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Description of key information

Two oral repeated toxicity studies in rats are available on Ethoxylated bisphenol A diacrylate : a 28-day and a 90-day studies.

In the 28-day toxicity study, the test item was administered daily by oral route to rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil. The No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the effects seen at 1000 mg/kg/day on mean blood cholesterol levels, which were observed in presence of non-adverse increase of mean liver weight and liver microscopic findings (hepatocellular hypertrophy).

In the 90-day toxicity study, the test item treatment at 1000 mg/kg/day induced adverse effects on mean body weight (males only), mean cholesterol concentration and on kidneys (increase creatinine and urea levels associated with increase of relative kidney weight and vacuolisation and dilatation of cortical tubules). The test item-related histopathological renal changes were considered to have contributed to the moribund status of one male which was prematurely euthanized. There were no adverse findings at 50 and 250 mg/kg/day. The No Observed Adverse Effect Level (NOAEL) was considered to be at 250 mg/kg/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
07 August 2012 - 06 December 2012
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)
Deviations:
no
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories France, l’Arbresle, France
- Age at study initiation: approximately 5 weeks old on the first day of treatment
- Mean body weight at study initiation: the males had a mean body weight of 183 g (range: 168 g to 203 g) and the females had a mean body weight of 157 g (range: 145 g to 172 g)
- Fasting period before study: no
- Housing: the animals were housed by five in polycarbonate cages with stainless steel lids
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: 8 days before the beginning of the study.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C (except rare increase up to 25.6°C)
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 17 August 2012 to 14 September 2012.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
The test item was administered as a suspension in the vehicle. It was mixed with the required quantity of vehicle. No correction factor was applied.
The frequency of dose formulation preparation was on a daily basis. The dose formulations were delivered to the study room at room temperature and protected from light.

VEHICLE
- Concentration in vehicle: 20, 60 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: HPLC-UV
Test item concentrations: within an acceptable range of variation compared to nominal values.
Homogeneity: homogenous
Duration of treatment / exposure:
4 weeks
Frequency of treatment:
Daily
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 animals per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose-levels were selected in agreement with the Sponsor based on the results of a previous 2-week toxicity study performed in the same species and strain. In this study, three groups of three males and three females received the test item by gavage at 100, 300 or 1000 mg/kg/day as a suspension in corn oil. Another group of three males and three females received the vehicle only. There was no mortality and the only test item-related clinical sign was ptyalism in all high-dose animals and one mid-dose male. There were no toxicologically significant effects on mean food consumptions or on mean body weights and no clear effects on mean body weight gains. At necropsy, there were no test item-related macroscopic findings.
Therefore, 1000 mg/kg/day was selected as the high-dose level for this 4-week study. Mid and low-dose levels were selected in order to cover approximately three fold intervals.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORTALITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day from the start of the treatment period, including weekends.

- CLINICAL SIGNS:
Each animal was observed once a day, at approximately the same time, for the recording of clinical signs.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all the animals once before the beginning of the treatment period and then once a week until the end of the study

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, and then at least once a week until the end of the study.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week until the end of the study.

NEUROBEHAVIOURAL EXAMINATION:
- Time schedule: each animal was evaluated in week 4.

HAEMATOLOGY, CLINICAL CHEMISTRY, URINALYSIS:
- Time schedule: at the end of the treatment period.
Sacrifice and pathology:
ORGAN WEIGHTS: see table below

GROSS PATHOLOGY:
Complete macroscopic post-mortem examination of all study animals.

HISTOPATHOLOGY:
- all tissues listed in the Tissue Procedure Table for the control and high-dose animals (groups 1 and 4) sacrificed at the end of the treatment period,
- liver and kidneys for the low and mid-dose animals (groups 2 and 3) sacrificed at the end of the treatment period,
- all macroscopic lesions from all low- and intermediate-dose animals (groups 2 and 3) sacrificed on completion of the treatment period.
Other examinations:
no
Statistics:
Citox software (version D.6, see § Study plan adherence) was used to perform the statistical analysis of body weight, hematology, blood biochemistry and urinalysis data. PathData software (version 6.2d2) was used to perform the statistical analysis of organ weight data (level of significance of 0.05 or 0.01).
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
All animals treated at 1000 mg/kg/day had ptyalism generally from week 2. This was considered to be related to the treatment with the test item but of minor toxicological importance.
Incidental findings included reflux at dosing, scabs, alopecia and chromorhynorrhea and were generally observed in isolated animals.
Mortality:
no mortality observed
Description (incidence):
There were no unscheduled deaths during the study.
Body weight and weight changes:
no effects observed
Description (incidence and severity):
There were a lower mean body weight gain in males treated at 1000 mg/kg/day and a dose-related higher mean body weight gain in females, compared to the control group. However, this was considered not to be of toxicological relevance as the changes were not statistically significant and this had no toxicological impact on the mean body weight at the end of the treatment period.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects on mean food consumption.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
In males, there was a trend towards slightly dose-related higher mean red blood cell parameters (count, hemoglobin and hematocrit/PCV) reaching statistical significance at 1000 mg/kg/day and a statistically significant lower mean reticulocyte percentage from 300 mg/kg/day, when compared with controls. In view of the low amplitude of differences from control values, the absence of similar findings in females and the lack of histopathological correlates, these findings were considered to be of limited toxicological importance.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
There was a dose-related higher mean cholesterol level from 100 mg/kg/day in males and from 300 mg/kg/day in females when compared with controls, together with a trend towards a slightly higher non-dose-related mean triglyceride blood concentration in males. A tendency towards higher mean bile acid level and ALAT activity was also noted in males from 300 mg/kg/day (bile acids) or at 1000 mg/kg/day (ALAT); the high bile acid level measured in females was due to one female only.
The effect on the cholesterol level was considered to be adverse at 1000 mg/kg/day in view of the amplitude of differences from control and historical control data. The effects observed on mean triglyceride and bile acid levels and on mean ALAT activity were considered to be of no toxicological importance as rare statistical level or dose-relationship and included in historical control data (triglyceride and bile acids) or in view of the very low amplitude of the modification (ALAT).
Females treated at 1000 mg/kg/day also had slightly higher mean urea blood concentration and a slightly lower mean alkaline phosphatase activity than controls. In view of the low severity of the changes and the fact that they were included in historical control data, they were considered not to be adverse and of minor toxicological importance.
The statistical significance observed for proteins, A/G ratio and chloride was considered to be fortuitous as there was no dose-relationship and/or the amplitudes of variations from controls were low.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
At 1000 mg/kg/day, mean urine pH of males was slightly lower than that of controls (6.0 vs. 6.9, p<0.01). As the severity of the differences from controls was low, this non adverse finding was considered to be of minor toxicological importance.
There were 4/10 animals at 1000 mg/kg/day with more calcium oxalate crystals in their urine than controls. As the incidence was not high and as calcium oxalate crystal is one of the most common urine crystals, a relationship with the test item treatment was considered to be doubtful.
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects at Functional Observation Battery and motor activity.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, statistically significant increase in mean liver weights was seen in females treated at 300 mg/kg/day (absolute weight), and in males (relative weight) and females (absolute and relative weights) treated at 1000 mg/kg/day.
Other organ weight changes were considered not to be related to the test item as they were small in amplitude, were not dose-related, had no gross or microscopic correlates and/or were not consistent between the sexes.
Gross pathological findings:
no effects observed
Description (incidence and severity):
There were no test item-related macroscopic findings.
The macroscopic findings that were observed had no histological correlates or correlated with common histological findings in control rats, and were considered to be incidental.
Specifically the 0.3 cm in diameter brown mass found in the para-renal region from a male treated at 300 mg/kg/day corresponded to an atrophic kidney (noted as agenesis at necropsy). The 0.3 cm in diameter white mass in the subcutis found in a female treated at 1000 mg/kg/day was a cyst containing hair (considered as a developmental abnormality). Both findings were considered as incidental.
Neuropathological findings:
no effects observed
Description (incidence and severity):
There were no toxicologically relevant effects at Functional Observation Battery and motor activity.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Non adverse microscopic findings were seen in the liver and kidneys.
. liver: Minimal to slight hepatocellular hypertrophy was seen in males and females treated at 1000 mg/kg/day. No changes were observed in males and females at 300 mg/kg/day. This correlated with the increased mean liver weights in both sexes.
There were no associated degenerative changes at any of the dose-levels.
. kidney: Minimal increased of vacuolation was seen in the proximal tubules from females treated at 300 mg/kg/day, and in males and females treated at 1000 mg/kg/day. The vacuoles were round, pale and located in the cytoplasm. They had various sizes (about 1-5 µm in diameter).
There were no associated degenerative or necrotic lesions. Consequently, this finding was considered as non adverse.

Other microscopic findings noted in treated animals were considered incidental changes, as they also occurred in controls, were of low incidence, and/or are common background findings for the Sprague-Dawley rat.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
clinical biochemistry
histopathology: non-neoplastic
organ weights and organ / body weight ratios
Critical effects observed:
not specified

Table 1: mean body weight and mean body weight changes (g)

 

Sex

Males

Females

Dose level (mg/kg /day)

0

100

300

1000

0

100

300

1000

Bodyweight change

Days 1 to 28

+209

+223

+205

+186

+72

+79

+83

+86

Difference to controls

/

+7%

-2%

-11%

/

+10%

+15%

+19%

Bodyweight

Day 1

184

182

187

180

154

158

159

159

Difference to controls

/

-1%

+2%

-2%

/

+3%

+3%

+3%

Day 28

392

404

392

366

226

237

242

244

Difference to controls

/

+3%

0

-7%

/

+5%

+7%

+8%

 

Table 2: Relevant differences in hematology

 

Sex

Males

Females

Dose level (mg/kg /day)

0

100

300

1000

0

100

300

1000

Red blood cell count (T/L)

7.90

8.15

8.33

8.40*

7.83

7.93

7.77

7.65

Hemoglobin (g/g/dL)

15.0

15.5

15.7

15.9*

14.7

14.9

15.0

14.6

Packed cell volume (L/L)

0.47

0.49

0.49

0.50*

0.44

0.45

0.46

0.45

Reticulocytes percentage (%)

2.87

2.49

2.00**

2.04**

1.76

1.64

2.08

2.20

Statistically significant from controls: * p<0.05, ** p<0.01

 

Table 3: Statistically relevant differences in blood biochemistry

 

Sex

Males

Females

Dose level (mg/kg /day)

0

100

300

1000

0

100

300

1000

Cholesterol (mmol/L)

1.6

2.1

2.6**

3.6**

1.8

1.9

2.4

4.3**

Triglycerides (mmol/L)

0.60

1.11*

0.97

0.99

0.30

0.32

0.39

0.39

Bile acids (µmol/L)

12.3

14.6

17.7*

17.0

15.2

13.1

14.7

18.2

Alanine aminotransferase (ALAT) (IU/L)

34

37

33

43*

29

32

31

33

Urea (mmol/L)

3.5

3.8

4.1

3.9

4.7

4.3

4.4

5.6*

Alkaline phosphatase (IU/L)

458

423

507

387

325

267

297

220**

Proteins (g/L)

59

63*

61

62*

63

60

60

66

A/G ratio

1.56

1.48

1.52

1.50

1.53

1.76**

1.65

1.60

Chloride (mmol/L)

104.5

102.3*

104.6

104.3

105.3

107.8

106.5

107.0

Statistically significant from controls: * p<0.05, ** p< 0.01

 

 

Table 4: Test item-related differences (expressed in %) noted between test item-treated and control animals in the absolute and relative organ weights

 

sex

male

female

Dose-level (mg/kg/day)

100

300

1000

100

300

1000

Exam.animals / Num. of animals

5/5

5/5

5/5

5/5

5/5

5/5

Body weight

+6

+2

-8

+4

+7

+5

Liver (absolute)

+13

+9

+5

+7

+19*

+40**

Liver (relative)

+6

+7

+14**

+3

+12

+34**

Statistically significant from controls: * p<0.05, ** p< 0.01

The significance concerned the organ weights values and not the percentages.

 

 

Table 5: Incidences and severity of microscopic findings on liver and kidneys

 

sex

 

 

Dose-level (mg/kg/day)

0

100

300

1000

0

100

300

1000

Liver : Hepatocellular hypertrophy

  • Minimal
  • Slight

-

-

-

-

-

-

2

1

-

-

-

-

-

-

4

-

Kidney: Increased vacualisation; proximal tubules

  • Minimal

-

-

-

4

-

-

3

5

-: no affected animal

Conclusions:
The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose levels of 100, 300 or 1000 mg/kg/day in corn oil.
Under the experimental conditions of this study, the No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the effects seen at 1000 mg/kg/day on mean blood cholesterol levels, which were observed in presence of non-adverse increase of mean liver weight and liver microscopic findings (hepatocellular hypertrophy).
Executive summary:

The objective of this study was to evaluate the potential of the test item, following daily oral administration (gavage) to rats for 4 weeks,according to OECD (No. 407, October 2008) and EC (No. 440/2008, B7, May 2008) guidelines.

The study was conducted in compliance with the principles of Good Laboratory Practice Regulations.

 

Methods

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a suspension in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

Test item concentrations were checked on formulations used in weeks 1 and 4.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. In addition, detailed clinical examinations were performed at least once weekly. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as food consumption. Towards the end of the dosing period, a Functional Observation Battery including motor activity measurement, and hematology, blood biochemistry and urinalysis were performed on all animals.

On completion of the treatment period, the animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and selected tissues were preserved. A microscopic examination was performed on selected tissues from control- and high-dose animals sacrificed at the end of the treatment period and on all macroscopic lesions.

Results

The test item concentrations in the administered dose formulations analyzed in weeks 1 and 4 were within the acceptance criteria.

 

There were no unscheduled deaths during the study. The only test item-related clinical sign was ptyalism in all animals treated at 1000 mg/kg/day and was considered to be of minor toxicological importance. There were no toxicologically relevant effects at Functional Observation Battery and motor activity, and on mean food consumption, mean body weight and mean hematology parameters.

 

At blood biochemistry analysis and when compared with controls, there was a dose-related higher mean cholesterol level from 100 mg/kg/day in males (2.1, 2.6 (p<0.01) and 3.6 (p<0.01) mmol/L at 100, 300 and 1000 mg/kg/day, respectively, vs. 1.6) and from 300 mg/kg/day in females (2.4 and 4.3 (p<0.01) at 300 and 1000 mg/kg/day, vs. 1.8). This effect was considered to be adverse at 1000 mg/kg/day. Females treated at 1000 mg/kg/day also had slightly higher mean urea (5.6 mmol/L vs. controls, p<0.05) blood concentration and a slightly lower mean alkaline phosphatase activity (220 IU/L vs. 325, p<0.01) than controls, which were considered not to be adverse and of minor toxicological importance.

At 1000 mg/kg/day, mean urine pH of males was slightly lower than that of controls (6.0 vs. 6.9, p<0.01) which was considered to be non adverse and of minor toxicological importance.

At pathology, mean liver weights were higher in females treated at 300 mg/kg/day, and in males and females treated at 1000 mg/kg/day. There were no test item-related macroscopic findings. Microscopic findings were seen in the liver (non adverse hepatocellular hypertrophy in males and females treated at 1000 mg/kg/day) and kidney (non adverse increased vacuolation in proximal tubules in females treated at 300 mg/kg/day and in males and females treated at 1000 mg/kg/day).

 

Conclusion

The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil.

The No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the effects seen at 1000 mg/kg/day on mean blood cholesterol levels, which were observed in presence of non-adverse increase of mean liver weight and livermicroscopic findings(hepatocellular hypertrophy).

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
06 February 2017 - 18 May 2017
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
21 September 1998
Deviations:
yes
Remarks:
Ophthalmological examinations were forgotten at the end of the study. Microscopic examination of the eye was therefore performed in all groups in order to compensate this deviation.
GLP compliance:
yes (incl. QA statement)
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: breeder: Charles River Laboratories Italia, Calco, Italy
- Age: at the beginning of the treatment period, the animals were 6 weeks old
- Mean body weight at the beginning of the treatment period: the males had a mean body weight of 186 g (range: 140 g to 201 g) and the females had a mean body weight of 168 g (range: 147 g to 190 g)
- Fasting period before study: no
- Housing: the animals were housed in twos, by sex and group, in polycarbonate cages with stainless steel lids (Tecniplast 2000P, 2065 cm²) containing autoclaved sawdust
- Diet: SSNIFF R/M-H pelleted diet (free access)
- Water: tap water filtered with a 0.22 µm filter (free access)
- Acclimation period: for a period of 8 days before the beginning of the treatment period.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): approximately 8 to 15 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12 h/12 h.

IN-LIFE DATES: 15 February 2017 to 18 May 2017.
Route of administration:
oral: gavage
Vehicle:
corn oil
Details on oral exposure:
PREPARATION OF DOSING FORMULATIONS:
- Emulsion in the vehicle
- Concentration in vehicle: 10, 50 and 200 mg/mL
- Amount of vehicle (if gavage): 5 mL/kg/day.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Type of method: High Performance Liquid Chromatography with UV detection (HPLC/UV)
Test item concentrations: within an acceptable range of -2.5% to +3.1% when compared to the nominal values (± 15% of the nominal concentrations required)
Homogeneity: the dose formulations containing the test item in corn oil at 2 and 200 mg/mL were found to be homogeneous. They are therefore considered to be suitable for routine administration in GLP Toxicological studies, based on a daily preparation.
Stability: no stability available, dose formulations prepared daily.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
250 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:
10 animals per sex and per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
The dose levels were selected by the Sponsor Representative based on the results of a previous 4-week toxicity study (OECD 407) and a previous OECD 421 study (5 weeks of treatment for males, about 6 weeks for females in total) performed in the same species and strain.
At the end of these studies, all animals treated up to 1000 mg/kg/day were in good clinical conditions. There were at 1000 mg/kg/day a few hematology, blood biochemistry and/or urine findings including adverse increase of mean cholesterol level (about x2.5 vs. controls), minimal to slight hepatocellular hypertrophy and/or vacuolation of Kupffer cells correlating with high liver weight (up to +40% vs. controls), and/or minimal increase of vacuolation in kidneys. At 250 and 300 mg/kg/day, some of these findings were observed to a lesser extend in one or both sexes. At 50 and 100 mg/kg/day, there were a few minor hematology and/or blood biochemistry findings in one sex generally.

- Rationale for animal assignment: computerized stratification procedure.
Positive control:
no (not required)
Observations and examinations performed and frequency:
MORTALITY / MORBIDITY:
- Time schedule: each animal was checked for mortality and morbidity once a day during the acclimation period and at least twice a day during the treatment period, including weekends and public holidays.

CLINICAL OBSERVATIONS:
- Time schedule: each animal was observed once a day, at approximately the same time on the days of treatment.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: detailed clinical examinations were performed on all animals once before the beginning of the treatment period and then once a week until the end of the study.

BODY WEIGHT:
- Time schedule: the body weight of each animal was recorded once before the beginning of the treatment period, on the first day of treatment, at least once a week until the end of the study and before premature euthanasia.

FOOD CONSUMPTION:
- Time schedule: the quantity of food consumed by the animals in each cage was recorded once a week during the study.

HAEMATOLOGY/COAGULATION:
- Time schedule for collection of blood: the parameters were determined for all surviving animals in Week 13.
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: Yes
- Parameters checked in table 1 were examined.

CLINICAL CHEMISTRY:
- Time schedule for collection of blood: the parameters were determined for all surviving animals in Week 13.
- Animals fasted: Yes
- Parameters checked in table 2 were examined.
Sacrifice and pathology:
ORGAN WEIGHTS: see table 3.
The body weight of each animal was recorded before euthanasia at the end of the treatment period. The organs specified in table 3 were weighed wet as soon as possible after dissection. The ratio of organ weight to body weight (recorded immediately before euthanasia) was calculated.

GROSS PATHOLOGY:
A complete macroscopic post-mortem examination was performed on all animals.

PRESERVATION OF TISSUES:
For all animals, the tissues specified in table 3 were preserved in 10% buffered formalin (except for the eyes and optic nerves and Harderian glands, and the testes and epididymides which were fixed in Modified Davidson's Fixative).

PREPARATION OF HISTOLOGICAL SLIDES:
All tissues required for microscopic examination were trimmed according to the RITA guidelines, when applicable (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004), embedded in paraffin wax, sectioned at a thickness of approximately four microns and stained with hematoxylin-eosin.

HISTOPATHOLOGY:
A microscopic examination was performed:
- on all tissues listed in table 3 for the control- and high-dose animals (groups 1 and 4) euthanized at the end of the treatment period,
- on eyes for all animals (groups 1 to 4) euthanized at the end of the treatment period,
- on liver, kidneys and thyroids for the low- and mid-dose animals (groups 2 and 3) euthanized at the end of the treatment period,
- on all tissues listed in the Tissue Procedure Table for the male euthanized prematurely,
- on all macroscopic lesions for all low- and intermediate-dose animals (groups 2 and 3) euthanized on completion of the treatment period.
Statistics:
CITOX software was used to perform the statistical analyses on body weight, food consumption, hematology and blood biochemistry data.
PATHDATA software was used to perform the statistical analysis of organ weight data.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
The only test item-related clinical sign was ptyalism noted in three animals given 250 mg/kg/day (for 5 to 22 days towards the end of the study) and in all animals treated at 1000 mg/kg/day (from Day 10 and for the most part of or the remaining study). This test item effect was considered non-adverse (minimal clinical sign).

The other clinical signs recorded in test item-treated groups (abnormal growth of teeth, alopecia/thinning of hair, scabs, reflux at dosing, chromorhynorrhea, nodosity, soiled head) were noted in single animals mostly or at comparable incidence to controls, not dose-related and/or common in this strain of rats under laboratory conditions.
Mortality:
mortality observed, treatment-related
Description (incidence):
There were no premature deaths at 0, 50 and 250 mg/kg/day.

At 1000 mg/kg/day, one male was prematurely sacrificed in Week 10 for humane reasons: the animal was thin for a few days before euthanasia and had on the day of euthanasia hunched posture, generalised pallor, piloerection and was cold to the touch. In the week before its euthanasia, the food consumption in its cage was reduced and the animal lost 15.5% of body weight. At necropsy, the spleen, thymus and seminal vesicles were reduced in size, and the kidney had an irregular color. The latter observation correlated with moderate dilatation and vacuolation of cortical tubules (changes also observed in some test item-treated rats at the end of treatment). These renal changes were considered to have contributed to the moribund status of this rat; therefore this death was considered to be related to the test item treatment.
There were no premature deaths in females at this dose.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
See table 1.
In males at 1000 mg/kg/day, mean body weights were statistically significantly lower than in controls from Week 4, down to -14% vs. controls in Week 10. This effect was considered to be test item-related and adverse. It correlated with statistically significantly lower mean body weight gain over the treatment period. At 250 mg/kg/day, the same tendencies were noted but to a lesser extent. They were therefore considered to be test item-related but non-adverse.
There were no test item-related effects in males treated at 50 mg/kg/day.

In females, there were no effects on mean body weight that could be considered as test item treatment related. The slightly statistically significant lower mean body weight gain over the treatment period at 1000 mg/kg/day vs. controls was considered to be test item related and non-adverse.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
There were no test item-related effects at 50 and 250 mg/kg/day.

None of the variations observed in mean food consumption at 1000 mg/kg/day when compared with controls were considered to be of toxicological significance as statistical significances were rare, and as variations from controls were < ± 20% (even < ± 15% except once in females) with the exception of the high mean food consumption noted in females in Week 9 due to a likely aberrant value in one cage.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
See table 2.
There were no effects at 50 and 250 mg/kg/day considered to be test item-related. The statistical significances noted in males at 50 mg/kg/day on red blood parameters when compared with controls were considered to be incidental in absence of dose-relationship.

At 1000 mg/kg/day when compared with controls, females had slightly and statistically significantly lower mean red blood cell count, mean hemoglobin concentration and mean PCV (hematocrit), as well as shortened mean prothrombin time. These effects were considered to be test item treatment-related, and non-adverse in view of the magnitude of differences from controls.
In males at the same dose and when compared with controls, despite the absence of dose-relationship in this sex and most individual values included in the historical control data, a slight test item effect on the statistically significantly lower mean red blood cell count was not excluded in view of the similarity with the female data.

The statistical significance reached at 250 mg/kg/day in females in mean platelet and reticulocyte concentrations were considered to be incidental in absence of similar effects at the high-dose and in the other sex, and/or in view of the magnitude of the change from controls.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
See table 3.
At 1000 mg/kg/day when compared with controls, there were statistically significant higher mean creatinine and urea concentrations in males and/or females, which were considered to be test item-related; they correlated with adverse renal findings seen at pathology.

At 250 and 1000 mg/kg/day, males and females had statistically significantly higher mean blood level of total cholesterol than in controls. Similar trend was observed at 50 mg/kg/day, in females especially.
Higher mean calcium level was also noted at both higher doses, in males and/or females.
These effects were ascribed to the test item, and considered as adverse for the cholesterol at 1000 mg/kg/day in view of the magnitude of differences from controls.

Statistically significant differences from controls in mean albumin, inorganic phosphorus, sodium and potassium levels at 1000 mg/kg/day were of low magnitude and considered to be not toxicologically significant.
The effects observed on mean triglyceride concentration were considered as not toxicologically significant: there was no dose-relationship in males, and the magnitude of difference from controls in females was low and almost all individual data were included in the min-max range of historical control data.
The lower statistically significant mean ALP or ASAT activity at 250 mg/kg/day vs. controls, noted without dose-relationship, in one sex only, with a low magnitude, was considered to be incidental.
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
See table 4.
The relative kidney weights were minimally increased in both sexes at the high-dose. It correlated with cortical tubular dilatation and vacuolation. At lower doses in males, although a minimal but statistically significant increase in relative to body weight values was present, the magnitude of the change was too low to be considered relevant.

Liver weights were slightly increased from the mid-dose in females and at the high-dose in males. It correlated histologically with centrilobular hypertrophy.

Higher adrenal weights in both sexes at the high-dose, and lower thymus weights in both sexes at all dose levels except in low-dose females did not have histological correlates. They were considered possibly related to a non-specific stress response to the test item (Everds et al., 2013). Given the low magnitude of these differences, they are considered of no toxicological significance and non-adverse.

Marginally but statistically significant higher absolute epididymides weights at the high dose was not accompanied by changes in the testes (organ weights or histology), and were considered a direct consequence of lower terminal body weights.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
See table 5.
Tan discoloration of the adrenal was observed in all groups, except in control females. In the absence of correlating histological change, and given that the high incidence in high-dose females (7/10) is comparable to the incidence in control males (5/10), this change is considered spurious and unrelated to the test item administration.
All other macroscopic observations were spontaneous in nature.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See table 6.
Test item treatment-related changes were observed in the liver, thyroid glands and kidney.

Minimal to moderate centrilobular hypertrophy in the liver occurred from the dose of 250 mg/kg/day in females, and at the high-dose in males. In some animals in these groups, it was accompanied by minimal to slight follicular cell hypertrophy of the thyroid. Follicular cell hypertrophy in the thyroid often accompanies hepatic centrilobular hypertrophy in rats, although this is not relevant to humans. Neither change was considered adverse, because there was no evidence of ill health resulting from the thyroid change, and because hepatocellular hypertrophy, often observed with enzyme inducers, is well-established as an adaptive and non-adverse change in the absence (as in this study) of histologic or clinical pathology alterations indicative of liver toxicity (Hall et al., 2012).

Vacuolation of Kupffer cells was observed in the liver in both sexes at the high-dose only. The origin of this change is unknown. Because of the macrophagic activity of these cells, it possibly represents accumulation of the test item or one of its metabolite. In the absence of associated clinical pathology changes, it is not considered adverse.

In the kidney, minimal to slight dilatation of cortical tubules was observed in both sexes at the high-dose. Minimal to slight vacuolation of cortical tubules was observed in high-dose males and to a lesser extend, because of background renal vacuolation in this sex, in high-dose females. The same changes at a higher severity were considered to have contributed to demise in the premature decedent high-dose male No. J28850. Moreover, they were associated with changes in clinical pathology (increases in urea and creatinine). They are therefore considered adverse.

There were no ocular changes considered to be test item-related at any dose level.

Other observations belonged to the spectrum of spontaneous changes in rats of this age and strain.
Histopathological findings: neoplastic:
not examined
Other effects:
not examined
Key result
Dose descriptor:
NOAEL
Effect level:
250 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
body weight and weight gain
clinical biochemistry
histopathology: non-neoplastic
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
urinary
Organ:
kidney
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
not specified

Table 1: Body weight

 

Sex

Male

Female

 

Dose level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Body weight

 

 

 

 

 

 

 

 

. Week 1

188

187

184 

184

169

168

165

169

 

 

-1

-2

-2

 

-1

-2

0

. Week 4

367

362

346

332*

234

232

227

230

 

 

-1

-6

-10

 

-1

-3

-2

. Week 10

517

510

486

445**

286

283

276

276

 

 

-1

-6

-14

 

-1

-3

-3

. End of Week 13

554

540

511

489**

297

292

283

281

 

 

-3

-8

-12

 

-2

-5

-5

Body weight change

 

 

 

 

 

 

 

 

. Weeks 1-13

+366

+354

+327 

+300**

+127

+124

+118

+112*

Statistically significant from controls: *: p<0.05; **: p<0.01; initalic: differences from control values (%).

Table 2: Haematology and coagulation

 

Sex

Male

Female

Dose level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Red blood cells (T/L)

9.55

8.93*

9.18

8.79**

8.07

8.07

8.05

7.42**

Hemoglobin (g/dL)

16.1

15.8

16.0

15.5

14.7

14.6

14.5

13.7*

Packed Cell Volume (PCV; L/L)

0.49

0.48

0.49

0.47

0.43

0.43

0.43

0.41*

Mean Cell Volume (MCV; fL)

51.7

53.7*

53.5

53.8*

53.8

53.7

53.8

55.1

Mean Cell Hemoglobin (MCH; pg)

16.9

17.7*

17.4

17.7*

18.2

18.0

17.9

18.4

Platelets (G/L)

859

921

867

783

868

881

725*

745

Reticulocytes (T/L)

0.18

0.16

0.16

0.16

0.16

0.14

0.13*

0.16

Prothrombin time (s)

21.3

21.9

21.3

22.0

22.4

22.1

21.7

20.6*

Statistically significant from controls: *: p<0.05; **: p<0.01.

Table 3: Blood biochemistry

 

Sex

Male

Female

Dose level (mg/kg/day)

0

50

250

1000

0

50

250

1000

Cholesterol (mmol/L)

1.62

1.71

2.18**

4.29**

1.79

2.23

3.41**

5.26**

Calcium (mmol/L)

2.63

2.59

2.67

2.83**

2.60

2.61

2.71**

2.93**

Urea (mmol/L)

4.1

3.9

4.3

4.8

5.1

5.2

4.8

6.9**

Creatinine (µmol/L)a

27.5

28.4

29.2

31.2*

34.8

34.6

34.9

40.1*

Sodium (mmol/L)a

144

143

144

143**

142

142

142

142

Potassium (mmol/L)

4.06

3.88

3.97

3.60**

3.28

3.19

3.38

3.24

Albumin (g/L)

39

38

38

37*

42

43

45

45

Inorganic phosphorus (mmol/L)

1.93

1.80

1.86

2.02

1.37

1.41

1.43

1.69**

Triglycerides (mmol/L)

0.70

0.79

1.08*

0.86

0.43

0.56

0.57

0.61*

ALP (U/L)

323

273

275

291

147

133

94*

113

ASAT (U/L)

72

65

61*

72

72

74

83

69

Statistically significant from controls: *: p<0.05; **: p<0.01;a:values rounded to three significant figures.

Table 4: Organ weights  

Sex

Males

Females

Dose level(mg/kg/day)

50

250

1000

50

250

1000

Number of animals

10

10

9

10

10

10

Final body weight

 

. absolute

-2

-8*

-13**

-2

-5

-7

Adrenal glands

 

 

 

 

 

 

. absolute

-5

-6

+14

0

-9

+10

. relative

-4

+1

+32

+2

-5

+19

Epididymides

 

 

 

 

 

 

. absolute

-6

-5

-12**

-

-

-

. relative

-5

+3

+1

-

-

-

Kidney

 

 

 

 

 

 

. absolute

+7

0

+5

-1

-4

+5

. relative

+8*

+8*

+20**

+1

+1

+14**

Liver

 

 

 

 

 

 

. absolute

0

-1

+10

+10

+27**

+41**

. relative

+2

+7*

+26**

+12

+34

+52

Thymus

 

 

 

 

 

 

. absolute

-21

-24*

-29*

-4

-14

-23*

. relative

-20

-17

-18

-3

-9

-17

Statistically significant from controls: *: p<0.05 or **: p<0.01 (based on actual values and not on the percentages presented in the table); -: not applicable

 

Table 5: Macroscopic post-mortem examination

 

Sex

Males

Females

Dose level(mg/kg/day)

0

50

250

1000

0

50

250

1000

Number of animals

10

10

10

9

10

10

10

10

Adrenal glands

 

. tan discoloration

5

4

4

6

0

1

4

7

Table 6: Microscopic examination

 

Sex

Males

Females

Dose level(mg/kg/day)

0

50

250

1000

0

50

250

1000

Number of animals

10

10

10

9

10

10

10

10

Kidney

 

Vacuolation, cortical tubules

 

. minimal

0

0

0

4

5

2

5

7

. slight

0

0

0

2

0

0

1

2

Dilatation, cortical tubules

 

. minimal

0

0

0

0

2

0

1

4

. slight

0

0

0

8

0

0

0

6

Liver

 

Vacuolation, Kupffer cells

 

. minimal

0

0

0

6

0

0

0

8

. slight

0

0

0

2

0

0

0

1

. moderate

0

0

0

1

0

0

0

0

Hypertrophy, centrilobular

 

. minimal

0

0

0

4

0

0

2

1

. slight

0

0

0

3

0

0

5

6

. moderate

0

0

0

0

0

0

0

3

Thyroid glands

 

Follicular cell hypertrophy

 

. minimal

0

0

0

6

0

0

1

4

. slight

0

0

0

1

0

0

0

3


 

 

Conclusions:
The test item was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats at the dose levels of 50, 250 or 1000 mg/kg/day in corn oil.

The test item treatment at 1000 mg/kg/day induced adverse effects on mean body weight (males only), mean cholesterol concentration and on kidneys (increase creatinine and urea levels associated with increase of relative kidney weight and vacuolisation and dilatation of cortical tubules). The test item-related histopathological renal changes were considered to have contributed to the moribund status of one male which was prematurely euthanized. There were no adverse findings at 50 and 250 mg/kg/day.

Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 250 mg/kg/day following to the adverse effects noted at 1000 mg/kg/day.
Executive summary:

The objective of this GLP study was to evaluate the potential toxicity of the test item following daily oral administration (gavage) to rats for 13 weeks.

 

Methods

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) administration for at least 13 weeks. The test item was administered at dose levels of 50, 250 and 1000 mg/kg/day as an emulsion in the vehicle (corn oil) under a constant dosage volume of 5 mL/kg/day. A control group of ten animals per sex received the vehicle, alone, under the same experimental conditions.

The concentration of the dose formulations was checked in Weeks 1, 5, 9 and 13.

The animals were checked at least twice daily during the dosing period for mortality and morbidity and once daily for clinical signs. Detailed clinical observations were performed once a week. Body weight was recorded once before the beginning of the treatment period, and then at least once a week during the study as well as food consumption.

In the last week of treatment,hematology and blood biochemistry were performed in all animals.

Animals were euthanized and submitted to a full macroscopic post-mortem examination. Designated organs were weighed (adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, uterus and thymus) and more tissues were preserved. A microscopic examination was performed on several tissues from animals of the control- and high-dose groups sacrificed at the end of the treatment period and on all macroscopic lesions.

 

Results

The test item concentrations in the administered dose formulations analyzed were within the acceptance criteria (± 15% from nominal concentrations) and no test item was detected in control formulations.

 

One male treated at 1000 mg/kg/day was prematurely sacrificed in Week 10 for poor health condition. Test item-related renal changes (moderate dilatation and vacuolation of cortical tubules) were considered to have contributed to the moribund status of this rat. Therefore this death was considered to be related to the test item treatment. There were no other premature deaths in the study.

The only test item-related clinical sign was ptyalism noted in a few animals given 250 mg/kg/day and in all animals treated at 1000 mg/kg/day.

 

Test item treatment induced effects on mean body weight in males treated at 1000 mg/kg/day (mean body weight down to -14% vs. controls, p<0.01; mean body weight change over the treatment period: +300 g vs. +366 g in controls, p<0.01).

Similar trend on mean body weight was observed at 250 mg/kg/day but to a lesser extend (<-10%).

There were no effects on mean body weight in females (only a slightly lower mean body weight gain over the treatment period at 1000 mg/kg/day: +112 g vs. +127 g in controls, p<0.05).

There were no toxicologically significant effects on mean food consumption.

 

Laboratory investigations revealed slight findings on hematology parameters in females treated at 1000 mg/kg/day: lowermean red blood cell count (7.42 T/L, vs. 8.07 T/L in controls, statistical significance p<0.01), mean hemoglobin concentration (13.7 g/dL vs. 14.7 in controls, p<0.05), and mean hematocrit (0.41L/L vs. 0.43 L/L in controls, p<0.05). Also, mean prothrombin time was shortened in females at this dose (20.6 vs. 22.4 s in controls, p<0.05).

In males at 1000 mg/kg/day, an effect of the test item treatment on mean red blood cell count could not be excluded (8.79 vs. 9.55 T/L in controls, p<0.01).

There were no hematological effects at 50 and 250 mg/kg/day considered to be test item-related.

There were also changes in blood biochemistry parameters at 1000 mg/kg/day, i.e. in mean creatinine (31.2 and 40.1 µmol/L vs. 27.5 and 34.8 µmol/L in control males and females, respectively, p<0.05) and urea (6.9 mmol/L vs. 5.1 mmol/L in females, p<0.01) concentrations in males and/or females which correlated with renal pathological findings.

At 250 and 1000 mg/kg/day, males and females had higher mean blood level of total cholesterol (1.62, 1.71, 2.18 and 4.29 mmol/L in males at 0, 50, 250 and 1000 mg/kg/day, respectively; 1.79, 2.23, 3.41 and 5.26 mmoL/L in females at 0, 50, 250 and 1000 mg/kg/day respectively; statistical significance p<0.01 at 250 and 1000 mg/kg/day only). Similar trend was observed at 50 mg/kg/day, in females especially.

Higher mean calcium level was also noted at both higher doses (controls: 2.63 and 2.83 mmol/L in males at 0 and 1000 mg/kg/day, respectively; 2.60, 2.71 and 2.93 mmoL/L in females at 0, 250 and 1000 mg/kg/day respectively; statistical significance p<0.01).

At histopathology, the test item treatment produced at 1000 mg/kg/day similar renal changes in surviving animals (both sexes) as in the prematurely dead male: vacuolisation and dilatation of cortical tubules. It also induced hepatic centrilobular hypertrophy in both sexes at 1000 mg/kg/day and in females at 250 mg/kg/day, accompanied by follicular cell hypertrophy in the thyroid. These changes were considered adaptive and probably related to enzyme inducing properties of the test item. Vacuolation of Kupffer cells in the liver was also observed in both sexes at 1000 mg/kg/day. In addition, there were decreased mean thymic weights in all test item-treated groups except in 50 mg/kg/day females, and increased mean adrenal weights in 1000 mg/kg/day males and females, which were secondary effects of test item treatment, stress-related.

 

All the above-mentioned effects were considered to be test item-related. The test item treatment at 1000 mg/kg/day was considered to induce adverse effects onmean body weight (males only), mean cholesterol concentration and on the kidneys (pathology findings).

 

Conclusion

The test item was administered daily for 13 weeks by gavage to male and female Sprague-Dawley rats at the dose levels of50, 250 or 1000 mg/kg/day in corn oil.

 

The test item treatment at 1000 mg/kg/day induced adverse effects on mean body weight (males only), mean cholesterol concentration and on kidneys (increase creatinine and urea levels associated with increase of relative kidney weight and vacuolisation and dilatation of cortical tubules). The test item-related histopathological renal changes were considered to have contributed to the moribund status of one male which was prematurely euthanized. There were no adverse findings at 50 and 250 mg/kg/day.

 

Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 250 mg/kg/day following to the adverse effects noted at 1000 mg/kg/day

Endpoint conclusion
Endpoint conclusion:
adverse effect observed
Dose descriptor:
NOAEL
250 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The 90-day study is considered to be reliable (study with a klimisch score of 1).

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

28 -day repeated toxicity studyby oral route in rats (Bentz 2013) :

Three groups of five male and five female Sprague-Dawley rats received the test item by daily oral administration for 28 days at dose-levels of 100, 300 or 1000 mg/kg/day. The test item was administered as a suspension in the vehicle (corn oil) at a constant dosage-volume of 5 mL/kg/day. A control group of five males and five females received the vehicle alone under the same experimental conditions.

There were no unscheduled deaths during the study. The only test item-related clinical sign was ptyalism in all animals treated at 1000 mg/kg/day and was considered to be of minor toxicological importance. There were no toxicologically relevant effects at Functional Observation Battery and motor activity, and on mean food consumption, mean body weight and mean hematology parameters.

At blood biochemistry analysis and when compared with controls, there was a dose-related higher mean cholesterol level from 100 mg/kg/day in males (2.1, 2.6 (p<0.01) and 3.6 (p<0.01) mmol/L at 100, 300 and 1000 mg/kg/day, respectively, vs. 1.6) and from 300 mg/kg/day in females (2.4 and 4.3 (p<0.01) at 300 and 1000 mg/kg/day, vs. 1.8). This effect was considered to be adverse at 1000 mg/kg/day. Females treated at 1000 mg/kg/day also had slightly higher mean urea (5.6 mmol/L vs. controls, p<0.05) blood concentration and a slightly lower mean alkaline phosphatase activity (220 IU/L vs. 325, p<0.01) than controls, which were considered not to be adverse and of minor toxicological importance. At 1000 mg/kg/day, mean urine pH of males was slightly lower than that of controls (6.0 vs. 6.9, p<0.01) which was considered to be non adverse and of minor toxicological importance.

At pathology, mean liver weights were higher in females treated at 300 mg/kg/day, and in males and females treated at 1000 mg/kg/day. There were no test item-related macroscopic findings. Microscopic findings were seen in the liver (non adverse hepatocellular hypertrophy in males and females treated at 1000 mg/kg/day) and kidney (non adverse increased vacuolation in proximal tubules in females treated at 300 mg/kg/day and in males and females treated at 1000 mg/kg/day).

The test item was administered daily for 4 weeks by oral route to male and female Sprague-Dawley rats at dose-levels of 100, 300 or 1000 mg/kg/day in corn oil. The No Observed Adverse Effect Level (NOAEL) was considered to be at 300 mg/kg/day based on the effects seen at 1000 mg/kg/day on mean blood cholesterol levels, which were observed in presence of non-adverse increase of mean liver weight and livermicroscopic findings(hepatocellular hypertrophy).

90 -day repeated toxicity studyby oral route in rats (Bentz 2017) :

Three groups of ten male and ten female Sprague-Dawley rats received the test item daily by oral (gavage) administration for at least 13 weeks. The test item was administered at dose levels of 50, 250 and 1000 mg/kg/day as an emulsion in the vehicle (corn oil) under a constant dosage volume of 5 mL/kg/day. A control group of ten animals per sex received the vehicle, alone, under the same experimental conditions.

One male treated at 1000 mg/kg/day was prematurely sacrificed in Week 10 for poor health condition. Test item-related renal changes (moderate dilatation and vacuolation of cortical tubules) were considered to have contributed to the moribund status of this rat. Therefore this death was considered to be related to the test item treatment. There were no other premature deaths in the study.

The only test item-related clinical sign was ptyalism noted in a few animals given 250 mg/kg/day and in all animals treated at 1000 mg/kg/day.

Test item treatment induced effects on mean body weight in males treated at 1000 mg/kg/day (mean body weight down to -14%vs.controls, p<0.01; mean body weight change over the treatment period: +300 g vs. +366 g in controls, p<0.01).

Similar trend on mean body weight was observed at 250 mg/kg/day but to a lesser extend (<-10%).

There were no effects on mean body weight in females (only a slightly lower mean body weight gain over the treatment period at 1000 mg/kg/day: +112 gvs.+127 g in controls, p<0.05).

There were no toxicologically significant effects on mean food consumption.

Laboratory investigations revealed slight findings on hematology parameters in females treated at 1000 mg/kg/day: lowermean red blood cell count (7.42 T/L,vs.8.07 T/L in controls, statistical significance p<0.01), mean hemoglobin concentration (13.7 g/dLvs.14.7 in controls, p<0.05), and mean hematocrit (0.41L/Lvs.0.43 L/L in controls, p<0.05). Also, mean prothrombin time was shortened in females at this dose (20.6vs.22.4 s in controls, p<0.05).

In males at 1000 mg/kg/day, an effect of the test item treatment on mean red blood cell count could not be excluded (8.79vs.9.55 T/L in controls, p<0.01).

There were no hematological effects at 50 and 250 mg/kg/day considered to be test item-related.

There were also changes in blood biochemistry parameters at 1000 mg/kg/day,i.e.in mean creatinine (31.2 and 40.1 µmol/Lvs.27.5 and 34.8 µmol/L in control males and females, respectively, p<0.05) and urea (6.9 mmol/Lvs.5.1 mmol/L in females, p<0.01) concentrations in males and/or females which correlated with renal pathological findings.

At 250 and 1000 mg/kg/day, males and females had higher mean blood level of total cholesterol (1.62, 1.71, 2.18 and 4.29 mmol/L in males at 0, 50, 250 and 1000 mg/kg/day, respectively; 1.79, 2.23, 3.41 and 5.26 mmoL/L in females at 0, 50, 250 and 1000 mg/kg/day respectively; statistical significance p<0.01 at 250 and 1000 mg/kg/day only). Similar trend was observed at 50 mg/kg/day, in females especially.

Higher mean calcium level was also noted at both higher doses (controls: 2.63 and 2.83 mmol/L in males at 0 and 1000 mg/kg/day, respectively; 2.60, 2.71 and 2.93 mmoL/L in females at 0, 250 and 1000 mg/kg/day respectively; statistical significance p<0.01).

At histopathology, the test item treatment produced at 1000 mg/kg/day similar renal changes in surviving animals (both sexes) as in the prematurely dead male: vacuolisation and dilatation of cortical tubules. It also induced hepatic centrilobular hypertrophy in both sexes at 1000 mg/kg/day and in females at 250 mg/kg/day, accompanied by follicular cell hypertrophy in the thyroid. These changes were considered adaptive and probably related to enzyme inducing properties of the test item. Vacuolation of Kupffer cells in the liver was also observed in both sexes at 1000 mg/kg/day. In addition, there were decreased mean thymic weights in all test item-treated groups except in 50 mg/kg/day females, and increased mean adrenal weights in 1000 mg/kg/day males and females, which were secondary effects of test item treatment, stress-related.

All the above-mentioned effects were considered to be test item-related. The test item treatment at 1000 mg/kg/day was considered to induce adverse effects on mean body weight (males only), mean cholesterol concentration and on the kidneys (pathology findings). Under the experimental conditions and results of this study, the No Observed Adverse Effect Level (NOAEL) was set at 250 mg/kg/day following to the adverse effects noted at 1000 mg/kg/day.

Justification for classification or non-classification

Based on the 28 -day and 90 -day repeated toxicity studies, no severe organ toxicity was showed in rats treated with Ethoxylated bisphenol A diacrylate. No classification is required for the repeated toxicity according to the Regulation EC n°1272/2008.