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Toxicological information

Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
migrated information: read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
November 1991 to May 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: well documented study performed according to OECD test guideline

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)
Version / remarks:
adopted May 12, 1981
GLP compliance:
yes
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Details on test material:
- Name of test material (as cited in study report): phthalic acid, di-n-octyl, n-decyl ester (DODP)
- Substance type: pure active substance
- Physical state: clear slightly yellow liquid
- Lot/batch No.: 900706
- Expiration date of the lot/batch: no data
- Storage condition of test material: no data

Test animals

Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: Charles River UK Ltd, Margate, Kent or Harlan-Olac Ltd, Bciester, Oxon
- Age at study initiation: 42 +/- 2 days
- Weight at study initiation: 137-166g (males); 91-116 (females)
- Fasting period before study: no data
- Housing: in groups of 2 animals (dose-range study: groups of 5) in polypropylene and stainless-steel grid-floored cages
- Diet (e.g. ad libitum): RM1 (E)SQC FG (ad libitum) supplied by Special Diets Services Ltd (Witham, Essex, UK)
- Water (e.g. ad libitum): tap water (ad libitum)
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19-24
- Humidity (%): 45-70
- Air changes (per hr): min. 15
- Photoperiod (hrs dark / hrs light): 12/12


IN-LIFE DATES: From: April 7, 1992 To: July 15, 1992

Administration / exposure

Route of administration:
oral: feed
Details on oral exposure:
DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Storage temperature of food: 2-6 °C
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
analyses of concentration, mixing efficiency and stability in the diet by GLC after extraction with acetone
Duration of treatment / exposure:
90 days
Frequency of treatment:
continuously
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0.1 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0.3 %
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
1.0 %
Basis:
nominal in diet
No. of animals per sex per dose:
10
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale:
A 14-day dose range finding study was carried out in 5 rats/sex/dose with dose levels of 0.1, 0.3 and 1.0 % in the diet resulting in lower mean body weights in males and increased liver weights in both sexes in the high dose group.

Examinations

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once weekly

BODY WEIGHT: Yes
- Time schedule for examinations: three days before start of treatment, at start of treatment (day 0) and subsequently twice weekly until the end of the treatment

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): YES
- 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

FOOD EFFICIENCY: No

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: during the week preceding the start of the study and during the last week of treatment
- Dose groups that were examined: control and high dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at necropsy
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: all aimals
- Parameters checked: total erythrocyte count, total leucocyte count, haemoglobin concentration, mean cell haemoglobin, mean cell haemoglobin concentration, hematocrit, platelet count, differential laucocyte count, reticulocyte count, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at necropsy
- Animals fasted: Yes
- How many animals: all aimals
- Parameters checked: concentrations of glycose, urea, total protein, albumin, creatinine, calcium, sodium, chloride, potassium, phosphorus and total bilirubin; alkaline phosphate activity (ALKP), alanine aminotransferase activity (ALAT), aspartate ainotransferase activity (ASAT).

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes ; examination on all animals; inspection of the thoracic and abdominal viscera; determination of weights of livers, kidneys, adrenals and testes

HISTOPATHOLOGY: Yes ; examination on all animals of control and high dose group; tissues examined: adrenal gland, artery (aorta), bladder (urinary), brain, caecum, colon, cervix uteri, diaphragm, duodenum, epididymis, eye, Haderian gland, heart, ileum, kidneys, liver, lungs, lymph nodes (axillary, cervical and mesenteric), mammary gland (inguinal region), nasal bone, nerve (sciatic taken together with surrounding muscle), oesophagus, ovaries, pancreas, rectum, salivary gland, seminal viscles, skeletal muscle, skin (inguinal region), spinal cord, spleen, stomach, testes, thymus, thyroid gland, tongue, trachea, uterine horns, vagina, vein (posterior vena cava). Aditionally, the lungs from animals of the mid dose group were examined on evidence of infection.
Statistics:
Body weights, organ weights, food intakes, biochemical, haematology and serum analysis data were compared by analysis of variance followed by least significant difference tests. Incidence data from microscopic examination was analysed by Chi square techiques or Fisher's exact test. A level of propability of less than 0.05 was taken to indicate statistical signifcance.

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):
no effects observed
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
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:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related changes in condition or behaviour were observed in male rats throughout the study. In female rats fur loss was a common observation in all groups and as such was not considered to be related to treatment.

BODY WEIGHT AND WEIGHT GAIN
Statistically significantly lower mean body weights were observed for males of all three dose groups. These differences were present from day 45 for the low dose group, from day 17 for the mid dose group and from day 14 for the high dose group. These findings persisted to the end of the study although they were not significant for the mid dose group at days 87 and 90. The mean weights for the low and mid dose group were similar for the second half of the study (from day 45) but were considerably lower for the high dose group. (see table 1)
In females isolated statistically significant differences in mean body weights were seen throughout the study in the low and high dose groups. In general, the mean body weights of the low dose group were slightly higher than controls and those of the high dose group were slightly lower.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
There were occasional statistically significant differences in the food intakes of both males and females when compared to the controls. Because of the sporadic nature of these effects and no clear dose-response relationship, they were not considered to be treatment-related.
Mean compound intakes were calculated for the respective treatment groups to be 79.6, 236.4 and 826.0 mg/kg bw/d for males and 102.3, 303.9 and 1027.7 mg/kg bw/d for females.

OPHTHALMOSCOPIC EXAMINATION
No treatment-related findings observed.

HAEMATOLOGY
In males only an increase of prothrombin and activated partial thromboplastin times were observed in the mid dose group. In female rats statistically significantly higher platelet counts were observed in the mid and high dose groups. In the high dose group additionally statistically significantly lower red blood cells, haemoglobin, haematocrit and neutrophil values as well as higher mean cell haemoglobin concentration (MCHC) values were observed. (see table 2)

CLINICAL CHEMISTRY
The main effect of treatment was seen in male rats of the high dose group showing significantly higher levels of ALKP, ALAT, ASAT and bilirubin. Higher levels of ALAT were also seen in mid dose males. Statistically significantly lower levels of creatinine, glycose, total protein and albumin were also observed in the high dose group when compared to controls. In female rats, the effects on liver enzymes were much less severe and although, compared with controls, slightly higher levels of ALKP and ASAT were seen in the high dose group, the only enzyme levels which showed statistically significant difference for this group was higher ALAT and lower bilirubin levels. A lower protein level observed in the mid dose group was not considered to be treatment-related. (see table 3)

ORGAN WEIGHTS
Male animals had statistically significantly lower absolute adrenals weights in the high dose group, liver weights in the low dose group and kidneys weights in the low and high dose group. None of these differences were seen for the weights relative to body weight. Relative testes weights were slightly higher in the mid and high dose groups.
In females statistically significantly higher absolute liver weights were observed in the low and high dose groups. Relative liver and kidney weights were statistically significantly increased in high dose females. (see table 4)

HISTOPATHOLOGY: NON-NEOPLASTIC

Test article related changes were seen in the livers of male rats of the mid and high dose group. In the high dose group the parenchymal cells were vacuolated and enlarged with marked individual cell necrosis associated with a diffuse inflammatory cell infiltrate. Bile duct hyperplasia was also present. Similar but less severe changes were present in the mid dose group with diffuse inflammatory infiltrate only in one rat and no evidence of bile duct hyperplasia. The lesions were not observed in males of the low dose group nor in any of the treated females. (see table 5)
Histopathological changes, which were not related to treatment, were seen in the epididymus, testes, heart, lungs and pancreas of male rats an in the kidneys, lungs, ovaries and uterus of female rats. Nematodes were present in one control male and two males of the high dose group.

HISTOPATHOLOGY: NEOPLASTIC (if applicable)
No treatment-related findings were observed.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
ca. 303.9 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: organ weights; haematology; clinical chemistry
Dose descriptor:
NOAEL
Effect level:
ca. 79.6 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: body weight; clinical chemistry; organ weights; histopathology

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: Body weights of males at selected time points (mean values and standard deviations)
Group Control 0.1 % 0.3 % 1.0 %
day 0 168.5 ± 9.76 165.2 ± 6.51 164.5 ± 10.32 165.4 ± 9.72
day 28 266.1 ± 9.50 254.8 ± 11.80 251.5 ± 24.14* 241.5 ± 10.34***
day 56 318.9 ± 11.46 301.9 ± 15.79* 303.3 ± 24.38* 284.4 ± 11.02***
day 90 359.1 ± 11.24 339.7 ± 16.18* 345.3 ± 24.74 314.6 ± 14.72***
*p ¿ 0.05; *** p ¿ 0.001
Table 2: Haematology findings in females (mean values and standard deviations)
Group Control 0.1 % 0.3 % 1.0 %
Platelet count (*103/µl) 595.6 ± 39.64 586.0 ± 51.12 667.9 ± 41.33** 670.4 ± 76.48**
Red blood cells (*103/µl) 7.926 ± 0.1691 7.763 ± 0.3601 7.725 ± 0.2557 7.540 ± 0.4791*
Haemaglobin (g/dl) 14.77 ± 0.206 14.59 ± 0.494 14.62 ± 0.290 14.34 ± 0.561*
Haematocrit (%) 48.27 ± 1.876 47.18 ± 2.617 46.20 ± 1.402 45.22 ± 3.500*
Neutrophils (*103/µl) 0.5856 ± 0.37660 0.5222 ± 0.27815 0.5590 ± 0.27404 0.3144 ± 0.17045*
MCHC (g/dl) 30.64 ± 0.873 31.01 ± 1.211 31.64 ± 0.737 31.79 ± 1.416
*p ¿ 0.05; ** p ¿ 0.01
Table 3: Clinical chemistry findings (mean values and standard deviations)
Group Control 0.1 % 0.3 % 1.0 %
Albumin (g/l) males 38.47 ± 1.387 38.46 ± 1.468 38.13 ± 1.238 36.58 ± 1.426**
ALKP (U/l) males  97.2 ± 6.32 101.6 ± 10.28 103.3 ± 5.66 165.5 ± 15.83***
females 92.3 ± 13.70 90.7 ± 5.38 89.7 ± 8.07 98.6 ± 9.94
ALAT (U/l) males 51.4 ± 4.12 48.3 ± 17.68 62.5 ± 8.81** 147.6 ± 19.15***
females 44.6 ± 17.48 52.8 ± 17.43 47.1 ± 7.59 63.3 ± 25.65*
ASAT (U/l) males 101.2 ± 12.42 100.5 ± 9.51 110.6 ± 11.76 268.6 ± 37.33***
females 114.1 ± 33.19 118.6 ± 18.95 111.7 ± 18.86 138.1 ± 52.49
Creatinine (µmol/l) males 65.6 ± 3.17 65.9 ± 4.15  66.1 ± 5.17  58.3 ± 1.89***
Glucose (mmol/l) males 10.23 ± 1.658 9.19 0.890 9.54 ± 1.403 7.42 ± 0.637***
Bilirubin (µmol/l) males 1.41 ± 0.228 1.21 ± 0.318 1.20 ± 0.283 1.72 ± 0.447*
females 1.10 ± 0.471 1.28 ± 0.239 1.27 ± 0.283 0.64 ± 0.299**
Protein (g/l) males 60.9 ± 2.77 61.0 ± 2.26 60.3 ± 1.83 57.1 ± 2.23***
females 59.8 ± 3.08 59.8 ± 3.29 56.8 ± 1.55* 58.8 ± 3.52
*p ¿ 0.05; ** p ¿ 0.01; p*** p ¿ 0.001
Table 4: Organ weights (mean values and standard deviations)
Group Control 0.1 % 0.3 % 1.0 %
Liver (absolute) males 8.458 ± 0.5993 7.820 ± 0.6136* 8.327 ± 0.8664 7.883 ± 0.5618
females 4.280 0.2508 4.582 0.3396* 4.428 0.1425 5.317 0.4863***
Liver (relative) males 2.534 ± 0.1490 2.469 ± 0.1216 2.704 ± 0.3595 2.689 ± 0.1390
females 2.488 0.1225 2.596 0.2496 2.554 0.0555 3.219 0.2656***
Kidneys (absolute) males 1.852 ± 0.1144 1.720 ± 0.1375* 1.758 ± 0.1027 1.659 ± 0.1239**
females 1.109 0.0987 1.162 0.0626 1.161 0.0523 1.130 0.0503
Kidneys (relative) males 0.555 ± 0.0201 0.543 ± 0.0261 0.572 ± 0.0660 0.566 ± 0.0308
females 0.644 0.0472 0.658 0.0454 0.670 0.0408 0.685 0.0316*
Adrenal (absolute) males 0.058 ± 0.0068 0.055 ± 0.0098 0.052 ± 0.0060 0.047 ± 0.0056**
Adrenal (relative) males 0.017 ± 0.0023 0.017 ± 0.0027 0.017 ± 0.0032 0.016 ± 0.0013
Testis (absolute) males 3.101 ± 0.1582 3.019 ± 0.1655 3.074 ± 0.0988 3.106 ± 0.1440
Testist (relative) males 0.931 ± 0.0414 0.957 ± 0.0582 1.000 ± 0.1062* 1.070 ± 0.0666***
*p ¿ 0.05; ** ¿ 0.01; p*** p ¿ 0.001
Table 5: Histopathological findings in males (liver)
Group Control 0.1 % 0.3 % 1.0 %
Vacuolation centrilobular 0 0 8*** 10***
Hypertrophy centrilobular 0 0 5* 10***
Diffuse inflammtory cell infiltration 0 0 1 10***
Bile duct hyperplasia 0 0 0 10***

Applicant's summary and conclusion

Executive summary:

Based on the hepatotoxic effects shown as histological changes in the liver and effects on liver enzymes the No Observed Advers Effect level (NOAEL) for males is considered to be 0.1 %, corresponding to a mean intake of 79.6 mg/kg bw/d of the structural related substance 1,2-Benzenedicarboxylic acid, di-C8-10-alkyl esters. For females the NOAEL is considered to be 0.3 % corresponding to a mean intake of 303.9 mg/kg bw/d on the basis of increased liver and kidney weights, effects on liver enzymes and haematological parameters.