Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
18/01/2012-23/07/2012
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study

Data source

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

Materials and methods

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

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): Brominated epoxy having epoxy equivalent of 400gr/eq.
- Physical state: pale yellow solid
- Analytical purity: 395.6 gr/eq epoqy equivalent
- Lot/batch No.: B21E-86
- Expiration date of the lot/batch: 30 May 2014
- Storage condition of test material: room temperature in the ark

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Harlan Laboratories U.K. Ltd., Oxon, UK.
- Age at study initiation: 6-8 weeks
- Weight at study initiation: males: 218-249g, females: 168-196g
- Fasting period before study: no
- Housing: in solid floor polypropylene cages with stainless steel mesh lids and softwood flake bedding (Datesand Ltd., Cheshire, UK).
- Diet (e.g. ad libitum):ad libitum
- Water (e.g. ad libitum):ad libitum
- Acclimation period:for seven days during which time their health status was assessed.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 +/- 2°C
- Humidity (%): 55 +/- 15°C
- Air changes (per hr): at least fifteen air changes per hour
- Photoperiod (hrs dark / hrs light): the low intensity fluorescent lighting was controlled to give twelve hours continuous light and twelve hours darkness

IN-LIFE DATES: death after 29 or 43 days

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

DIET PREPARATION
- Rate of preparation of diet (frequency): twice during the study. The formulations were stable for at least twenty one days.
- Storage temperature of food: 4°C

VEHICLE
- Concentration in vehicle: 7.5, 75, 250 mg/ml
- Amount of vehicle (if gavage): 4ml/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The concentration of Brominated Epoxy having Epoxy Equivalent of 400gr/eq in the test item formulations was determined by high performance liquid chromatography (HPLC) using an external standard technique. The test item formulations were extracted with acetonitrile to give a final, theoretical test item concentration of approximately 0.1 mg/ml. Standard solutions of test item were prepared in acetonitrile at a nominal concentration of 0.1 mg/ml.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Daily
Doses / concentrationsopen allclose all
Dose / conc.:
30 mg/kg bw/day (nominal)
Dose / conc.:
300 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose levels were chosen based on the results of previous toxicity work (Harlan Laboratories Ltd., Project Number 41104821

- Post-exposure recovery period in satellite groups: Recovery group animals were maintained for a further fourteen days treatment-free period following termination of treatment.
Positive control:
no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: prior to the start of the treatment and on days 7, 14, 21, 26
- Cage side observations checked in table [2,3,4] were included.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed daily.

BODY WEIGHT: Yes
- Time schedule for examinations:immediately before dosing, up to thirty minutes post dosing and one and five hours after dosing during the working week. Animals were observed immediately before and after dosing and one hour after dosing at weekends. During the treatment-free period, animals were observed daily.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No data


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: daily

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42)
- Anaesthetic used for blood collection: No
- Animals fasted:No
- How many animals: all
- Parameters checked in Addendum 2 were examined. (Haemoglobin, Erythrocyte count, Haematocrit, Erythrocyte indices, Total Leucocyte count, Platelet count, Reticulocyte count, Prothrombin time, Activated partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment period (Day 28) and on all recovery group animals at the end of the treatment-free period (Day 42)
- Animals fasted: No
- How many animals: all
- Parameters examined: urea, glucose, total protein, albumin, albumin/globulin ration, sodium, potaasium, chloride, calcium, inorganic phosphorus, gamma glutamyltranspeptidase, aspartate aminotransferase, alkaline phosphatase, creatinine, triglycerides, total cholesterol, total bilirubin, bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: week 4 (non-recovery test and control group) and week 6 (all recovery group animals)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters examined: volume, specific gravity, pH, protein, glucose, ketones, bilirubin, urobilinogen, blood

NEUROBEHAVIOURAL EXAMINATION: yes
- Time schedule for examinations:Prior to the start of treatment and on Days 7, 14, 21 and 26, all animals were observed for signs of functional/behavioural toxicity.
- Dose groups that were examined:all
- Battery of functions tested: sensory activity / grip strength / motor activity

Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 14, 15)
HISTOPATHOLOGY: Yes (see Appendix 18)
Statistics:
Where considered appropriate, quantitative data was subjected to statistical analysis to detect the significance of intergroup differences from control; statistical significance was achieved at a level of p<0.05. Statistical analysis was performed on the following parameters:
Grip Strength, Motor Activity, Body Weight Change, Haematology, Blood Chemistry, Urinalysis (Volume and Specific Gravity), Absolute Organ Weights, Body Weight-Relative Organ Weights
Data were analysed using the decision tree from the ProvantisTM Tables and Statistics Module as detailed below:
Where appropriate, data transformations were performed using the most suitable method. The homogeneity of variance from mean values was analysed using Bartlett’s test. Intergroup variance was assessed using suitable ANOVA, or if required, ANCOVA with appropriate covariates. Any transformed data were analysed to find the lowest treatment level that showed a significant effect, using the Williams Test for parametric data or the Shirley Test for non-parametric data. If no dose response was found, but the data shows non-homogeneity of means, the data were analysed by a stepwise Dunnett’s (parametric) or Steel (non-parametric) test to determine significant difference from the control group. Where the data were unsuitable for these analyses, pair-wise tests were performed using the Student t-test (parametric) or the Mann-Whitney U test (non-parametric).
Probability values (p) are presented as follows:
p<0.01 **
p<0.05 *
p≥0.05 (not significant)

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
A reduction in body weight gains was evident for males treated with 1000 mg/kg bw/day when compared to controls, during the treatment period. Statistical significance was achieved for these males during Weeks 1, 3 and 4.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
.
Food efficiency:
effects observed, treatment-related
Description (incidence and severity):
Food efficiency was reduced for males treated with 1000 mg/kg bw/day.
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Increase in alanine aminotransferase levels. Bile acids were also elevated. Increase in aspartate aminotransferase.
Urinalysis findings:
no effects observed
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):
Higher liver weight of males treated with 1000 mg/kg bw/day
Gross pathological findings:
no effects observed
Neuropathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Minimal centrilobular hypertrophy was evident in all males treated with 1000 and 300 mg/kg bw/day and in three females each treated with 1000 and 300 mg/kg bw/day
Histopathological findings: neoplastic:
no effects observed
Details on results:
Clinical signs: no toxicologically significant clinical signs detected in treated animals

Mortality: there were no unscheduled deaths during the study

Body weight: A reduction in body weight gains was evident for males treated with 1000 mg/kg bw/day when compared to controls, during the treatment period. Statistical significance was achieved for these males during Weeks 1, 3 and 4.
No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 30 mg/kg bw/day.

Food consumption: No adverse effect on food consumption was detected. Food efficiency was however reduced for males treated with 1000 mg/kg bw/day.

Water consumption and compound intake: No toxicologically significant effect on water consumption was detected.

Haematology:There were no toxicologically significant effects detected in the haematological parameters examined.

Clinical chemistry: Animals of either sex treated with 1000 and 300 mg/kg bw/day showed a statistically significant increase in alanine aminotransferase levels (P<0.05 males, P<0.01 females) when compared to controls. Bile acids were also elevated (P<0.01) for animals of either sex treated with 1000 mg/kg bw/day when compared to controls, with the effect extending into the 300 mg/kg bw/day female dose group. Females treated with 1000 and 300 mg/kg bw/day showed an increase in aspartate aminotransferase (P<0.05). Although the changes in these parameters did not demonstrate a true dose related response a number of the individual values were outside the normal expected ranges. Recovery 1000 mg/kg bw/day males continued to show a statistically significant increase in alanine aminotransferase and recovery 1000 mg/kg bw/day females continued to show a statistically significant increase in bile acid following fourteen days without treatment.
No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.

Urinalysis: There were no toxicologically significant effects detected in the urinalytical parameters measured.

Gross pathology: There were no toxicologically significant effects detected in the urinalytical parameters measured.

Organ weights: Males treated with 1000 mg/kg bw/day showed a statistically significant increase (P<0.05) in relative liver weight. The mean relative liver weight was 3% greater than the mean control value. A statistically significant reduction in absolute weight (P<0.05) was however also evident. The effect on liver weight extended to males treated with 300 mg/kg bw/day (P<0.05).
No such effects were detected in females treated with 1000 or 300 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Histopathology: Males treated with 1000 mg/kg bw/day showed a statistically significant increase (P<0.05) in relative liver weight. The mean relative liver weight was 3% greater than the mean control value. A statistically significant reduction in absolute weight (P<0.05) was however also evident. The effect on liver weight extended to males treated with 300 mg/kg bw/day (P<0.05).
No such effects were detected in females treated with 1000 or 300 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Effect levels

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
ca. 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No observed effects at highest dose tested.
Key result
Dose descriptor:
NOAEL
Effect level:
300 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
body weight and weight gain
food efficiency
clinical biochemistry
organ weights and organ / body weight ratios
histopathology: non-neoplastic

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

See study report

Applicant's summary and conclusion

Conclusions:
The oral administration of Brominated Epoxy having Epoxy Equivalent of 400gr/eq to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment-related effects in animals of either sex treated with 1000 and 300 mg/kg bw/day.
The effect on body weight gain detected in 1000 mg/kg bw/day males was considered to represent an adverse effect of treatment. The microscopic liver changes (identified as centrilobular hypertrophy) and metabolic blood chemical changes (identified as increases in alanine aminotransferase, aspartate aminotransferase or bile acids) at 1000 and 300 mg/kg bw/day however were a consequence of treatment, but were considered not to represent an adverse health effect. As such the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day for females and 300 mg/kg bw/day for males.
Executive summary:

Introduction: The study was designed to investigate the systemic toxicity of the test item.

Methods: The test item was administered by gavage to three groups, each of five male and five female Wistar Han™:RccHan™:WIST strain rats, for twenty-eight consecutive days, at dose levels of 30, 300 and 1000 mg/kg bw/day. A control group of five males and five females was dosed with vehicle alone (Arachis oil BP). Two recovery groups, each of five males and five females, were treated with the high dose (1000 mg/kg bw/day) or the vehicle alone for twenty-eight consecutive days and then maintained without treatment for a further fourteen days.

Clinical signs, body weight change and food and water consumption were monitored during the study. Haematology, blood chemistry and urinalysis were evaluated for all non-recovery group animals at the end of the treatment period and for all recovery group animals at the end of the treatment-free period.

Results:

Mortality. There were no unscheduled deaths during the study.

Clinical Observations. There were no toxicologically significant clinical signs detected in treated animals.

Behavioural Assessment. There were no toxicologically significant changes in the behavioural parameters measured.

Functional Performance Tests. There were no toxicologically significant changes in functional performance.

Sensory Reactivity Assessments. There were no treatment-related changes in sensory reactivity.

Body Weight. A reduction in body weight gains was evident for males treated with 1000 mg/kg bw/day when compared to controls, during the treatment period. No such effects were detected in females treated with 1000 mg/kg bw/day or animals of either sex treated with 300 or 30 mg/kg bw/day.

Food Consumption. No adverse effect on food consumption was detected. Food efficiency was however reduced for males treated with 1000 mg/kg bw/day.

Water Consumption. There were no toxicologically significant effects on water consumption.

Haematology. There were no toxicologically significant effects detected in the haematological parameters examined.

Blood Chemistry. Animals of either sex treated with 1000 and 300 mg/kg bw/day showed an increase in alanine aminotransferase levels. Bile acids were also higher for animals of either sex treated with 1000 mg/kg bw/day with the effect extending into the 300 mg/kg bw/day female dose group. Females treated with 1000 and 300 mg/kg bw/day showed an increase in aspartate aminotransferase. Recovery 1000 mg/kg bw/day males continued to show an increase in alanine aminotransferase and recovery 1000 mg/kg bw/day females continued to show an increase in bile acid. No such effects were detected in animals of either sex treated with 30 mg/kg bw/day.

Urinalysis: There were no toxicologically significant effects detected in the urinalytical parameters measured.

Necropsy. There were no toxicologically significant macroscopic abnormalities detected.

Organ Weights. Males treated with 1000 and 300 mg/kg bw/day showed a reduction in absolute liver weight. Relative liver weights for these animals were however increased. No such effects were detected for females treated with 1000 or 300 mg/kg bw/day or in animals of either sex treated with 30 mg/kg bw/day.

Histopathology. The following treatment-related microscopic findings were detected:

Liver: Minimal centrilobular hypertrophy was evident in all males treated with 1000 and 300 mg/kg bw/day and in three females each treated with 1000 and 300 mg/kg bw/day. At 1000 mg/kg bw/day, associated minimal increased glycogen deposits was also evident in either sex.

Conclusion. The oral administration of Brominated Epoxy having Epoxy Equivalent of 400gr/eq to rats by gavage, at dose levels of 30, 300 and 1000 mg/kg bw/day, resulted in treatment-related effects in animals of either sex treated with 1000 and 300 mg/kg bw/day.

The effect on body weight gain detected in 1000 mg/kg bw/day males was considered to represent an adverse effect of treatment. The microscopic liver changes (identified as centrilobular hypertrophy) and metabolic blood chemical changes (identified as increases

in alanine aminotransferase, aspartate aminotransferase or bile acids) at 1000 and 300 mg/kg bw/day however were a consequence of treatment, but were considered not to represent an adverse health effect. As such the No Observed Adverse Effect Level (NOAEL) was considered to be 1000 mg/kg bw/day for females and 300 mg/kg bw/day for males.