Registration Dossier

Administrative data

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1991-03-25 to 1991-11-12
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP guideline study

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Report date:
1991

Materials and methods

Test guideline
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(daily application over a period of 30 days)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
EC Number:
407-410-2
EC Name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
Molecular formula:
C28H40O8
IUPAC Name:
2-[({5-[(2-carboxycyclohexanecarbonyloxy)methyl]tricyclo[5.2.1.0²,⁶]decan-8-yl}methoxy)carbonyl]cyclohexane-1-carboxylic acid; 2-[({8-[(2-carboxycyclohexanecarbonyloxy)methyl]tricyclo[5.2.1.0²,⁶]decan-4-yl}methoxy)carbonyl]cyclohexane-1-carboxylic acid
Constituent 2
Reference substance name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
IUPAC Name:
4,7-methanooctahydro-1H-indene-diyldimethyl bis(2-carboxybenzoate)
Test material form:
solid: crystalline
Details on test material:
- Name of test material (as cited in study report): TCD-Emulgator
- Physical state: solid
- Analytical purity: 95.7 %
- Lot/batch No.: 90003
- Expiration date of the lot/batch: 1992-01-08
- Storage condition of test material: room temperature, protected from direct light

Test animals

Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Winkelnmann, Borchen, Germany
- Age at study initiation: 6-8 weeks
- Weight at study initiation: male: 141-164 g, female: 111-140 g
- Housing: Makrolon cage typ II
- Diet (e.g. ad libitum): Altromin 1324 pellets, ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2 °C
- Humidity (%): ca. 50 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From 1991-03-18 to 1991-05-07

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
polyethylene glycol
Remarks:
(Lutrol)
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Lutrol (polyethylene glycol 400) was used as vehicle.
- Concentration in vehicle: 0, 12.5, 62.5 and 312.5 mg/mL vehicle
- Amount of vehicle (if gavage): 2 mL/kg bw
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Stability:
The stability of the test item in the vehicle was tested over a period of seven days.
Formulation analysis:
Analysis of the dose formulations were conducted once during the study.
Duration of treatment / exposure:
30 days
Frequency of treatment:
Daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 25, 125 and 625 mg/kg bw
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale:
In a pre-test 5 females and 5 males received the test substance once orally per gavage at a concentration of 2500 and 1000 mg/kg bw. The development of the body weight was affected and in certain animals a reduction in body weight was observed. Based on these observations, the actual doses were selected
- Rationale for animal assignment (if not random): Animals were randomly assigned
- Post-exposure recovery period in satellite groups: 20 days
Positive control:
N.A.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: at least twice a day

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least twice a day. A detailed clinical observation on the individual animal was conducted once a week.

BODY WEIGHT: Yes
- Time schedule for examinations: before the treatment started and afterwards once a week until week 4 (main group) and week 7 (recovery group). Additionally, prior to sacrifice

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

WATER CONSUMPTION: Yes, once a week

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: in week 5 from all animals of the main group, in week 8 from all animals of the recovery group
- Anaesthetic used for blood collection: ether
- Animals fasted: No
- How many animals: all animals
- Parameters examined: see table 2

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: in week 5 from all animals of the main group, in week 8 from all animals of the recovery group
- Animals fasted: No
- How many animals: all animals
- Parameters examined: see table 2

URINALYSIS: Yes
- Time schedule for collection of urine: in week 4 from all animals of the main group, in week 7 from all animals of the recovery group
- Metabolism cages used for collection of urine: no data
- Animals fasted: Yes (food)
- Parameters examined: see table 3

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, see table 4
HISTOPATHOLOGY: Heart, kidney, adrenals and spleen from control group and high dose group animals and from all animals the liver
Other examinations:
Organ weights:
Brain, heart, testicles, liver, kidney, spleen, ovaries and adrenal glands were weighed before fixation
Statistics:
Wilcoxon-Mann-Whitney test

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
effects observed, treatment-related
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Clinical biochemistry findings:
effects observed, treatment-related
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment related clinical signs were observed.
There were no test-item related deaths. One female from the 25 mg/kg bw dose group died during blood sampling without any explanation. One male of the 625 mg/kg recovery dose group was found dead within the first week. Gross and histopathological changes of the liver were observed. But, as no other animal died at 625 mg/kg, no animal showed clinical symptoms the death of this one male was considered to be incidental
BODY WEIGHT AND WEIGHT GAIN
No effect on the body weight was seen up to a dose of 125 mg/kg bw in both sexes. At 625 mg/kg bw the body weight gain of males was affected at the end of the treatment period by 12% (main group) and 13% (recovery group). After the recovery period of 14 days the value dropped from 13% to 9%, showing an trend for reversibility. For the female dose group no significant effect was noticed.
FOOD CONSUMPTION
Food consumption was not affected during the treatment period. During the recovery period the food consumption related to the body weight was slightly increased for the males (13%).
WATER CONSUMPTION
At 125 mg/kg bw and above the water consumption in relation to the body weight was for the males at 125 mg/kg 14% and at 625 mg/kg bw 19%. For the females the water consumption in relation to the body weight was increased by 17% (625 mg/kg bw). Also during the recovery period was the water consumption increased by 20% (males) and 17% (females) in relationship to the body weight.
HAEMATOLOGY
Up to 125 mg/kg bw no effect on erythrocytes parameters were noticed in both sexes (count, mean cell volume, mean cell hemoglobin and mean cell hemoglobin concentration). After treatment with 625 mg/kg bw the values for mean cell hemoglobin concentration, hematocrit, mean cell hemaglobin and mean cell volume decreased. Some values were out of the range of the historical data. The effects were reversible. Only the value for the mean cell hemoglobin was still statistically significantly increased after recovery.
No effects were seen for erythrocyte morphology, thrombocyte count and prothrombin time. The leucocyte count was not increased/decreased in the females, but in males at 625 mg/kg an increase was observed, which was reversible.
CLINICAL CHEMISTRY
No effects were noticed for enzymatic activity, except a slight increase in alkaline phosphatase activity in females after treatment with 625 mg/kg bw. Moreover, in both sexes was the triglyceride concentration in blood at 625 mg/kg bw decreased. Within the male high dose group was also the cholesterin concentration decreased. Additionally, in the 625 mg/kg bw group (both sexes) was an increase of the bilirubin concentration and of the quotient of albumin/globulin noticed. The results after the end of the recovery period showed no toxicological relevant differences to control values.
URINALYSIS
No effects were noticed in any dose group.
ORGAN WEIGHTS
An increase of the absolute liver weight of 10% (male) and 19% (female) was notice for the 625 mg/kg bw group. The relative liver weight of this dose group was increased by 27% (male) and 28% (female). Females showed also after the recovery period increased liver weights. The significant difference in kidney weight in comparison to the untreated control is evaluated as not toxicological relevant as no dose-related effects were noticed and as the relative kidney weights were statistically not significantly different to control samples. Moreover, no damage of the kidney was observed by histopathology
GROSS PATHOLOGY
In one male of the control (main group) it was in both cerebral ventricles an increased volume of a clear water like liquid observed. One male of the high dose group (recovery) showed a colour change of both kidneys.
HISTOPATHOLOGY: NON-NEOPLASTIC
Up to 125 mg/kg bw no treatment related effects were observed in the organs. A hypertrophy of hepatocytes was observed in both sexes in the 625 mg/kg bw dose group. The male animals of this dose group showed additionally a starting degeneration of liver cells. The histopathology of the liver from the recovery animals revealed no adverse effects. Additionally, a strong congestion of the red splenic pulp was observed in males from the high dose group.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
125 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects noticed
Dose descriptor:
LOAEL
Effect level:
625 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Effects on hematologic parameters and on the liver

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

FORMULATION ANALYSIS:

Under the experimental conditions described, the concentration of formulations at 3125 mg/10 mL (625 mg/kg bw), 625 mg/10 mL (125 mg/kg bw) and 125 mg/mL (25 mg/kg bw) of TCD Emulgator showed an deviation of <6% from the nominal concentration. Moreover, no test item was detected in the vehicle.

STABILITY:

the formulations were tested stable over 7 days.

Applicant's summary and conclusion

Conclusions:
The NOAEL of TCD Emulsifier after a daily administration over a period of 30 days by oral gavage is 125 mg/kg bw/day based on effects on hematologic parameters in the liver at 625 mg/kg/d.
Executive summary:

A repeated oral dose toxicity study [OECD 407] was conducted using male and female Wistar rats at doses of 0, (vehicle: polyethylene glycol), 25, 125, and 625 mg/kg/day of TCD Emulsifier (95.7% a.i.). The animals received the test item daily over a period of 30 days. The test substance had no effect on mortality, food consumption and clinical signs. Effects were noted for the body weight in the 625 mg/kg bw male dose group. Moreover, in the female and male 625 mg/kg bw dose group significant hematologic effects were observed. An increased liver weight together with a hypertrophy of hepatocytes were noted in the 625 mg/kg bw dose group for both female and male rats. Based on the results reported the NOAEL for orally administered TCD Emulsifier is considered to be 125 mg/kg bw/day for both sexes.

This study in rats is acceptable and satisfies the principle requirement for a repeated oral dose toxicity study according to OECD 407 in rats.