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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
11 July - 16 August 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:
2013

Materials and methods

Test guidelineopen allclose all
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
Limit test:
no

Test material

Constituent 1
Test material form:
liquid: viscous
Details on test material:
- Name of test material (as cited in study report): 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-trimethylcyclohexylamine
- Substance type: Clear colourless viscous liquid
- Physical state: liquid
- Purity: 100% (UVCB)
- Lot/batch No.: WA 522
- Expiration date of the lot/batch: 01 January 2014
- Storage condition of test material: At room temperature in the dark
- Purity/composition correction factor required No
- Hygroscopic No
- Reactivity: Reactive to moisture
- Test substance handling: No specific handling required
- Density: 1.02 g/cm3 (20°C)
- Stability in vehicle Polyethylene glycol: Not indicated
- Solubility in vehicle Polyethylene glycol: Not indicated

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
- Source: Charles River Deutschland, Sulzfeld, Germany..
- Age at study initiation: Young adult animals. Approximately 6 weeks (control and 500 mg/kg) or approximately 7 weeks (150 and 1000 mg/kg.
- Weight at study initiation: Body weight variation was within +/- 20% of the sex mean (males: 149 - 218 grams; females: 118 - 166 grams).
- Housing: Group housing of 5 animals per cage in labeled Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany).
- Water: Free access to tap water.
- Acclimation period: At least 5 days

ENVIRONMENTAL CONDITIONS
Environmental controls for the animal room were set to maintain 18 to 24°C, a relative humidity of 40 to 70%, approximately 15 room air changes/hour, and a 12-hour light/12-hour dark cycle. Any variations to these conditions were maintained in the raw data and had no effect on the outcome of the study.
Deviations from the daily mean relative humidity occurred. Evaluation: Laboratory historical data do not indicate an effect of the deviations.

IN-LIFE DATES: From: 11 July - 16 Augustus 2012

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenized to a visually acceptable level. Adjustment was made for specific gravity of the vehicle and the density of the test substance. No correction was made for the purity of the test substance.

VEHICLE
- Justification for use and choice of vehicle (if other than water): Based on trial formulations performed at WIL Research Europe and on information from the sponsor.

DOSE VOLUME:
5 mL/kg body weight. Actual dose volumes were calculated weekly according to the latest body weight.

Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No chemical analysis of formulations were performed in the 28-day range finding study.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 d/w.
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 150, 500, 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
3
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
Dose level of group 2 is based on an acute oral toxicity study (Project 500254). Dose levels of group 3 and 4 were amended based on the results of group 1 and 2.

Initially all animals at 500 mg/kg were dosed too low on 09 August 2012. The additional amount was administered short after (within maximally 20 minutes).
Evaluation: The dose levels were correct after the additional amount was administered. Dosing twice on one day occurred only once and is considered not to have affected the study results.
Positive control:
Not required.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS:
- Time schedule: At least twice daily. Animals showing pain, distress or discomfort, which was considered not transient in nature or was likely to become more severe, were sacrificed for humane reasons based on OECD guidance document on humane endpoints (ENV/JM/MONO/ 2000/7). The time of death was recorded as precisely as possible.

DETAILED CLINICAL OBSERVATIONS:
- Time schedule: At least once daily from start of treatment onwards, detailed clinical observations were made in all animals, at 0-15 minutes, 1 hour (±15 minutes) and 3 hours (± 30 minutes) after dosing. The time of onset, grade and duration of any observed signs were recorded.

BODY WEIGHT:
- Time schedule for examinations:Weekly.

FOOD CONSUMPTION:
- Weekly.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day

FOOD EFFICIENCY: yes

WATER CONSUMPTION: No
- Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

CLINICAL CHEMISTRY: Yes / No / No data
- Time schedule for collection of blood: immediately prior to scheduled post mortem examination
- Anaesthetic used for blood collection: isoflurane
- Animals fasted: yes
- How many animals: all animals
- Parameters checked: According to test guidelines

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

- All animals were fasted overnight with a maximum of 24 hours prior to planned necropsy, but water was provided. Animals surviving to scheduled necropsy and all moribund animals were deeply anaesthetised and subsequently exsanguinated.
- Dose groups that were examined: all groups
- Tissues/organs checked: According to test guidelines

ORGAN WEIGHTS: Yes
Organs checked according to test guidelines.

HISTOPATHOLOGY: Yes
According to test guidelines

No terminal body weight was determined in females at 150 mg/kg.
Evaluation: Although relative organ weights cannot be determined in this study, sufficient information is available for interpretation of the results of this study.
Statistics:
A descriptive statistical analysis was performed (mean, SD, median).A descriptive statistical analysis was performed (mean, SD, median).

Results and discussion

Results of examinations

Clinical signs:
effects observed, treatment-related
Mortality:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not specified
Water consumption and compound intake (if drinking water study):
no effects observed
Description (incidence and severity):
Based on subjective appraisal.
Ophthalmological findings:
not specified
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
not specified
Behaviour (functional findings):
not specified
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Histopathological findings: neoplastic:
no effects observed
Details on results:
MORTALITY AND CLINICAL SIGNS
- All rats treated at 1000 mg/kg died spontaneously or were killed moribund on Days 4 and 6 of the study. No mortality occurred in other dose groups.
- At 1000 mg/kg the following clinical signs were noted during the observation period prior to death:
Lethargy, hunched posture, abnormal gait, rales, swelling of the abdomen, piloerection, chromodacryorrhoea on the snout, pale or dehydrated appearance, ptosis, yellow urine and/or hypothermia.

All animals at 500 mg/kg showed hunched posture and piloerection. The males at this dose levels showed additionally lethargy, laboured respiration, rales and ptosis during the observation period.

Piloerection was noted in males at 150 mg/kg on several days during the observation period.

No clinical signs of toxicity were noted in females at 150 mg/kg and control animals.

The slight degree of salivation shown among the animals at 150, 500 and 1000 mg/kg is often noted in rats of this age and strain following oral gavage and may be related to irritant properties or taste of the test substance. Therefore salivation was not considered to be of toxicological relevance.

BODY WEIGHT AND WEIGHT GAIN
Body weights in males at 500 mg/kg are considered to be slightly lower than controls over the 4-week study period. Body weights from animals treated at 150 mg/kg were within the normal range of variation for this strain and comparable to the control animals.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
In accordance with slightly lower body weights in males treated at 500 mg/kg, food consumption in these animals is considered to be slightly lower than controls over the 4-week study period. No effects on food consumption were noted in animals treated at 150 mg/kg.


HAEMATOLOGY
The following changes in haematology parameters distinguished animals at 500 mg/kg from control animals. Although the changes are within normal range levels encountered for rats of this age and strain these findings together suggest a toxicological relevance:
• Higher reticulocyte count females
• Lower heamoglobin level in females
• Lower heamatocrit level in males and females
• Lower mean corpuscular volume (MCV) in females
• Higher mean corpuscular haemoglobin concentration (MCHC) in males
• Higher platelet count in males and females
• Higher prothrombin time (PT) in males
• Lower activated partial thromboplastin time (APTT) in males and females

All values in animals treated at 150 mg/kg remained in the range expected for rats of this age and strain.

CLINICAL CHEMISTRY
The following changes in clinical biochemistry parameters distinguished animals at 500 mg/kg from control animals and from normal range levels encountered for rats of this age and strain:
• Higher alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT) in males and females
• Lower total protein and albumin levels in males and females
• Lower glucose level in males

All other values in the animals treated at 500 mg/kg and all values in animals treated at 150 mg/kg remained in the range expected for rats of this age and strain.

ORGAN WEIGHTS
No toxicological relevant changes were noted in absolute and relative organ weights in animals treated at 150 and 500 mg/kg.

Heart, thymus and spleen weight appeared to be lower in males at 500 mg/kg compared to normal. However, the organ:body weight ratios of these organs were considered to be similar to those of control animals. Therefore this was considered to be related to the lower body weights.

No relative organ weights could be calculated in females at 150 mg/kg. Despite the missing information, sufficient information is available for evaluation of the absolute and relative organ weights.


GROSS PATHOLOGY
Necropsy at the end of the observation period did not reveal any toxicologically relevant alterations in animals at 150 and 500 mg/kg.

The animals treated at 1000 mg/kg, which were all found death or sacrificed in moribund condition, showed advanced autolysis, discolouration of the stomach, foci in the stomach, gelatinous contents of the small intestines and caecum and/or reduced size of the thymus.

HISTOPATHOLOGY
There were minor treatment-related microscopic findings in the liver of animals at 500 mg/kg. These findings included: minimal single cell necrosis in one male and one female, minimal micro-vesicular vacuolation in one female and minimal increased severity of inflammatory cell foci, lymphocytic (noted in all animals at grades minimal up to slight).

No treatment related findings were present in the livers of the animals at 150 mg/kg.

Effect levels

Dose descriptor:
NOAEL
Effect level:
500 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: see 'Remark'

Target system / organ toxicity

Critical effects observed:
not specified

Applicant's summary and conclusion

Conclusions:
Based on the changes seen in body weight gain, haematology and clinical biochemistry and
microscopic findings in the liver 4,4’-Isopropylidenediphenol, oligomeric reaction products with 1-
chloro-2,3-epoxypropane, reaction products with butan-1-ol and 3-aminomethyl-3,5,5-
trimethylcyclohexylamine is considered to induce toxicological changes at 500 and 1000 mg/kg.