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Toxicological information

Repeated dose toxicity: dermal

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Administrative data

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Well documented study report, comparable to OECD guideline study with acceptable restrictions (no data on analytical purity, only 2 dose levels tested, open instead of semi-occlusive testing)
Cross-reference
Reason / purpose for cross-reference:
reference to same study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
1986
Report date:
1986
Reference Type:
study report
Title:
Unnamed
Year:
1985
Report date:
1985
Reference Type:
study report
Title:
Unnamed
Year:
1987
Report date:
1987
Reference Type:
secondary source
Title:
Diesters Category of the Aliphatic Esters Chemicals (Test Plan and Robust Summaries for Substances in the HPV Test Plan)
Author:
US-EPA (American Chemistry Council's Aliphatic Esters Panel)
Year:
2010
Bibliographic source:
High Production Volume (HPV) Chemical Challenge Program (201-16837A and 201-16837B)

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Version / remarks:
(adopted 1981)
Deviations:
yes
Remarks:
(only two doses tested instead of three, open instead of semi-occlusive application, no ophthalmological examination as recommended by the OECD guideline)
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Bis(tridecyl) adipate
EC Number:
241-029-0
EC Name:
Bis(tridecyl) adipate
Cas Number:
16958-92-2
Molecular formula:
C32H62O4
IUPAC Name:
ditridecyl adipate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Details on the strain: Rat/crl COBS CD[SD] BR/Charles River, Lakeview, New Jersey
- Source: Charles River, Lakeview, New Jersey
- Age at study initiation: 49 days
- Weight at study initiation: males: 161.9 - 166.8 g; females: 149.2 - 150.5 g
- Housing: individually, in suspended, stainless steel cages, with wire mesh bottoms and fronts
- Diet: Purina Certified Lab Chow #5002 in pellet form; ad libitum
- Water: tap water, delivered by an automatic watering syste; ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21
- Humidity (%): 50
- Photoperiod (hrs dark / hrs light): 12 / 12

Administration / exposure

Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: on the backs of the animals, beginning at the scapula and continuing laterally and posteriorly
- Type of wrap if used: no wrap. Animals were fitted with cardboard "Elisabethan" collars to minimize ingestion of test material.
- Time intervals for shavings or clipplings: approx. 24 h before the start of dosing, hair was then reclipped as necessary, but at least once per week

REMOVAL OF TEST SUBSTANCE
- Washing: approx. 24 h after the last dose each week, as much residual test substance as practical was wiped off with gauze pads.

TEST MATERIAL
- Constant volume or concentration used: yes
Analytical verification of doses or concentrations:
no
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
24 h, 5 days/week
Doses / concentrationsopen allclose all
Dose / conc.:
800 mg/kg bw/day (nominal)
Dose / conc.:
2 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
10
Control animals:
yes, concurrent no treatment
Details on study design:
- Dose selection rationale:
The dose levels were chosen on the basis of data obtained in thirteen-week studies of other petroleum-based oils previously conducted in the testing laboratory and on practical considerations. The high dose was the maximum amount that could routinely be applied to the backs of the rats without some of the material running off.

Examinations

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

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Parameters: appearance, behaviour, excretory function and discharges

DERMAL IRRITATION: Yes
- Time schedule for examinations: weekly
- Erythema and edema at the site of application were graded using the Draize scales. the skin was also examined and graded for chronic deterioration: flaking, thickening, stiffening, cracking, and sloughing.

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

HAEMATOLOGY: Yes
- Time schedule for collection of blood: weeks 5, 9, 13
- Anaesthetic used for blood collection: Yes (diethyl ether)
- Animals fasted: Yes
- How many animals: all
- Parameters:
- red blood cell (RBC) morphology,
- white blood cell (WBC) differentials,
- hematocrit (HTC), hemoglobin (HGB),
- mean corpuscular volume (MCV),
- mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH),
- WBC count, RBC count, platelet count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: weeks 5, 9, 13
- Animals fasted: Yes
- How many animals: all
- Parameters:
- glucose,
- alanine aminotransferase, aspartate aminotransferase, alkaline phosphatase,
- total protein, albumin, globulin,
- A/G ratio,
- urea nitrogen, uric nitrogen,
- creatinine,
- total bilirubin,
- sodium, potassium, chloride, phosphorus, calcium,
- cholesterol, triglycerides,
- iron,
- lactate dehydrogenase.

URINALYSIS: Yes
- Time schedule for collection of urine: weeks 5, 9, 13
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters:
- pH,
- specific gravity,
- blood,
- protein,
- bilirubin, urobilinogen,
- glucose,
- ketones.

Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals were subjected to gross necropsy after sacrifice or spontaneous death.

The following tissues were removed and preserved in 10% neutral buffered formalin:
- adrenals,
- bone with marrow (sternum, rib),
- brain,
- epididymides,
- oesophagus,
- eyes and optic nerves,
- Harderian glands,
- head (entire),
- heart and aorta,
- large intestine (cecum, colon, and rectum); small intestine (duodenum, jejunum, and ileum),
- kidneys,
- lacrimal glands,
- liver (part of median and right lateral lobes),
- lungs and bronchi,
- lymph nodes (cervical, mesenteric, draining if abnormal),
- mammary gland (with skin),
- ovaries,
- pancreas,
- pituitary,
- prostate and seminal vesicles,
- salivary glands (major),
- skeletal muscle and sciatic nerve,
- treated skin,
- spinal cord (cervical, thoracic),
- spleen,
- stomach (glandular and squamous),
- testes,
- thymus,
- thyroid and parathyroids,
- tongue,
- trachea,
- urinary bladder,
- uterus (cervix, corpus, and horns),
- vagina,
- gross lesions.

The following organs were weighed from all animals at terminal sacrifice:
- adrenals, brain, epididymides, gonads, heart, kidneys, liver, prostate, spleen, thymus, thyroid/parathyroid (weighed after fixation), uterus.

HISTOPATHOLOGY: Yes
The following tissues from the control and high-dose animals were processed for microscopic examination. Sections for examination were stained with hematoxylin and eosin.
- adrenal, bone and marrow (sternum), brain, eye and optic nerve (left), gonad, small intestine (duodenum) and large intestine (colon), kidney, liver (median lobe), lung (left lobe), pancreas, treated skin (2 sections), spleen, stomach, thymus, thyroid, urinary bladder, gross lesions.

SPERM MORPHOLOGY: Yes
- The effect of the test substance on sperm morphology was assessed by examining cauda epididymal sperm from the first five control males and the first five high-dose males sacrificed. After the epididymides were weighed, the right epididymis was fixed for possible histopathologic examination and the left was used for analysis of sperm morphology.
Statistics:
Body weight data were analyzed for normality and homogeneity of variances, and then by analysis of variance and Duncan's Multiple Range Test. Organ weights were evaluated using Analysis of Variance and Student-Newman-Keuls' test (p<0.05).

Results and discussion

Results of examinations

Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
chronic deterioration of the skin manifested as flaking in all treated animals (no dose-response relationship)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
2000 mg/kg bw/day: slight, but statistically significant reduction in body weight gains in males
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Dose-related decreases of globulin concentrations in females.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Dose-related increases in urinary protein concentrations in males and females.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Reduction in thymus weight was observed in females in both dose groups, however, when the weights were adjusted for differences in body weight, the differences were not statistically significant.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
Mild hyperplasia of sebaceous glands of both sexes at 2000 mg/kg bw/day.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related deaths occurred during the course of the study.
There were no indications of systemic toxicity during the study.
In general, most clinical signs were local effects from the collars (e.g. lesions around the neck, reddish nasal discharge, chromodacryorrhea) or the ocular bleedings (e.g. corneal opacities).

BODY WEIGHT AND WEIGHT GAIN
The rate of body weight gain by the high-dose males was less than that of the controls during the first half of the study. Although always within 10% of the control means, the differences from controls were statistically significant (p<0.05) from day 36 until the end.
The low-dose males and both groups of females had slight reductions in the rate of body weight gain for the first five weeks of dosing. After day 36 the gains in body weight kept pace with their respective control groups, so that there was little percent change for the remainder of the study. Statistically significant differences from control values (p<0.05) were seen sporadically in all three groups between day 29 and 57, but not thereafter.

SKIN IRRITATION
Chronic deterioration of the skin (CDS), manifested only as flaking, was observed equivalently in the males of the low- and high-dose groups (mean scores 0.0 - 1.0). Very slight erythema was seen in a few high-dose males (mean scores: 0.0 - 0.2), mostly at the beginning of the study.
Both groups of females reacted equivalently to the substance, showing the same amount of CDS as the males (mean scores: 0.0 - 1.3), but slightly more erythema (mean scores: 0.0 - 0.5).
In the first part of the study, scabbing was observed on the backs of most of the animals in both treated groups of both sexes, with the males being more affected. The scabs were usually small and were found mostly on the anterior portion. No dose-effect relationship was apparent in either sex and scabbing was rarely seen later in the study.
The skin of the control rats of both sexes was normal throughout the study.

HAEMATOLOGY
No consistent differences among the groups were found that would suggest that the substance had an effect on any of the hematologic parameters examined.

CLINICAL CHEMISTRY
A number of statistically significant effects were observed during weeks five and nine of the study; however, most of the effects disappeared by week thirteen. Apparent effects shown earlier on six out of eight serum components in males and on four out of seven serum components in females were not observed at the termination of the study. Those serum components showing statistically significant effects of the substance following 13 weeks of treatment were: glucose (-13 to -24%), alkaline phosphatase (+39 to +41%) and inorganic phosphorus (+11%) in males and glucose (-12 to
- 14%), albumin/globulin ratio (-15%), globulin (+10%) and iron (-25 to -39%) in females. A dose-effect relationship at a 95% confidence level was observed only in female serum globulin among all the affected serum components.


URINALYSIS
There were dose-related increases in urinary protein concentrations in the females during weeks 5, 9, and 13, and males during weeks 5 and 9. A slight increase in protein concentration, but not dose-related, was seen in the males at week 13.
There were also slight, dose-related increases in urinary ketone concentrations seen sporadically in both males and females. High values in the controls of both sexes at week 13 and the variability of the effect make interpretation of this finding difficult.
All other changes in urinalysis parameters were infrequent and minor.

SPERM MORPHOLOGY
No differences in sperm head or general sperm morphology were observed between control and high-dose rats.

ORGAN WEIGHTS
LIVER: Relative to body weight, the livers were mildly enlarged (p<0.05) in exposed rats (both sexes at 2000 mg/kg bw/day, only in males at 800 mg/kg bw/day). This change was considered treatment-related with no observable microscopic changes (see below). It is regarded as an adaptive response to the test material.
KIDNEY: The mean absolute kidney weight was mildly large in males and females at 2000 mg/kg bw/day and slightly large in males at 800 mg/kg bw/day by approx. 28, 31.4, and 18.2%, respectively. Relative to body weight the kidneys were larger than controls in both sexes at both dose levels. These differences from control were all statistically significant. It is concluded that the mild enlargement of kidneys in rats at 2000 mg/kg bw/day and slight enlargement in males at 800 mg/kg bw/day was treatment-related, with no treatment-related microscopic changes (see below). This may be an adaptive response.
THYMUS: Compared to controls the mean thymus weight was smaller (p<0.05) in exposed females of both the low and high dose levels, by approx. 19 and 22%, respectively. In males, thymus weights were slightly lower than controls, but not statistically significantly different.
OTHER ORGANS: No treatment-related changes.

GROSS PATHOLOGY
LIVER: No gross lesions were seen at necropsy.
KIDNEY: No treatment-related gross lesions were seen at necropsy.
THYMUS: No abnormalities.
OTHER ORGANS: No treatment-related changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
SKIN: During microscopic examination the treated skin of exposed animals was compared with that of the sham controls. This revealed mild hyperplasia of sebaceous glands of both sexes at 2000 mg/kg bw/day. This change is treatment-related.
OTHER: The adrenals, brain, eyes and optic nerves, femur, gonads, heart, intestine (small and large), lung, pancreas, salivary gland (submaxillary), spleen, sternum, thyroids and urinary bladder did not show any treatment-related microscopic changes.

Effect levels

Key result
Dose descriptor:
NOAEL
Effect level:
2 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: (highest dose tested)

Target system / organ toxicity

Key result
Critical effects observed:
no

Any other information on results incl. tables

Table 1: Mean body weights (g)

 

 

Dose [mg/kg bw/day]

Sex

Day

800

2000

0.0

 

 

 

 

 

Males

1

200.1

204.6

199.1

 

8

237.2

238.6

242.8

 

15

277.2

279.0

287.3

 

22

305.8

303.4

319.0

 

29

327.6

326.6

344.6

 

36

337.5*

335.8*

358.3

 

43

358.4*

352.3*

388.5

 

50

379.2

387.4*

394.3

 

57

390.1

383.9*

412.6

 

64

391.1

391.9*

416.8

 

71

409.1

399.2*

435.4

 

78

420.3

409.8*

442.8

 

85

433.7

424.9*

458.9

 

91

441.1

433.4*

468.0

Females

1

164.5

163.8

168.8

 

8

177.6

178.7

182.9

 

15

185.8

196.3

203.3

 

22

198.7

198.5

209.9

 

29

207.8*

210.5

220.3

 

36

213.0*

214.6*

226.1

 

43

221.4

222.1

234.4

 

50

228.0*

228.4

240.8

 

57

230.8*

231.7*

247.1

 

64

232.3

235.8

249.9

 

71

239.9

238.5

253.9

 

78

242.8

245.8

258.8

 

85

244.5

248.5

262.8

 

91

247.8

253.0

268.2

* significantly different from control (p<0.05)

Table 2: Selected urinalysis parameters [mg/dL]

 

Sex

Group

Week 5

Week 9

Week 13

 

 

Protein

Ketone

Protein

Ketone

Protein

Ketone

Male

Control

50

2.2

38

1.1

38

4.4

 

Low dose

85

1.0

92

0.5

72

5.0

 

High dose

122

1.7

100

2.5

76

7.5

Female

Control

10

0.8

31

0.8

28

2.0

 

Low dose

34

1.0

41

1.0

18

3.0

 

High dose

65

2.0

48

0.5

69

4.5

 

Table 3: Selected clinical chemistry parameters after 13 weeks of treatment [percent deviation from control]

 

Sex

Group

Glucose

Alkaline phosphatase

Inorganic phosphorus

Albumin / globulin ratio

Globulin

Iron

Male

Low dose

-13%*

+39%*

+3%

-

-

-

 

High dose

-24%*

+41%*

+11%*

-

-

-

Female

Low dose

-12%*

-

-

-4%

+3%

-25%*

 

High dose

-14%*

-

-

-16%*

+10%*

-39%*

* significantly different from control (p<0.05)

 

DISCUSSION

 

The test substance, when applied dermally under open conditions at 800 or 2000 mg/kg bw/day, five days per week for thirteen weeks, caused minor effects in rats of both sexes at both dose levels.

 

Small reductions in body weights of the males and females were seen. The reductions in body weight, except in the high-dose males, were considered to be of minimal toxicological importance because they were of very small magnitudes and inconsistent statistical significance. There was no indication that the test substance was causing a prolonged effect on the body weights of either group of either sex.

According to the author of the study report, the basis for the decreases in weight gain in the high-dose males was not known. As possible causes for the observed decreases in body weight gain, the author offered less-than-efficient use of nutrients, an increased metabolic rate, decreased food consumption, or any combination of these.

 

Skin irritation was never severe. The mild reactions that were observed were considered not to be of toxicologic importance because of the extreme conditions employed in the present study (i.e., the large amount of material applied, the fact that it was not removed on a daily basis).

 

Increased urine protein and ketone concentrations also occurred in both sexes.

 

Gross and histopathological examination revealed mild enlargement of the kidneys in males and females at 2000 mg/kg bw/day and in males at 800 mg/kg bw/day. Microscopically, treatment-related lesions were not found in females and males, treated at 2000 mg/kg bw/day. The liver was mildly enlarged in both sexes at the high dose and in males at the low dose. Microscopic examination did not reveal treatment-related changes in the livers. The kidney and liver enlargement were considered adaptive responses.

In exposed females at both dose levels, statistically significant thymus growth reduction was observed and considered treatment-related. No microscopic abnormalities were observed. Therefore, this effect is not considered adverse.

The skin showed treatment-related mild hyperplasia of sebaceous glands in both sexes from the high dose group.

 

All effects in the low-dose groups were borderline as far as their biological significance is concerned. Their importance resides in the fact that they are part of the dose-response relationship which supports the findings in the high-dose groups.

In general, none of the described effects are considered to be adverse effects, thus, the dermal NOAEL was set at 2000 mg/kg bw/d, which was the highest dose level tested.

Applicant's summary and conclusion