Registration Dossier

Data platform availability banner - registered substances factsheets

Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.

The new ECHA CHEM database has been released by ECHA, and it now contains all REACH registration data. There are more details on the transition of ECHA's published data to ECHA CHEM here.

Diss Factsheets

Toxicological information

Carcinogenicity

Currently viewing:

Administrative data

Description of key information

Key studies. Read across approach. In two long term carcinogenicity studies, the analogues diisononyl phthalate (DINP) and diisodecyl phthalate (DIDP) were not found carcinogenic at dietary levels up to 0.6% (approximately 340 mg/kg/day) or 8000 ppm (17.37 mg/kg/day in males or 13.36 mg/kg/day in females) respectively. Based on read across approach, DUP is not condsidered carcinogenic and the NOAEL is determined to be 385.57 mg/kg/day.

Key value for chemical safety assessment

Carcinogenicity: via oral route

Link to relevant study records

Referenceopen allclose all

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)
GLP compliance:
not specified
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratory (Stoneridge, New York)
- Age at study initiation: 6 weeks old
- Weight at study initiation: 109 g (male) and 89 g (female)
- Fasting period before study: not specified
- Housing: individually except during the first week of acclimation. Cages: suspended stainless steel
with indirect bedding
- Diet (e.g. ad libitum): ad libitum. Fresh diet presented weekly. Purina Certified Rodent Chow 5002
- Water (e.g. ad libitum): ad libitum. Automatic watering system
- Acclimation period: not specified.

Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): weekly
- Mixing appropriate amounts with (Type of food): DINP was added to Purina Certified Rodent Chow 5002 on a fixed weight percentage (w/w) basis and mixed thoroughly to ensure homogeneity. The stability of DINP in the test diets was demonstrated periodically.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
PRETREATMENT:
Extraction: Weighed aliquots of feed samples were extracted with n-hexanol
Filtration: extracted samples were filtered before analysis.

IDENTIFICATION AND QUANTIFICATION OF TEST SUBSTANCE/PRODUCT
- Separation method (e.g. HPLC, GC): Homogeneity and stability of test chemical in the diets were verified analytically by HPLC.
- Detection limits (LOD, LOQ) (indicate method of determination/calculation): The lower limit of detection, based on 2-g aliquots of feed, was 0.004 wt% DINP.
- Extraction recovery (indicate if results are corrected or not for recoveries): Analysis for uniformity of mixing was conducted on all test material containing batches prepared for use on Weeks 1, 2, 27,
and 53 of the study and was determined to be within ±10% of the target concentration.
Duration of treatment / exposure:
24 months
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
0 other: %
Remarks:
nominal in diet
Dose / conc.:
0.03 other: %
Remarks:
Nominal in diet.
Equivalent to mean daily intake of 15 (male) and 18 (female) mg/kg bw/day
Dose / conc.:
0.3 other: %
Remarks:
Nominal in diet.
Equivalent to mean daily intake of 152 (male) and 184 (female) mg/kg bw/day
Dose / conc.:
0.6 other: %
Remarks:
Nominal in diet.
Equivalent to mean daily intake of 307 (male) and 375 (female) mg/kg bw/day
No. of animals per sex per dose:
110
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale: the high dose selected was limited to 0.6% DINP because higher doses of DINP might have jeopardized survival due to kidney lesions (Bucher et al., 1996; Ettlin et al., 1994). The mid dose of 0.3% was selected to produce lesser effects on the liver and kidney. The low dose o f 0.03% was selected as an anticipated NOEL based on 13-week data and pharmacokinetic considerations in F344 rats.
Positive control:
N/A
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly

BODY WEIGHT: Yes
- Time schedule for examinations: weekly on the same day of the week.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): yes
- Time schedule for examinations: weekly on the same day of the week.

FOOD EFFICIENCY: No

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at sacrified for animals scheduled for interim sacrifices and at termination for the rest of animals.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: overnight fast (16 hr)
- How many animals: 10 rats/sex/group scheduled for interim sacrifices and on 20 rats/sex/group sacrificed at termination.
- Parameters examined: red blood count (RBC), hematocrit, hemoglobin, leukocyte count (total and differential), mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentrations, platelets, and reticulocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at sacrified for animals scheduled for interim sacrifices and at termination for the rest of animals.
- Anaesthetic used for blood collection: Not specified
- Animals fasted: overnight fast (16 hr)
- How many animals: 10 rats/sex/group scheduled for interim sacrifices and on 20 rats/sex/group sacrificed at termination.
- Parameters examined: albumin/globulin ratio, albumin, blood urea nitrogen, calcium, cholesterol, creatinine, electrolytes (Na +, K +, Cl-), globulin, glucose, serum glutamic oxaloacetic transaminase (SGOT or AST), serum glutamic pymvic transaminase (SGPT or ALT), serum osmolality, total bilirubin, total protein, and triglycerides.

URINALYSIS: Yes
- Time schedule for collection of urine: One week prior to each interim sacrifice and 2 weeks prior to terminal sacrifice, urine samples were collected during a 16-hr period for qualitative and quantitative analyses.
- Metabolism cages used for collection of urine: No data
- Animals fasted: No
- Parameters checked:
Quantitative analyses: urine volume, glucose, protein, creatinine, renal epithelial cell counts (REC), sodium, potassium, and osmolality.
Qualitative analyses: pH, specific gravity, ketones, bilirubin, and occult blood.

NEUROBEHAVIOURAL EXAMINATION: No

IMMUNOLOGY: No


Sacrifice and pathology:
GROSS PATHOLOGY: Yes
Preselected groups of 10 rats/sex/group were scheduled for interim sacrifice after 6, 12, and 18 months on study. The animals remaining on study were sacrificed at 24 months. Complete necropsies were performed on all animals.
Organ weighed: adrenals, brain, heart, kidney, liver, ovaries, spleen, testes, and thyroid/parathyroids.

HISTOPATHOLOGY: Yes
Tissues were preserved in 10% neutral buffered formalin and processed (paraffin technique) for light microscopic examination. Microscopic examination of hematoxylin- and eosin-stained sections was performed on tissues from all major organs in the high-dose and control groups plus gross lesions and target tissues (liver, kidney) from the mid- and low-dose groups.
All tissues were examined from animals that died or were sacrificed moribund during the study except when precluded by autolysis.
Tissues examined at study termination: adrenals, aorta, brain, epididymis, esophagus, eyes with optic nerve, femoral muscle with sciatic nerve, Harderian glands, heart, kidneys, large intestines (colon, cecum), liver, lungs (infused with fixative), mammary gland, mesenteric lymph nodes, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, seminal vesicles, skin, small intestines (duodenum, jejunum, ileum), spinal cord (cervical, midthoracic, lumbar), spleen, sternum with marrow, stomach, testes, thyroid/parathyroids, trachea, urinary bladder, uterus, vagina, all gross lesions, and all tissue masses.
Tissues examined from the animals sacrificed at 6, 12, and 18 months: all gross lesions, all tissue masses, epididymides, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, and thyroid.
Other examinations:
Morphological examination of liver: Livers of two rats/sex/dose group including controls were examined by electron microscopy for evidence of hepatic peroxisome induction at study termination. The evaluation was conducted using an identical protocol as that used in the 13-week dietary study on DINP in F344 rats (Bird et aL, 1987).
Statistics:
Bartlett's test of homogeneity of variance was used to determine if the groups had equivalent variances (Snedecor and Cochran, 1971). If the variances were equal, the testing for significance was done using a parametric method, a standard one-way ANOVA. If significant differences among the means were indicated, Dunnett's test was used (Dunnett, 1964) to determine which treatment groups differed from controls. If the groups did not have equivalent variances, the test of equality of means was performed using the Kruskall-Wallis test (Hollander and Wolfe, 1973). If the means were different, Dunn's summed rank test was used to determine which treatment groups differ significantly from control (Hollander and Wolfe, 1973). The test for equal variance was conducted at the 1% level of significance. All other tests were conducted at the 5 and 1% levels of significance.
The survival analysis used two methods: (1) the Weibull estimate of median survival and (2) the Kaplan-Meier method with a comparison between groups using both the generalized Kruskal-Wallis test and Cox's test (Kaplan and Meier, 1958).
Three methods of statistical analyses were used to assess tumor incidence data in the chronic study: (1) life table analyses (Mantel and Haenszel, 1959; Cox, 1972; Tarone, 1975), (2) incidental tumor analysis (Haseman, 1984), and (3) unadjusted analysis for linear dose response. The last method consists of a pairwise comparison of each dose group with the control by the Fisher exact test and a test for linear trend (Armitage, 1971; Gartet al., 1979).
Clinical signs:
no effects observed
Description (incidence and severity):
The incidence of clinical in-life observations was relatively low throughout the 24-month test period. The most frequently noted observation included alopecia and scabs on the skin which appeared unrelated to treatment.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
A slight decrease in overall survival was noted in the mid- and high-dose groups of both sexes compared to controls, but whether or not this decrease was statistically significant depended on the statistical method employed. No difference was observed using the Weibull technique. The Cox and generalized Kruskall-Wallis tests indicated a significant difference in survival in mid- and high-dose female rats.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
When compared with controls, the high-dose males had a statistically significant, dose-related decrease (4-7%) in body weight at 12, 18 and 24 months of treatment. There were no statistically significant changes in females.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Compared to the controls, slight decreases were observed.
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
Description (incidence and severity):
In general, the mean hematologic values were within normal limits for both sexes treated with DINP for 6, 12, and 18 months. At study termination, the red blood cell count and haemoglobin and hematocrit mean values were slightly lower for mid- and high-dose males and females compared to control values. However, only the high-dose males showed a statistically significant decrease in RBC, hemoglobin, and hematocrit compared with controls. The high-dose males and females also exhibited an increased frequency of occurrence of nucleated RBC, polychromatophilic red cells, and reticulocytes when compared with control animals.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
An evaluation of the serum chemistry values at 6, 12, and 18 months revealed no remarkable findings. Some parameters were statistically increased (BUN, A/G ratio, creatinine) but were judged unlikely to be of biological significance due to a lack of dose response.
The following parameters usually associated with liver function were weakly increased in the mid and high-dose males at all time intervals in the study: alkaline phosphatase, SGOT, and SGPT. In general, these responses were statistically significant and may be related to treatment. No statistically significant effects were observed on total bilirubin.
The results of the female serum chemistry profile revealed no statistically significant treatment-related effects on liver function parameters.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The qualitative urine chemistry parameters were non remarkable at all time intervals for both males and females.
The quantitative urine chemistry parameters were unaffected in females at all time intervals, but the males showed slight but statistically significant changes in some of the parameters throughout the study. The high-dose males exhibited increased urine volumes when compared to controls at all time intervals. In the mid- or high-dose males, potassium and glucose showed a statistically significant increase at 6, 12, and 18 months, but not at 24 months. A statistically significant increase in excretion of renal epithelial cells was noted in high-dose males at 6 months but not at later treatment periods.
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Both males and females of the mid- and high dose groups had a statistically significant, dose-related increase in relative kidney and liver weights throughout most of the treatment period; the absolute liver and kidney weights demonstrated a similar trend. At study termination, the mid- and high-dose males and high-dose females exhibited a statistically significant increase in absolute and relative spleen weight compared to controls. Absolute and relative adrenal weights were slightly but significantly increased in high-dose female rats after 6 and 12 months of treatment. In addition, at study termination, relative but not absolute adrenal weights were slightly but significantly increased in high-dose rats of both sexes. No treatment-related changes were observed in the absolute or relative organ weights for ovaries, testes, brain, heart, or thyroid/parathyroid.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
No grossly observable and treatment-related abnormalities were apparent at each of the interim sacrifices. At study termination, however, the mid- and high-dose males and high-dose females exhibited an increased incidence of splenic enlargement when compared with control animals.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects on tissues were observed in rats sacrificed after 6 and 12 months of treatment.
At the 18-month sacrifice nonneoplastic lesions were observed in the liver and kidney of treated rats. Minimal to slight centrilobular to midzonaI hepatocellular enlargement was present in 9/10 male and 10/10 female rats treated with 0.6% DINP; this effect was absent in controls. In the kidneys, a minimal increase in tubular cell pigment was noted in the tubular epithelium of high dose male rats.
At terminal sacrifice, a variety of spontaneous disease lesions and incidental findings of the expected type and incidence occurred in control and treated rats without relationship to treatment with the exception of liver lesions and mononuclear cell leukemia (MNCL). Slight centrilobular to midzonal hepatocellular enlargement was present in a small number of rats and was comparable to the findings at the 18-month sacrifice.
DINP failed to produce any treatment-related effects on the testes.
Histopathological findings: neoplastic:
effects observed, treatment-related
Description (incidence and severity):
No treatment-related effects on tissues were observed in rats sacrificed after 6, 12 and 18 months of treatment.
The most common causes of the unscheduled deaths and/or morbidity were mononuclear cell leukemia (MNCL) and, to a lesser extent, pituitary neoplasia. Tubular cell pigmentation in the kidney was increased in severity in animals with advanced MNCL.
MNCL occurred at a statistically significant increased incidence in the mid- and high-dose groups of both sexes when compared with the control groups and with a significantly increasing trend over time.
The MNCL was associated with a variety of hepatic alterations (nonneoplastic lesions) which included regenerative nodules, focal necrosis, spongiosis hepatis, and "hepatopathy associated with leukemia".
DINP produced no treatment-related preneoplastic or neoplastic lesions of the liver.
Transitional cell carcinoma and tubular cell carcinoma were found in 3 mid-dose and 2 high-dose male rats respectively, although they were not was not statistically significant when compared to control animals.
There was no evidence of any treatment-related preneoplastic renal lesions.
DINP also failed to produce any treatment-related effects on the testes. As expected, the control animals displayed a high incidence of testicular tumors.
Other effects:
no effects observed
Description (incidence and severity):
Morphological Examination of Liver: Ultrastructural examination of liver specimens from representative rats of each sex from the four groups did not reveal any treatment-related peroxisomal proliferation.
Relevance of carcinogenic effects / potential:
DINP was not considered a liver carcinogen in F344 rats at a dietary level up to 0.6% DINP. Morphologic evaluations also indicated that hepatic peroxisome proliferation was not induced in this chronic study.
At study termination, DINP produced statistically significant increases in MNCL in the mid- and high-dose groups of F344 rats. MNCL in F344 rats is associated with secondary effects on organ weights (splenic enlargement), hematological parameters (decreases in hemoglobin and hematocrit), clinical chemistry parameters (liver function indices such as ALT, AST, bilirubin, etc.), as well as liver lesions such as necrosis (Stromberg et al., 1983a,b,c).
The prevailing data indicate that the histogenic tumor type (MNCL) in F344 rats does not occur in humans (Swaen and Van Heerde, 1987). Moreover, this leukemia in F344 rats appears to arise from the spleen, but this does not appear to be the case for humans where leukemia originates from affects on the bone marrow (Iatropoulos, 1983). Thus, the study suggests that MNCL is a common finding in aging F344 rats and that increased incidence of MNCL is not relevant to humans.
Key result
Dose descriptor:
NOEL
Effect level:
0.03 other: % diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Biological effects found in the mid-dose group
Remarks on result:
other: Equivalent to ca. 17 mg/kg bw/day
Key result
Dose descriptor:
NOAEL
Effect level:
0.6 other: % in diet
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: Equivalent to ca. 340 mg/kg bw/day
Key result
Critical effects observed:
no

Table 1: Body Weights (g) for F344 Rats in a Chronic DINP Study (Mean ± SD)

Dietary concentration of DINP

(wt %)

Sex

Month

Nº examined

per dose

0

0.03

0.3

0.6

Male

Pretest

110

109±5

109±5

109±5

109±5

6

110

329±22

332±23

330±20

324±23

12

100

386±23

385±21

380±22

371±25a

18

78-83

404±24

406±24

401±19a

388±23a

24

46-56

391±21

379±32

377±29a

364±27a

Female

Pretest

110

89±3

89±3

89±3

89±3

6

110

188±11

190±10

192±10

190±10

12

99-100

223±15

226±15

228±16

223±19

18

76-83

266±22

269±19

272±21

263±19

24

44-63

281±27

283±18

292±23a

280±21

a Statistically significant from control group mean at 5% level.

Table 2. Survival for F344 Rats in a Chronic DINP Study at Terminal Sacrifice

DINP dose

groups (wt %)

No. in group

No. of

unscheduled deaths

No. of survivors at terminal sacrifice

Percentage survivors

Males

 

 

 

 

0

81

20

61

75

0.03

80

25

55

69

0.3

80

30

50

62

0.6

80

29

51

64

Females

 

 

 

 

0

81

16

65

80

0.03

81

24

57

70

0.3

80

31

49

61a

0.6

80

26

54

68b

a Statistically significant from control group at 1% level based on Kruskall-Wallis test and Cox's test, but not statistically significant by the Weibull technique.

b Statistically significant from control group at 5% level based on Kruskall-Wallis test and Cox's test.

Table 3. Organ Weights as a Percentage of Body Weight for Male F344 Rats in a 2-Year DINP Study (Mean ± SD)

Dietary concentration of DINP (wt %)

Observation

Month

(N)

0

0.03

0.3

0.6

Liver

6

10

2.4±0.1

2.5±0.1

2.8±0.1a

3.1±0.2"

12

10

2.6±0.1

2.6±0.1

2.9±0.1a

3.1±0.1a

18

10

2.7±0.3

2.7±0.1

3.1±0.5

3.1±0.2b

24

50-61

3.2±0.6

3.4±0.8

3.8±0.8a

4.2±0.8a

Kidney

6

10

0.66±0.03

0.67±0.03

0.71±0.02a

0.75±0.036a

12

10

0.64±0.03

0.65±0.02

0.70±0.03a

0.73±0.03a

18

10

0.72±0.07

0.69±0.03

0.76±0.05

0.78±0.04b

24

50-61

0.81±0.10

0.87±0.12

0.89±0.10a

0.97±0.14a

Spleen

6

9-10

0.19±0.01

0.19±0.01

0.19±0.01

0.20±0.01

12

10

0.18±0.02

0.17±0.01

0.18±0.01

0.19±0.02

18

9-10

0.23±0.06

0.22±0.02

0.35±0.34

0.25±0.04

24

50-61

0.46±0.37

0.54±0.76

0.74±0.87a

0.74±0.76a

a Statistically significant from control group mean at 1% level.

b Statistically significant from control group mean at 5% level.

Table 4. Organ Weights as a Percentage of Body Weight for Female F344 Rats in a 2-Year DINP Study (Mean ± SD)

Dietary concentration of DINP (wt %)

Observation

Month

(N)

0

0.03

0.3

0.6

Liver

6

10

2.6±0.1

2.7±0.2

2.9±0.1a

3.3±0.1a

12

10

2.6±0.1

2.6±0.1

2.9±0.1a

3.3±0.1a

18

9-10

2.6±0.1

2.5±0.2

2.9±0.3

3.3±0.4a

24

48-65

3.1±0.5

3.2±0.7

3.6±0.8a

4.0±0.7a

Kidney

6

10

0.77±0.03

0.76±0.03

0.80±0.03

0.86±0.04a

12

10

0.71±0.05

0.72±0.04

0.78±0.04a

0.81±0.03a

18

9-10

0.70±0.04

0.69±0.03

0.77±0.07b

0.76±0.05b

24

48-65

0.81±0.13

0.80±0.09

0.87±0.15a

0.89±0.10a

Spleen

6

10

0.25±0.02

0.26±0.02

0.25±0.02

0.25±0.01

12

10

0.21±0.01

0.21±0.02

0.21±0.02

0.23±0.03

18

9-10

0.22±0.04

0.24±0.18

0.28±0.17

0.23±0.06

24

48-65

0.42±0.70

0.54±1.02

0.44±0.67

0.66±0.99b

a Statistically significant from control group mean at 1% level.

b Statistically significant from control group mean at 5% level.

Table 5. Hematologic Values of F344 Rats Fed 2 Years at Dietary Concentrations of DINP (Mean ± SD)

Dietary concentration of DINP (wt %)

Sex

Hematology parameter a

0

0.03

0.3

0.6

Male

White blood cell (x 103/mm3)

5.1±1.2

4.8±1.9

21.7±58.6

32.7±64.8

Red blood cell (x 106/mm3)

7.4±0.9

7.4±1.5

7.2±1.8

6.4±1.7b

Hemoglobin (g%)

16.3±1.8

15.3±3.2

15.0±3.1

13.2±2.9c

Hematocrit (%)

47.0±5.6

44.7±9.8

43.2±8.7

38.2±8.0c

MCV

60±2

61±6

61±5

61±8

Female

White blood cell (x 103/mm3)

3.1±0.9

4.6±4.1

5.0±3.2b

8.4±17.2

Red blood cell (x 106/mm3)

7.3±0.8

7.0±1.8

6.3±1.6

6.3±2.0

Hemoglobin (g%)

16.3±1.7

15.5±3.5

13.9±3.5

14.1±3.7

Hematocrit (%)

46.1±4.7

43.8±10.4

39.6±10.0

40.1±11.0

MCV

63±2

64±5

63±5

65±7

a Nineteen to 20 rats were examined at each concentration.

b Statistically significant from control group mean at 5% level.

c Statistically significant from control group mean at 1% level.

Table 6. Selected Serum Chemistry Values for Rats in a Chronic DINP Study (Mean ± SD)

Dietary concentration of DINP (wt %)

Serum chemistry parameter

Month a, b

Sex

0

0.03

0.3

0.6

AST (IU/liter)

6

M

68±8

71±15

102±32c

137±95c

12

M

102±17

101±22

145±36c

185±86c

18

M

69±7

68±12

113±76

146±105c

24

M

92±42

93±72

112±58

206±220

24

F

83±35

120±147

110±90

185±249

ALT (IU/liter)

6

M

37±8

38±7

81±52

128±145c

12

M

71±13

70±21

103±56

158±101

18

M

42±10

39±7

69±39

128±126c

24

M

42±10

45±23

89±76d

74±68

24

F

55±23

59±46

71±53

135±154

Alkaline phosphatase (IU/liter)

6

M

56±10

54±6

59±6

72±16d

12

M

49±9

51±13

54±13

70±19

18

M

49±6

41±6

65±48

63±18

24

M

41±14

47±38

65±47c

116±132d

24

F

47±20

65±85

73±53

78±63

a Group size at 6, 12, and 18 months was 9-10 rats examined for each parameter.

b Group size at 24 months was 20 rats examined for each parameter.

c Statistically significant from control group mean at 5% level.

d Statistically significant from control group mean at 1% level.

Table 7. Incidence of Nonneoplastic Liver Lesions in Rats Fed DINP for 2 Years

Dietary concentration of DINP (wt %)

0

0.03

0.3

0.6

0

0.03

0.3

0.6

Sex

Males

Females

Number examined

81

80

80

80

81

81

80

80

Liver

 

 

 

 

 

 

 

 

  Focal necrosis

10

9

16

26

13

11

19

21

  Spongiosis hepatis

24

24

51

62

4

1

3

4

  Sinusoid ectasia

16

16

24

33

9

4

6

10

  Hepatopathy associated with leukemia

22

17

34

33

16

18

24

33

  Hepatocellular enlargement

1

1

1

9

1

0

0

11

  Foci of vacuolated hepatocytes

15

14

1

0

17

16

8

5

  Diffuse fatty changes

12

4

4

0

10

9

3

3

  Cholangiectasis

52

25

16

20

10

6

3

5

  Regenerative nodules

3

3

6

6

5

7

10

9

Table 8. Incidence of selected Neoplastic and Preneoplastic Lesions in Rats fed DINP for 2 years

Dietary concentration of DINP (wt %)

0

0.03

0.3

0.6

0

0.03

0.3

0.6

Sex

Males

Females

Number examined

81

80

80

80

81

81

80

80

           Liver

 

 

 

 

 

 

 

 

Neoplastic nodules (1)

3

1

1

1

0

2

0

1

Hepatocellular cancer (2)

0

0

0

3

1

0

0

1

Combined (1 and 2)

3

1

1

4

1

2

0

2

Basophilic foci

53

62

48

42

72

64

64

54

Eosinophilic foci

58

50

45

51

59

45

42

32

             Kidney

 

 

 

 

 

 

 

 

Transitional cell carcinoma

0

0

3

0

0

0

0

0

Transitional cell adenoma

0

0

0

0

0

0

0

0

Tubular cell carcinoma

0

1

0

2

0

0

0

0

Tubular cell adenoma

0

0

 

0

0

0

0

0

Hyperplasia of transitional cell epithelium

21

23

31

25

32

39

34

32

Testes:  Bilateral interstitial cell tumours

71

64

70

72

-

-

-

-

Hematopoietic system:
Mononuclear cell leukemia

33

28

48a

51a,c

22

20

30c

43a,b,c

a Statistically significant from control group at 5% level using the Fisher exact test.

b Statistically significant from control group at 5% level using the incidental tumor test.

c Statistically significant from control group at 5% level using the life table test.

Conclusions:
Under the conditions of this 2-year diet study in Fischer 344 rats, DINP did not show clear evidence of any carcinogenic effects except for a marginal increase in the incidence of MNCL in mid- and high-dose rats which is not considered a relevant risk for humans. A NOEL was demonstrated to be 0.03 wt% or approximately 17 mg/kg/day.
Executive summary:

A 2-year repeated dose toxicity study (diet route) was performed on diisononyl phthalate (DINP) according to a method similar to OECD guideline 453. Groups of 110 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 0.03, 0.3, and 0.6 wt% for periods up to 2 years. Interim sacrifices of 10 predesignated rats/sex/dose were at 6, 12, and 18 months with surviving animals sacrificed at 24 months. At study termination, survival was higher than 60% for every group. At the mid or high dose, the following biological effects were noted: slight decreases in food consumption and body weight; slight increase in mortality; a dose-related increase in relative organ weights of liver and kidney; and some slight effects on urinalysis, hematologic, and clinical chemistry parameters. No peroxisome induction was observed in livers of treated rats compared with controls. No clear treatment-related nonneoplastic or neoplastic lesions were found. However, mononuclear cell leukemia (MNCL) and changes known to be associated with an increased incidence of MNCL were seen in the mid-dose and high dose groups. Published literature suggests that MNCL is a common finding in aging F344 rats and that this increased incidence in rats treated with DINP is not relevant to man. Thus, DINP was not considered a liver carcinogen in F344 rats at a dietary level up to 0.6% or approximately 340 mg/kg/day. A clear NOEL was demonstrated for all biological end points at a dietary level of 0.03 wt% or approximately 17 mg/kg/day of DINP.

Endpoint:
carcinogenicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 451 (Carcinogenicity Studies)
GLP compliance:
not specified
Remarks:
(this information was not reported)
Species:
rat
Strain:
Fischer 344
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Japan Inc.
- Age at study initiation: 6 weeks old
- Weight at study initiation: Not specified
- Fasting period before study: not specified
- Housing: Rats were housed two per cage. The cages were changed twice weekly, with the racks changed every 2weeks.
- Diet (e.g. ad libitum): ad libitum. Purina Certified Rodent Chow 5002.
- Water (e.g. ad libitum): ad libitum.
- Acclimation period: 10-14 days.

ENVIRONMENTAL CONDITIONS:
- Temperature (°C): 23 ± 1ºC
- Humidity (%): 55 ± 5%
- Photoperiod (hrs dark / hrs light): 12 hours light/dark cycle


Route of administration:
oral: feed
Vehicle:
unchanged (no vehicle)
Details on exposure:
- DIET PREPARATION
- Rate of preparation of diet (frequency): monthly
- Mixing appropriate amounts with (Type of food): DINP was added to Purina Certified Rodent Chow 5002 on a fixed weight percentage (w/w) basis and mixed thoroughly to ensure homogeneity.
- Storage temperature of food: 4ºC.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
IDENTIFICATION AND QUANTIFICATION....
- Separation method (e.g. HPLC, GC): Homogeneity and stability of test chemical in the diets were verified analytically by HPLC.
Duration of treatment / exposure:
24 months
Frequency of treatment:
Daily
Post exposure period:
No
Dose / conc.:
0 ppm
Dose / conc.:
400 ppm
Remarks:
equivalent to mean daily intake of 0.85 (male) and 0.53 (female) mg/kg bw/day
Dose / conc.:
2 000 ppm
Remarks:
equivalent to mean daily intake of 4.13 (male) and 3.03 (female) mg/kg bw/day
Dose / conc.:
8 000 ppm
Remarks:
equivalent to mean daily intake of 17.13 (male) and 13.36 (female) mg/kg bw/day
No. of animals per sex per dose:
52
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The maximum tolerated dose of DIDP (9000 ppm) was determined in a preliminary 13-week repeated dose toxicity study (Hong et al., 1999).

- Rationale for selecting satellite groups: Experiment for Assessment of peroxisome proliferating activity: For assessment of peroxisomal proliferation, a total of 50 male F344 rats were fed diets containing a vehicle control, 400, 2,000, 8,000 ppm DIDP or 12,000 ppm di(2-ethylhexyl) phthalate (DEHP). The rats were sacrificed at 12 or 32 weeks after the treatment for the analysis of enzyme catalase. Liver samples were taken for western blotting, enzyme activity assay and immunohistochemistry.

Positive control:
DEHP was included as a postive control for induction of peroxisome proliferation
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for moribundity and mortality.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly through week 13, and every 2 weeks thereafter until terminal sacrifice.

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
- Time schedule for examinations: weekly through week 13, and every 2 weeks thereafter until terminal sacrifice.

FOOD EFFICIENCY: No
WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No
OPHTHALMOSCOPIC EXAMINATION: No
HAEMATOLOGY: No
CLINICAL CHEMISTRY: No
URINALYSIS: No
NEUROBEHAVIOURAL EXAMINATION: No
IMMUNOLOGY: No

OTHER:
Assessment of peroxisome proliferating activity. Liver samples were taken for western blotting, enzyme activity assay, and immunohistochemistry.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, performed on all animals. The following organs were weighed at the interim and terminal sacrifices: adrenal glands, brain, heart, kidneys, liver, ovaries, spleen and testes. All organs and tissues were examined for grossly visible lesions.

HISTOPATHOLOGY: Yes, performed on all animals. All major tissues were fixed and preserved in 10% neutral buffered formalin, processed and trimmed, embedded in paraffin, sectioned to a thickness of 4–6m and stained with hematoxylin and eosin for microscopic examination.

Statistics:
The probability of survival was estimated using the product-limit procedure of Kaplan and Meier (1958). Statistical analyses for possible dose-related survival effectswere conducted using Cox’s (1972) method and Tarone’s (1975) life table test for testing two groups for equality and to identify dose-related trends, respectively. Feed consumption, as well as organ and body weights data were analyzed using the parametric multiple comparison procedures of Dunnett (1955) and Williams (1972). Extreme values were identified using the outlier test of Dixon and Massey (1951). Average severity values were analyzed for significance using the Mann–Whitney Utest. The Poly-k test (Bailer and Portier, 1988; Portier and Bailer, 1989) was used to assess the prevalence of neoplastic and non-neoplastic lesions. The catalase assay data were compared using the Dunnett t-test after an ANOVA analysis. For all comparisons, P values less than 5% (P < 0.05) were considered statistically significant.
Clinical signs:
no effects observed
Description (incidence and severity):
There were no clinical findings related to DIDP exposure.
Dermal irritation (if dermal study):
not examined
Mortality:
mortality observed, treatment-related
Description (incidence):
A significant decrease in overall survival was noted in both sexes of the high-dose groups compared with the vehicle control group (males: vehicle control 44/52, low dose 38/52, mid dose 43/52, high dose 19/52; females control 44/52, low dose 39/52, mid dose 39/52, high dose 29/52). The survival in the control groups was 85%; similar to that observed in chronic feeding studies on F344 rats sponsored by the National Toxicology Program (Cameron et al., 1985; Solleveld et al., 1984).
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
The mean body weights of rats exposed to 8,000 ppm were generally less than those of the other treatment groups throughout the study.
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:
not examined
Haematological findings:
not examined
Clinical biochemistry findings:
not examined
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
The relative kidney and liver weights of both males and females exposed to 8,000 ppm were significantly increased compared to those of the control animals. No treatment-related changes were observed in the relative organ weights for the spleen, testes, ovary, brain, adrenal glands, and heart.
Gross pathological findings:
effects observed, treatment-related
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Males: The incidence of animals with altered cell foci in the liver was significantly decreased in rats exposed to 2,000 and 8,000 ppm DIDP, in a dose-dependent manner. The fatty change in the liver decreased in the 8,000 ppm group. Oval cell hyperplasia, hypertrophy, necrosis and peliosis of the liver were increased in the 8,000 ppm group. Microgranuloma and spongiosis hepatis of the liver were increased in all treatment groups compared to control. Medullary hyperplasia of the adrenal glands was increased in the 400 and 2,000 ppm exposed males. Mineralization and interstitial nephritis of the kidneys were increased in the high dose. Degeneration and inflammation of the prostate and hyperplasia were increased in the 400 ppm, and 400 and 2,000 ppm exposed males, respectively. C-cell hyperplasia of the thyroid gland was decreased in the 2,000 ppm group.

Females: The incidence of altered cell foci in the liver was generally decreased in the exposed females, with those decreases significant at 8,000 ppm. Inflammation of the liver and necrosis were increased in the 400 and 2,000 ppm and the 8,000 ppm groups, respectively. Microgranuloma of the liver was decreased with the high dose. Hyaline cast, interstitial nephritis and chronic progressive nephropathy of the kidney were decreased in the high dose. Inflammation of the kidney was increased in the 400 and 2,000 ppm treatment groups. The incidence of extramedullary hematopoiesis was decreased in the 2,000 and 8,000 ppm groups. C-cell hyperplasia in the thyroid gland was increased in the 400 and 2,000 ppm groups.
Histopathological findings: neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
DIDP produced no treatment-related neoplastic lesions of the liver in either sex. However, the incidences of mononuclear cell leukemia (MNCL) in the males and females exposed to 8,000 ppm were significantly increased compared with the vehicle control, but were within historical ranges in the controls. The C-cell adenomas of the thyroid gland were significantly decreased in the males exposed to 400 ppm and females exposed to 2,000 and 8,000 ppm compared with the vehicle control, but were again within the NTP historical ranges.
Other effects:
effects observed, non-treatment-related
Description (incidence and severity):
CELLULAR CATALASE EXPRESSION LEVELS AND ACTIVITY
Western blot, catalase assay and immunohistochemical staining for the H2O2-degrading enzyme catalase contained within the peroxisome were performed. After 12 weeks of treatment, the levels of catalase in the high dose DIDP and 12,000 ppm DEHP treated livers were significantly increased compared to control. However, after 32 weeks of treatment, no significant differences were found in the expression and activity of catalase protein among DIDP treated liver tissues. A significant increase in the expression and activity of catalase protein was seen with the positive control. Similar results were obtained when the catalase protein was investigated via immunohistochemistry. Moreover, 104 weeks of DIDP treatment caused no significant changes in the expression of catalase.
Relevance of carcinogenic effects / potential:
DIDP produced statistically significant increases in MNCL in the 8000-ppm exposed groups. The incidence of MNCL was similar to the results of a DINP carcinogenicity study (Lington et al., 1997). MNCL is a common neoplasm in F344 rats, and the increased incidence of MNCL in F344 rats following chronic exposure to certain substances is likely to be a species-specific effect. Thus, the increased incidence ofMNCL observed in F344 rats exposed to certain alkyl phthalateswas likely to be a strain-specific
effect, with little or no relevance to humans. The characterization of these chemicals as carcinogens, based on increased MNCL in F344 rats, has no scientific support (Caldwell, 1999).
Key result
Dose descriptor:
NOAEL
Effect level:
8 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Key result
Critical effects observed:
no

Table 1: Final body weights and relative organ weights for F344 rats exposed to DIDP for 2 years a

Observation

No. of rats

Dietary concentration of DIDP (ppm)

control

400

2000

8000

Males

 

 

 

 

 

Body weight

52

350.43 ± 46.72

344.19 ± 56.97

357.31 ± 60.80

301.49 ± 68.25*

Kidney

52

7.94 ± 1.60

8.63 ± 2.79

8.12 ± 1.53

10.46 ± 2.96*

Liver

52

31.81 ± 11.26

36.29 ± 20.31

35.04 ± 21.24

44.41 ± 16.02*

Spleen

52

6.89 ± 10.26

10.81 ± 17.01

10.31 ± 17.41

13.19 ± 15.00

Testis

52

14.19 ± 5.38

13.53 ± 5.91

14.36 ± 5.16

14.87 ± 8.10

Females

 

 

 

 

 

Body weight

52

269.68 ± 36.04

272.68 ± 69.07

268.24 ± 66.46

221.29 ± 50.15*

Kidney

52

8.07 ± 1.43

8.51 ± 3.05

8.94 ± 3.72

10.18 ± 2.77*

Liver

52

31.02 ± 8.33

31.75 ± 11.00

32.14 ± 7.66

46.17 ± 13.89*

Spleen

52

5.45 ± 9.67

6.88 ± 9.43

7.48 ± 12.84

11.71 ± 16.28

Ovary

52

0.71 ± 2.47

0.36 ± 0.27

0.57 ± 0.86

0.49 ± 0.53

*Significantly different (P < 0.01) from the vehicle control group by Williams’ or Dunnett’s test.

a Relative organ weights are given as mg organ weight/g body weight (Mean ± SD).

Table 2. Incidence of neoplastic lesions in rats exposed to DIDP for 2 years

Parameter

Dietary concentration of DIDP (ppm)

control

400

2000

8000

NTP a

Males

 

 

 

 

 

All sites

50b

50

50

50

1354

Mononuclear cell leukemia

10c ((20.0)d)

16(32.0)

14(28.0)

23** (46.0)

32–74%

Thyroid gland

49

49

49

40

1347

C-cell adenoma

10(20.4)

4* (8.2)

11(22.4)

6(15.0)

2–35%

Females

 

 

 

 

 

All sites

48

50

49

49

1351

Mononuclear cell leukemia

11(23.0)

7(14.0)

11(22.4)

22* (44.9)

14–52%

Thyroid gland

49

47

47

40

1347

C-cell adenoma

8(16.3)

5(10.6)

2* (4.3)

0** (0.0)

4–22%

*,**Significantly different (P < 0.05 and P < 0.01) from the vehicle control group by the poly-3 test.

a Tumor incidences in control F344 rats from NTP carcinogenicity studies (Haseman et al., 1998).

b Number of animals examined microscopically.

c Number of animals with lesion.

d Values are expressed as percentage.

Table 3. Incidence of non-neoplastic lesions in male rats exposed to DIDP for 2 years

Lesions

Dietary concentration of DIDP (ppm)

control

400

2000

8000

Number examined

49

48

49

39

Adrenal glands

 

 

 

 

Cortical hyperplasia

3a ((6.1)b)

2(4.2)

0* (0.0)

0* (0.0)

Medullary hyperplasia

0(0.0)

10** (20.8)

6** (12.2)

0(0.0)

Kidney 

 

 

 

 

Mineralization 

0 (0.0)

1(2.1)

1(2.0)

13** (33.3)

Interstitial nephritis

2(4.1)

2(4.2)

5(10.2)

7** (17.9)

Liver 

 

 

 

 

Fatty change

4(8.2)

6(12.5)

1(2.0)

0* (0.0)

Altered cell foci

27(55.1)

19(39.6)

18* (36.7)

3** (7.7)

Oval cell hyperplasia

1(2.0)

3(6.3)

2(4.1)

6* (15.4)

Hypertrophy 

0(0.0)

0(0.0)

1(2.0)

4* (10.3)

Microgranuloma 

1(2.0)

5* (10.2)

6* (12.2)

4* (10.3)

Necrosis 

3(6.1)

7(14.6)

5(10.2)

8* (20.5)

Peliosis 

1(2.0)

0 (0.0)

2(4.1)

4* (10.3)

Spongiosis hepatis

0(0.0)

3* (6.3)

3* (6.1)

5** (12.8)

Prostate 

 

 

 

 

Degeneration 

10(20.4)

20* (41.7)

16(32.7)

8(20.5)

Hyperplasia 

4(8.2)

11* (22.9)

12* (24.5)

6(15.4)

Inflammation 

5(10.2)

7(14.6)

11* (22.4)

8(20.5)

Spleen 

 

 

 

 

Extramedullary hematopoiesis

9(18.4)

5(10.4)

5(10.2)

2* (5.1)

Red pulp hyperplasia

3(6.1)

1(2.1)

0* (0.0)

1(2.6)

Thyroid gland

 

 

 

 

C-cell hyperplasia

14(28.6)

8(16.7)

7* (14.3)

11(28.2)

*,**Significantly different (P < 0.05 and P < 0.01) from the vehicle control group by the poly-3 test.

a Number of animals with lesion.

b Values are expressed as percentage.

Table 4. Incidence of non-neoplastic lesions in female rats exposed to DIDP for 2 years

Lesions

Dietary concentration of DIDP (ppm)

control

400

2000

8000

Number of examined

49

47

47

40

Kidney 

 

 

 

 

Hyaline cast

6a ((12.2)b)

11(23.4)

8(17.0)

1* (2.4)

Inflammation 

0(0.0)

4* (8.5)

4(8.5)*

0(0.0)

Interstitial nephritis

6(12.2)

3(6.4)

3(6.4)

1* (2.5)

Chronic progressive nephropathy

9(18.4)

4(8.5)

10(21.3)

0* (0.0)

Liver 

 

 

 

 

Altered cell foci

31(63.3)

26(55.3)

27(57.4)

17* (42.5)

Inflammation 

2(4.1)

8* (17.0)

11** (23.4)

3(7.5)

Microgranuloma 

10(20.4)

6(12.8)

12(25.5)

3* (7.5)

Necrosis 

2(4.1)

4(8.5)

6(12.8)

9** (20.9)

Spleen 

 

 

 

 

Extramedullary hematopoiesis

15(30.6)

11(23.4)

3** (6.4)

5* (12.5)

Thyroid gland

 

 

 

 

C-cell hyperplasia

15(30.6)

24* (51.1)

25* (53.2)

10(25.0)

*,**Significantly different (P < 0.05 and P < 0.01) from the vehicle control group by the poly-3 test.

a Number of animals with lesion.

b Values are expressed as percentage.

Conclusions:
Under the conditions of this study, DIDP was not carcinogenic to male or female rats at doses up to 8,000 ppm. A marginal increase of MNCL in the 8000 ppm groups was observed, although it was not considered relevant for humans.
Executive summary:

A 2-year carcinogenicity study was conducted to characterize the toxic responses in rats exposed to DIDP in their diet. Groups of 52 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 400, 2000 and 8000 ppm for 2 years. Cage side observations, clinical findings, body weights and feed consumptions were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. Also, the peroxisome proliferating activity was assessed through the analysis of the enzyme catalase in a satellite group of rats fed with DIDP or di(2-ethylhexyl) phthalate (DEHP) as positive control. Liver samples were taken for western blotting, enzyme activity assay and immunohistochemistry. Significant decreases in the overall survival and body weights, and increases in the relative weights of kidneys and liver were noted in both sexes of the highest dose groups. DIDP produced no treatment-related neoplastic lesions of the liver in either sex. However, the incidences of mononuclear cell leukemia (MNCL) in the males and females exposed to 8000ppm were significantly increased compared with the vehicle control, but were within historical ranges in the controls. No significant changes were observed in cellular catalase expression levels and activity by DIDP treatment. In conclusion, there was no evidence of carcinogenicity in male or female F344 rats exposed to DIDP, with the exception of a marginal increase of MNCL in the 8000ppm exposed groups, which was not considered a relevant risk for humans.

Endpoint:
carcinogenicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DIDP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Description (incidence and severity):
There were no clinical findings related to DIDP exposure.
Key result
Dose descriptor:
NOAEL
Effect level:
8 502.47 ppm
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: read-across from an analogue for which NOAEL = 8000 ppm
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach from the analogue diisodecyl phthalate (DIDP), diundecyl phthalate is not considered carcinogenic to male or female rats up to a concentration in diet equivalent to ca. 8,500 ppm.
Executive summary:

A 2-year carcinogenicity study was conducted to characterize the toxic responses in rats exposed to DIDP in their diet. Groups of 52 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 400, 2000 and 8000 ppm for 2 years. Cage side observations, clinical findings, body weights and feed consumptions were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. Also, the peroxisome proliferating activity was assessed through the analysis of the enzyme catalase in a satellite group of rats fed with DIDP or di(2-ethylhexyl) phthalate (DEHP) as positive control. Liver samples were taken for western blotting, enzyme activity assay and immunohistochemistry. Significant decreases in the overall survival and body weights, and increases in the relative weights of kidneys and liver were noted in both sexes of the highest dose groups. DIDP produced no treatment-related neoplastic lesions of the liver in either sex. However, the incidences of mononuclear cell leukemia (MNCL) in the males and females exposed to 8000ppm were significantly increased compared with the vehicle control, but were within historical ranges in the controls. No significant changes were observed in cellular catalase expression levels and activity by DIDP treatment. In conclusion, there was no evidence of carcinogenicity in male or female F344 rats exposed to DIDP, with the exception of a marginal increase of MNCL in the 8000 ppm exposed groups, which was not considered a relevant risk for humans. Based on these results, the read-across approach was applied and DUP is not considered as carcinogenic up to a concentration in diet equivalent to ca. 8,500 ppm.

Endpoint:
carcinogenicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
REPORTING FORMAT FOR THE ANALOGUE APPROACH
The target substance (DUP) belongs to the OECD HPV category High Molecular Weight Phthalate Ester (HMWPE) which consists of esters with an alkyl carbon backbone with 7 carbon (C) atoms or greater. The source substance (DINP) although is not formally part of this category as it was assessed previously, it satisfies the category definition as its backbone length is C7 or above and also produces similar effects of developmental or reproductive toxicity. Thus, these substances share the same functional groups and also have comparable environmental and toxicological properties.
See attached the reporting format.
Reason / purpose for cross-reference:
read-across source
Key result
Dose descriptor:
NOEL
Effect level:
19.28 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
other: Read-across from an analogue for which biological effects were found in the mid-dose group.
Remarks on result:
other: read-across from an analogue for which NOEL = 0.03% in diet (Equivalent to ca. 17 mg/kg bw/day)
Key result
Dose descriptor:
NOAEL
Effect level:
385.57 mg/kg bw/day (actual dose received)
Based on:
other: Read across from an analogue
Sex:
male/female
Basis for effect level:
histopathology: neoplastic
Remarks on result:
other: read-across from an analogue for which NOAEL = 0.6% in diet (Equivalent to ca. 340 mg/kg bw/day)
Key result
Critical effects observed:
no
Conclusions:
Based on the read-across approach from the analogue DINP assessed in a 2-year chronic toxicity study with rats, DUP is not considered as carcinogenic and the NOEL was determined to be 19.28 mg/kg/day.

Executive summary:

A 2-year repeated dose toxicity study (diet route) was performed on diisononyl phthalate (DINP) according to a method similar to OECD guideline 453. Groups of 110 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 0.03, 0.3, and 0.6 wt% for periods up to 2 years. Interim sacrifices of 10 predesignated rats/sex/dose were at 6, 12, and 18 months with surviving animals sacrificed at 24 months. At study termination, survival was higher than 60% for every group. At the mid or high dose, the following biological effects were noted: slight decreases in food consumption and body weight; slight increase in mortality; a dose-related increase in relative organ weights of liver and kidney; and some slight effects on urinalysis, hematologic, and clinical chemistry parameters. No peroxisome induction was observed in livers of treated rats compared with controls. No clear treatment-related nonneoplastic or neoplastic lesions were found. However, mononuclear cell leukemia (MNCL) and changes known to be associated with an increased incidence of MNCL were seen in the mid-dose and high dose groups. Published literature suggests that MNCL is a common finding in aging F344 rats and that this increased incidence in rats treated with DINP is not relevant to man. Thus, DINP was not considered a liver carcinogen in F344 rats at a dietary level up to 0.6% or approximately 340 mg/kg/day. A clear NOEL was demonstrated for all biological end points at a dietary level of 0.03 wt% or approximately 17 mg/kg/day of DINP. Based on these results, the read-across approach was applied and DUP is not considered as carcinogenic up to approximately 385.57 mg/kg/day and the NOEL was determined to be 19.28 mg/kg/day.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
385.57 mg/kg bw/day
Study duration:
chronic
Species:
rat
Quality of whole database:
Two experimental studies are available with a Klimisch score of 2.

Carcinogenicity: via inhalation route

Endpoint conclusion
Endpoint conclusion:
no study available

Carcinogenicity: via dermal route

Endpoint conclusion
Endpoint conclusion:
no study available

Justification for classification or non-classification

Based on the available results, diundecyl phthalate should not be considered as a potential human carcinogen in accordance with CLP Regulation (EC) no 1272/2008.

Additional information

Key studies from experimental results with analogues:

Diisodecyl phthalate (DIDP): A 2-year carcinogenicity study was conducted to characterize the toxic responses in rats exposed to DIDP in their diet. Groups of 52 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 400, 2000 and 8000 ppm for 2 years. Cage side observations, clinical findings, body weights and feed consumptions were recorded periodically. Complete necropsies and microscopic examinations were performed on all animals. Also, the peroxisome proliferating activity was assessed through the analysis of the enzyme catalase in a satellite group of rats fed with DIDP or di(2-ethylhexyl) phthalate (DEHP) as positive control. Liver samples were taken for western blotting, enzyme activity assay and immunohistochemistry. Significant decreases in the overall survival and body weights, and increases in the relative weights of kidneys and liver were noted in both sexes of the highest dose groups. DIDP produced no treatment-related neoplastic lesions of the liver in either sex. However, the incidences of mononuclear cell leukemia (MNCL) in the males and females exposed to 8000ppm were significantly increased compared with the vehicle control, but were within historical ranges in the controls. No significant changes were observed in cellular catalase expression levels and activity by DIDP treatment. In conclusion, there was no evidence of carcinogenicity in male or female F344 rats exposed to DIDP, with the exception of a marginal increase of MNCL in the 8000 ppm exposed groups, which was not considered a relevant risk for humans. Based on these results, the read-across approach was applied and DUP is not considered as carcinogenic up to a concentration in diet equivalent to ca. 8,500 ppm.

Diisononyl phthalate (DINP): A 2-year repeated dose toxicity study (diet route) was performed on diisononyl phthalate (DINP) according to a method similar to OECD guideline 453. Groups of 110 Fischer 344 rats/sex were fed test substance at dietary levels of 0, 0.03, 0.3, and 0.6 wt% for periods up to 2 years. Interim sacrifices of 10 predesignated rats/sex/dose were at 6, 12, and 18 months with surviving animals sacrificed at 24 months. At study termination, survival was higher than 60% for every group. At the mid or high dose, the following biological effects were noted: slight decreases in food consumption and body weight; slight increase in mortality; a dose-related increase in relative organ weights of liver and kidney; and some slight effects on urinalysis, hematologic, and clinical chemistry parameters. No peroxisome induction was observed in livers of treated rats compared with controls. No clear treatment-related nonneoplastic or neoplastic lesions were found. However, mononuclear cell leukemia (MNCL) and changes known to be associated with an increased incidence of MNCL were seen in the mid-dose and high dose groups. Published literature suggests that MNCL is a common finding in aging F344 rats and that this increased incidence in rats treated with DINP is not relevant to man. Thus, DINP was not considered a liver carcinogen in F344 rats at a dietary level up to 0.6% or approximately 340 mg/kg/day. A clear NOEL was demonstrated for all biological end points at a dietary level of 0.03 wt% or approximately 17 mg/kg/day of DINP. Based on these results, the read-across approach was applied and DUP is not considered as carcinogenic up to approximately 385.57 mg/kg/day and the NOEL was determined to be 19.28 mg/kg/day.