Reaction product of 2,2'-oxydiethanol and 2-hydroxyethyl acrylate and 2-hydroxyethyl methacrylate and hexan-6-olide and trimethylhexa-1,6-diyl diisocyanate

Endpoint summary

Description of key information

Oral (OECD 407), rat, 28-day study: NOAEL systemic = 1000 mg/kg bw/day

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

Type of information:

experimental study

Adequacy of study:

key study

Study period:

13 Feb - 18 Apr 2019

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 Oct 2008

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Version / remarks:

adopted 31 May 2008

Qualifier:

according to guideline

Guideline:

other: EPA OPPTS 870.3050 (28-Day Oral Toxicity in Rodents)

Version / remarks:

adopted July 2000

GLP compliance:

yes (incl. QA statement)

Remarks:

OGYEI National Institute of Pharmacy and Nutrition, Budapest, Hungary

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Han:WIST

Sex:

male/female

Details on test animals or test system and environmental conditions:

- Source: Toxi-Coop Zrt., Budapest, Hungary
- Females nulliparous and non-pregnant: yes
- Age at study initiation: less than 9 weeks
- Weight at study initiation: 233 - 252 g (males) and 164 - 190 g (females)
- Housing: Up to 3 animals of the same sex in type III polypropylene/polycarbonate cages embedded with certified laboratory wood bedding
- Diet: ssniff® SM R/M-Z+H complete diet (ssniff Spezialdiäten GmbH, Soest, Germany), ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 days

DETAILS OF FOOD AND WATER QUALITY: The food was considered not to contain any contaminants at levels 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 National Public Health and Medical Officer Service. The water was considered not to contain any contaminants at levels that could reasonably be expected to affect the purpose or integrity of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 ± 3
- Humidity (%): 30-70
- Air changes (per hr): 8-12
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 07 Feb To: 27 Mar 2019

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS: The test item was formulated in vehicle at concentrations of 10, 30 and 100 mg/mL. Formulations were prepared beforehand for not longer than 3 days and were stored in a refrigerator (5 ± 3 °C) until use.

- VEHICLE
- Concentration in vehicle: 10, 30 and 100 mg/mL
- Amount of vehicle: 10 mL/kg bw
- Lot no.: 17/154012

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Dosing formulations were analysed for homogeneity and concentration of the test item twice during the study. The analyses of the formulations on the first and last week of treatment demonstrated homogeneity of the dose formulations with achieved concentrations in the range of 94 % to 108 % of nominal concentrations.

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 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:

5 (main groups)
5 (recovery period: controls and high-dose group)

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 14-day dose range finding study in rats (Toxi-Coop study no. 673-400-4231). The test item was formulated in polyethylene glycol and administered at doses of 100, 300 and 1000 mg/kg bw/day by oral gavage to five rats per group and sex for a period of 14 days. There was no mortality and no clinical signs of toxicity noted up to the highest dose tested. Body weight development, food consumption, haematology and clinical chemistry were as well unaffected and gross necropsy revealed no abnormalities.
- Rationale for selecting satellite groups: Assessment of reversibility, persistence or delayed occurrence of potential toxicological effects
- Post-exposure recovery period in satellite groups: 14 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily (at the beginning and end of each working day)
- Parameters examined: clinical signs, morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once daily
- Parameters examined on general appearance: Skin, fur, eyes and mucous membranes; special attention was directed towards the observation of tremors, convulsions, salivation, diarrhea, lethargy, sleep and coma.
- Parameters examined on autonomic activity: lachrymation, piloerection, pupil size, respiratory pattern, occurrence of secretions and excretions
- Parameters on battery of functions: circulatory and central nervous system, somatomotor activity and behavior pattern, changes in gait, posture and response to handling

BODY WEIGHT: Yes
- Time schedule for examinations: Day 0 (prior to first treatment), once weekly during the treatment and recovery periods and immediately prior to sacrifice.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No, food consumption was evaluated and reported by weekly interval for each group.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the acclimatisation period and prior to test termination
- Dose groups that were examined: control and the high dose groups (1000 mg/kg bw/day)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: one day after the last treatment and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane CP)
- Animals fasted: Yes
- How many animals: all animals of all dose groups
- Parameters examined for haematology: white blood cell count (WBC), red blood cell count (RBC), haemoglobin concentration (HGB), haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration MCHC), platelet count (PLT), mean platelet volume (MPV) and reticulocytes (RET)
- Parameters examined for blood coagulation: activated partial thromboplastin time (APTT) and prothrombin time (PT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: one day after the last treatment and at the end of the recovery period
- Anaesthetic used for blood collection: Yes (Isoflurane CP)
- Animals fasted: Yes
- How many animals: all animals of all dose groups
- Parameters examined: alanine aminotransferase activity (ALT), aspartate aminotransferase activity (AST), gamma glutamyltransferase activity (GGT), alkaline phosphatase activity (ALP), total bilirubin concentration (TBIL), creatinine concentration (CREA), urea concentration, glucose concentration (GLUC), cholesterol concentration (CHOL), inorganic phosphate concentration (Pi), calcium concentration (Ca++), sodium concentration (Na+), potassium concentration (K+), chloride concentration (Cl-), albumin concentration (ALB), total protein concentration (TPROT) and albumin/globulin ratio (A/G ratio)

URINALYSIS: No

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
All animals underwent gross pathological examination at termination of the treatment treatment, on Day 28 (main groups) and at the end of the recovery period on Day 42 (recovery groups). The external appearance (surface of the body, all orifices) was examined, cranium, thoracic and abdominal cavities were opened and the appearance of the tissues and organs was observed macroscopically for each animal. All observations were recorded with details of the location, color, shape and size.
The following organs were weighed: Liver, kidneys, testes, epididymides, prostate and seminal vesicles with coagulating glands as a whole, uterus and fallopian tubes, thymus, spleen, brain and heart, adrenal glands and ovaries. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
The following organs/tissues were preserved for histopathological examination: adrenal glands, aorta (thoracic and abdominal), bone with joint and marrow (femur), brain (cerebrum, cerebellum, pons and medulla oblongata), esophagus, eyes (lachrymal gland with Harderian glands), gonads (testes with epididymides, ovaries, uterus with vagina, fallopian tubes), gross lesions, heart, kidneys, large intestine (caecum, colon, rectum, including Peyer’s patches), liver, lungs (with main stem bronchi), lymph nodes (submandibular and mesenteric), mammary gland, muscle (quadriceps), nasal turbinates, pancreas, pituitary, prostate, salivary glands (submandibular), sciatic nerve, seminal vesicles with coagulating gland, skin, small intestine (duodenum, ileum jejunum), spinal cord (at cervical, mid-thoraic and lumbar level), spleen, sternum, stomach, thymus, thyroids and parathyroid, trachea, urinary bladder
Histopathological examination was performed on the preserved organs and tissues of the control animals and the animals from the high dose group including recovery animals.

Statistics:

Statistical analysis was performed on body weight, food consumption, haematology, blood coagulation, clinical chemistry and organ weight. The heterogeneity 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 was carried out. If the obtained result was positive, Duncan's Multiple Range test was used to assess the significance of inter-group differences.
Where significant heterogeneity was found, the normal distribution of data was examined by Kolmogorov-Smirnov test. In case of a non-normal distribution, the non-parametric method of Kruskal-Wallis One-Way analysis of variance was used. If there was a positive result, the inter-group comparisons were performed using the Mann-Whitney U-test.
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 clinical signs, pathological and histopathological findings by sex and dose was calculated.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Soft faeces were noted among all groups from study Day 9 until the end of the treatment period, and within the first two days of the recovery period (study Days 28-29). The faeces of the animals were normal from the third day of the recovery period. The findings were attributed to the vehicle and no related to the test item.

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In males there was a trend toward reduced body weight and body weight gain at all doses. However, statistically significant changes were only noted in the high dose during the first week of treatment (30.3 g versus 37.3 g in the control in males). At the end of the treatment period mean body weights were reduced by 1.1, 3.5 and 4.2% at 100, 300 and 1000 mg/kg bw/day dose levels, respectively. Although this reduction in final body weights was dose-related it was not considered of toxicological relevance due to the slight degree of the effect.
In females of the high dose group, the mean body weight on study Day 7 was significantly reduced when compared to controls (188.6 g versus 195.0 g in the control). Similar to the observation in males, the mean body weight gain in females during the first week was significantly reduced (9.4 g versus 16.3 g in the control). These slight changes had no influence on the overall body weight gain of these animals therefore it was judged to be without any toxicological relevance.

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

During the treatment period, a statistically significant decrease of the mean food consumption was observed in male animals at 100 and 1000 mg/kg bw/day during the first week, in male animals at 300 and 1000 mg/kg bw/day during the third week and in male animals at 1000 mg/kg bw/day during the last week of the treatment period. In females, a statistically significant decrease of the mean food consumption was observed at 1000 mg/kg bw/day during the first and last week of the treatment period. These occasionally fluctuations were considered to be without toxicological meaning.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

There were no test item-related adverse changes in the examined haematological parameters at any dose group. At the end of the treatment period, a number of adverse effects was observed in males:
100 mg/kg bw/day: statistically significant decrease in the mean HCT volume and statistically significant increases in the MCHC and in MPV
300 mg/kg bw/day: biologically significant decrease of HGB, HCT volume and statistically significant decrease in the mean RBC and statistically significant increase in the MCHC
1000 mg/kg bw/day: biologically significant decrease in the mean RBC, HGB concentration, HCT volume and biologically significant increase in the MCHC
Although the mentioned differences in RBC, HGB, HCT and MCHC between control and treated groups reached statistical significance, they were of minor degree and mostly remained within the historical control ranges (refer to Table No. 1 under “Any other information in results incl. tables”). The statistically significant increase in MPV at the lowest dose group was considered to be without a toxicological meaning. All alterations in haematology parameters in males were fully reversible and recovered to those of control levels during the recovery period.
At the end of the recovery period, there was a statistically significant increase in the mean percentage of reticulocytes at 1000 mg/kg bw/day. The finding was judged to be toxicologically not relevant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of statistically significant alterations was noted in males and females among all dose groups, but the changes were considered not test item-related and not toxicologically relevant:
100 mg/kg bw/day: slightly lower mean K+ levels in males and decreased AST activity in females after 28 days of treatment
300 mg/kg bw/day: decreased CHOL in females after 28 days of treatment
1000 mg/kg bw/day: increased mean A/G ratios in males and decreased CHOL in females after 28 days of treatment; decreased CREA after 14 days of recovery in females

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 28 days of treatment, the absolute mean heart weight was statistically significantly reduced in males at 300 mg/kg bw/day (-13%) and 1000 mg/kg bw/day (-11%) when compared to control animals. There were no significant alterations of the relative heart weight. At 300 and 1000 mg/kg bw/day, mean relative testes weights (relative to body and/or brain weights) were significantly increased. Absolute testes weight was similar in all groups. The alterations were considered as significant differences without biological meaning in the absence of any histopathological findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 28 days of treatment, bilateral pyelectasia was observed in 2/5 males of the 1000 mg/kg bw/day group and in 1/5, 2/5 and 1/5 females of the control, 100 and 300 mg/kg bw/day groups, respectively. There were no inflammatory or other pathological signs related to this finding. Pyelectasia is a common finding in rats of this strain, thus the findings were not attributed to treatment.
After the recovery period, 2/5 females of the control group and 2/5 females of the high dose group showed slight or moderate hydrometra. Hydrometra is a frequent observation in experimental rats. Due to the lack of inflammatory or other pathological signs and the absence of a dose-response relationship the finding was judged to be toxicologically not relevant and not related to the test item.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There were no treatment-related microscopical lesions observed. A number of histopathological abnormalities was occasionally observed in control animals and animals of the high dose group:
Alveolar emphysema was noted in the lungs of 1/5 recovery control males, in 1/5 recovery control females, in 1/5 males and 1/5 females at 1000 mg/kg bw/day and in 1/5 recovery 1000 mg/kg bw/day females.
Hyperplasia of bronchus associated lymphoid tissue (without inflammatory lesions) was noted in 1/5 control males, 1/5 recovery control males and in 1/5 males at 1000 mg/kg bw/day. The finding represents a physiological immunomorphological phenomenon without pathological significance.
Uni- or bilateral pyelectasia was noted in1/5 females and 2/5 males at 1000 mg/kg bw/day. The finding was without degenerative, inflammatory or other histopathological lesions and is commonly found in experimental rats, thus the observation was considered without toxicological significance.
Dilatation of the uterus horns was observed in 1/5 control females, 2/5 recovery control females and in 2/5 females of the recovery 1000 mg/kg bw/day group. The finding was related to the prooestrus phase of the inner genital organs and not related to treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

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 adverse effects observed

Key result

Critical effects observed:

no

Table 1: Body weight gain in male and female rats

Dose Group [mg/kg bw/day]	Body weight gain (g, mean ± SD) between days
	Treatment period				Recovery period
	0-7	7-14	14-21	21-27	0-27	27-34	34-41	27-41
MALES
Control	37.3 ± 6.3	36.1 ± 7.4	25.5 ± 6.4	11.3 ± 5.7	110.2 ± 16.1	15.8 ± 6.2	15.2 ± 9.9	31.0 ± 11.5
100	36.0 ± 6.0	32.6 ± 5.2	24.2 ± 6.6	15.4 ± 4.5	108.2 ± 15.9	nd	nd	nd
300	34.6 ± 4.2	33.4 ± 5.5	18.8 ± 4.0	12.2 ± 7.5	99.0 ± 9.0	nd	nd	nd
1000	30.3 ± 4.8*	37.4 ± 7.1	19.1 ± 8.4	8.3 ± 5.9	95.1 ± 16.4	19.2 ± 4.7	13.8 ± 6.8	33.0 ± 6.6
FEMALES
Control	16.3 ± 4.6	14.5 ± 4.8	3.1 ± 8.6	9.3 ± 9.0	43.2 ± 12.7	-0.2 ± 7.4	5.0 ± 3.2	5.2 ± 8.8
100	16.8 ± 5.2	11.4 ± 6.9	7.0 ± 7.5	5.2 ± 7.2	40.4 ± 11.1	nd	nd	nd
300	14.0 ± 3.8	12.2 ± 3.3	6.2 ± 10.6	6.2 ± 8.8	38.6 ± 9.4	nd	nd	nd
1000	9.4 ± 7.3*	16.2 ± 6.8	4.5 ± 7.3	6.9 ± 7.2	37.0 ± 11.0	2.0 ± 4.0	6.2 ± 4.5	6.0 ± 3.4
± %: Percent deviation versus control; * = p < 0.05; ** p < 0.01; nd: no data Number of animals: control animals and 1000 mg/kg bw/day group: 10 male + 10 female during treatment, 5 male + 5 female during recovery; 100 and 300 mg/kg bw/day: 5 male + 5 female during treatment, no recovery animals

Table 2: Haematology parameters

Group (mg/kg bw/day)	RBC (10^12/L)	HCD range (10^12/L)	HGB (g/L)	HCD range (g/L)	HCT (L/L)	HCD range (L/L)	MCHC (g/L)	HCD range (g/L)
Control	8.67	7.4 - 9.9	163.4	142 - 184	0.50	0.39 - 0.52	328.20	350 - 375
100	8.31		159.2		0.47		340.40
300	8.17		156.6		0.46		342.00
1000	7.99		154.0		0.44		346.60

RBC: red blood cells; HCD: historical control data; HGB: haemoglobin; HCT: hematocrit; MCHC: mean corpuscular haemoglobin concentration

Conclusions:

Based on the results of this study, the NOAEL for systemic toxicity was 1000 mg/kg bw/day, which corresponds to the highest dose tested.

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 an adequate and reliable (Klimisch score 1) study performed with the registered substance, and is thus sufficient to fulfil the standard information requirements set out in Annex VIII, Item 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 subacute oral toxicity study was performed in male and female Wistar rats according to OECD guideline 407 and in compliance with GLP (Stahl, 2019). The test item was dissolved in polyethylene glycol and administered daily via oral gavage. Dose levels for this study were selected based on the results of a foregoing 14-days doserange-finding study. Doses of 100, 300 and 1000 mg/kg bw/day were administered to groups of five rats per sex. A similar constituted group received the vehicle only and served as control group. In addition, five rats per sex were included as recovery animals for the control and the high dose group. Animals of the recovery group were treated for 28 days in the same manner as the animals of the main group, but observed for an additional 14-day recovery period before terminal sacrifice. 

The animals were observed twice daily for mortality, morbidity and clinical signs. Detailed clinical examinations were performed once daily and included the general appearance, autonomic activity and battery of functions. Individual body weights were recorded prior to treatment start, once weekly during the treatment period and before terminal sacrifice. Food consumption was recorded at weekly intervals. Ophthalmological examinations were performed prior to treatment initiation and before termination of the study. At study termination, blood was collected and analysed for haematological and clinical chemistry parameters. All animals underwent gross pathological examination including organ weight analysis of selected organs and histopathological examination.

There was no mortality in any group during the 28-day treatment or 14-day recovery periods. Clinical signs of toxicity comprised soft faeces among all groups from study Day 9 until the end of the treatment period, and within the first two days of the recovery period (study Days 28-29). The observation was also made in the control group and attributed to the vehicle but not related to treatment with the test item. Functional observations did not reveal test item related changes and the physical condition, behavior and reaction to different type of stimuli were judged to be normal in all animals.

Statistically significant reduction in body weight and/or body weight gain were observed in males and females of the high dose groups only during the first week of treatment. At the end of the treatment period, mean body weights in males were were significantly reduced. All changes were slight (1.1 -4.2%) and had no influence on the overall body weight gain of these animals, therefore they were judged to be without any toxicological relevance.

Food consumption was found to be occasionally significantly decreased in males of all dose groups in females of the high dose group within different weeks of the study. These occasional fluctuations were considered to be without toxicological meaning. During the recovery period, the mean daily food consumption was comparable in the control and 1000 mg/kg bw/day groups.

Analysis of haematological parameters revealed occasionally statistically significant fluctuations in red blood cells, haemoglobin, haematocrit and mean corpuscular haemoglobin concentration in male rats among all dose groups. Hence, the observations were not attributed to treatment and considered toxicologically not relevant. Increased mean platelet volumes in males at 100 mg/kg bw/day were considered to be without toxicological meaning. All alterations in haematology parameters were fully reversible and recovered to those of control levels during the 14-day recovery period. There were no statistically or biologically significant alterations in treated females.

Clinical chemistry revealed as well no treatment-related findings. A number of statistically significant alterations was noted in males and females among all dose groups and comprised slightly reduced potassium levels in males, decreased aspartate aminotransferase levels in females, decreased cholesterol in females, increased albumin to globulin ratios in males and decreased creatinine in females. However, there were no dose-response relationship and no histopathological abnormalities observed, thus the findings were considered toxicologically not relevant.

After 28 days of treatment, the mean absolute heart weight was significantly reduced in males at 300 and 1000 mg/kg bw/day. In addition, the mean relative testes weight (relative to body weight and/or brain weight) was significantly increased in males of the same dose groups. In the absence of any histopathological abnormalities, both findings were considered to be without biological meaning.

At gross necropsy, pyelectasia was noted in males and females among all treatment and control groups and hydrometa was noted in females of the control and high dose groups. The observations represent common findings in rats of this strain and therefore not related to treatment. Histopathological examination revealed as well no test item-related findings. Alveolar emphysema, hyperplasia of bronchus associated lymphoid tissue and uni- or bilateral pyelectasia were ocassionally observed, but the findings were without degenerative, inflammatory or other histopathological lesions. In the absence of any dose-response relationship, the findings were considered not related to treatment.

Based on the results of the study and under the experimental conditions mentioned, a NOAEL of 1000 mg/kg bw/day was derived for male and female Wistar rats.

Justification for classification or non-classification

The available data on repeated dose toxicity with the registered substance do not meet the criteria for classification according to Regulation (EC) No. 1272/2008, and are therefore conclusive but not sufficient for classification.