Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
07 August 2012 - 06 December 2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Compliant to GLP and testing guidelines; adequate consistence between data, comments and conclusions.

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
2012

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity in Rodents)
Deviations:
no
Qualifier:
according to
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
no
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Type:
Constituent
Type:
Constituent
Test material form:
other: liquid
Details on test material:
- Name of test material: Ethoxylated bisphenol A diacrylate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals 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.

Administration / exposure

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
Doses / concentrations
Remarks:
Doses / Concentrations:
100, 300 and 1000 mg/kg/day
Basis:
actual ingested
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)

Examinations

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).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
for clinical signs but no mortality
Mortality:
mortality observed, treatment-related
Description (incidence):
for clinical signs but no mortality
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
effects observed, treatment-related
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
not examined
Details on results:
MORTALITY:
There were no unscheduled deaths during the study.

CLINICAL SIGNS:
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.

BODY WEIGHT (GAIN):
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:
There were no toxicologically relevant effects on mean food consumption.

NEUROBEHAVIOURAL EXAMINATION:
There were no toxicologically relevant effects at Functional Observation Battery and motor activity.

HAEMATOLOGY:
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 CHEMISTRY:
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:
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.

ORGAN WEIGHTS:
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 PATHOLOGY:
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.

HISTOPATHOLOGY: NON-NEOPLASTIC:
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.

Effect levels

Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

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

Applicant's summary and conclusion

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).