4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Repeated dose toxicity: oral

Administrative data

Endpoint:

repeated dose toxicity: oral, other

Remarks:

OECD 422

Type of information:

experimental study

Adequacy of study:

key study

Study period:

05 September 2017 to 11 April 2018?????

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2018

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Developmental Toxicity Screening Test. EPA 712-C-00-368, July 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

EC Number:

281-468-5

EC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Cas Number:

83950-14-5

Molecular formula:

C24H27N2O.C2H3O2

IUPAC Name:

4-[2-[4-[benzylmethyl(ethyl)amino]phenyl]vinyl]-1-(2-hydroxyethyl)pyridinium acetate

Test material form:

liquid

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain: Wistar rats, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 14-15 weeks
- Weight at study initiation: males: 344-420 g; females: 222 - 258 g
- Fasting period before study: none
- Housing:
5 animals / sex / cage in type IV polysulphone cages or in double decker IVC cages during the premating period for both males and females and during post-mating period for males. During mating period males and females were housed together in ratio 1:1 (male to female).
After the confirmation of mating, females were kept individually during gestation/lactation period in type III H, polysulphone cages
- Diet: Altromin 1324 maintenance diet for rats and mice ad libitum
- Water: tap water, sulphur acidified to a pH of approximately 2.8 ad libitum
- Acclimation period: at least 5 days

DETAILS OF FOOD AND WATER QUALITY: Certificates of food, water and bedding are filed for two years at BSL Munich and afterwards archived at Eurofins Munich

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 55±10%
- Air changes (per hr): 10/hour
- Photoperiod (hrs dark / hrs light): 12/12

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Remarks:

aqua ad injectionem

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was weighed into a tared plastic vial on a suitable precision balance and the vehicle was added to give the appropriate final concentration of the test item.
The test item formulations were prepared at least once every 10 days (within stability time frame as given by Eurofins Munich Study No. 173717). The prepared formulation was stored at room temperature. Formulates were kept under magnetic stirring during the daily administration.

- VEHICLE : water (aqua ad injectionem)
-Dosing volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

HPLC Conditions
Column: XBridge, C18, 3.5 µm, 1 x 50 mm; (Art. Nr: 186003021, Waters z.B. BSL Säule Nr. 32)
Precolumn: C18, 4 x 2.0 mm, Art.Nr. AJ0-4286, Phenomenex
Injection volume: 15 µL
Sample storage temperature: 22°C
Oven temperature: 30°C
Flow rate: 0.5 mL/min
Solvent A: 3.8539 g/L Ammoniumacetate in water;Solvent B: Acetonitrile
Gradient: Time [min] A [%] B [%]
0.00 85 15
0.01 85 15
8.00 5 95
10.00 5 95
12.00 85 15
14.00 85 15
Run time: 14 min
Retention time: Approx. 5.4 min, main component
Detection: 254 nm

Calibration: 0.02-0.52 mg/mL linear r= 0.99996 (accuracy 99.1-101.1% of spiked)
QC samples (11.2, 21.4 and 50.3 mg/mL): 97.5-100.8% of nominal (COV 0.40-2.47%)
Stability (11.2 and 50.3 mg/mL):: 90.3-100.2% and 92.8-99.2% (stability measured over 6 hours, RT; 12 days, RT; 12 days, 2-8°C ;12 days, -15 to -35°C)
Homogeneity (11.2 and 50.3 mg/mL mg/mL): 100% (COV 0.7%) and 97.5% (COV 0.2%)

Accuracy of concentrations applied (see attached document)
control: NA
10 mg/mL: 100.5 ± 0.4%
20 mg/mL: 100.2 ± 0.9%
50 mg/mL: 100.9 ± 0.8%

Duration of treatment / exposure:

females maximum exposure of 63 days in total (at least 14 days pre-mating, up to 14 days mating, approximately 22 days of gestation and up to post-natal day 12).
males minimum of two weeks prior to mating, during the mating period and up to two weeks post-mating

Frequency of treatment:

daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Doses / concentrations 2

Dose / conc.:

100 mg/kg bw/day (nominal)

Doses / concentrations 3

Dose / conc.:

250 mg/kg bw/day (nominal)

No. of animals per sex per dose:

10/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Dose selection rationale: based on a dose range finding study (see below)

On the basis of this dose range finding reproduction/ developmental toxicity screening test with C.I. Basic Orange 60 in male and female Wistar rats (3/sex/dose) with dose levels of 100, 300 and 800 mg/kg body weight day the following conclusions can be made:
Due to worsened health and mortality both the mid dose and the high dose were lowered for survivors on day 8 to 200 mg/kg bw.
After this reduction there were slight effects on body weight and food consumption in females during lactation treated at 200 mg/kg bw. At 300/200 and 800/200 mg/kg bw orange discoloration of feaces was reported from day 6 throughout the study period. There were no adverse clinical symptoms observed during the treatment period. At necropsy, there were no macroscopic findings and no effects on organ weights.
There was a slightly lower number of pups and implantation sites and increased preimplantation loss in 1/3 females at 200 mg/kg bw. Other post-natal parameters including the number of corpora lutea were not affected. There were no other litter parameters including the sex ratio, total litter weight, male litter and female litter weight affected.
Based on the generated data the dose levels 50, 100 and 250 mg/kg bw were considered for the main OECD 422 study with C.I. Basic Orange 60.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes /
- Time schedule: clinical observations at least once a daily, mortality twice daily

DETAILED CLINICAL OBSERVATIONS: Yes in a standard arena
- Time schedule: weekly starting pre-test: spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour

BODY WEIGHT: Yes
- Time schedule for examinations: weekly in males and females premating; females on (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Time schedule for examinations: weekly in males and females premating; females on (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), on PND 4 and PND 13

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: Yes as part of the functional observations pre-treatment and in the last week of the treatment for 5 males and 5 (lactating) females/dose

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in the last week of the treatment for 5 males and 5 (lactating) females/dose from abdominal aorta (control 4 females, 50 mg/kg bw 3 females)
- Anaesthetic used for blood collection: ketamine/xylazine
- Animals fasted: Not specified
- Parameters checked: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono), eosinophils (Eos), basophils (Baso), large unstained cells (Luc), prothrombin time (PT) and activated partial thromboplastin time (aPTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in the last week of the treatment for 5 males and 5 (lactating) females/dose from abdominal aorta (50 mg/kg bw 4 females)
- Anaesthetic used for blood collection: ketamine/xylazine
- Animals fasted: Not specified;
- Parameters checked: alanine aminotransferase (ALAT); aspartate-aminotransferase (ASAT); alkaline phosphatase (AP); creatinine (Crea); total protein (TP); albumin (Alb) ;urea; total bile acids (TBA); total cholesterol (Chol); glucose (Gluc); sodium (Na); potassium (K)

URINALYSIS: Yes
- Time schedule for collection: in the last week of the treatment for 5 males and 5 (lactating) females/dose qualitatively with Henry Schein Urine Stripes
- Animals fasted: Not specified
- Parameters checked:color, appearance, specific gravity, nitrite, pH-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: pre-treatment and in the last week of the treatment for 5 males and 5 (lactating) females/dose
- Battery of functions tested: Sensory reactivity to different modalities, grip strength and motor activity (2 minutes) assessment and other functional observations as well as rearing (supported and not supported), urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy

IMMUNOLOGY: Yes
- Time schedule for examinations: in the last week of the treatment for all males and (lactating) females/dose
- Dose groups that were examined: males only
- Parameters checked: thyroxine (T4)

OTHER: estrous cycle
Estrous cycles were monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Sacrifice and pathology:

ORGAN WEIGHTS: Yes from 5 males and 5 females per dose groups
thymus, thyroid/parathyroid glands (from all adult males and females (weighed after fixation (complete weight)), liver, kidneys (paired weight), spleen, adrenals (paired weight), brain, pituitary gland, heart
Reproductive organs (testes, epididymides, prostate with seminal vesicles and coagulating glands, uterus with cervix and ovaries) were weighed from all animals.

GROSS PATHOLOGY: Yes (see table)

HISTOPATHOLOGY: Yes (see table) A full histopathology was carried out on the preserved organs and tissues of all animals of the control and high dose groups which were sacrificed at the end of the treatment period and the animal that died.
Ovaries, uterus with cervix, vagina, testes, epididymides, accessory sex organs (prostate, seminal vesicles with coagulating glands as a whole), the thyroid/parathyroid glands and all organs showing macroscopic lesions of all adult animals were preserved in 4 % neutral-buffered formaldehyde, except for testes and epididymides which were preserved in modified Davidson’s Solution for 24 hours and then transferred to 70 % ethanol.

Other examinations:

estrous cycle:: monitored before treatment initiation to select for the study females with regular estrus cyclicity. Vaginal smears were also examined daily from the beginning of the treatment period until evidence of mating.

Statistics:

The evaluation included the relationship between the dosing of the test item and the presence or absence, incidence and severity of abnormalities, including gross lesions, identified target organs, infertility, clinical abnormalities, affected reproductive and litter performance, body weight changes, effects on mortality and any other toxic effects.
Toxicology and pathology data were captured either on paper according to appropriate SOPs or using the validated computerised system Ascentos® System (version 1.1.3, Pathology Data Systems Ltd.).
A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The main findings included diarrhoea, discoloured faeces, moving of bedding and increased salivation that increased in incidence with dose
see overview table

Mortality:

mortality observed, non-treatment-related

Description (incidence):

Four female animals died during the treatment period. Animal no. 52 in the LD group was found dead on premating day 5, animal no. 62 in the MD group on mating day 1 and in the HD group animal no. 71 on premating day 2 and animal no. 80 on premating day 10. All remaining animals survived their scheduled study period.
Histopathologically, there was no morphological indicator for toxicity in any organ or tissue examined. However, due to the detection of alveolar fluid in the lungs of one descendent animal at histopathological evaluation (animal no. 80) and findings seen at necropsy (animal no. 52 and 62), it is considered that gavage error might have caused death in these female animals.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

see table

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

see table

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

checked under functional observations in the animals used for behavioural assessment (5/sex/dose) pre-test and during the week of termination. Data only available in individual tables

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males: at 250 mg/kg bw non-sign decreased aPTT (-32%)
females: no treatment related effects

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

males: non significant effects on ALAT, ASAT, ALP, urea and bile acids (see overview table)
females: at 50 mg/kg bw ASAT sign decrease (32%); at 250 mg/kg bw ASAT sign decrease (29%) within historical control values and without dose effect relationship
non-significant effects on ALAT and bile acids (see overview table)

(Historical control data range- Male- ALAT: 14.8- 126.5 U/L, ASAT: 42.1 -129.3 U/L; Female- ALAT: 4.6- 113.7 U/L, ASAT: 30.3 -148.1 U/L).

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

only table with individual data available (see table)
High protein levels in male and females of all groups including control group.
Slightly elevated levels of bilirubin in one male animal of the HD group and urobilinogen in one female HD animal are considered to be without toxicological relevance as there were no correlating clinical chemical or histopathological findings observed

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

No relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups. (see table)
Only individual tables available for most parameters

Immunological findings:

no effects observed

Description (incidence and severity):

There was no test item related effect on serum T4 level of terminally sacrificed male animals (see table)

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Significantly increased abs and/or rel liver weights in males and females at mid dose and high dose (15 and 14% compared to controls), no histopathological findings and no dose response
Incidental effects within historical control values (see overview table)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

deaths
For female animal no. 52, which was found dead on premating day 5, the liver showed dark red discolouration and the lung was filled with orange fluid, for animal no. 62 in the female MD group the thoracic segment was filled with test item on mating day 1. In the HD group two female animals were found dead during the premating period (animal no. 71 on day 2, animal no. 80 on day 10). Animal no. 71 showed general autolysis and gas filled ileum at necropsy. The stomach and intestine (duodenum, jejunum, ileum, cecum) were filled with test item. Additionally, the lung was red coloured. A gas filled stomach was recorded for animal no. 80.

survivors:no reatment related effects

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no histological findings that distinguished controls from test item-treated animals.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

Pre-mating estrous cycle in days: 4.00, 4.00, 4.00 and 4.17 d for control low dose, mid dose and high dose

Details on results:

No mortality occurred in the male control or any of the male dose groups during the treatment period of the study. Four female animals died during the treatment period most likely due to gavage errors. Clinical signs that could be related to treatment include increased salivation, discoloration of the faeces, diarrhea and abnormal breathing. In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. In addition there were no effects on body weight and food consumption. Effects on blood were limited to a decreased aPTT in high dosed males. Clinical biochemistry findings included a significant decrease of ASAT in females at 50 and 250 mg/kg bw , that was within historical control data. Parameters measured during urine analysis were within normal ranges. In survivors there were no treatment related macroscopic effects. Liver weights in females at 100 and 250 mg/kg bw were significantly increased but there were no histopathological findings in these livers. In absence of a clear dose response this effect was considered not related to treatment. Other effects on organ weights were considered incidental and not related to treatment. There were no histological findings that distinguished controls from test item-treated animals.

Effect levels

Dose descriptor:

NOAEL

Effect level:

250 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related effects observed

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

Dose	0		50		100		250		Treatment related
Endpoint	M	F	M	F	M	F	M	F
Mortality	0/10	0/10	0/10	1/10	0/10	1/10	0/10	2/10	No
Clinical signs -diarrhoea -discoloured faeces -moving of bedding -increased salivation -abnormal breathing			10 4	9 1	10 10	9 9 9 1	5 10 10 10 2	1 8 8 10 4	Yes
Body weight (gain)	NTRE								No
Food consumption	NTRE								No
Behavioral effects (pre-test +post-mating/lact)	NTRE								No
Motoractivity (during 2 min)	NTRE								No
Estrous cycle (pre-test)	NTRE (4 days)								No
Pre-coital interval		2.5		2.6		2.4		3.5
Thyroxine (T4) (males)	NTRE								No
Copulation index (%)		100		100		100		100	No
Fertility index (%)		100		100		88.9		100	No
Gestation length	NTRE (22-23 days)								No
Delivery index (%)		100		100		100		100	No
Live litters		10		9		8		8	No
Corpora lutea		13.4		13.7		12.9		13.5	No
Implementation sites (mean)		13.2		13.6		12.9		13.3	No
Litter size (mean)		12.2		11.6		12.4		12.1	No
Haematology								aPTT ↓(32% ns)	Within historical controls
Clinical biochemistry			ALAT↑ (25% ns) ALP ↑ (16% ns)	ALAT↓ (29% ns) ASAT ↓ (32%) Bile ac↓ (29% ns)	ALAT↑ (23% ns) ASAT ↓ (8% ns) ALP↑ (15% ns)	ALAT↓ (18% ns) ASAT ↓ (20% ns)	ALAT↑ (38% ns) ASAT ↓ (12 % ns) ALP↑ (10% ns) Urea↑ (9% ns) Bile ac↓ (10 % ns)	ALAT↓ (15% ns) ASAT ↓ (29%) Bile ac↓ (36% ns)	No
Urinalysis	NTRE								no
Organ weights (values rel. to bw)			Kidneys↓ (12%)	Thymus↓ (13% ns) Adrenals ↑ (7% ns)		Thymus↓ (20% ns) Adrenals ↑ (26% ns) Liver ↑ (15%)	Spleen↑(11% ns)	Thyroid↑ (23% ns) Thymus↓ (28% ns) Adrenals ↑ (20% ns) Liver↑ (14%)	No
Marcoscopy (survivors)	NTRE								No
Histopathology	NTRE								No

NTRE= no treatment related effects

↑/↓= increased/decreased l

% compared to controls

Functional Observations – Males – Summary

Group		Body Temperature (°C)		Rearing Supported (Count)		Rearing not Supported (Count)		Urination (Count)		Defecation (Count)
		Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment
C	Mean	38.2	38.0	4.4	6.2	0.4	0.2	0.6	0.2	1.6	1.6
	SD	0.2	0.2	0.9	1.5	0.9	0.4	0.9	0.4	1.7	1.1
	N	5	5	5	5	5	5	5	5	5	5
LD	Mean	38.1	38.1	4.4	6.6	0.6	0.4	0.4	0.0	1.4	1.0
	SD	0.3	0.3	1.7	1.9	0.9	0.5	0.9	0.0	1.7	1.0
	N	5	5	5	5	5	5	5	5	5	5
MD	Mean	38.2	38.3	4.4	6.6	0.0	0.6	1.2	0.4	1.2	0.8
	SD	0.3	0.2	0.9	1.9	0.0	0.5	1.3	0.9	0.8	1.3
	N	5	5	5	5	5	5	5	5	5	5
HD	Mean	38.2	38.4	6.0	6.2	0.4	0.4	0.6	0.4	1.8	0.8
	SD	0.4	0.3	0.7	1.3	0.5	0.5	0.9	0.9	1.8	0.8
	N	5	5	5	5	5	5	5	5	5	5

 

 

Functional Observations – Females – Summary

Group		Body Temperature (°C)		Rearing Supported (Count)		Rearing not Supported (Count)		Urination (Count)		Defecation (Count)
		Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment	Before Treatment	Last Week of Treatment
C	Mean	38.3	38.1	4.0	4.8	0.4	0.2	0.4	1.8	0.6	1.4
	SD	0.2	0.2	0.7	1.8	0.9	0.4	0.5	1.3	0.9	1.7
	N	5	5	5	5	5	5	5	5	5	5
LD	Mean	38.3	38.1	4.2	5.2	0.4	0.4	0.6	1.0	0.6	2.2
	SD	0.2	0.3	0.8	1.5	0.9	0.5	0.9	0.7	0.9	0.8
	N	5	5	5	5	5	5	5	5	5	5
MD	Mean	38.3	38.2	5.0	5.6	0.4	0.4	0.2	0.4	0.6	1.8
	SD	0.1	0.3	1.0	1.3	0.9	0.9	0.4	0.5	0.9	1.5
	N	5	5	5	5	5	5	5	5	5	5
HD	Mean	-	38.2	4.4	5.2	0.4	0.6	0.4	0.2	1.2	1.8
	SD	-	0.2	1.1	2.3	0.9	0.9	0.5	0.4	1.3	0.8
	N	-	5	5	5	5	5	5	5	5	5

 

 

Mean Body Weight (g) – Males

Group		Premating			Mating and Postmating		Terminal Sacrifice
		Day 1	Day 7	Day 14	Day 7	Day 14
C	Mean	371.90	380.00	390.30	396.70	405.00	408.20
	SD	20.04	20.55	22.90	26.77	30.67	30.61
	N	10	10	10	10	10	10
LD	Mean	368.20	377.60	386.80	391.30	398.10	401.50
	SD	16.46	14.65	17.51	19.05	21.91	20.87
	N	10	10	10	10	10	10
	%	99	99	99	99	98	98
MD	Mean	371.00	380.10	389.60	392.70	396.40	399.60
	SD	25.32	27.08	27.72	31.42	35.06	35.71
	N	10	10	10	10	10	10
	%	100	100	100	99	98	98
HD	Mean	375.70	381.00	382.00	387.60	396.00	400.40
	SD	20.49	27.65	33.18	32.57	34.29	35.39
	N	10	10	10	10	10	10
	%	101	100	98	98	98	98

 

Mean Body Weight (g) – Females

Group		Premating			Gestation				Lactation
		Day 1	Day 7	Day 14	Day 0	Day 7	Day 14	Day 20	Day 0	Day 4	Day 13
C	Mean	236.40	236.90	242.80	242.70	267.80	293.50	359.50	278.00	292.00	302.90
	SD	7.38	10.28	10.42	13.66	15.31	16.67	21.38	14.03	18.21	17.57
	N	10	10	10	10	10	10	10	10	10	10
LD	Mean	238.80	241.44	248.33	248.22	273.78	299.22	361.00	286.22	295.22	306.22
	SD	10.72	8.60	10.61	10.80	13.18	17.10	18.68	23.83	21.06	9.87
	N	10	9	9	9	9	9	8	9	9	9
	%	101.0	101.9	102.3	102.3	102.2	101.9	100.4	103.0	101.1	101.1
MD	Mean	237.40	238.40	244.80	240.33	265.57	287.14	355.29	288.25	284.71	297.38
	SD	9.47	9.30	11.02	8.26	10.42	10.37	11.34	19.59	9.30	15.54
	N	10	10	10	6	7	7	7	8	7	8
	%	100.4	100.6	100.8	99.0	99.2	97.8	98.8	103.7	97.5	98.2
HD	Mean	238.10	236.56	247.50	247.38	268.88	297.13	369.63	282.38	301.50	311.38
	SD	8.25	11.75	7.29	8.26	9.58	7.30	10.36	6.52	7.98	7.07
	N	10	9	8	8	8	8	8	8	8	8
	%	100.7	99.9	101.9	101.9	100.4	101.2	102.8	101.6	103.3	102.8

 

Individual Urinalysis – Males

Group

Animal No.

Ery

UBG

BIL

Protein

Nitrite

Ket

Glucose

pH

Specific Gravity

Leuc

Colour / Appearance

Units

cells/µL

mg/dL

mg/dL

mg/dL

-

mg/dL

mg/dL

-

-

cells/µL

-

C

1

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

2

ca.10

norm.

neg.

30

neg.

neg.

20

7

1.005

ca.25

colourless

5

ca.10

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

colourless

8

ca.10

norm.

neg.

500

neg.

25

neg.

8

1.005

ca. 500

colourless

10

ca.10

norm.

neg.

30

neg.

neg.

neg.

9

1.000

ca. 25

colourless

LD

11

ca.10

norm.

neg.

100

neg.

neg.

neg.

7

1.005

ca.25

colourless

12

ca.10

norm.

neg.

30

neg.

neg.

neg.

9

1.005

ca.25

colourless

13

ca.10

norm.

neg.

100

neg.

25

20

8

1.015

ca.25

yellowish

17

ca.10

norm.

neg.

30

neg.

neg.

neg.

7

1.005

ca.25

yellowish

18

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

MD

21

neg.

norm.

neg.

100

neg.

25

neg.

8

1.005

ca.25

yellowish

23

ca.10

norm.

neg.

100

neg.

neg.

neg.

8

1.005

ca.25

colourless

24

ca.10

norm.

neg.

30

neg.

neg.

neg.

9

1.005

ca.25

colourless

27

ca.10

norm.

neg.

30

neg.

neg.

neg.

8

1.005

neg.

colourless

28

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

i.s.

HD

32

ca.10

norm.

neg.

100

neg.

neg.

20

7

1.015

ca.25

yellowish

34

ca.10

norm.

1

100

neg.

25

neg.

9

1.025

ca.25

colourless

38

ca.10

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

colourless

39

neg.

norm.

neg.

30

neg.

neg.

neg.

9

1.005

ca.25

colourless

40

neg.

norm.

neg.

100

neg.

neg.

neg.

8

1.010

ca.25

colourless

neg. = negative; norm. = normal; i.s. = insufficient sample volume; n.a. = no data availible

 

 

Individual Urinalysis – Females

Group

Animal No.

Ery

UBG

BIL

Protein

Nitrite

Ket

Glucose

pH

Specific Gravity

Leuc

Colour / Appearance

Units

cells/µL

mg/dL

mg/dL

mg/dL

-

mg/dL

mg/dL

-

-

cells/µL

-

C

42

ca. 10

norm.

1

30

neg.

neg.

neg.

8

1.000

ca. 25

colourless

43

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

44

neg.

norm.

neg.

100

neg.

neg.

neg.

8

1.005

neg.

yellowish

45

ca.10

norm.

2

30

neg.

neg.

neg.

7

1.000

neg.

colourless

49

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

LD

51

ca.10

norm.

1

30

neg.

neg.

neg.

9

1.000

neg.

colourless

52*

-

-

-

-

-

-

-

-

-

-

-

53

neg.

norm.

neg.

500

neg.

neg.

neg.

8

1.005

neg.

yellow

57

ca.50

norm.

1

30

neg.

neg.

neg.

8

1.005

ca. 25

yellowish

59

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

yellowish

MD

63

ca.10

norm.

1

30

neg.

neg.

neg.

7

1.005

ca. 75

colourless

65

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

67

neg.

norm.

1

30

neg.

25

neg.

8

1.005

ca.25

yellowish

68

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

69

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

n.a.

HD

74

neg.

norm.

neg.

100

neg.

neg.

neg.

9

1.005

ca.25

yellowish

76

ca.50

2

neg.

30

neg.

25

neg.

8

1.010

ca.25

colourless

77

neg.

norm.

neg.

100

neg.

neg.

neg.

8

1.005

ca.25

yellowish

78

ca.50

norm.

neg.

30

neg.

neg.

neg.

9

1.005

ca.25

colourless

79

neg.

norm.

neg.

30

neg.

neg.

neg.

8

1.005

ca.25

yellowish

neg. = negative; pos. = positive; norm. = normal; n.a. = no data available; * = found dead

 

Mean Thyroxine (T4) Analysis – Males

Group		Male- Thyroxine (T4)
	Units	nmoL/L
C	Mean	99.20
	SD	14.16
	N	10
LD	Mean	103.18
	SD	8.84
	N	10
MD	Mean	95.94
	SD	18.85
	N	10
HD	Mean	93.20
	SD	17.19
	N	10

 

 

Applicant's summary and conclusion

Conclusions:

Based on the absence of treatment related adverse effects the NOAEL is 250 mg/kg bw.

Executive summary:

In a test according to OECD 422 the substance was administered to male and female Wistar rats at 0, 50, 100 and 250 mg/kg bw (in water) during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. During the period of administration, the animals were observed each day for signs of toxicity, body weight and food consumption were measured. Functional observations were performed for all animals before treatment and in five males and females in the last week of treatment. Hematological, clinical biochemistry and urine investigation were performed on selected males and females from each group. Blood samples from the adult males were assessed for serum levels for thyroid hormones (T4). After 14 days of treatment, animals were mated (1:1) for a maximum of 14 days. The males were sacrificed after completion of the mating period on treatment day 29 and the females were sacrificed on post natal day 13. A full histopathological evaluation of the preserved tissues was performed on high dose and control animals and dead animals. These examinations were not extended to animals of all other dosage groups as treatment-related changes were not observed in any organ/tissues of the high dose group. For the testes, a detailed qualitative examination was made; taking into account the tubular stages of the spermatogenic cycle at evaluation of additional hematoxylin-PAS (Periodic Acid Schiff) stained slides. All gross lesions macroscopically identified were examined microscopically in all animals.
No mortality occurred in the male control or any of the male dose groups during the treatment period of the study. Four female animals died during the treatment period most likely due to gavage errors. Clinical signs that could be related to treatment include increased salivation, discoloration of the faeces, diarrhea and abnormal breathing. In males and females, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. In addition there were no effects on body weight and food consumption. Effects on blood were limited to a decreased aPTT in high dosed males. Clinical biochemistry findings included a significant decrease of ASAT in females at 50 and 250 mg/kg bw, that was within historical control data. Parameters measured during urine analysis were within normal ranges. In survivors there were no treatment related macroscopic effects. Liver weights in females at 100 and 250 mg/kg bw were significantly increased but there were no histopathological findings in these livers. In absence of a clear dose response this effect was considered not related to treatment. Other effects on organ weights were considered incidental and not related to treatment. There were no gross lesions or histological findings that could be attributed to treatment with the test item. There were no histological findings on reproductive organs that distinguished controls from test item-treated animals.

The NOAEL of the substance in this study for general toxicity is considered to be 250 mg/kg bw/day.