Registration Dossier

Toxicological information

Repeated dose toxicity: oral

Currently viewing:

Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across based on grouping of substances (category approach)
Adequacy of study:
key study
Study period:
24 Nov 1992 - 05 Mar 1993
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions

Data source

Reference
Reference Type:
study report
Title:
Unnamed
Year:
1993
Report Date:
1993

Materials and methods

Test guideline
Qualifier:
equivalent or similar to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Deviations:
yes
Remarks:
there was no satellite group. Some clinical chemistry, gross necropsy and hitopathological data as well as urinalysis are missing.
GLP compliance:
yes

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): Dimer Acid
- Physical state: Yellow liquid
- Analytical purity: no data
- Source: Unichema, Gouda; Holland

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River (UK) Ltd.
- Age at study initiation: 4-5 weeks
- Weight at study initiation: 114.5-155.5 g (male); 104.6-142.0 g (female)
- Housing: 5 animals per cage
- Diet (e.g. ad libitum): ESL modified AIN-76A (MODAIN) purified diet, ad libitum
- Water (e.g. ad libitum): tap water, ad libitum
- Acclimation period: 1 week


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 ± 2°C
- Humidity (%): 55 ± 10%
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: 30 Nov 1992 To: 01-05 Mar 1993

Administration / exposure

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly from 26 Nov 1992 to 10 Dec 1992; every 2 weeks from 17 Dec 1992 to the end of the study
- Mixing appropriate amounts with (Type of food): ESL modified AIN-76A (MODAIN) purified diet
- Storage temperature of food: 4°C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
-Stability and homogeneity in diet
The stability of Dimer Acid at concentrations of 0.1%, 1% and 5% in diet was determined over periods of 7 and 14 days when stored at 4°C or in an animal room.
The diets were analysed to confirm Dimer Acid was distributed homogeneously in the diet. After mixing the diets, five samples were taken for analysis from the top, the middle and bottom centre, and left and right centre of the mixing bowl.
Diet samples were extracted into propan-2-ol and centrifuged to remove particulate matter. An aliquot was concentrated to dryness, then redissolved and analysed by HPLC on a 5µ Lichrosorb Diol column, detection by a light scattering detector. Quantitation was achieved by comparison of peak areas with external standards of Dimer Acid.
Separation on the HPLC sytem was based on the interaction of the carboxylic acid of Dimer Acid with free hydroxyl groups at the surface of the diol phase. Thus, interaction increased with the number of carboxylic acid groups. Dimer Acid contains mixtures of mono, di and polyacids. In the assay preparation based on two peaks, di-acid denoted dimmer and tri or greater (poly) acid denoted trimer.
Dimer Acid was shown to be stable in diet over 14 days. The results for 0.1% (w/w) Dimer Acid in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.
Using the methods described, Dimer Acid was shown to be mixed homogeneously in the diet at a concentration of 0.1%, 1% and 5% (w/w).

-Confirmation of achieved concentration
Diets containing 5%, 1% and 0.1% Dimer Acid prepared on 26 Nov 1992, 07 Jan 1993 and 18 Feb 1993 were analysed for the achieved concentration of Dimer Acid after first passing the UV lock. The methodology was the same as that used for the determination of stability.
Analysis of the diets prepared on Week 1 and 13 confirmed the nominal concentration had been achieved within the expected experimental error of the analytical method. The results for 0.1% (w/w) Dimer Acid in diet showed more variation that would normally be accepted, but the level was very close to the limits of detection of the analytical method.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 0.1, 1, 5% (w/w) corresponding to ca. 0, 74.1, 740.9, 3591.2 mg/kg bw/day for males and ca. 0, 90.5, 854.9, 4085.5 mg/kg bw/day for females (average dose weeks 0-13)
Basis:
nominal in diet
No. of animals per sex per dose:
20
Control animals:
yes, plain diet

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were checked at least twice each day (once on Saturdays and Sundays) for signs of ill-health or reaction to treatment.


BODY WEIGHT: Yes
- Time schedule for examinations: Animals were weighed on the firts day and subsequently at weekly intervals throughout the study.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No. For each cage of five animals, food and water intakes were recorded twice-weekly throughout the study and weekly values were calculated.
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No


FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: Water intake was recorded twice-weekly throughout the study and weekly values were calculated.


OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: In the week prior to the start of the study all animals were given an ophthalmoscopic examination. In the week prior to the end of the study all animal were re-examined.
- Dose groups that were examined: all


HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the test period
- Anaesthetic used for blood collection: Yes (Halothane)
- Animals fasted: No
- How many animals: 20 per sex per dose
- Parameters checked in table 1 were examined.


CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the test period
- Animals fasted: No
- How many animals: 20 per sex per dose
- Parameters checked in table 2 were examined.

URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (see table 3)
HISTOPATHOLOGY: Yes (see information on materials and methods)
Statistics:
For body weights, food and water intakes, food conversion efficieny, clinical pathology and organ weights (incuding the ration of organ weight to body weight at terminal kill), a statistical analysis was conducted. Initially the data were examined to see if parametric or non-parametric analysis was appropriate.
If the data were parametric, a one way analysis of variance was used to see if any of the treatments differed. A t-test versus control was used to show any significant differences between control group and any of the other treatment levels at the 5%, 1%, and 0.1% probability levels. A multiple T test was used for pairwise comparisons between groups.

Results and discussion

Results of examinations

Details on results:
CLINICAL SIGNS AND MORTALITY
There were no decedents during the 13 week treatment period.
The clinical signs observed during the course of the study were not considered to be treatment-related. These generally involved scabs, alopecia, excoriation, nasal discharge and ocular discharge.

BODY WEIGHT AND WEIGHT GAIN
There were no treatment-related changes in body weight during the 13-week dosing period. Statistically significant changes in body weight were few, minor, randomly distributed and of no biological significance.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Food intake was significantly lower during the first four weeks of the study in male and female rats fed 5.0% Dimer Acid in diet, which may refelct an initial reluctance of the rats to eat the diet.

FOOD EFFICIENCY
Food conversion efficiency was statistically significantly higher in females fed 5.0% Dimer Acid during the first four weeks of the study.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study)
Statistically significant changes in water intake occured but no clear treatment-related pattern in water intake was observed. Accumulated water intake did not differ significantly between groups.

OPHTHALMOSCOPIC EXAMINATION
On ophthalmoscopic examination at the end of the study a persistent pupillary membrane was recorded for one male animal in the 0.1% treatment group and ocular opacity was recorded for one female animal in the 5.0% treatment group. These findings were not considered treatment related.

HAEMATOLOGY
See remarks on results.

CLINICAL CHEMISTRY
See remarks on results.

ORGAN WEIGHTS
See remarks on results.

GROSS PATHOLOGY
Treatment with Dimer Acid has no effects on bodily condition as assessed by estimation of abdominal fat reserves at necropsy.
Mesenteric lymph nodes were slightly or moderately enlarged in a proportion of rats from all groups fed Dimer Acid.
The colour of caecal contents was affected by feeding with Dimer Acid. Caecal contents were predominantly yellow in rats fed 5.0% Dimer Acid and predominantly yellow-green in rats given 1.0% Dimer Acid, compared with green or gray-green in rats fed 0.1% Dimer Acid or the control animals. Dimer Acid is a yellow liquid.
The incidence of uterine fluid distension was slightly increased in rats fed 5.0% Dimer Acid.
All other macroscopic findings were considered to be incidental and within the range expected for this age and strain of rat.

HISTOPATHOLOGY: NON-NEOPLASTIC
On microscopic examination treatment-related findings were observed in mesenteric lymph nodes, in the spleen, the liver, the adrenal glands and thyroidglands (in females). Of these effects, only those in the mesenteric lymph node and spleen extended down to the group fed 0.1% Dimer Acid.

-Mesenteric lymph nodes:
Aggregations of macrophages, some of them containing golden brown pigment, were observed in the paracortex and in the medulary cords in all rats fed 5.0% Dimer Acid, the majority of rats fed 1.0% Dimer Acid and a proportion of rats fed 0.1% Dimer Acid. The incidence and the amount of aggregations were dose-related, with only a few aggregations present in rats fed 0.1% Dimer Acid. The histological findings in the mesenteric lymph nodes correlated with the lymph node enlargement noted at necropsy.

-Spleen:
Macrophages containing golden/dark brown pigment were seen in the red and white pulp in all rats fed 5.0% Dimer Acid, the majority of rats fed 1.0% Dimer Acid and a proportion of female rats fed 0.1% Dimer Acid. The incidence and amount of macrophages showed a dose relationship in both male and female rats. The effect was more pronounced in female rats.

-Liver:
The incidence of bile duct proliferation was increased, as was incidence of sclerosis of the bile ducts, in male rats fed 5.0% Dimer Acid. The sclerosis was associated with a minimal mixed inflammatory cell infiltration.There was a very slight increase in incidence of bile duct proliferation in female rats fed 5.0% Dimer Acid. Periportal cytoplasmic vacuolation was decreased in both male and female rats fed 1.0% or 5.0% Dimer Acid.

-Adrenals:
Cortical vacuolation was observed in the adrenal gland of female rats fed 5.0% or 1.0% Dimer Acid. One female rats fed 0.1% Dimer Acid had trace levels of vacuolation, which is not considered toxicologically important as vacuolation may occasionally be seen in control females. Cytoplasmic rarefaction was decreased in female rats fed 5.0% Dimer Acid.
Cortical extramedullary haemopoiesis was absent in female rats fed 5.0% Dimer Acid. In rats fed 1.0% or 0.1% Dimer Acid extramedullary haemopoiesis was slightly reduced in incidence in the females. However, since the incidence of this finding generally varies considerably among groups of untreated rats this was not considered of toxicological importance.

-Thyroids:
Follicular epithelial hypertrophy was slightly increased in female rats fed 5.0% Dimer Acid.

-Spontaneous pathology:
Microscopic examination of uteri with macroscopic fluid distension revealed this to be due to a variety of different reasons - luminal dilatation or dilated/cystic endometrial glands. In view of this, and in view of the variation in uterine size with different phases of the oestrous cycle, this finding is not thought to be of any toxicological importance.
The incidence of retinal folding/atrophy was higher in rats fed Dimer Acid. However, as the overall incidence was very low and there was no dose relationship, these lesions were not considered to be of any toxicological importance.
There were slightly more lesions in the nasal passage in rats fed 5.0% Dimer Acid compared with controls.These lesions were minor in nature and included focal epithelial hypertrophy or hyperplasia associated with mucosal inflammatory cells or a luminal inflammatory exudate. Lesions of this nature are a common finding in control rats, and, while it is possible that they could be exacerbated by inhalation of diet containing irritant test material, the incidence in treated rats was still within the normal range.
A variety of spontaneous changes was recorded in animals from all dose groups with no evidence of a treatment-related distribution. These findings were within the spectrum of spontaneous lesions commonly encountered in laboratory rats of this age and strain and were considered to be unrelated to the feeding of Dimer Acid.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
10 000 mg/kg diet
Sex:
male/female
Dose descriptor:
NOAEL
Effect level:
ca. 855 mg/kg bw/day (nominal)
Sex:
female
Basis for effect level:
other: clinical chemistry; histopathology. Average dose calculated on the basis of the reported body weight and food intake data.
Dose descriptor:
NOAEL
Effect level:
ca. 741 mg/kg bw/day (nominal)
Sex:
male
Basis for effect level:
other: clinical chemistry; histopathology. Average dose calculated on the basis of the reported body weight and food intake data.

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Doses

Based on compund concentrations in diet as well as data on body weight and food intake over the 13 -weeks study period, averege doses in mg/kg bw/day were calculated as presented in the table below.

 

body weight

 

mean
body weight (g)

food intake (g)

food
intake (g/day)

compund
intake(g/day)

dosis
(mg/kg/day)

 

week 0

week 13

week 0-13

week 0-13

week 0-13

week 0-13

week 0-13

Male

 

 

 

 

 

 

 

5%

133,7

541,5

337,6

2182,3

24,2

1,21

3591,2

1%

135,1

533,5

334,3

2229,1

24,8

0,25

740,9

0,1%

135,8

560,8

348,3

2321,6

25,8

0,03

74,1

0%

133,3

545,5

339,4

2215,3

24,6

0,00

0,0

 

 

 

 

 

 

 

 

Female

 

 

 

 

 

 

 

5%

120,2

317,5

218,85

1609,4

17,9

0,89

4085,5

1%

123,6

335,9

229,75

1767,7

19,6

0,20

854,9

0,1%

120,2

317,7

218,95

1782,7

19,8

0,02

90,5

0%

119,2

317,5

218,35

1693,8

18,8

0,00

0,0

Haematology

Statistically significant changes by Student's t-test and by multiple t-test in haematology parameters are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

Dimer Acid (% w/w)

Haematological change Control 0.1 1.0 5.0
Increase in mean cell haemoglobin

M

Increase in prothrombin time 

F

M/F

The changes observed were slight and unlikely to be of any toxicological significance. A number of other changes, for instance a decrease in neutrophil count in females fed 5.0% or 1.0% Dimer Acid, were statistically significant by Student's t-test but did not trigger the multiple t-test as significant.

In the absence of any treatment-related changes in either the red or white cell count it was considered unnecessary to examine the bone marrow smears.

Clinical chemistry

Statistically significant changes by Student's t-test and by multiple t-test in clinical chemistry parameters are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

Dimer Acid (% w/w)

Clinical chemistry change Control

0.1

1.0

5.0
Decrease in plasma calcium F F M/F
Increase in alkaline phosphatase M/F M/F
Decrease in 5'-nucleotidase M M/F
Increase in alanine aminotransferase

M/F

Increase in aspartate aminotransferase F F
Increase in bilirubin M  M 
Decrease in total cholesterol M/F M/F
Decrease in triglycerides M M/F
Decrease in glucose

F

Decrease in total serum protein

M/F

Decrease in serum albumin

M/F

Decrease in beta globulin fraction M M
Increase in albumin/globulin ratio M M

The changes in plasma electrolyte levels were slight. The increase in plasma alkaline phosphatase were marked, the level being more than double that of the control group in rats fed 5.0% Dimer Acid. Other fluctuations in plasma enzyme levels were not so marked.

Serum triglyceride and cholesterol levels were decreased in rats fed 5.0% or 1.0% Dimer Acid in diet. Serum protein levels were also decreased in rats fed 5.0% Dimer Acid.

It should be noted that while a very small increase in plasma bilirubin was observed in male rats fed Dimer Acid, the levels measured were below the sensitivity of the method and must be viewed with caution.

Statistically significant changes in plasma glucose appear to be random and not part of a treatment-related pattern. Plasma levels of pseudocholinesterase were statistically significantly increased in males fed 5.0% Dimer Acid but decreased in females.

Organ weights

Statistically significant changes by Student's t-test and by multiple t-test in organ weights are summarised in the table below. In this table the symbols M and F are used to indicate a significant change in a particular parameter and whether this is seen in male (M) or female (F) rats.

Dimer Acid (% w/w)

Organ weight change Control 0 .1 1.0 5.0
Decrease in spleen weight M  M 
Decrease in relative spleen weight M  M
Decrease in kidney weight

F

Decrease in liver weight F M  M
Decrease in relative liver weight F M/F M/F

Relative kidney weight was significantly lower in rats fed 5.0% or 1.0% Dimer Acid by Student's t-test though not by multiple t-test. A dose-related trend in this parameter was evident.

Applicant's summary and conclusion

Conclusions:
Based on clinical chemistry parameters and histopathological findings, 1.0 % (w/w) test material in diet can be considered a no-observed-adverse-effect-level (NOAEL), corresponding to a dose of 741 and 855 mg/kg bw/day for males and females, respectively.