Hexamethylene diisocyanate, trimers, reaction products with 2-hydroxyethyl acrylate

Endpoint summary

Description of key information

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

NOAEL = 1000 mg/kg bw/day for males and 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:

10 May - 17 Jul 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

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3650 (Combined Repeated Dose Toxicity with the Reproduction/Developmental Toxicity Screening Test)

Version / remarks:

adopted in 2000

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: 98 - 103 days (males and females)
- Weight at study initiation: 367 - 439 g (males), 221 - 267 g (females); The weight variation did not exceed ± 20% 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: 48 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
- 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. The weighed amount of the test item was heated up to 60°C in a water bath (protected from light) and filled up to the final volume with the vehicle. Formulations were prepared in the formulation laboratory of the Test Facility not longer than three days before 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 200 mg/mL
- Amount of vehicle: 5 mL/kg bw
- Lot/batch no.: 16I284004

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 97% to 102% 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 96 and 99% at ~1 and ~200 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 96% of starting concentrations at 1 mg/mL and 99% at 200 mg/mL) and at 5 ± 3°C for 3 days (recovery was 104% of starting concentrations at 1 mg/mL and 100% at 200 mg/mL).

Duration of treatment / exposure:

54 - 55 days (males, control and test groups)
54 - 56 days (females, control and test groups, depending on the effectiveness of mating)
54 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 0, 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 (Day 54; control and test groups), at the end of the 14 days post-treatment period (recovery Day 14; 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 54) 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 54.

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 Days 54, 55; dams not selected for toxicology examinations: on post-partum days 14 - 20 or shortly thereafter (Days 55, 56 or 57); dams selected for toxicology examinations: shortly after post-partum days 15 - 18 (Day 54); recovery animals after 14-day post-treatment observation period (Day 68). Examination of the external appearance and the appearance of the tissues and organs was performed. 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 one non-pregnant female animal and male animal this female cohabited with at 300 mg/kg bw/day, and in four dams 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 organs with macroscopic findings (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 from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: alopecia on the skin of the left forelimb in 1/12 animal

FEMALES
Control, 100, 300 and 1000 mg/kg bw/day: soft stool from Day 3 up to termination of treatment and on the first day of the recovery period (control and high dose)
Control: dyspnea in 1/17 animal in the control on Day 34 (gestation day 19; animal died on the same day)
100 mg/kg bw/day: alopecia in 1/12 animal between lactation days 0 and 12

Mortality:

mortality observed, non-treatment-related

Description (incidence):

FEMALES
Control: 1/12 animal died on gestation day 19 (Day 34)

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

FEMALES
100 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant decreased mean body weight gain on week 2; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
300 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant slightly increased mean body weight gain between lactation days 4 and 13
1000 mg/kg bw/day: statistically significant increased mean body weight gain during the first week of the treatment period; statistically significant increased summarised body weight gain during the pre-mating observation period; statistically significant slightly increased mean body weight on lactation day 13; statistically significant increased summarized body weight gain in the recovery group; statistically significant slightly decreased mean body weight gain during the second week of the recovery period

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

FEMALES
100 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
300 mg/kg bw/day: statistically significant slightly increased food consumption during the second week of the gestation period
1000 mg/kg bw/day: statistically significant slightly increased food consumption during the first 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, 300 and 1000 mg/kg bw/day: statistically significant increased percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes
1000 mg/kg bw/day: statistically significant increased mean percentage of neutrophil granulocytes and decreased mean percentage of lymphocytes at the end of the recovery period; statistically significant increased mean percentage of reticulocytes at the end of the recovery period

FEMALES
100, 300 and 1000 mg/kg bw/day: slightly increased percentage of neutrophil granulocytes (not statistically significant)
1000 mg/kg bw/day: statistically significant slightly decreased activated partial thromboplastin time at the end of the recovery period

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
100 mg/kg bw/day: statistically significant decreased mean activity of aspartate aminotransferase and sodium concentration
300 mg/kg bw/day: statistically significant decreased sodium concentration
1000 mg/kg bw/day: statistically significant decreased mean urea and sodium concentration; statistically significant slightly decreased sodium concentration at the end of the recovery period

FEMALES
100 mg/kg bw/day: statistically significant slightly decreased mean 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 increased mean liver weight (relative to body and brain weight) at the end of the recovery period

FEMALES
100 mg/kg bw/day: statistically significant increased mean liver weights (absolute and relative to body and brain weights)
300 mg/kg bw/day: statistically significant decreased brain weight relative to body weight; statistically significant increased body weight relative to brain weight; statistically significant decreased kidney weights (relative to body weight)
1000 mg/kg bw/day: statistically significant decreased kidney and spleen weights (relative to body weight)

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Control: pyelectasia of both kidneys in 1/12 animal; thymic hemorrhage in 2/5 animals at the end of the recovery period
1000 mg/kg bw/day: thymic hemorrhage in 1/5 animal at the end of the recovery period

FEMALE
Control: hydrometra in 1/12 animal; hemorrhage in the lungs in 1/12 animal (animal died on Day 34); hydrometra in 1/5 animal at the end of the recovery period
300 mg/kg bw/day: hydrometra in 6/12 animal
1000 mg/kg bw/day: hydrometra in 2/5 animals at the end of the recovery period

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

MALES
Control: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; hyperplasia of bronchus associated lymphoid tissue in1/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; hyperplasia of bronchus associated lymphoid tissue in1/5 animal and in 1/5 animal at the end of the recovery period

FEMALES
Control: dilatation of uterine horns in 1/11 animal and in 1/5 animal at the end of the recovery period; pulmonary alveolar emphysema of minimal degree in1/5 animal and in 1/5 animal at the end of the recovery period; alveolar emphysema (moderate degree) and acute hemorrhage (moderate degree) in the lung in one animal which dies on Day 34 (gestation day 19)
300 mg/kg bw/day: dilatation of uterine horns in 5/5 animal
1000 mg/kg bw/day: pulmonary alveolar emphysema of minimal degree in1/5 animal; acute hemorrhage in1/5 animal; dilatation of uterine horns in 2/5 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

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 considered to be caused by the vehicle. Alopecia is a common spontaneous finding in this strain of experimental rats and is seen also in untreated rats, which occurred in a single animal of low dose group in this study. This was considered to be an individual sign and not related to the test item.

MORTALITY
Histological examination of the dead female of the control group revealed alveolar emphysema and acute hemorrhage (moderate degree) in the lungs, as the cause of death, in connection with a probably shock. No other lesions were observed in the investigated organs of this animal.

BODY WEIGHT AND BODY WEIGHT GAIN
The differences in body weight and body weight gain observed in females as associated with increased food and was not judged to be of toxicological relevance. The differences in the recovery groups were of minor degree and judged to be toxicologically not relevant.

HAEMATOLOGICAL FINDING
Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males of all dose groups at the end of the treatment period were related to doses but were not proportional to doses. The differences with respect to the control were of minor degree and all values remained within the historical control range. Moreover, the changes were not accompanied with changes in white blood cell count and therefore were considered to have little or no biological significance. Effects on the percentage of neutrophil granulocytes observed infemale animals at all dose groups at the end of the treatment period were not related to doses. Effects on the percentage of neutrophil granulocytes and lymphocytes observed in males at 1000 mg/kg bw/day at the end of the recovery period was partially originated from the relative low value of the control group (13.74 ± 5.5% vs. 19.2 ± 6.5% of historical control mean). The increased mean percentage of reticulocytes in the male animals at 1000 mg/kg bw/day at the end of the recovery period was of minor degree and was not observed at the end of the treatment period; therefore, was considered to have no toxicological relevance. The slightly lower activated partial thromboplastin time in females at 1000 mg/kg bw/day at the end of the recovery period is a result of a relatively high control mean value. This difference was not observed at the end of the treatment period and was considered to have no toxicological relevance.

CLINICAL BIOCHEMISTRY FINDINGS
The slight, but statistically significant differences with respect to their controls in aspartate aminotransferase activity, urea and sodium concentration observed in males were considered to have little or no toxicological importance. Lower enzyme activity of aspartate aminotransferase and creatinine concentration was only seen in the low dose group of males and females, respectively, and has no toxicological meaning. Thus, the slight, statistically significant differences with respect to control were considered toxicologically not relevant due to the minor degree of changes or due to the lack of dose relevance. All values were within the historical control ranges.

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. The pyelectasia was an individual change in a control animal. Therefore, these macroscopic changes were considered to be independent from the treatment with the test item.

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
The dilatation of uterine horns observed in females of the control and mid-dose group is a slight neuro-hormonal phenomenon and was in connection with the normal sexual cycle (pro-estrus phase) of uterus without pathological significance. Pulmonary alveolar emphysema and acute hemorrhage observed in males and females of control and high-dose groups were considered as consequence of hypoxia, dyspnea and circulatory disturbance developed during exsanguination procedure. Hyperplasia of bronchus associated lymphoid tissue is a physiological immune-morphological phenomenon, without toxicological significance.

Dose descriptor:

NOAEL

Effect level:

>= 1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment-related effects at highest dose tested

Critical effects observed:

no

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	10/11	12/12	11/11	12/12
Soft stool	11/11	12/12	11/11	12/12
Dyspnea	1/11	0/12	0/11	0/12
Died	1/11	0/12	0/11	0/12
Lactation period
Normal	10/10	11/12	11/11	12/12
Soft stool	10/10	12/12	11/11	12/12
Skin: Alopecia	0/10	1/12	0/11	0/12
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 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	48 - 53	0 – 53
Control	Mean	-0.1	7.6	7.5	2.0	9.2	2.6	0.6	1.6	3.4	24.5
	SD	4.5	6.6	5.7	2.1	5.0	4.4	7.9	7.8	7.2	5.0
	n	17	17	17	5	5	5	5	5	5	6
100 mg/kg bw/day	Mean	3.8*	2.3*	6.1	-	-	-	-	-		-
	SD	6.9	8.2	8.3	-	-	-	-	-		-
	n	12	12	12	-	-	-	-	-		-
300 mg/kg bw/day	Mean	8.5**	6.3	14.8**	-	-	-	-	-		-
	SD	5.2	5.2	4.9	-	-	-	-	-		-
	n	12	12	12	-	-	-	-	-		-
1000 mg/kg bw/day	Mean	7.6**	8.1	15.7**	0.8	8.6	-0.6	4.8	4.2	2.4	35.8
	SD	3.8	4.1	4.3	5.1	5.0	5.3	5.0	5.8	5.0	2.3
	n	17	17	17	5	5	5	5	5	5	5
		DN	NS	DN	NS	NS	NS	NS	NS	NS	**

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 4: 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	-1.2	3.6	2.4
	SD	2.6	2.3	1.1
	n	5	5	5
1000 mg/kg bw/day	Mean	1.4	-1.4	0.0
	SD	7.4	4.2	4.6
	n	5	5	5
		NS	*	NS

* p < 0.05

NS = Not Significant

 

Table 5: 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 – 53	Day 0 – 6	Day 6 – 13
Control	Mean	15.7	15.7	17.4	17.5	17.1	16.8	16.5	17.2	17.5	18.0
	SD	1.70	0.86	0.17	0.45	0.44	0.67	1.03	0.35	1.36	0.15
	n	8	8	2	2	2	2	2	2	2	2
100 mg/kg bw/day	Mean	16.3	15.5	-	-	-	-	-	-	-	-
	SD	1.67	1.73	-	-	-	-	-	-	-	-
	n	6	6	-	-	-	-	-	-	-	-
	±%	4	-1	-	-	-	-	-	-	-	-
300 mg/kg bw/day	Mean	17.3	16.9	-	-	-	-	-	-	-	-
	SD	1.07	0.88	-	-	-	-	-	-	-	-
	n	6	6	-	-	-	-	-	-	-	-
	±%	11	7	-	-	-	-	-	-	-	-
1000 mg/kg bw/day	Mean	17.4*	16.9	18.1	18.2	18.4	18.9	17.3	19.2	18.4	18.4
	SD	1.30	1.18	1.20	1.35	1.94	2.12	1.41	2.22	1.87	1.08
	n	8	8	2	2	2	2	2	2	2	2
	±%	11	8	4	4	8	13	5	12	6	2
		DN	NS	NS	NS	NS	NS	NS	NS	NS	NS

* p < 0.05

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 6: Summary of hematology

Group		Neutrophil [%]	Lymphocyte [%]	Reticulocyte [%]	Activated partial thromboplastin time [sec]
MALES - TREATMENT PERIOD
Control	Mean	18.26	78.00	3.11	20.94
	SD	2.25	2.29	0.69	1.54
	n	5	5	5	5
100 mg/kg bw/day	Mean	24.60**	70.82**	2.94	20.96
	SD	1.87	1.94	0.43	1.14
	n	5	5	5	5
	±%	35	-9	-5	0
300 mg/kg bw/day	Mean	27.14*	67.06*	3.24	20.18
	SD	7.86	8.84	0.44	1.70
	n	5	5	5	5
	±%	49	-14	4	-4
1000 mg/kg bw/day	Mean	28.96**	66.34**	3.28	21.76
	SD	6.07	7.11	0.10	1.58
	n	5	5	5	5
	±%	59	-15	5	4
		U	U	NS	NS
MALES - RECOVERY PERIOD
Control	Mean	13.74	81.94	2.58	26.96
	SD	5.05	5.84	0.33	4.48
	n	5	5	5	5
1000 mg/kg bw/day	Mean	21.76	72.90	3.09	29.30
	SD	4.47	5.43	0.12	5.04
	n	5	5	5	5
	±%	58	-11	20	9
		*	*	*	NS
FEMALES - TREATMENT PERIOD
Control	Mean	17.78	79.00	8.14	20.70
	SD	7.08	6.90	2.82	1.63
	n	5	5	5	5
100 mg/kg bw/day	Mean	25.36	70.80	8.37	21.22
	SD	11.96	12.43	1.76	1.27
	n	5	5	5	5
	±%	43	-10	3	3
300 mg/kg bw/day	Mean	22.00	74.18	8.33	19.32
	SD	3.69	4.36	2.92	0.50
	n	5	5	5	5
	±%	24	-6	2	-7
1000 mg/kg bw/day	Mean	22.60	74.00	6.79	20.26
	SD	10.57	10.89	1.54	0.81
	n	5	5	5	5
	±%	27	-6	-17	-2
		NS	NS	NS	NS
FEMALES - RECOVERY PERIOD
Control	Mean	16.08	79.60	3.73	28.76
	SD	3.28	3.09	0.49	3.13
	n	5	5	5	5
1000 mg/kg bw/day	Mean	14.82	81.44	3.40	23.92
	SD	3.62	4.34	0.67	1.09
	n	5	5	5	5
	±%	-8	2	-9	-17
		NS	NS	NS	*

* p < 0.05, **p < 0.01

NS = Not Significant

U = Mann-Whitney U-test vs. Control

 

Table 7: Summary of clinical biochemistry findings

Group		Aspartate aminotransferase activity [U/L]	Urea [mmol/L]	Creatinine [µmol/L]	Sodium [mmol/L]
MALES - TREATMENT PERIOD
Control	Mean	117.36	7.88	26.44	142.20
	SD	12.24	1.58	1.65	1.10
	n	5	5	5	5
100 mg/kg bw/day	Mean	97.58*	7.67	25.50	140.40*
	SD	11.21	1.02	2.57	1.14
	n	5	5	5	5
	±%	-17	-3	-4	-1
300 mg/kg bw/day	Mean	126.70	7.54	29.30	139.40**
	SD	15.05	0.82	2.65	0.89
	n	5	5	5	5
	±%	8	-4	11	-2
1000 mg/kg bw/day	Mean	123.74	6.13*	27.92	138.00**
	SD	6.36	0.96	2.43	1.00
	n	5	5	5	5
	±%	5	-22	6	-3
		DN	DN	NS	DN
MALES - RECOVERY PERIOD
Control	Mean	102.94	6.65	25.00	142.20
	SD	24.99	1.29	1.12	0.45
	n	5	5	5	5
1000 mg/kg bw/day	Mean	98.82	6.66	25.82	141.20
	SD	6.72	1.12	3.44	0.84
	n	5	5	5	5
	±%	-4	0	3	-1
		NS	NS	NS	*
FEMALES - TREATMENT PERIOD
Control	Mean	127.00	11.30	26.06	135.80
	SD	18.29	1.35	2.89	1.30
	n	5	5	5	5
100 mg/kg bw/day	Mean	120.28	12.48	21.86*	135.00
	SD	24.17	1.86	1.81	1.22
	n	5	5	5	5
	±%	-5	10	-16	-1
300 mg/kg bw/day	Mean	115.62	12.23	28.26	135.00
	SD	15.47	3.29	1.73	0.71
	n	5	5	5	5
	±%	-9	8	8	-1
1000 mg/kg bw/day	Mean	101.04	11.42	24.92	134.80
	SD	13.17	2.09	2.71	1.30
	n	5	5	5	5
	±%	-20	1	-4	-1
		NS	NS	DN	NS
FEMALES - RECOVERY PERIOD
Control	Mean	88.68	7.29	30.36	141.20
	SD	6.05	1.13	3.71	1.48
	n	5	5	5	5
1000 mg/kg bw/day	Mean	92.96	7.75	31.98	140.20
	SD	18.69	1.01	4.83	1.10
	n	5	5	5	5
	±%	5	6	5	-1
		NS	NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

 

Table 8: Summary of organ weights of males

MALES – MAIN GROUP
		Body weight [g]	Organ weight [g]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	450.6	2.16	10.20	2.62	0.65
	SD	38.40	0.11	0.99	0.21	0.08
	n	12	12	5	5	5
100 mg/kg bw/day	Mean	449.0	2.17	10.37	2.59	0.66
	SD	23.69	0.08	0.92	0.17	0.05
	n	12	12	5	5	5
	±%	0	0	2	-1	1
300 mg/kg bw/day	Mean	449.4	2.15	10.81	2.63	0.63
	SD	28.72	0.15	0.65	0.22	0.09
	n	12	12	5	5	5
	±%	0	0	6	0	-3
1000 mg/kg bw/day	Mean	451.7	2.16	11.11	2.67	0.65
	SD	27.77	0.09	1.22	0.21	0.05
	n	12	12	5	5	5
	±%	0	0	9	2	-1
		NS	NS	NS	NS	NS
MALES – RECOVERY GROUP
		Body weight [g]	Organ weight [g]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	464.4	2.24	11.41	2.54	0.73
	SD	27.55	0.08	1.01	0.14	0.07
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	476.4	2.21	12.84	2.77	0.78
	SD	26.28	0.15	1.15	0.23	0.06
	n	5	5	5	5	5
	±%	3	-1	13	9	7
		NS	NS	NS	NS	NS
MALES – MAIN GROUP
			Organ weight relative to body weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	NA	0.482	2.302	0.592	0.148
	SD	NA	0.041	0.101	0.024	0.014
	n	NA	12	5	5	5
100 mg/kg bw/day	Mean	NA	0.485	2.360	0.591	0.150
	SD	NA	0.037	0.151	0.040	0.011
	n	NA	12	5	5	5
	±%	NA	1	2	0	1
300 mg/kg bw/day	Mean	NA	0.480	2.409	0.585	0.141
	SD	NA	0.042	0.153	0.038	0.014
	n	NA	12	5	5	5
	±%	NA	0	5	-1	-5
1000 mg/kg bw/day	Mean	NA	0.480	2.462	0.593	0.144
	SD	NA	0.034	0.178	0.041	0.015
	n	NA	12	5	5	5
	±%	NA	0	7	0	-2
			NS	NS	NS	NS
MALES – RECOVERY GROUP
			Organ weight relative to body weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	NA	0.483	2.455	0.547	0.157
	SD	NA	0.029	0.132	0.016	0.015
	n	NA	5	5	5	5
1000 mg/kg bw/day	Mean	NA	0.464	2.692	0.581	0.165
	SD	NA	0.029	0.137	0.036	0.018
	n	NA	5	5	5	5
	±%	NA	-4	10	6	5
			NS	*	NS	NS
MALES – MAIN GROUP
		Body weight relative to brain weight [%]	Organ weight relative to brain weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	20874.9	NA	468.94	120.48	30.06
	SD	1701.64	NA	38.24	6.99	3.14
	n	12	NA	5	5	5
100 mg/kg bw/day	Mean	20747.8	NA	482.91	120.64	30.65
	SD	1577.94	NA	50.77	10.34	2.98
	n	12	NA	5	5	5
	±%	-1	NA	3	0	2
300 mg/kg bw/day	Mean	20963.0	NA	502.37	122.02	29.37
	SD	1773.58	NA	57.17	13.79	4.36
	n	12	NA	5	5	5
	±%	0	NA	7	1	-2
1000 mg/kg bw/day	Mean	20925.5	NA	518.51	124.96	30.25
	SD	1509.23	NA	65.83	16.95	3.30
	n	12	NA	5	5	5
	±%	0	NA	11	4	1
		NS		NS	NS	NS
MALES – RECOVERY GROUP
		Body weight relative to brain weight [%]	Organ weight relative to brain weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	20763.1	NA	509.21	113.55	32.53
	SD	1309.90	NA	32.90	6.56	2.73
	n	5	NA	5	5	5
1000 mg/kg bw/day	Mean	21619.1	NA	582.79	125.87	35.64
	SD	1244.43	NA	55.10	12.42	4.72
	n	5	NA	5	5	5
	±%	4	NA	14	11	10
		NS		*	NS	NS

* p < 0.05

NS = Not Significant

NA = Not Applicable

 

Table 9: Summary of organ weights of females

FEMALES – MAIN GROUP
		Body weight [g]	Organ weight [g]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	263.8	1.97	8.73	1.88	0.66
	SD	14.34	0.06	0.59	0.15	0.07
	n	5	5	5	5	5
100 mg/kg bw/day	Mean	272.4	1.96	10.02*	1.87	0.71
	SD	20.84	0.05	1.07	0.04	0.11
	n	5	5	5	5	5
	±%	3	-1	15	-1	7
300 mg/kg bw/day	Mean	284.8	1.92	8.81	1.84	0.62
	SD	11.58	0.11	0.56	0.11	0.05
	n	5	5	5	5	5
	±%	8	-2	1	-2	-7
1000 mg/kg bw/day	Mean	277.6	1.94	8.95	1.79	0.57
	SD	12.78	0.08	0.55	0.10	0.09
	n	5	5	5	5	5
	±%	5	-2	3	-5	-14
		NS	NS	DN	NS	NS
FEMALES – RECOVERY GROUP
		Body weight [g]	Organ weight [g]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	262.2	2.05	7.74	1.71	0.59
	SD	6.38	0.04	1.10	0.16	0.05
	n	5	5	5	5	5
1000 mg/kg bw/day	Mean	266.4	2.01	7.83	1.70	0.56
	SD	12.03	0.07	0.24	0.06	0.05
	n	5	5	5	5	5
	±%	2	-2	1	-1	-6
		NS	NS	NS	NS	NS
FEMALES – MAIN GROUP
			Organ weight relative to body weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	NA	0.750	3.315	0.714	0.252
	SD	NA	0.056	0.251	0.067	0.019
	n	NA	5	5	5	5
100 mg/kg bw/day	Mean	NA	0.723	3.676*	0.689	0.260
	SD	NA	0.051	0.252	0.049	0.036
	n	NA	5	5	5	5
	±%	NA	-4	11	-4	3
300 mg/kg bw/day	Mean	NA	0.676*	3.094	0.647*	0.218
	SD	NA	0.038	0.197	0.019	0.021
	n	NA	5	5	5	5
	±%	NA	-10	-7	-9	-13
1000 mg/kg bw/day	Mean	NA	0.698	3.226	0.646*	0.206*
	SD	NA	0.024	0.158	0.020	0.025
	n	NA	5	5	5	5
	±%	NA	-7	-3	-10	-18
			DN	DN	DN	DN
FEMALES – RECOVERY GROUP
			Organ weight relative to body weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	NA	0.780	2.951	0.653	0.226
	SD	NA	0.012	0.417	0.050	0.017
	n	NA	5	5	5	5
600 mg/kg bw/day	Mean	NA	0.758	2.940	0.639	0.209
	SD	NA	0.049	0.059	0.024	0.017
	n	NA	5	5	5	5
	±%	NA	-3	0	-2	-7
			NS	NS	NS	NS
FEMALES – MAIN GROUP
		Body weight relative to brain weight [%]	Organ weight relative to brain weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	13393.6	NA	442.45	95.26	33.73
	SD	945.10	NA	21.58	5.54	3.80
	n	5	NA	5	5	5
100 mg/kg bw/day	Mean	13883.8	NA	510.19*	95.22	36.02
	SD	1006.61	NA	46.31	1.40	4.98
	n	5	NA	5	5	5
	±%	4	NA	15	0	7
300 mg/kg bw/day	Mean	14830.5*	NA	459.23	95.99	32.42
	SD	820.56	NA	44.96	6.08	4.45
	n	5	NA	5	5	5
	±%	11	NA	4	1	-4
1000 mg/kg bw/day	Mean	1434.1	NA	463.02	92.53	29.50
	SD	493.85	NA	34.50	2.05	3.73
	n	5	NA	5	5	5
	±%	7	NA	5	-3	-13
		DN		DN	NS	NS
FEMALES – RECOVERY GROUP
		Body weight relative to brain weight [%]	Organ weight relative to brain weight [%]
Group			Brain	Liver	Kidney	Spleen
Control	Mean	12815.8	NA	378.06	83.72	28.90
	SD	196.57	NA	52.61	6.77	2.11
	n	5	NA	5	5	5
1000 mg/kg bw/day	Mean	13245.5	NA	389.19	84.55	27.62
	SD	885.93	NA	22.07	3.07	2.35
	n	5	NA	5	5	5
	±%	3	NA	3	1	-4
		NS		NS	NS	NS

* p < 0.05, **p < 0.01

NS = Not Significant

DN = Duncan´s multiple range test

NA = Not Applicable

 

Table 10: 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	3/5	12/12	12/12	12/12	4/5
Thymus	Hemorrhages	0/12	2/5	0/12	0/12	0/12	1/5
Kidney	Pyelectasia – both sides	1/12	0/5	0/12	0/12	0/12	0/5
FEMALES - DAMS
	No macroscopic finding	10/10	4/5	12/12	6/11	12/12	3/5
Uterus	Hydrometra	0/10	1/5	0/12	5/11	0/12	2/5
FEMALES – NON-PREGNANT
Uterus	Hydrometra	1/1	NA	NA	1/1	NA	NA
FEMALES – DEAD ANIMAL
Lungs	Hemorrhages	1/1	NA	NA	NA	NA	NA

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

NA = not applicable

 

Table 11: 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	Dead animal	Recovery group			Main group	Recovery group
MALES
Kidneys	Pyelectasia (both side)	1/5	NA	0/5	-	-	0/5	0/5
Lungs	Alveolar emphysema	1/5	NA	0/5	-	-	1/5	1/5
	Acute hemorrhage	1/5	NA	0/5	-	-	0/5	0/5
	Hyperplasia of BALT	1/5	NA	0/5	-	-	1/5	1/5
Thymus	Acute hemorrhage	0/5	NA	1/5	-	-	0/5	1/5
FEMALES
Lungs	Alveolar emphysema	1/5	1/1	1/5	-	-	1/5	0/5
	Acute hemorrhage	0/5	1/1	0/5	-	-	1/5	0/5
	Hyperplasia of BALT	0/5	0/1	0/5	-	-	0/5	1/5
Uterus	Dilatation	1/11	0/1	1/5	-	5/5	0/12	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 and female Hsd.Han: Wistar rats was 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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.

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 54 or 55 days). Females were additionally exposed through the gestation period and up to lactation days 13 - 19, i.e. up to the day before necropsy (altogether for 54, 56 or 57 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 parent 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. 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.

One female animal of the control group died on gestation day 19 (Day 34). Histological examination revealed alveolar emphysema and acute hemorrhage (moderate degree) in the lungs, as the cause of death, in connection with a probably shock.

There were no test item related changes in clinical signs, body weight and body weight gain, food consumption, hematology, clinical chemistry parameters, macroscopic alterations at necropsy, organ weights and histopathology. 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 54 – 57 days is 1000 mg/kg bw/day in male and female Wistar rats.

Justification for classification or non-classification

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.