7,7,9(or 7,9,9)-trimethyl-4,13-dioxo-3,14-dioxa-5,12-diazahexadecane-1,16-diyl bismethacrylate

Endpoint summary

Description of key information

Combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, oral (OECD 422), rat:

NOAEL = 100 mg/kg bw/day for males

NOAEL = 300 mg/kg bw/day for females

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Remarks:

screening study (OECD 422)

Type of information:

experimental study

Adequacy of study:

key study

Study period:

12 Apr - 20 Jun 2017

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline 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:

adopted in 2016

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Országos Gyógyszerészeti és Élelmezés-egészségügyi Intézet, Budapest, Hungary

Limit test:

no

Species:

rat

Strain:

other: Hsd.Han: of Wistar origin

Details on species / strain selection:

The rat is regarded as suitable species for reproduction studies and the test guideline is designed to use the rat. The Wistar rat was selected due to large experience with this strain of rat in reproduction toxicity studies and known fertility.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Toxi-Coop Zrt.Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: 83 - 85 days (males), 83 - 85 days (females)
- Weight at study initiation: 330 - 384 g (males), 208 - 246 g (females); The weight variation did not exceed ± 20 per cent of the mean weight.
- Housing: 2 animals of the same sex per cage (before mating), 1 male and 1 female per cage (mating), individually (pregnant females), 2 animals per cage (males after mating), 2 or 3 animals of the same sex per cage (recovery animals) in Type III polypropylene/polycarbonate (Size: 22 x 32 x 19 cm (width x length x height)); certified laboratory wood bedding (Lignocel® Hygienic Animal Bedding, J. Rettenmaier & Söhne GmbH+Co.KG, Rosenberg, Germany) suitable as nesting material
- Diet: ssniff® SM R/M-Z+H complete diet for rats and mice – breeding and maintenance (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 20 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants that could reasonably be expected to affect the purpose or integrity of the study. The supplier provided an analytical certificate of the standard diet for the batch used. Water quality control analysis and microbiological assessment are performed once in every six months by Government Office of Capital Budapest Department of Public Health and Medical Officer Service (Budapest,Hungary).


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70%
- Air changes (per hr): > 10
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Details on route of administration:

The route of application was selected in compliance with international guidelines. The oral route is the anticipated route of human exposure to the test item.

Vehicle:

polyethylene glycol

Remarks:

polyethylene glycol 400 (PEG 400)

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test material was formulated in the vehicle at concentrations of 20, 60 and 200 mg/mL in the formulation laboratory of the Test Facility beforehand not longer than for three days before the administration and was stored in a refrigerator until use.

VEHICLE
- Justification for use and choice of vehicle: The suitability of the chosen vehicle for the test item at the intended concentrations was analytically verified up front.
- Concentration in vehicle: 20, 60 and 20 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 15I040501 (batch number 1), 16I284004 (batch number 2)

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical control of dosing formulations (control of concentration) was performed twice during the study. Five aliquots of 1 mL of each formulation) (20, 60 and 200 mg/mL) and five aliquots of 1 mL control substance (vehicle) were taken and analyzed. The samples were stored at 5 ± 3 °C before the analysis. Concentration of the test item in the dosing formulations varied in the range of 101% to 105% in comparison to the nominal values. The formulation samples were homogenous at both analytical occasions. Recovery of the test material from formulations in the vehicle was 102 and 99% at ~1 and ~300 mg/mL concentrations, respectively. A sufficient stability and homogeneity in the chosen vehicle was verified over the range of relevant concentrations at the appropriate frequency of preparation in a separate analytical report. The test material proved to be stable at room temperature for 1 day (recovery was 106% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL) and at 5 ± 3°C for 3 days (recovery was 102% of starting concentrations at 1 mg/mL and 105% at 300 mg/mL).

Duration of treatment / exposure:

56 days (males, control and test groups)
56, 57 or 64 days (females, control and test groups, depending on the effectiveness of mating)
55 days and 14 day post-exposure observation period (recovery groups)

Frequency of treatment:

once daily at similar time (± 2 h), 7 days/week

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:

Treatment period: 12/sex (control and test groups)
Treatment and recovery period: 5/sex (control group and 1000 mg/kg bw/day)

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were chosen on the basis of the results of a preliminary toxicity screening test with the tets material in rats. The high and mid-high doses were chosen with the aim of inducing toxic effects but no mortality or severe suffering of animals. The low dose was chosen to induce no toxic effect. The mid dose was interpolated geometrically.
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily and once daily
- Cage side observations included: morbidity and mortality (twice daily), general clinical observations (once daily)

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at the times of weekly weighing, prior to and during the mating until necropsy; weekly during the recovery period

BODY WEIGHT: Yes
- Time schedule for examinations: on the first day of dosing and weekly thereafter and on the day of necropsy (parental males); on the first day of dosing and weekly thereafter and on gestation days 0, 7, 14 and 21 and on days 0 (within 24 h after parturition), 4 and 13 post-partum (parental females); on day of necropsy (females subjected to organ weighing); weekly during treatment and post-treatment observation period (recovery groups)

FOOD CONSUMPTION:
The food consumption was determined weekly by reweighing the non-consumed diet during the treatment period except mating phase (pre-mating days 7, 13, gestation days 0, 7, 14 and 21, lactation days 0, 4 and 13). Food consumption of male animals was also determined by weekly interval during post-mating period. The food consumption of animals assigned to the recovery groups were weighed by weekly interval during the treatment and post-treatment observation period.

FOOD EFFICIENCY: No

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Anaesthetic used for blood collection: Yes (Isofluran)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: white blood cell (leukocyte) count, red blood cell (erythrocyte) count, hemoglobin concentration, hematocrit (relative volume of erythrocytes), mean corpuscular (erythrocyte) volume, mean corpuscular (erythrocyte) hemoglobin, mean corpuscular (erythrocyte) hemoglobin concentration, platelet (thrombocyte) count, reticulocytes, differential white blood cell count (neutrophil, monocyte, lymphocyte, basophil, eosinophil), blood coagulation (activated partial thromboplastin time, prothrombin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of necropsy (control and test groups), at the end of the 14 days post-treatment period (recovery groups)
- Animals fasted: Yes
- How many animals: 5 males and 5 females randomly selected from each group (control and test groups), all animals (recovery groups)
- Parameters examined: alanine aminotransferase activity, aspartate aminotransferase activity, total bilirubin concentration, creatinine concentration, urea concentration, glucose concentration, cholesterol concentration, bile acids, sodium concentration, potassium concentration, albumin concentration, total protein concentration

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: during the last exposure week (Day 56) but before the blood sampling
- Dose groups that were examined: 5 male and 5 female animals randomly selected from each group
- Battery of functions tested: sensory activity / grip strength / motor activity / other: modified Irwin test

IMMUNOLOGY: No

OTHER: Determination of serum levels of thyroid hormones (T4) from all parental male animals at termination on Day 56.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
Terminally (one day after the last treatment), animals were euthanized by exsanguination after verification of deep narcosis by Isofluran and were subjected to gross necropsy (parental male animals: after the optionally extended post-mating period on Day 56; dams not selected for toxicology examinations: on post-partum days 14 - 22 or shortly thereafter (Days 57 or 64); dams selected for toxicology examinations: shortly after post-partum days 15 - 21 (Day 56); recovery animals after 14-day post-treatment observation period (Day 69). Examination of the external appearance and the appearance of the tissues and organs. Special attention was paid to the organs of the reproductive system. The number of implantation sites was recorded. The uterus with cervix, vagina, testes, epididymides (total and cauda), prostate, and seminal vesicles with coagulating glands, ovaries of all adult animals were preserved. Testes and epididymides were preserved in modified Davidson solution, all other organs in 4 % buffered formaldehyde solution. Thyroid gland was preserved from all adult males and females for the intended subsequent histopathological examination. Thyroid and parathyroid were preserved together with larynx.
All organs showing macroscopic lesions and the following organs were preserved in 4% buffered formaldehyde solution (except testes and epididymides; see above) for five male and five female animals randomly selected from each group: adrenal glands, aorta, bone with bone marrow and joint (femur), brain (representative regions: cerebrum, cerebellum and pons and medulla oblongata), eyes (lachrymal gland with Harderian glands), female mammary gland, gonads (testes with epididymides, ovaries, uterus with fallopian tube and vagina), gross lesions, heart, kidneys, large intestines (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi; inflation with fixative and then immersion), lymph nodes (submandibular, mesenteric), muscle (quadriceps), esophagus, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicle with coagulating gland, skin, small intestines (representative regions: duodenum, ileum, jejunum), spinal cord (at three levels: cervical, mid-thoracic and lumbar), spleen, sternum, stomach, thymus, thyroid + parathyroid, trachea, urinary bladder.
The following organs of male adults were weighed: brain, testes, epididymides and prostate and seminal vesicles with coagulating glands as a whole. In addition, for 5 males and females randomly selected from each group, and for recovery animals, adrenals, brain, heart, kidneys, liver, spleen and thymus were weighed.


HISTOPATHOLOGY: Yes
Detailed histological examination was performed on the ovaries, uterus, vagina, testes, epididymides, prostate and seminal vesicles with coagulating gland in all animals of control and high dose groups with special emphasis on stages of spermatogenesis in the male gonads and histopathology of interstitial testicular cell structure and on the ovaries covering the follicular, luteal, and interstitial compartments of the ovary, as well as the epithelial capsule and ovarian stroma. Additionally, these organs were processed and examined histologically in not mated male animals (1/17 at 100 mg/kg bw/day and 1/17 at 300 mg/kg bw/day), in non-pregnant female and its male pair at 100 mg/kg bw/day and in one dam at 300 mg/kg bw/day based on macroscopic observation. Full histopathology examinations were performed on the preserved organs and tissues of the randomly selected animals in the control and high dose including recovery groups. Histological examinations were performed on the liver in all animals and on organs with macroscopic findings (thymus, uterus) in the low and mid dose groups.

Statistics:

The statistical evaluation of appropriate data was performed with the statistical program package SPSS PC+4.0.
The homogeneity of variance between groups was checked by Bartlett’s homogeneity of variance test. Where no significant heterogeneity was detected a one-way analysis of variance (ANOVA) is carried out. If the obtained result was significant Duncan Multiple Range test was used to access the significance of inter-group differences. Getting significant result at Bartlett’s test, the Kruskal-Wallis analysis of variance was used and the inter-group comparisons were performed using Mann-Whitney U-test. Chi2 test was performed if feasible. For evaluation of data obtained during the recovery period, the homogeneity of variance between groups was checked by F-test. Depending on the result pooled or separate variance estimate of the Two-Sample t-test was performed. Frequency of toxic response, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment

FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool caused by the vehicle from Day 3 up to the termination of treatment
Control: alopecia on the forelimbs and on the abdomen in 1/12 animal from gestation Day 9 and gestation Day 21, respectively, up to the termination of the treatment
300 mg/kg bw/day: alopecia on the forelimbs in 1/12 animal between lactation days 4 and 14
1000 mg/kg bw/day: alopecia on the abdomen in 1/11 dams between lactation day 0 and 17

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
1000 mg/kg bw/day: statistically significant slightly increased mean body weight gain comparing to their control during the first week of the recovery period; slightly increased summarized body weight gain during the post-treatment observation period (not statistically significant)

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

MALES
1000 mg/kg bw/day: statistically significant slightly decreased mean daily food consumption during the second week of the premating period

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
100 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value; statistically significant shorter mean activated thromboplastin time
300 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes, mean concentration of hemoglobin and mean hematocrit value
1000 mg/kg bw/day: statistically significant slightly decreased mean percentage of basophil granulocytes; statistically significant slightly decreased mean percentage of lymphocytes at the end of the recovery period

FEMALES
1000 mg/kg bw/day: statistically significant decreased mean percentage of neutrophil granulocytes, statistically significant increased percentage of lymphocytes and mean platelet count

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
100 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
300 mg/kg bw/day: statistically significant slightly decreased mean concentration of albumin
1000 mg/kg bw/day: statistically significant slightly decreased mean activity of alanine aminotransferase; statistically significant slightly decreased mean concentration of potassium in the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased mean urea concentration
1000 mg/kg bw/day: statistically significant slightly increased mean urea and creatinine concentration

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):

MALES
1000 mg/kg bw/day: statistically significant slightly increased absolute kidney weight at the end of the recovery period

FEMALES
300 mg/kg bw/day: statistically significant slightly increased absolute liver weight and liver weight relative to brain weight

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

MALES
Control: thymic hemorrhage in 1/12 animal; 3 mm cavity at the renal medulla in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: pale liver in 1/12 animals, thymic hemorrhage in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: pale liver in 4/12 animals, thymic hemorrhage in 1/12 animal (non-treatment-related)

FEMALES
Control: thymic hemorrhage in 1/12 animal, hydrometra in 2/12 animals, alopecia on the skin of forelimbs and abdomen in 1/12 animal
300 mg/kg bw/day: hydrometra in 1/12 animal (non-treatment-related), dilatation of cecum in 1/12 animal (non-treatment-related), alopecia on the skin of forelimbs in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: pale liver in 3/11 dams, thymic hemorrhage in 1/11 dam (non-treatment-related), alopecia on the abdomen in 1/11 dam (non-treatment-related), slight hydrometra in 1/11 dam (non-treatment-related); slight hydrometra in 1/1 non-pregnant female (non-treatment-related)

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

MALES
Control: acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal; alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period; renal cyst in 1/5 animal
300 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; acute thymic hemorrhage in 1/5 animal (non-treatment-related)
1000 mg/kg bw/day: centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 7/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); alveolar emphysema of minimal degree in 1/5 animal at the end of the recovery period (non-treatment-related)

FEMALES
Control: dilatation of uterine horns in 2/12 animals; alveolar emphysema in 1/5 animal; acute thymic hemorrhage in 1/5 animal; hyperplasia of bronchus associated lymphoid tissue in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: dilatation of uterine horns in 1/12 animal (non-treatment-related)
1000 mg/kg bw/day: dilatation of uterine horns in 1/12 animal at the end of the treatment period and in 2/5 animals at the end of the recovery period (non-treatment-related); centrilobular microvesicular vacuolation in hepatocytes/ hepatic lipidosis in 3/12 animals; alveolar emphysema in 1/5 animal (non-treatment-related); acute thymic hemorrhage in 1/5 animal (non-treatment-related); hyperplasia of bronchus associated lymphoid tissue in 1/5 animal (non-treatment-related)

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Description (incidence and severity):

There were no statistically significant differences in the thyroid hormone (free T4) level in parental male animals compared to the control.

Details on results:

CLINICAL SIGNS
Soft stool was observed in all animals at all dose groups and was attributed to the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats. The effect occurred in single animals of control, mid and high dose groups in this study. Therefore, it was considered to be an incidental finding and not related to the treatment with the test item.

BODY WEIGHT AND BODY WEIGHT GAIN
Statistical significant slightly increased mean body weight gain was observed in males during the first week of the recovery period at the highest dose group. This resulted in a slightly higher summarized body weight gain during the post-treatment observation period and caused no significant difference with respect to the control in the mean body weight. This difference was not considered treatment-related.

FOOD CONSUMPTION
The slightly lower mean daily food consumption in males of the high dose group (1000 mg/kg bw/day) compared to controls was with minor degree and was transient and therefore was judged to be toxicologically not relevant.

HAEMATOLOGICAL FINDINGS
The differences in several haemotological parameters with respect to control were judged to be indicative of biological variation and toxicologically not relevant. Changes in basophil granulocytes, hemoglobin, hematocrit and activated thromboplastin time in treated male animals were of minor degree and not related to the doses at the end of the treatment period. Moreover, all values were well within the historical ranges and statistical significances were mainly originated from the relative high value of the control group (hemoglobin, hematocrit and activated thromboplastin time). Slight changes in the neutrophil granulocytes, lymphocytes and platelet count were only observed in female animals at the end of the post-treatment observation period but not at the end of the treatment period.

CLINICAL BIOCHEMISTRY FINDINGS
Higher concentrations of urea and creatinine were indicative of altered renal function in in female animals at 300 or 1000 mg/kg bw/day. Changes in concentrations of creatinine and urea in serum were considered to be signs of adaptation of the organ to the altered demands. Without supporting histopathological changes, these effects were judged to be toxicologically not relevant. The slight, but statistically significant differences compared to the controls with respect to albumin and potassium concentration, were considered to have little or no toxicological relevance. Decrease in enzyme activity of alanine aminotransferase has no toxicological relevance without tissue damage. Albumin concentrations changed only in the lower dose groups but not at the high dose.

GROSS PATHOLOGICAL FINDINGS
Hemorrhage in the thymus was due to circulatory disturbance developed during the exsanguination procedure. Hydrometra, related to the female sexual cycle, is a frequent observation in experimental rats. Cecum dilatation was an individual change probably due to an increased water accumulation as there were no accompanying histological findings. Alopecia on the skin is a common observation in this strain of experimental rats, which occurred with similar incidence in the control and test item treated female animals (300 and 1000 mg/kg bw/day) in this study. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.

ORGAN WEIGHTS
The slight increases in liver weights in females at 300 mg/kg bw/day were not considered to be toxicologically relevant due to the small degree and in the lack of associated histopathological alterations as well as in the absence of similar changes in the high dose group.

HISTOPATHOLOGICAL FINDINGS: NON-NEOPLASTIC
The dilatation of uterine horns observed in females is not associated with inflammatory or other pathological lesions and is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance.
The treatment related observed hepatic lipidosis was of minimal or mild degree and and is considered to be a slight reversible liver injury and it might be in connection with a disturbance of energy metabolism of affected hepatocytes.
The acute changes in the lungs (alveolar emphysema) and thymus (hemorrhage) in control or high dose treated animals were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguinations.
Hyperplasia of bronchus associated lymphoid tissue as observed in males and females of the control as well as of the highest dose group is a physiological immune-morphological phenomenon, without toxicological significance.

Dose descriptor:

NOAEL

Effect level:

100 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

gross pathology

histopathology: non-neoplastic

Critical effects observed:

yes

Lowest effective dose / conc.:

300 mg/kg bw/day (actual dose received)

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

not specified

Table 1: Summary of clinical signs in males (pre-mating, mating and post-mating periods)

Observations	Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
	Treatment period*	Recovery period			Treatment period*	Recovery period
Normal	17/17	5/5	12/12	12/12	17/17	5/5
Soft stool	17/17	5/5	12/12	12/12	17/17	5/5

Frequency of observations: number of animals (cage) with observation/number of animals (cage) examined

* Including animals of recovery group

 

Table 2: Summary of clinical signs in females

Observations	Control	100 mg/kg bw/day	300 mg/kg bw/day	600 mg/kg bw/day
Pre-mating and mating periods
Normal	12/12	12/12	12/12	12/12
Soft stool	12/12	12/12	12/12	12/12
Post-mating periods
Normal	-	1/1	-	1/1
Soft stool	-	1/1	-	1/1
Gestation period
Normal	11/12	11/11	12/12	11/11
Soft stool	12/12	11/11	12/12	11/11
Skin: Alopecia	1/12	0/11	0/12	0/11
Lactation period
Normal	11/12	11/11	11/12	10/11
Soft stool	12/12	11/11	12/12	11/11
Skin: Alopecia	1/12	0/11	1/12	1/11
Recovery animals
Normal	5/5	5/5	5/5	5/5
Soft stool	5/5	5/5	5/5	5/5

 

Table 3: Summary of body weight gain in males

Group		Body weight gain (g) between
		Pre-mating days			Mating and post-mating days						Total
		0 – 7	7 – 13	0 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	48 - 55	0 – 55
Control	Mean	17.9	17.2	35.1	14.5	11.3	10.2	7.9	10.9	11.5	101.4
	SD	4.5	4.1	6.1	4.7	4.2	6.7	5.1	3.5	4.5	17.2
	n	17	17	17	17	17	17	17	17	17	17
100 mg/kg bw/day	Mean	18.3	15.6	33.9	15.7	10.8	11.0	8.4	10.9	12.1	102.8
	SD	7.7	5.6	10.1	4.6	5.2	4.0	4.8	8.4	7.1	18.9
	n	12	12	12	12	12	12	12	12	12	12
300 mg/kg bw/day	Mean	15.8	16.6	32.4	10.8	12.7	10.2	10.0	13.6	10.8	100.4
	SD	6.2	4.1	9.1	8.5	6.8	4.3	4.0	4.7	3.7	22.2
	n	12	12	12	12	12	12	12	12	12	12
1000 mg/kg bw/day	Mean	16.3	15.0	31.3	12.2	11.5	13.1	7.4	11.2	8.0	94.6
	SD	5.6	4.3	8.1	7.5	4.6	5.4	3.4	4.9	4.3	20.0
	n	17	17	17	17	17	17	17	17	17	17
		NS	NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

Table 4: Summary of body weight gain in males of the recovery group

Group		Body weight gain (g) between
		Recovery days
		0 – 6	6 – 13	0 – 13
Control	Mean	4.8	0.8	5.6
	SD	3.8	3.1	6.5
	n	5	5	5
1000 mg/kg bw/day	Mean	11.0*	3.6	14.6
	SD	4.4	4.6	7.8
	n	5	5	5
		*	NS	NS

* p < 0.05

 

Table 5: Summary of body weight gain in females

Group		Body weight gain (g) between
		Pre-mating days			Treatment days					Total
		0 – 7	7 – 13	0 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	0 – 55
Control	Mean	8.2	7.2	15.4	6.0	2.6	7.0	4.0	1.8	39.6
	SD	6.4	7.1	6.8	4.0	7.6	4.5	2.6	4.9	10.0
	n	17	17	17	5	5	5	5	5	5
100 mg/kg bw/day	Mean	11.3	6.4	17.8	-	-	-	-	-	-
	SD	9.1	7.7	4.5	-	-	-	-	-	-
	n	12	12	12	-	-	-	-	-	-
300 mg/kg bw/day	Mean	13.3	5.1	18.3	-	-	-	-	-	-
	SD	7.1	5.0	9.0	-	-	-	-	-	-
	n	12	12	12	-	-	-	-	-	-
1000 mg/kg bw/day	Mean	10.1	8.4	18.5	8.8	-2.2	9.6	0.6	5.0	39.4
	SD	9.6	6.4	9.5	2.3	6.9	4.2	4.5	1.2	9.4
	n	17	17	17	5	5	5	5	5	5
		NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

Table 6: Summary of body weight gain in females of the recovery group

Group		Body weight gain (g) between
		Recovery days
		0 – 6	6 – 13	0 – 13
Control	Mean	0.8	0.0	0.8
	SD	7.7	1.9	7.3
	n	5	5	5
1000 mg/kg bw/day	Mean	6.6	-1.0	5.6
	SD	6.66	3.9	3.0
	n	5	5	5
		NS	NS	NS

NS = Not Significant

 

 

Table 7: Summary of food consumption in males

Group		Daily mean food consumption (g/animal/day)
		Pre-mating days		Post-mating days					Recovery period
		0 – 7	7 – 13	20 – 27	27 – 34	34 – 41	41 – 48	48 – 55	Day 0 – 6	Day 6 – 13
Control	Mean	25.9	27.4	24.9	24.9	24.5	25.0	25.1	24.4	24.6
	SD	1.93	1.46	1.59	1.86	1.25	1.26	1.54	0.55	0.35
	n	8.00	8.00	8.00	8.00	8.00	8.00	8.00	2.00	2.00
100 mg/kg bw/day	Mean	26.0	27.1	24.8	24.9	24.9	25.1	24.9	-	-
	SD	1.52	2.03	1.09	0.85	0.73	1.66	1.66	-	-
	n	6	6	5	5	6	6	6	-	-
	±%	0	-1	0	0	1	0	-1	-	-
300 mg/kg bw/day	Mean	25.8	26.2	24.4	24.6	24.6	25.1	25.1	-	-
	SD	2.22	1.31	1.38	1.16	0.94	1.27	1.16	-	-
	n	6	6	6	6	6	6	6	-	-
	±%	-1	-4	-2	-1	0	1	0	-	-
1000 mg/kg bw/day	Mean	24.7	25.2*	24.0	24.2	24.0	24.3	23.6	25.1	25.6
	SD	2.30	1.80	2.16	1.91	1.65	1.54	1.3	1.73	2.54
	n	8	8	8	8	8	8	8	2	2
	±%	-5	-8	-4	-3	-2	-3	-6	3	4
		NS	DN	NS	NS	NS	NS	NS	NS	NS

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 8: Summary of food consumption in females

Group			Daily mean food consumption (g/animal/day)
		Pre-mating days		Post-mating days						Recovery period
		0 – 7	7 – 13	13 – 20	20 – 27	27 – 34	34 – 41	41 – 48	48 – 55	Day 0 – 6	Day 6 – 13
Control	Mean	19.7	19.4	17.7	19.3	18.3	17.7	17.3	17.0	16.9	17.3
	SD	2.36	1.06	1.94	0.12	0.99	0.62	0.84	0.00	1.77	0.61
	n	8	8	2	2	2	2	2	2	2	2
100 mg/kg bw/day	Mean	19.8	19.0	-	-	-	-	-	-	-	-
	SD	1.53	1.26	-	-	-	-	-	-	-	-
	n	6	6	-	-	-	-	-	-	-	-
	±%	0	-2	-	-	-	-	-	-	-	-
300 mg/kg bw/day	Mean	18.9	18.8	-	-	-	-	-	-	-	-
	SD	1.18	1.69
	n	6	6	-	-	-	-	-	-	-	-
	±%	-4	-3	-	-	-	-	-	-	-	-
1000 mg/kg bw/day	Mean	18.6	19.0	17.4	16.5	17.8	16.6	17.5	16.8	18.6	18.9
	SD	0.96	2.00	0.62	1.92	1.03	1.16	0.45	0.13	0.12	0.02
	n	8	8	2	2	2	2	2	2	2	2
	±%	-6	-2	-2	-14	-3	-6	1	-1	10	10
		NS	NS	NS	NS	NS	NS	NS	NS	NS	NS

NS = Not Significant

 

 

Table 9: Summary of hematology

Group		Basophil [%]	Hemoglobin [g/L]	Hematocrit [L/L]	Neutrophil [%]	Lymphocyte [%]	Mean platelet count [×10E9/L]	Activated partial thromboplastin time [sec]
MALES – TREATMENT PERIOD
Control	Mean	0.16	175.40	0.47	23.80	72.40	891.60	23.60
	SD	0.05	9.34	0.03	5.06	5.74	118.00	2.60
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	0.04*	161.60**	0.43**	25.08	70.62	901.40	20.48*
	SD	0.09	4.72	0.01	5.03	5.26	88.05	0.96
	n	5	5	5	5	5	5	5
	±%	-75	-8	-9	5	-2	1	-13
300 mg/kg bw/day	Mean	0.02**	163.80**	0.44*	20.96	74.20	897.40	21.72
	SD	0.04	2.95	0.00	3.74	4.21	137.05	1.91
	n	5	5	5	5	5	5	5
	±%	-88	-7	-6	-12	2	1	-8
1000 mg/kg bw/day	Mean	0.04**	169.00	0.45	17.64	77.98	796.60	22.22
	SD	0.05	3.67	0.01	6.23	7.44	54.62	1.43
	n	5	5	5	5	5	5	5
	±%	-75	-4	-5	-26	8	-11	-6
		DN	DN	U	NS	NS	NS	DN
MALES – RECOVERY PERIOD
Control	Mean	0.08	176.20	0.47	16.44	78.94	823.80	19.48
	SD	0.04	3.11	0.01	3.44	3.44	83.96	3.49
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	0.02	107.40	0.46	22.28	72.40*	833.40	20.24
	SD	0.04	5.81	0.01	5.06	4.94	42.55	1.85
	n	5	5	5	5	5	5	5
	±%	-75	-3	-2	36	-8	1	4
		NS	NS	NS	NS	*	NS	NS
FEMALES – TREATMENT PERIOD
Control	Mean	0.10	159.20	0.43	25.68	69.78	1016.20	23.44
	SD	0.22	8.32	0.01	7.76	7.54	151.26	1.11
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	0.04	169.00	0.45	16.70	79.02	1079.80	23.94
	SD	0.09	9.67	0.02	3.57	2.94	181.70	2.09
	n	5	5	5	5	5	5	5
	±%	-60	6	4	-35	13	6	2
300 mg/kg bw/day	Mean	0.10	152.00	0.41	21.18	75.50	1064.20	22.12
	SD	0.10	15.02	0.03	9.78	10.17	195.41	1.62
	n	5	5	5	5	5	5	5
	±%	0	-5	-4	-18	8	5	-6
1000 mg/kg bw/day	Mean	0.10	162.80	0.43	21.90	73.34	996.20	21.80
	SD	0.10	5.67	0.01	4.39	5.74	79.97	0.70
	n	5	5	5	5	5	5	4
	±%	0	2	1	-15	5	-2	-7
		NS	NS	NS	NS	NS	NS	NS
FEMALES – RECOVERY PERIOD
Control	Mean	0.14	160.20	0.44	18.94	74.84	744.40	21.76
	SD	0.13	9.86	0.02	2.23	3.40	75.68	1.33
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	0.04	164.20	0.45	15.18*	80.86*	852.00*	20.30
	SD	0.09	5.45	0.01	1.92	2.28	67.62	1.18
	n	5	5	5	5	5	5	5
	±%	-71	2	3	-20	8	14	-7
		NS	NS	NS	*	*	*	NS

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

DN = Duncan´s multiple range test

 

Table 10: Summary of clinical biochemistry findings

Group		Albumin [g/L]	Alanine aminotransferase activity [U/L]	Urea [mmol/L]	Creatinine [µmol/L]	Potassium [mmol/L]
MALES – TREATMENT PERIOD
Control	Mean	37.16	70.98	8.10	28.04	4.71
	SD	1.08	18.19	0.99	3.22	0.54
	n	5	5	5	5	5
100 mg/kg bw/day	Mean	35.04**	56.78	8.22	31.24	4.67
	SD	1.33	13.38	0.90	2.22	0.15
	n	5	5	5	5	5
	±%	-6	-20	1	11	-1
300 mg/kg bw/day	Mean	35.74*	56.76	8.20	29.90	4.74
	SD	0.69	8.28	1.12	3.21	0.22
	n	5	5	5	5	5
	±%	-4	-20	1	7	1
1000 mg/kg bw/day	Mean	36.70	49.66*	8.68	30.50	4.57
	SD	0.76	6.45	1.23	1.47	0.45
	n	5	5	5	5	5
	±%	-1	-30	7	9	-3
		DN	DN	NS	NS	NS
MALES – RECOVERY PERIOD
Control	Mean	36.36	60.22	8.04	27.28	4.79
	SD	0.92	7.23	0.63	1.19	0.19
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	35.60	69.60	1.72	26.40	4.42*
	SD	0.69	11.83	0.31	3.50	0.19
	n	5	5	5	5	5
	±%	-2	16	11	-3	-8
		NS	NS	NS	NS	*
FEMALES – TREATMENT PERIOD
Control	Mean	34.24	53.30	7.87	27.56	4.39
	SD	0.46	12.42	1.32	1.44	0.47
	n	5	5	5	5	5
100 mg/kg bw/day	Mean	35.02	50.46	8.90	30.34	4.13
	SD	1.04	4.34	0.90	2.34	0.18
	n	5	5	5	5	5
	±%	2	-5	13	10	-6
300 mg/kg bw/day	Mean	34.26	64.36	10.07*	30.42	4.65
	SD	1.23	5.54	1.12	2.01	0.38
	n	5	5	5	5	5
	±%	0	21	28	10	6
1000 mg/kg bw/day	Mean	33.48	54.94	10.35*	32.30*	4.39
	SD	0.37	19.24	1.97	3.78	0.20
	n	5	5	5	5	5
	±%	-2	3	32	17	0
		NS	NS	DN	DN	NS
FEMALES – RECOVERY PERIOD
Control	Mean	39.22	53.32	7.66	31.86	4.19
	SD	1.70	14.32	0.81	4.40	0.31
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	39.04	62.18	8.24	32.98	3.95
	SD	1.69	20.47	0.66	4.23	0.15
	n	5	5	5	5	5
	±%	0	17	8	4	-6
		NS	NS	NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 11: Summary of necropsy findings

Organs	Observations	Frequency of observations per group
		Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
		Main group	Recovery group			Main group	Recovery group
MALES
	No macroscopic finding	11/12	4/5	12/12	10/12	8/12	5/5
Thymus	Hemorrhages	1/12	0/5	0/12	1/12	1/12	0/5
Kidney	3 mm cavity at the renal medulla	0/12	1/5	0/12	0/12	0/12	0/5
Liver	Pale	0/12	0/5	0/12	1/12	4/12	0/5
FEMALES - DAMS
	No macroscopic finding	8/12	5/5	11/11	9/12	6/11	3/5
Thymus	Hemorrhages	1/12	0/5	0/11	0/12	1/11	0/5
Uterus	Hydrometra	2/12	0/5	0/11	1/12	1/11	2/5
Cecum	Dilatation	0/12	0/5	0/11	0/12	3/11	0/5
Liver	Pale	0/12	0/5	0/11	0/12	3/11	0/5
Skin	Alopecia	1/12	0/5	0/11	1/12	1/11	0/5
FEMALES – NON-PREGNANT
	No macroscopic finding	NA	NA	1/1	NA	0/1	NA
Uterus	Hydrometra	NA	NA	0/1	NA	1/1	NA

Frequency of observation =number of animals with observations / number of animals examined

NA = not applicable

 

Table 12: Summary of organ weights

		Body weight [g]	Organ weight [g]		Organ weight relative to body weight [%]		Organ weight relative to brain weight [%]
Group			Liver	Kidney	Liver	Kidney	Liver	Kidney
MALES – TREATMENT PERIOD
Control	Mean	448.4	11.31	2.70	2.604	0.622	518.31	123.81
	SD	24.45	1.05	0.17	0.179	0.037	40.16	7.90
	n	12	5	5	5	5	5	5
100 mg/kg bw/day	Mean	443.5	11.65	2.62	2.632	0.595	533.25	120.14
	SD	29.02	1.86	0.22	0.235	0.039	70.18	7.46
	n	12	5	5	5	5	5	5
	±%	-1	3	-3	1	-4	3	-3
300 mg/kg bw/day	Mean	444.8	12.43	2.77	2.740	0.610	578.38	129.37
	SD	34.75	0.84	0.32	0.201	0.057	43.08	19.33
	n	12	5	5	5	5	5	5
	±%	-1	10	3	5	-2	12	4
1000 mg/kg bw/day	Mean	430.7	12.07	12.82	2.768	0.647	555.54	129.84
	SD	34.57	1.25	0.30	0.263	0.082	43.19	14.08
	n	12	5	5	5	5	5	5
	±%	-4	7	4	6	4	7	5
		NS	NS	NS	NS	NS	NS	NS
MALES – RECOVERY PERIOD
Control	Mean	448.8	11.68	2.54	2.599	0.566	436.09	116.69
	SD	23.21	1.19	0.19	0.193	0.018	45.94	7.51
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	458.2	11.93	2.82*	2.607	0.618	544.92	129.01
	SD	27.07	0.94	0.13	0.187	0.049	41.69	9.65
	n	5	5	5	5	5	5	5
	±%	2	2	11	0	9	2	11
		NS	NS	*	NS	NS	NS	NS
FEMALES – TREATMENT PERIOD
Control	Mean	248.8	7.92	1.78	3.183	0.716	392.68	88.44
	SD	7.29	0.45	0.12	0.137	0.052	15.47	7.80
	n	5	5	5	5	5	5	5
100 mg/kg bw/day	Mean	263.6	8.29	1.83	3.142	0.695	406.12	89.58
	SD	8.88	0.63	0.09	0.152	0.026	40.55	4.22
	n	5	5	5	5	5	5	5
	±%	6	5	3	-1	-3	3	1
300 mg/kg bw/day	Mean	270.8*	9.45*	1.88	3.481	0.695	469.07*	93.36
	SD	14.20	1.23	0.11	0.295	0.049	61.20	6.89
	n	5	5	5	5	5	5	5
	±%	9	19	6	9	-3	19	6
1000 mg/kg bw/day	Mean	264.0	9.00	1.94	3.404	0.737	447.20	96.67
	SD	13.11	1.05	0.13	0.288	0.063	40.25	5.75
	n	5	5	5	5	5	5	5
	±%	6	14	9	7	3	14	9
		DN	DN	NS	NS	NS	DN	NS
FEMALES – RECOVERY PERIOD
Control	Mean	255.6	6.99	1.56	2.738	0.614	350.45	78.40
	SD	18.20	0.86	0.12	0.309	0.057	47.29	6.87
	n	5	5	5	5	5	5	5
1000 mg/kg bw/day	Mean	264.6	7.17	1.67	2.705	0.630	371.57	86.39
	SD	18.70	0.81	0.12	0.118	0.021	33.63	4.98
	n	5	5	5	5	5	5	5
	±%	4	3	7	-1	3	6	10
		NS	NS	NS	NS	NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 13: Summary of histopathological findings

Organs	Observations	Incidence of observations per group
		Control		100 mg/kg bw/day	300 mg/kg bw/day	1000 mg/kg bw/day
		Main group	Recovery group			Main group	Recovery group
MALES
Kidneys	Cyst	0/5	1/5	-	-	0/5	0/5
Liver	Centrilobular vacuolation in hepatocytes	0/12	0/5	0/12	3/12	7/12	0/5
Lungs	Alveolar emphysema	0/5	1/5	-	-	1/5	1/5
	Hyperplasia of BALT	1/5	1/5	-	-	0/5	0/5
Thymus	Acute hemorrhage	1/5	0/5	-	1/1	1/5	0/5
FEMALES
Kidneys	Cyst	0/5	0/5	-	-	0/5	0/5
Liver	Centrilobular vacuolation in hepatocytes	0/12	0/5	0/11	0/11	3/12	0/5
Lungs	Alveolar emphysema	1/5	0/5	-	-	1/5	0/5
	Hyperplasia of BALT	0/5	1/5	-	-	1/5	0/5
Thymus	Acute hemorrhage	1/5	0/5	-	-	1/5	0/5
Uterus with cervix	Dilatation	2/12	0/5	0/1	1/1	1/12	2/5

Incidence of observation =number of animals with observations / number of animals examined

BALT = bronchus associated lymphoid tissue

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for systemic toxicity after oral administration via gavage of the test substance to male Hsd.Han: Wistar rats was 100 mg/kg bw/day. The NOAEL for systemic toxicity after oral administration via gavage of the test substance to female Hsd.Han: Wistar rats was 300 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable (Klimisch score 1) and consistent studies, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

System:

hepatobiliary

Organ:

liver

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

A reliable screening study regarding repeated dose toxicity is available for the test substance.

The potential toxicity of the test substance was assessed in a combined repeated dose toxicity study with the reproductive/developmental toxicity screening test in Hsd.Han: Wistar rats performed according to OECD Guideline 422 and in compliance with GLP. Three groups of 12 male and 12 female rats received the test substance in polyethylene glycol as vehicle at doses of 100, 300 or 1000 mg/kg bw/day orally via gavage. A control group of 12 animals/sex received the vehicle only. In addition, 5 animals/sex were added to the control and high dose group to assess the reversibility of any effects observed at the high dose level (recovery group). All animals were dosed prior to mating (14 days) and throughout mating. In addition, males received the test item or vehicle after mating up to the day before necropsy (altogether for 56 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 21, i.e. up to the day before necropsy (altogether for 56, 57 or 64 days). Observations and examinations included mortality, clinical signs, food consumption and body weight. Blood samples were collected for determination of serum levels of thyroid hormones (T4) from all parental male animals at termination. All parental animals were subjected to gross pathology one day after the last treatment. The body weight, brain weight and weight of the testes and epididymides, and prostate and seminal vesicles with coagulating gland (as a whole) of all adult male animals were determined. Five dams and the male mating partners were randomly selected from each group to examine further signs of toxicity such as functional observations, blood analysis (hematology and clinical chemistry), gross necropsy, organ weighing and histopathology. Based on necropsy observations, histological examination was performed on the liver of all animals and on the thymus and uterus with macroscopic findings in the low and mid dose groups. Animals allocated to the recovery group were observed for additional 14 days after termination of treatment and subjected to clinical pathology and gross pathology examinations, organ weighing and full histological examinations.

At the lowest dose (100 mg/kg bw/day) no treatment-related changes in any of the parameters were observed.

An oral dose of 300 mg/kg bw/day induced pale liver in one male animal. Histopathological examination revealed centrilobular microvesicular vacuolation in the hepatocytes (hepatic lipidosis) in male animals at the termination of treatment.

At the highest dose of 1000 mg/kg bw/day pale liver with high incidence at necropsy as well as hepatic lipidosis at histopathalogical examination was observed in male and female animals. These test item related hepatic and hepatocellular changes were considered reversible as these were not seen at the end of the 14-day post treatment observation period.

There were no test item related changes in body weight and body weight gain, food consumption, hematology, organ weights and clinical chemistry parameters. The T4 serum levels were similar between control and treatment groups. Thus, under the conditions of this study, the systemic NOAEL of the test substance following oral administration via gavage for 56 days is 100 mg/kg bw/day in male Wistar rats. The corresponding NOAEL in female Wistar rats following oral administration via gavage for 56, 57 or 64 days is 300 mg/kg bw/day.

Justification for classification or non-classification

The lowest observed adverse effect level (LOAEL) in male animals is 300 mg/kg bw/day. According to Annex I of Regulation (EC) No 1272/2008 classification as STOT RE Category 2 is applicable, when significant toxic effects observed in a 90-day repeated-dose study conducted in experimental animals are seen to occur within the guidance value ranges of 10 < C ≤ 100 mg/kg bw/day. These guidance values can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration, using dose/exposure time extrapolation similar to Haber's rule for inhalation, which states essentially that the effective dose is directly proportional to the exposure concentration and the duration of exposure. The assessment shall be done on a case-by- case basis; for a 28-day study the guidance value is increased by a factor of three. The available repeated dose toxicity study was conducted in combination with the reproductive/developmental toxicity screening test. Male animals were exposed to the test substance for 56 days. Thus, the guidance value is increased by a factor of 1.6 leading to a guidance value range of 16 < C ≤ 160 mg/kg bw/day for a classification as STOT RE Category 2. The LOAEL of 300 mg/kg/bw/day in the present study is above the guidance value for a classification with regard to repeated exposure. Thus, the available data on oral repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) No 1272/2008, and is therefore conclusive but not sufficient for classification.