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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: This study is rated a "1" because it applied GLP and used appropriate testing procedures.

Data source

Referenceopen allclose all

Title:
Peroxisome Induction studies on Seven Phthalate Esters
Author:
Barber ED, Astill BD, Moran E J, Schneider BF, Gray TJB, Lake BG and Evans JG
Year:
1987
Bibliographic source:
Toxicology and Industrial Health 3(2):7-22
Reference Type:
study report
Title:
Unnamed
Year:
1986

Materials and methods

Principles of method if other than guideline:
Method: other: not specified
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
IUCLID4 Test substance: other TS: DIDP with different CAS#

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Olac 1976 Ltd., Bicester, Oxon, UK
- Age at study initiation:
- Weight at study initiation:
- Fasting period before study:
- Housing: individually
- Diet (e.g. ad libitum):
- Water (e.g. ad libitum):
- Acclimation period: 7 days (males) 8 days (females)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 45-65
- Air changes (per hr): at least 15/hr with no recirculation-filtered air (0.5um)
- Photoperiod (hrs dark / hrs light): 12 hours dark/12 hours light)

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:


DIET PREPARATION
- Rate of preparation of diet (frequency): The mixtures were prepared once for the whole study.
- Mixing appropriate amounts with (Type of food): Rat and Mouse No.1., Special Diet Services, Witham, Essex)
- Storage temperature of food: 2-6 deg C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Mixing efficiency and stability tests were performed.
Duration of treatment / exposure:
21 days
Frequency of treatment:
daily
Doses / concentrations
Remarks:
Doses / Concentrations:
0.3, 1.2, 2.5% (approx. 280, 1100, and 2000 mg/kg/day)
Basis:

No. of animals per sex per dose:
5 rats/sex/dose
Control animals:
yes, concurrent no treatment
Details on study design:
Post-exposure period: no

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations included variations in behavior or condition, and once weekly more detailed examination occurred during weighing.


DETAILED CLINICAL OBSERVATIONS: Yes / No / No data
- Time schedule:


BODY WEIGHT: Yes / No / No data
- Time schedule for examinations: Three days before the start of treatment, on the day treatment began, and subsequently twice weekly until the end of the treatment period.


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: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Prior to autopsy
- Anaesthetic used for blood collection: No
- Animals fasted: Yes
- How many animals: All animals
- Parameters examined: Triglycerides and total cholesterol


CLINICAL CHEMISTRY: No


URINALYSIS: No


NEUROBEHAVIOURAL EXAMINATION: No


OTHER:
Sacrifice and pathology:
On the day of sacrifice, each animal was weighed and killed by exsanguination from the dorsal aorta under diethyl ether anaesthesia. Any abnormalities of the external condition and of the thoracic or abdominal viscera were noted. The liver, the left and right kidneys and, from the males, the left and right testes were weighed, and samples were preserved in 10% neutral buffered formalin. Samples of other tissues seen to be abnormal were also preserved.

For electron microscopy, two thin slices of liver, one from the left lobe, the other from the median lobe, were each cut into four or five pieces and fixed for two hours with continuous agitation. The remainder of the liver was used for biochemical analysis
Other examinations:
Whole homogenates of liver were prepared for each animal. These were assayed for protein and cyanide-insensitive palmitoyl-CoA. Microsomal lauric acid hydroxylation was also determined.
Statistics:
Data from the control and test substance treated groups were subject to analysis of variance, and if this was significant the treated groups were compared with the controls using the Least Significant Difference test.
The data from the controls and DEHP groups were compared using a two sample or pooled Student t test with Welch's correction. These methods were applied to the following data: body weights, food intakes, organ weights, serum analysis, and liver biochemistry. In all cases a probability level of p<0.05 was taken to indicate statistical significance.

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 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:
not examined
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
No animals died during the study. No variations in behavior were observed which could be considered to be related to treatment; however, two of the male rats administered 2.5% test substance showed piloerection.

BODY WEIGHT AND WEIGHT GAIN
The male rats fed 2.5% test substance lost weight during the first three days of treatment and remained significantly lighter than the controls (69-82% of control) throughout the treatment period. The difference from the controls increased as the study progressed. Males given 1.2% test substance faild to gain as much weight as the controls but the difference was significant on day 17 only. The female rats showed the same trends but to a lesser extent. Females given 2.5% test substance were significantly lighter than the controls from day 10 onwards (83-87% of control) but no significant weight reductions were observed in the other treated female groups.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
Male rats fed 1.2% and 2.5% test substance consumed significantly less diet than the controls during the first 3 days of treatment, but only in the 2.5% group was the food intake significantly reduced throughout the treatment period. Rats given 1.2% test substance showed a gradual recovery in food intakes as treatment continued, returning to normal levels by the end of the treatment period. The females showed a similar trend but to a lesser extent. During the first 3 days of treatment, only the female rats fed 2.5% test substance consumed significantly less food than the controls. Food intake in all female groups returned to normal levels by the end of the treatment period.

The intake of DIDP by each rat was calculated over each food intake interval using the analyzed dietary concentrations. Calculated intakes for males were 304, 1134, 2100, and 1077 mg/kg/day for the 0.3, 1.2, and 2.5% dietary levels, respectively. Calculated intakes for females were 264, 1042, 1972, and 1022 mg/kg/day for the 0.3, 1.2, and 2.5% dietary levels, respectively.


OBSERVATIONS POST-MORTEM
Enlarged livers were reported in one male rat given 1.2% test substance and one male given 2.5%. Areas of pale tissue were seen in the liver of one male control rat, and a female rat given 2.5% test substance had a raised area on the median liver lobe which was of normal color and texture.

CLINICAL CHEMISTRY
Triglyceride and Cholesterol:
Apart from the isolated low mean value for cholesterol concentration in rats fed 0.3% test substance (78% of control), the serum triglycerides and cholesterol concentraitons of the female rats were not significantly influenced by treatment. In males the triglyceride values were 66% of the controls in the groups fed 1.2 and 2.5%. In the male 1.2% treatment group, the cholesterol level was 75% of the control value. These three differences were statistically significant.

Hepatic Enzyme Levels:
The hepatic palmitoyl CoA oxidation was significantly increased in both sexes after treatment with 1.2 or 2.5 %. At the 1.2% dose level, values were 1014 and 748% of control and at the 2.5% level they were 1553 and 1287% of control in males and females, respectively. In males there was a small though not statistically signifiant increase after feeding with 0.3%.

In both sexes treated with the test substance, there was an increase in the 11- and 12-hydroxylation of lauric acid. The males were more sensitive than the females in this respect. For example, there were statistically significant increases of 12-hydroxylation at all dose levels in the males but at only the highest dose in females, while 11-hydroxylation was elevated to a statistically significant extent at all dose levels in the males but did not reach significance in females. In the males the increase in 12-hydroxylation (400-1138% of control) was greater than that of 11-hydroxylation (183-350% of control).

Hepatic Protein Levels:
Total hepatic protein concentrations were significantly higher in both sexes of rats fed 1.2% (119 (males) and 120% (females) of control) and 2.5% (118 (males) and 121% (females) of control). Microsomal protein levels did not show any obvious change in any of the treated groups.


ORGAN WEIGHTS
In both sexes there was a significant increase in the absolute and relative weight of the liver at dose levels of 1.2 and 2.5%. In the males, absolute weights were 186 and 172% of control for levels of 1.2 and 2.5% respectively, while relative liver weights for these groups were 201 and 254% of control. In the females absolute weights for the same levels were 160 and 192% of control and relative weights were 176 and 238% of control, respectively. In the males there were also significantly higher liver weights in rats given 0.3% test substance (121% of control for absolute and relative weights).

Males given the two lower doses of test substance had heavier kidneys than the controls (111%) while in both sexes given 2.5% test substance the kidney weights were lower than the controls (81% and 88% for the males and females, respectively). Relative kidney weights were significantly higher than the controls in all the treated groups except the females given 0.3%.

Values were 110% of control in males given 0.3% test substance and 119% in those given 1.2 or 2.5%. in females at the same levels they were 109% of control.

The testes from the males given 2.5% test substance were slightly but significantly lighter than the controls. However, when expressed relative to the low body weight, the value was significantly greater.

HISTOPATHOLOGY: NON-NEOPLASTIC
There was a marked increase in peroxisomes in male rats fed 2.5% test substance with some degree of variation between moderate and marked proliferation. There was a variable but always a very marked increase in peroxisomes in female rats fed 2.5% test substance.

There was a reduction of the normal cytoplasmic basophilia in the livers of both sexes of rats fed 1.2% or 2.5% diets. In liver sections from rats given 2.5%, this was associated with an increase in cytoplasmic eosinophilia.

All treated groups showed a reduction of neutral lipid but there was no obvious dose relationship in the magnitude of the response.

No testicular atrophy was seen after treatment and there was no effect on the kidney which could be associated with treatment. The test substance caused liver enlargement, with a reduction of the normal cytoplasmic basophilia. This was associated with an increase in cytoplasmic eosinophilia in rats given 2.5% test substance in the feed. The degree of the reduction of the histochemically demonstrable neutral fat in the livers of treated rats was similar in both sexes, but the effect was not clearly dose-related.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 280 mg/kg bw/day (nominal)
Sex:
male/female
Dose descriptor:
LOAEL
Effect level:
ca. 1 100 mg/kg bw/day (nominal)
Sex:
male/female

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

RS-Freetext:
Absolute and relative LIVER WEIGHTS and relative KIDNEY
WEIGHTS were INCREASED in animals given 1.2 or 2.5% DIDP.
Both sexes given 2.5% DIDP showed INCREASES IN PEROXISOME
NUMBERS AND SIZE, with the females showing a greater
response. INCREASES IN PALMITOYL CoA OXIDATION and TOTAL
HEPATIC PROTEIN LEVELS were also observed at these doses.

Applicant's summary and conclusion

Conclusions:
Under the conditions of this study, the test substance induces a marked proliferation of peroxisomes in both male and female rats at a dose of 2.5% which was accompanied by changes in measurements associated with peroxisome stimulation. Rats administered 1.2 or 2.5% showed reduced food intake and body weights, increased absolute and relative liver and kidney weights, and decreased hepatocyte cytoplasmic basophilia, serum cholesterol and triglycerides. Therefore the NOAEL = 0.3% (280 mg/kg/day) and LOAEL = 1.2% (1100 mg/kg/day).
Executive summary:

Di-isodecyl phthalate (the test substance) was fed to groups of five male and five female F344 rats at dietary levels of 0, 0.3, 1.2, or 2.5% for 21 days. A further group of five rats of each sex, fed 1.2% DEHP, served as a study control. Male rats given 1.2 or 2.5% showed decreased body weights as compared to control; this was seen in the females but to a lesser extent. Food intakes were reduced initially in both sexes given 1.2 or 2.5%, the effect persisting throughout treament in males given 2.5%. Absolute and relative liver weights and relative kidney weights were increased in both sexes given 1.2 or 2.5% test substance in the feed. In males given 2.5%, relative testis weights were significantly greater and no lesions were seen histologically. There was a reduction in hepatocyte cytoplasmic basophilia in rats fed 1.2 or 2.5% and in the latter group this was associated with an increase in eosinophilia. Lower periportal lipid levels were seen but this was not dose related. Serum tyiglycerides and cholesterol levels were reduced in males given 1.2 or 2.5% test substance, but no dose relationship was apparent. Both sexes given 2.5% in feed showed a marked but variable increase in peroxisome numbers and size, the females showing a greater response. Cyanide-insensitive palmitoyl-CoA oxidation was significantly increased in all treated animals except those given 0.3% in feed. There was a significant increase in the 11- and 12- hydroxylation of lauric acid in all treated males, but in the females the only significant increase was in the 12- hydroxylase level in those given 2.5% of the test substance. Therefore, the NOAEL = 0.3% (280 mg/kg/day) and LOAEL = 1.2% (1100 mg/kg/day).