Reaction mass of 5,8,11,14-Tetraoxaoctadecane and 5,8,11,14,17-Pentaoxaheneicosane

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

combined repeated dose and reproduction / developmental screening

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2011-11-09 to 2013-05-24

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP Guideline study

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2012

Report date:

2013

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Principles of method if other than guideline:

Health Effects guidelines, OPPTS 870.3650, Combined repeated dose Toxicity study with the Reproduction/ developmental Toxicity
screening Test. EPA 712-C-00-368, July 2000.
Commission Regulation (EC) No. 440/2008, L 142, Annex Part B, May 30, 2008
‘OECD Series on principles of Good Laboratory Practice and compliance monitoring’ Document No 13 ENV/JM/MONO (2002) 9.

GLP compliance:

yes (incl. QA statement)

Remarks:

(Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, München, Germany)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

EC Number:

940-417-3

Molecular formula:

C14H30O4 C16H34O5

IUPAC Name:

5,8,11,14,17-pentaoxahenicosane; 5,8,11,14-tetraoxaoctadecane

Test material form:

other: liquid

Details on test material:

CAS No.: 31885-97-9
Chemical Name: Poly(oxy-1,2-ethanediyl),a-butyl-w-butoxy-
pH: 6-7 (20 oC, 100 g/l)
Physical State at RT: liquid
Density: 0.92 g/cm3 (20°C)
Colour: colourless
Storage Conditions: at room temperature
Safety Precautions: The routine hygienic procedures were sufficient to assure personnel health and safety.

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test System
Species/strain: Wistar rats, Crl: WI(Han) (Full Barrier)
Source: Charles River, 97633 Sulzfeld, Germany
Sex: male and female; the female animals were non-pregnant and nulliparous.
Age at the start of the treatment period: males: 9-10 weeks old, females: 8-9 weeks old.
Body weight at the allocation of the animals to the experimental groups: Females: 171 to 202 g, (mean: 186.50 g, ± 20%= 37.30 g)
Males: 237 to 285 g, (mean: 264.21 g, ± 20%= 52.84 g)
The animals were derived from a controlled full-barrier maintained breeding system (SPF). According to Art. 9.2, No. 7 of the German Act on Animal Welfare the animals were bred for experimental purposes.

Housing and Feeding Conditions
- Full barrier in an air-conditioned room
- Temperature: 22 +/- 3°C
- Relative humidity: 55 +/- 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control,
microbiological controls at regular intervals)
- The animals were kept individually in IVC cages (except during the mating period when one female will be paired with one male),
type III H, polysulphone cages on Altromin saw fibre bedding
- Certificates of food, water and bedding are filed at BSL BIOSERVICE
- Adequate acclimatisation period (at least 5 days) under laboratory conditions

Preparation of the Animals
Prior to the start of the treatment period a detailed clinical observation outside the home cage was made.
Before the first administration all animals used for the study were weighed and assigned to the experimental groups
with achieving a most homogenous variation in body weight throughout the groups of males and females.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

The test item was weighed into a tared plastic vial on a precision balance and was suspended by adding the required volume of corn oil and
further vortexing it for 2-3 minutes. Homogeneity of the test item in the vehicle was maintained by vortexing the prepared suspension
thoroughly before every dose administration. The test item formulation was prepared freshly on each administration day before the
administration procedure. The vehicle was used as control item.

The following doses were evaluated:
Control: 0 mg/kg bw
LD: 100 mg/kg bw
MD: 250 mg/kg bw
HD: 750 mg/kg bw

The highest dose level was chosen with the aim of inducing toxic effects, but no death or severe suffering.
Thereafter, a descending sequence of dose levels was selected with a view to demonstrate any dosage related response and NOAEL.
The doses were selected on the basis of data from a Dose Range Finding Study.
The animals in the control group were handled in an identical manner to the test group subjects and received the vehicle
using the same dose volume.

Dose volumes were adjusted individually based on weekly body weight measurements. The administration volume was 4 mL/kg body weight.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Each dosing concentration was analysed for nominal concentration and homogeneity of the test item in the vehicle at various intervals.
Stability analysis was performed on samples collected from the top and bottom dose levels.
Samples for the nominal concentration verification was taken in study week 1 (first week of pre mating period), study week 3 (first week of mating),
study week 5 (gestation) and study week 7 (gestation/lactation).
Samples for homogeneity were taken from the top, middle and bottom of the high dose and low dose preparation in study week 1 and 5.
Samples for stability analysis was taken from top and bottom dose level in study week 1 at 0 hr and 6 hrs.
All concentration samples were stored frozen (approximately -20°C) until the analysis was performed.
The dose formulation analysis was performed at GLP-certified contract laboratory IBACON GmbH, Arheilger Weg 17, D-64380 Rossdorf,
Germany.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a period of 54 days,
i.e. during 14 days of pre-mating and 14 days of mating in both males and females, during the gestation period and
up to post-natal day 3 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

No. of animals per sex per dose:

96 animals (12 females and 12 males /group) were included in the study.

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
Once before the first exposure, and once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and on lactation days in 5 randomly selected females (only lactating females were evaluated) outside the home cage using a functional observational battery of tests.

BODY WEIGHT: Yes
- Time schedule for examinations: The body weight was recorded once before the assignment to the experimental groups, on the first day of administration and weekly during the treatment period as well as at the end of the study. During pregnancy, females were weighed on gestation days (GD) 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum) as well as day 4 post-partum along with pups.

FOOD CONSUMPTION:
- Food consumption was measured weekly on the corresponding days of the body weight measurements after the beginning of the
dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: yes

HAEMATOLOGY: Yes
The haematological examinations (Haematocrit, haemoglobin, erythrocyte count, total and differential leucocyte count, platelet count and blood clotting time) were made in all males (Control and HD groups), six selected males (LD and MD groups) and six selected females from Control, LD, MD and
HD groups. Blood samples were taken from abdominal aorta as a part of the procedure for killing the animals.




CLINICAL CHEMISTRY: Yes
Clinical biochemistry determinations to investigate major toxic effects in tissues and, specifically, effects on kidney and liver were performed on blood samples obtained from all males (Control and HD groups), six selected males (LD and MD groups) and six selected females of each groups. Investigations of serum included sodium, potassium, glucose, total cholesterol, urea, creatinine, total protein and albumin, two enzymes indicative of hepatocellular effects (such as alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase).


URINALYSIS: Yes
Urinalysis was performed on the samples collected from six randomly selected males at the terminal sacrifice.


NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: in the week before the first treatment and during the last week of the treatment
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The males were sacrificed after the completion of the mating period (after a dosing period of 28 days) except half of the males in control and HD
groups, which were further kept for 14 days recovery post 28 days dose administration and then scheduled for necropsy. Females were subjected to necropsy on the respective post-natal day 4. Males and females were sacrificed using an anesthesia (e.g. ketamine/xylazin). The pups were killed by decapitation on post-natal day 4.
Dead pups and pups sacrificed on day 4 post-partum were carefully examined externally for gross abnormalities.
Non-pregnant females (animal no. 54, 60 and 95) were sacrificed on study day 26 from the day of sperm-positive vaginal smear as an evidence of
mating.
All animals were subjected to a detailed gross necropsy, which includes careful examination of the external surface of the body, all orifices and the
cranial, thoracic and abdominal cavities and their contents. Special attention was paid to the organs of the reproductive system.
The number of implantation sites and corpora lutea was recorded for each parental female at necropsy.

The reproductive organs of all adult animals were weighed. The wet weight of the organs (liver, kidneys, adrenals, testes, epididymides, prostate,
seminal vesicles and coagulating glands, ovaries, uterus with cervix, thymus, thyroid/parathyroid glands, spleen, brain, pituitary gland, heart))
of all males of control and high dose groups (including the animals subjected to recovery), six randomly selected males of low and mid dose groups and six randomly selected females from each group were recorded as soon as possible. Paired organs were weighed separately.


The following tissues (brain (cerebrum, cerebellum and pons), ovaries (females), spinal cord, uterus with cervix (females),
liver, vagina (females), kidneys, testes (males), adrenal glands, epididymides (males), stomach, prostate and seminal vesicles
with coagulating glands as a whole (males), small and large intestines (including Peyer´s patches), urinary bladder,
thymus, lymphnodes (mesentric and axillary), Thyroid, peripheral nerve (e.g. sciatic nerve) with skeletal muscle,
spleen, bone with bone marrow (sternum), lung and trachea pituitary gland, mammary glands, oesophagus, heart, gross lesions)
of the same selected animals from each group were preserved in 10% neutral buffered formalin except eyes, testes and epididymides
that were fixed in Modified Davidson’s Fixative for approximately 24 hours before they were transferred to 10% neutral buffered formalin.

HISTOPATHOLOGY: Yes

All organs and tissues listed were evaluated from all males of the control and high dose group assigned to main or recovery necropsy,
as well as from the following six randomly selected females from the control and high dose group (assigned to main necropsy):
Females Nos.: 51, 53, 55, 56, 58, 59, 85, 87, 89, 90, 92, 94.
In addition, liver, kidney, spleen and thyroid gland were also evaluated from the following six randomly selected males and females from the
low dose and medium dose groups: Males Nos.: 13, 15, 19, 21, 22, 24, 27, 28, 29, 32, 33, 36; Females Nos.: 63, 64, 66, 68, 71, 72, 73, 74, 75,
80, 81, 83.

Reproductive organs (ovary, uterus, cervix, vagina, testis, epididymis, prostate gland, seminal vesicle and coagulating gland) and
macroscopic changes were evaluated in all study animals.

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.

Histological processing of tissues to microscope slides was performed at the GLP-certified contract laboratory Propath UK Ltd.
(test site for tissue processing), Willow Court, Netherwood Road, Hereford HR2 6JU, England. Histopathological evaluation was
performed at the GLP-certified contract laboratory KALEIDIS – Consultancy in Histopathology (test site for histopathology),
6 rue du Gers, 68300 Saint-Louis, France. Blocking, embedding, cutting, H&E staining and scientific slide evaluation were
performed according to the corresponding SOP’s of the test sites.

Statistics:

For statistical analysis one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test was carried out to reveal any differences between control- and test groups. The student-t test was used for the comparison of recovery control and recovery high dose groups.
These statistics were performed with GraphPad Prism V.5 software (p<0.05 was considered as statistical significant).

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Effect levels

Dose descriptor:

NOAEL

Effect level:

750 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: see 'Remark'

Target system / organ toxicity

Critical effects observed:

not specified

Any other information on results incl. tables

Table 1. Body weight development [g]
Observation Parameter		Dose
		Control	100 mg/kg	250 mg/kg	750 mg/kg
Males	Initial weight	299 ± 16 (n = 12)	298 ± 15 (n = 12)	296 ± 16 (n = 12)	298 ± 16 (n = 12)
	After premating phase	336 ± 23 (n = 12)	339 ± 20 (n = 12)	332 ± 26 (n = 12)	325 ± 19 (n = 12)
	After mating phase	361 ± 28 (n = 12)	361 ± 26 (n = 12)	356 ± 29 (n = 12)	350 ± 22 (n = 12)
	After recovery phase	380 ± 40 (n = 6)	-	-	359 ± 22 (n = 6)
Females	Initial weight	190 ± 10 (n = 10)	196± 8 (n = 12)	196± 8 (n = 12)	197± 8 (n = 11)
	After premating phase	203± 9 (n = 10)	211± 12 (n = 12)	209± 7 (n = 12)	206 ± 10 (n = 11)
	Gestation day 0	207± 8 (n = 10)	216± 13 (n = 12)	213± 9 (n = 12)	213 ± 10 (n = 11)
	Gestation day 20	316± 10 (n = 10)	320± 30 (n = 12)	317± 22 (n = 12)	312 ± 21 (n = 11)
	Lactation day 0	230± 12 (n = 10)	247± 20 (n = 12)	240± 18 (n = 12)	240 ± 13 (n = 11)
	Lactation day 4	242± 11 (n = 10)	259± 20 (n = 12)	248± 15 (n = 12)	250 ± 13 (n = 11)

 

Table 2. Hematology
Observation Parameter	At the treatment termination (one day after last treatment)				After 14 days of recovery
	Control	100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
WBC [10^3/µL]	4.66 ± 0.46 (n = 6)	4.74 ± 0.73 (n = 6)	5.47 ± 0.65 (n = 5)	5.50 ± 1.33 (n = 5)	4.82 ± 0.85 (n = 6)	5.64 ± 1.25  (n = 6)
RBC [10^6/µL]	9.02 ± 0.25 (n = 6)	8.60 ± 0.34 (n = 6)	8.10 ± 0.49 * (n = 5)	8.00 ± 0.61 * (n = 5)	8.82 ± 0.24 (n = 6)	8.79 ± 0.42 (n = 6)
HGB [g/dL]	15.72 ± 0.66 (n = 6)	15.43 ± 0.27 (n = 6)	14.78 ± 0.49 (n = 5)	14.42 ± 1.03* (n = 5)	15.38 ± 0.26 (n = 6)	15.45 ± 0.55 (n = 6)
HCT [%]	45.87 ± 1.46 (n = 6)	44.68 ± 1.29 (n = 6)	42.60 ± 1.61 * (n = 5)	42.00 ± 2.82 * (n = 5)	45.42 ± 1.43 (n = 6)	46.35 ± 2.88 (n = 6)
MCV [fl]	50.83 ± 0.45 (n = 6)	52.03 ± 1.56 (n = 6)	52.68± 1.34 (n = 5)	52.56± 0.92 (n = 5)	51.48 ± 1.41 (n = 6)	52.75 ± 2.30 (n = 6)
MCH [pg]	17.40 ± 0.28 (n = 6)	17.98 ± 0.47 (n = 6)	18.26 ± 0.73 * (n = 5)	18.06 ± 0.23 (n = 5)	17.47 ± 0.59 (n = 6)	17.62 ± 0.43 (n = 6)
MCHC [g/dL]	34.25 ± 0.40 (n = 6)	34.60 ± 0.51 (n = 6)	34.66 ± 0.52 (n = 5)	34.32 ± 0.18 (n = 5)	33.92 ± 0.91 (n = 6)	33.40 ± 1.64 (n = 6)
PLT [10^3/µL]	857 ± 46 (n = 6)	802 ± 195 (n = 6)	871 ± 90 (n = 5)	961± 50 (n = 5)	856 ± 124 (n = 6)	854 ± 74 (n = 6)
PT [sec]	17.20 ± 0.56 (n = 6)	18.87 ± 7.67 (n = 6)	15.78 ± 0.89 (n = 5)	12.00 ± 0.44 (n = 5)	16.72 ± 0.60 (n = 6)	17.30 ± 0.72 (n = 6)
aPTT [sec]	19.62 ± 5.38 (n = 6)	26.57 ± 12.47 (n = 6)	32.52 ± 18.66 (n = 5)	51.52 ± 18.65 ** (n = 4)	26.47 ± 8.91 (n = 6)	39.58 ± 29.46 (n = 6)
Females
WBC [10^3/µL]	3.66 ± 0.30 (n = 6)	3.50 ± 1.19 (n = 6)	3.06 ± 0.45 (n = 6)	3.85 ± 0.60 (n = 6)	-	-
RBC [10^6/µL]	6.51 ± 0.41 (n = 6)	6.17 ± 0.55 (n = 6)	6.45 ± 0.37 (n = 6)	6.20 ± 0.35 (n = 6)	-	-
HGB [g/dL]	12.42 ± 0.83 (n = 6)	11.93 ± 0.65 (n = 6)	12.48 ± 0.66 (n = 6)	12.00 ± 0.56 (n = 6)	-	-
HCT [%]	37.12 ± 2.21 (n = 6)	35.67 ± 2.13 (n = 6)	36.70 ± 1.49 (n = 6)	36.47 ± 0.87 (n = 6)	-	-
MCV [fl]	57.08 ± 1.21 (n = 6)	58.02 ± 3.70 (n = 6)	56.98 ± 1.64 (n = 6)	58.95 ± 2.31 (n = 6)	-	-
MCH [pg]	19.08 ± 0.55 (n = 6)	19.40 ± 1.26 (n = 6)	19.38 ± 0.57 (n = 6)	19.37 ± 0.42 (n = 6)	-	-
MCHC [g/dL]	33.43 ± 0.49 (n = 6)	33.43 ± 0.66 (n = 6)	34.02 ± 0.91 (n = 6)	32.85 ± 0.92 (n = 6)	-	-
PLT [10^3/µL]	1269 ± 179 (n = 6)	1251± 149 (n = 6)	1239 ± 85 (n = 6)	1331 ± 186 (n = 6)	-	-
PT [sec]	16.76 ± 0.46 (n = 5)	16.62 ± 0.44 (n = 6)	16.56 ± 0.85 (n = 5)	17.05 ± 0.32 (n = 6)	-	-
aPTT [sec]	24.61 ± 12.35 (n = 5)	19.02 ± 4.81 (n = 5)	29.66 ± 14.11 (n = 5)	31.93 ± 10.37 (n = 6)	-	-

Table 3. Clinical Biochemistry
Observation Parameter	At the treatment termination (one day after last treatment)					After 14 days of recovery
	Control		100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
ASAT [IU/L]	18.60± 5.17 (n = 6)		26.07± 6.50 (n = 6)	22.83± 6.19 (n = 6)	20.60± 5.51 (n = 5)	29.90 ± 5.32 (n = 6)	32.30 ± 23.19 (n = 6)
ALAT [IU/L]	18.58± 2.15 (n = 6)		24.52± 2.66* (n = 6)	23.40± 2.97 (n = 6)	24.62± 5.51* (n = 5)	19.27 ± 3.26 (n = 6)	35.02 ± 41.81 (n = 6)
ALP [IU/L]	126.17± 27.69 (n = 6)		194.50± 82.28 (n = 6)	161.17± 27.20 (n = 6)	142.75± 55.78* (n = 4)	139.17 ± 35.27 (n = 6)	133.83 ± 25.44 (n = 6)
GLU [mmol/L]	16.99 ± 1.82 (n = 6)		14.50 ± 4.85 (n = 6)	13.90 ± 1.08 * (n = 6)	15.40 ± 3.61 * (n = 4)	14.79 ± 2.97 (n = 6)	16.40 ± 5.15 (n = 6)
UREA [mmol/L]	5.97 ± 0.67 (n = 6)		6.17 ± 0.63 (n = 6)	5.40± 0.67 (n = 6)	5.49± 0.52 (n = 5)	6.25 ± 0.46 (n = 6)	6.11 ± 1.50 (n = 5)
CREA [µmol/L]	27.63 ± 9.05 (n = 6)		25.14 ± 5.41 (n = 6)	24.50 ± 3.0 (n = 5)	27.24 ± 5.75 (n = 5)	24.10 ± 6.07 (n = 6)	25.65 ± 12.29 (n = 5)
CHOL [mmol/L]	0.62 ± 0.11 (n = 6)		0.81 ± 0.11 * (n = 6)	0.90 ± 0.16 * (n = 5)	0.82 ± 0.09 * (n = 5)	0.60 ± 0.16 (n = 6)	0.74 ± 0.08 (n = 6)
TP [g/L]	43.63 ± 3.28 (n = 6)		55.08 ± 1.66 * (n = 6)	51.62 ±1.91 * (n = 6)	48.30 ± 1.89 * (n = 5)	56.82 ± 2.76 (n = 6)	58.23 ± 4.77 (n = 6)
Na [mmol/L]	134.83 ± 3.54 (n = 6)		139.33 ± 1.37 * (n = 6)	140.80 ±1.48 * (n = 5)	139.00 ± 1.73 * (n = 5)	140.67 ± 1.97 (n = 6)	141.50 ± 3.39 (n = 6)
K [mmol/L]	4.84 ± 0.53 (n = 5)		4.50 ± 0.28 (n = 6)	4.24 ± 0.18 (n = 5)	4.75 ± 0.29 (n = 3)	4.67 ± 0.51 (n = 6)	5.74 ± 2.55 (n = 6)
ALBB [g/L]	32.12 ± 2.02 (n = 6)		33.80 ± 1.10 (n = 6)	33.22 ± 2.19 (n = 5)	32.22 ±1.93 (n = 5)	32.02 ± 1.27 (n = 6)	32.48 ± 2.14 (n = 5)
TBA [µmol/L]	10.84 ± 3.31 (n = 5)		30.27 ± 21.24 (n = 6)	16.19 ± 2.52 (n = 5)	13.46 ± 6.44 (n = 5)	25.03 ± 12.03 (n = 6)	14.84 ± 6.39 (n = 6)
Females
ASAT [IU/L]	27.87 ± 7.27 (n = 6)	32.57 ± 9.37 (n = 6)		26.58 ± 5.07 (n = 6)	31.28 ± 8.59 (n = 6)	-	-
ALAT [IU/L]	22.78 ± 6.29 (n = 6)	22.93 ± 4.85 (n = 6)		20.48 ± 5.67 (n = 6)	27.43 ± 12.30 (n = 6)	-	-
ALP [IU/L]	201.17 ± 120.65 (n = 6)	194.83 ± 82.27 (n = 6)		164.67 ± 71.03 (n = 6)	157.17 ± 71.74 (n = 6)	-	-
GLU [mmol/L]	7.57 ± 1.40 (n = 6)	7.66 ± 1.96 (n = 6)		7.68 ± 1.70 (n = 6)	7.48 ± 1.25 (n = 6)	-	-
UREA [mmol/L]	11.19 ± 1.28 (n = 6)	12.07 ± 2.42 (n = 6)		10.73 ± 1.47 (n = 6)	11.39 ± 0.91 (n = 6)	-	-
CREA [µmol/L]	29.58 ± 4.41 (n = 5)	24.80 ± 9.02 (n = 5)		20.33 ± 4.12 (n = 5)	27.07 ± 1.99 (n = 5)	-	-
CHOL [mmol/L]	0.73 ± 0.15 (n = 6)	0.83 ± 0.07 (n = 6)		0.81 ± 0.23 (n = 6)	0.90 ± 0.23 (n = 6)	-	-
TP [g/L]	56.00 ± 3.66 (n = 6)	58.00 ± 4.54 (n = 6)		58.05 ± 4.91 (n = 6)	56.70 ± 3.64 (n = 6)	-	-
Na [mmol/L]	144.83 ± 3.60 (n = 6)	150.67 ± 6.02 (n = 6)		148.00 ± 4.77 (n = 6)	149.17 ± 6.68 (n = 6)	-	-
K [mmol/L]	4.83 ± 0.34 (n = 6)	5.46 ± 0.85 (n = 6)		4.99 ± 0.30 (n = 6)	5.17 ± 0.35 (n = 6)	-	-
ALBB [g/L]	37.77 ± 2.81 (n = 6)	38.13 ± 7.21 (n = 6)		34.50 ± 7.56 (n = 6)	38.25 ± 2.51 (n = 6)	-	-
TBA [µmol/L]	8.71 ± 8.73 (n = 6)	8.70 ± 6.31 (n = 6)		12.02 ± 7.11 (n = 6)	14.59 ± 9.78 (n = 6)	-	-

Table 4. Terminal body weight and liver weight [g]
Observation Parameter	At the treatment termination (one day after last treatment)				After 14 days of recovery
	Control	100 mg/kg	250 mg/kg	750 mg/kg	Control	750 mg/kg
Males
Terminal Body weight [g]	357 ± 18 (n = 6)	366 ± 25 (n = 12)	360 ± 31 (n = 12)	357 ± 19 (n = 6)	387 ± 40 (n = 6)	371 ± 23  (n = 6)
Liver weight [g]	11.80 ± 0.78 (n = 6)	13.12 ± 1.62 (n = 6)	14.43 ± 2.44 * (n = 6)	16.15± 1.60* (n = 6)	13.17 ± 1.18 (n = 6)	14.46 ± 1.59 (n = 6)
Females
Terminal Body weight [g]	242 ± 11 (n = 10)	259 ± 20 (n = 12)	248 ± 15 (n = 12)	250 ± 13 (n = 11)	-	-
Liver weight [g]	9.24 ± 0.74 (n = 6)	11.17 ± 1.40 * (n = 6)	11.56 ± 0.96 * (n = 6)	13.31 ± 1.22 * (n = 6)	-	-

Applicant's summary and conclusion

Conclusions:

The repeated dose toxicity of the registration substance was investigated according to the OECD Guideline 422. The registration substance induced hemolysis at 750 mg/kg bw at 750 mg/kg bw and liver enlargement in dose-dependent manner in all treated animals. These findings were reversible and there was no indication of functional impairment of blood system and liver. The NOAEL of 750 mg/kg bw was obtained.
Based on the findings obtained in this study, no classification is assigned to the registration substance.

Executive summary:

The repeated dose toxicity of the registration substance was investigated according to the OECD Gudieline 422. Twelve males and females each were treated at doses of 0, 100, 250 and 750 mg/kg bw via gavage. The study included the investigation of the recovery effect in males.Six rats of control and 750 mg/kg bw groups were allowed to recover for 14 days.

At 750 mg/kg bw, the hematology values were altered in males. Together with histopathological findings of extramedullary hemopoiesis and golden-brown pigments in spleens, these findings were considered as indication of hemolysis. Due to the limited degree of the hematology changes and due to the observed reversibility, no adversity was assigned.

Further, liver enlargement was found in dose-dependent manner both for males and females, which were accompanied with slightly changed values of clinical biochemistry and histopathological findings of glycogen storage. As there was no indication of impairment of liver funtion and as comparable values for liver weight were obtained from the animals of the recovery group, the liver enlargement was interpreted as of adaptive nature.

The NOAEL of 750 mg/kg bw was obtained for both males and females.