or spontaneous in origin and without any relation to treatment.

The one animal, sacrificed in a moribund state, showed hyperkeratosis and squamous cell hyperplasia in the forestomach, and decreased cellularity in the spleen (white pulp), thymus (cortical) and bone marrow of the sternum. All these findings were consistent with the moribund condition of the animal.

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

High-dose group (250 mg/kg bw/d):
- significantly decreased TSH values in males (regarded as incidental and not treatment-related, since the mean was within the historical control range (TSH 4.81 - 9.80 μg/L))

Dose descriptor:

NOAEL

Effect level:

75 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

water consumption and compound intake

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: no adverse effects observed up to and including the highest tested dose

Critical effects observed:

no

Table 3: Summary water consumption per animal and day

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 25 mg/kgbw/d	Test Group 2/M 75 mg/kgbw/d	Test Group 3/M 250 mg/kgbw/d
Pre-mating	d 0 -> 3	Mean [g]	17.0 n	19.1	19.5	18.3
		S.d.	5.9	3.6	4.5	5.5
		N	10	10	10	10
		Deviation Vs Control [%]		12.7	14.8	7.7
	d 7 -> 10	Mean [g]	16.8 n	20.1	19.3	20.8 *
		S.d.	2.8	3.4	3.6	3.1
		N	9	10	10	10
		Deviation Vs Control [%]		19.4	14.9	23.9

Females			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 25 mg/kgbw/d	Test Group 2/F 75 mg/kgbw/d	Test Group 3/F 250 mg/kgbw/d
Pre-mating	d 0 -> 3	Mean [g]	15.8 n	16.8	16.8	13.0
		S.d.	2.2	2.8	3.1	2.7
		N	10	10	10	10
		Deviation Vs Control [%]		6.0	6.6	-17.9
	d 7 -> 10	Mean [g]	16.6 n	17.6	18.1	14.2
		S.d.	3.1	2.7	3.6	3.3
		N	10	10	10	10
		Deviation Vs Control [%]		6.2	9.1	-14.6
Gestation	d 0 -> 1	Mean [g]	17.4 n	17.6	17.5	13.8
		S.d.	2.8	3.3	4.8	3.2
		N	9	8	9	8
		Deviation Vs Control [%]		1.0	0.5	-20.6
	d 6 -> 7	Mean [g]	23.6 n	26.8	27.0	25.3
		S.d.	2.0	4.7	5.5	5.8
		N	9	8	9	8
		Deviation Vs Control [%]		13.3	14.3	7.2
	d 13 -> 14	Mean [g]	30.3 n	31.6	33.9	36.6
		S.d.	7.3	4.9	6.5	9.8
		N	9	8	9	8
		Deviation Vs Control [%]		4.3	11.9	20.8
	d 19 -> 20	Mean [g]	32.4 n	33.4	31.0	36.5
		S.d.	2.7	5.6	4.3	7.4
		N	9	8	9	8
		Deviation Vs Control [%]		3.1	-4.3	12.7
Lactation	d 1 -> 2	Mean [g]	34.4 n	31.5	33.6	30.0
		S.d.	3.9	4.7	3.7	5.3
		N	9	8	9	8
		Deviation Vs Control [%]		-8.4	-2.1	-12.8
	d 3 -> 4	Mean [g]	46.2 n	41.2	42.6	35.7 **
		S.d.	5.0	7.3	7.6	6.5
		N	9	8	9	8
		Deviation Vs Control [%]		10.9	-7.8	-22.8
	d 6 -> 7	Mean [g]	51.4 n	46.9	42.9 *	42.2 *
		S.d.	5.6	5.8	5.1	7.8
		N	9	8	9	8
		Deviation Vs Control [%]		-8.6	-16.5	-17.8
	d 9 -> 10	Mean [g]	58.8 n	54.8	55.0	48.6 *
		S.d.	8.0	5.4	4.7	9.0
		N	9	8	9	8
		Deviation Vs Control [%]		-6.8	-6.5	-17.4
	d 12 -> 13	Mean [g]	63.0 n	62.2	57.7	52.2
		S.d.	7.6	7.6	9.1	12.1
		N	9	8	9	8
		Deviation Vs Control [%]		-1.2	-8.4	-17.1

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

d = day; n=DUNNETT

Table 4: Summary food consumption per animal and day

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 25 mg/kgbw/d	Test Group 2/M 75 mg/kgbw/d	Test Group 3/M 250 mg/kgbw/d
Pre-mating	d 0 -> 7	Mean [g]	19.6 n	22.1	22.1	20.7
		S.d.	6.3	1.5	2.6	1.6
		N	10	10	10	10
		Deviation Vs Control [%]		12.4	12.7	5.5
	d 7 -> 13	Mean [g]	21.7 n	23.0	22.9	23.1
		S.d.	1.5	1.9	2.8	1.2
		N	9	10	10	10
		Deviation Vs Control [%]		6.3	5.9	6.8
	d 0 -> 13	Mean [g]	21.6 n	22.5	22.5	21.8
		S.d.	1.5	1.6	2.6	1.3
		N	9	10	10	10
		Deviation Vs Control [%]		4.1	4.1	1.0

Females			Test Group 0/F 0 mg/kgbw/d	Test Group 1/F 25 mg/kgbw/d		Test Group 2/F 75 mg/kgbw/d		Test Group 3/F 250 mg/kgbw/d
Pre-mating	d 0 -> 7	Mean [g]	15.5 n	15.1		15.1		14.1 **
		S.d.	1.2	0.9		0.9		0.9
		N	10	10		10		10
		Deviation Vs Control [%]		-2.8		-2.5		-9.4
	d 7 -> 13	Mean [g]	16.3 n	16.1		16.0		14.6 *
		S.d.	1.6	1.9		1.2		1.0
		N	10	10		10		10
		Deviation Vs Control [%]		-0.9		-1.6		-10.3
	d 0 -> 13	Mean [g]	15.9 n	15.6		15.5		14.3 *
		S.d.	1.3	1.3		1.0		0.9
		N	10	10		10		10
		Deviation Vs Control [%]		-1.9		-2.1		-9.8
Gestation	d 0 -> 7	Mean [g]	20.3 n		19.2		19.3	18.4 *
		S.d.	1.4		1.0		1.8	1.3
		N	9		8		9	8
		Deviation Vs Control [%]			-5.4		-5.2	-9.1
	d 7 -> 14	Mean [g]	21.9 n		21.1		21.6	20.7
		S.d.	1.4		1.2		1.7	2.0
		N	9		8		9	8
		Deviation Vs Control [%]			-3.6		-1.5	-5.7
	d 14 -> 20	Mean [g]	22.9 n		23.1		22.9	22.2
		S.d.	1.6		1.9		1.9	2.1
		N	9		8		9	8
		Deviation Vs Control [%]			1.1		0.3	-2.7
	d 0 -> 20	Mean [g]	21.6 n		21.0		21.2	20.4
		S.d.	1.4		1.2		1.8	1.7
		N	9		8		9	8
		Deviation Vs Control [%]			-2.7		-2.1	-5.9
Lactation	d 1 -> 4	Mean [g]	35.6 n	32.9			33.5	27.5 **
		S.d.	2.5	2.7			2.8	6.8
		N	9	8			9	8
		Deviation Vs Control [%]		-7.7			-6.0	-22.7
	d 4 -> 7	Mean [g]	43.8 n	40.7			40.2	35.6 **
		S.d.	2.1	2.5			2.3	8.7
		N	9	8			9	8
		Deviation Vs Control [%]		-7.1			-8.2	-18.7
	d 7 -> 10	Mean [g]	52.4 n	51.7			50.0	45.4
		S.d.	3.2	3.1			3.9	12.4
		N	9	8			9	8
		Deviation Vs Control [%]		-1.4			-4.6	-13.3
	d 10 -> 13	Mean [g]	58.3 n	57.8			55.6	49.6
		S.d.	4.2	3.8			5.0	13.1
		N	9	8			9	8
		Deviation Vs Control [%]		-0.9			-4.7	-14.9
	d 1 -> 13	Mean [g]	47.5 n	45.8			44.8	39.6 *
		S.d.	2.5	2.6			3.2	9.8
		N	9	8			9	8
		Deviation Vs Control [%]		-3.7			-5.7	-16.8

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

d = day; n=DUNNETT

Table 5: Summary body weight changes [g]

Males			Test Group 0/M 0 mg/kgbw/d	Test Group 1/M 25 mg/kgbw/d	Test Group 2/M 75 mg/kgbw/d	Test Group 3/M 250 mg/kgbw/d
Pre-mating	d 0 -> 7	Mean	-1.3 n	5.4	6.3	2.4
		S.d.	30.3	4.3	4.8	5.3
		N	10	10	10	10
	d 7 -> 13	Mean	8.0 n	8.9	10.2	11.0
		S.d.	3.7	3.2	6.1	4.7
		N	9	10	10	10
	d 0 -> 13	Mean	16.3 n	14.4	16.5	13.3
		S.d.	5.9	6.7	7.5	7.6
		N	9	10	10	10
Mating	d 7 -> 14	Mean	8.6 n	7.4	12.2	7.3
		S.d.	2.7	3.6	5.8	4.9
		N	9	10	10	10

Females					Test Group 0/F 0 mg/kgbw/d		Test Group 1/F 25 mg/kgbw/d		Test Group 2/F 75 mg/kgbw/d	Test Group 3/F 250 mg/kgbw/d
Pre-mating	d 0 -> 7			Mean	0.3 n		0.9		-1.7	-1.0
				S.d.	4.8		5.2		4.2	6.6
				N	10		10		10	10
	d 7 -> 13			Mean	7.1 n		8.4		11.9	7.7
				S.d.	7.9		3.7		4.8	5.1
				N	10		10		10	10
	d 0 -> 13			Mean	7.4 n		9.3		10.2	6.7
				S.d.	5.9		5.3		4.0	7.8
				N	10		10		10	10
Gestation		d 0 -> 7	Mean			26.1 n	25.1	22.1		23.0
			S.d.			6.6	4.4	2.8		4.0
			N			9	8	9		8
		d 7 -> 14	Mean			25.7 n	21.6	26.5		24.6
			S.d.			3.3	3.7	5.4		5.8
			N			9	8	9		8
		d 14 -> 20	Mean			59.4 n	58.2	61.2		51.0
			S.d.			7.4	10.4	7.0		17.9
			N			9	8	9		8
		d 0 -> 20	Mean			111.3 n	105.0	109.8		98.5
			S.d.			13.7	11.4	9.8		23.3
			N			9	8	9		8
Lactation		d 0 -> 4	Mean			11.9 n	8.8	7.7		-1.5 *
			S.d.			11.9	9.0	8.2		10.3
			N			9	8	9		8
		d 4 -> 7	Mean			5.9 n	7.1	4.9		4.8
			S.d.			7.5	9.1	7.2		7.4
			N			9	8	9		8
		d 7 -> 10	Mean			10.0 n	12.1	12.7		12.4
			S.d.			4.7	2.9	5.4		3.2
			N			9	8	9		8
		d 10 -> 13	Mean			5.1 n	8.6	5.1		3.7
			S.d.			5.2	4.4	6.4		4.8
			N			9	8	9		8
		d 0 -> 13	Mean			32.9 n	36.6	30.4		19.4 *
			S.d.			6.1	9.5	12.1		13.1
			N			9	8	9		8

Statistic Profile = Dunnett test (two-sided), * p<=0.05, ** p <=0.01, X = Group excluded from statistics

d = day; n=DUNNETT

Conclusions:

The no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.

Executive summary:

The test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 75 and 250 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.

The oral administration of the test substance resulted in signs of systemic toxicity in females at the highest dose of 250 mg/kg bw/d. In high-dose F0 females, adverse findings consisted of a reduction in water and food consumption and a decrease in body weight change. Male animals showed no adverse signs at any tested dose.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

75 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

In a Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test (according to OECD TG 422), the test substance was administered daily as an aqueous preparation to groups of 10 male and 10 female Wistar rats (F0 animals) by gavage at doses of 25, 75 and 250 mg/kg body weight/day (mg/kg bw/d). Control animals (10 male and 10 female Wistar rats) were dosed daily with the vehicle only (deionized water). The duration of treatment covered a 2-weeks premating and mating period in both sexes (mating pairs were from the same test group), 3 days postmating in males, the entire gestation and approximately 3 weeks of lactation period in females and 4-weeks postmating for sperm negative females up to the day of scheduled sacrifice of the animals.

The oral administration of the test substance resulted in signs of systemic toxicity in females at the highest dose of 250 mg/kg bw/d. Adverse findings consisted of a reduction in water consumption

during premating (up to 18% below control), gestation (GD 0-1: 21%) and lactation (up to 23% below control, statistically significant) and, concomitantly, a statistically significant reduction in food consumption during premating, gestation and lactation (up to 10%, 9% and 23% below control, respectively). Furthermore, in high-dose females, a body weight loss and a decreased body weight change during lactation were observed.

High-, mid- and low dose (250, 75 and 25 mg/kg bw/d) males and mid- and low-dose females showed no adverse findings in any of the examined parameters.

Thus, the no observed adverse effect level (NOAEL) for general systemic toxicity was the highest dose of 250 mg/kg bw/d for male and the mid-dose of 75 mg/kg bw/d for female Wistar rats.

Justification for classification or non-classification

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. As a result the substance is not considered to be classified for specific target organ toxicity under Regulation (EC) No. 1272/2008, as amended for the 13th time in Regulation (EU) 2018/1480.