Diisodecyl adipate

Endpoint summary

Description of key information

A subchronic oral toxicity study similar to OECD 408 was performed with the read-across substance, bis(2-ethylhexyl) adipate (CAS 103-23-1), in Fischer 344 rats and B6C3F1 mice at dose levels of 1600, 3100, 6300, 12500 and 25000 ppm for a period of 90 days (NTP, 1982). Ten animals per sex and dose received the test substance daily via diet, whereas a similar constituted control group was administered the plain diet. No signs of toxic effects and no mortality were observed in any of the animals during the study period. In mice, an adverse decrease in body weight gain compared to controls was noted starting at 3100 ppm in males and at 6300 or 25000 ppm in females, respectively. In rats, body weight gain was adversely reduced in males at 12500 and 25000 ppm. Average food consumption was not altered between treated and control groups of both genders and species. No adverse effects were noted at histopathological examination in rats and mice. Clinical chemistry and haematological parameters were not reported in this study. Based on these results, a NOAEL of 1600 ppm was derived for male B6C3F1 mice, corresponding to an actual ingested dose of 200 mg/kg bw/day. In male rats, the NOAEL was set at 6300 ppm, which was equivalent to a dose of 630 mg/kg bw/day. In female rats, the NOAEL was set at 25000 ppm, which was equivalent to a dose of 2187 mg/kg bw/day.

The subchronic dermal toxicity of read-across substance, bis(tridecyl) adipate, was investigated in a 90-day study similar to OECD guideline 411 and in compliance with GLP (Lane, 1985 and 1986).

The undiluted test substance was applied once daily for 5 days/week to the clipped skin of 10 Sprague Dawley rats per sex and group at dose levels of 800 and 2000 mg/kg bw/day under open conditions. A similar constituted group of animals remained untreated and served as controls. During the study, no treatment-related mortalities and no signs of systemic toxicity were observed. In general, most clinical signs involved local effects from the collars (e.g. lesions around the neck, reddish nasal discharge, chromodacryorrhea) or ocular bleedings (e.g. corneal opacities). Chronic deterioration of the skin (CDS), manifested only as flaking, was observed in males and females treated with 800 and 2000 mg/kg bw. Very slight erythema occurred in animals of both sexes at 2000 mg/kg bw/day and mostly at the beginning of the study. During the first part of the study, scabbing was observed on the backs of most animals of both treatment groups, with males being more affected than females. However, no dose-dependent relationship was apparent in either sex and scabbing was rarely seen later in the study. A slight, statistically significant decrease in body weights compared to controls was noted in treated males from Day 36 to the end of the study at 2000 mg/kg bw/day. Urinalysis revealed a dose-dependent increase in protein concentrations in females during Weeks 5, 9, and 13, and males during Weeks 5 and 9. Furthermore, the concentration of urinary ketone was slightly and dose-relatedly increased in both sexes. No toxicologically relevant changes in parameters of haematology were observed at any sampling interval. At study termination, a dose-dependent (correlation at a 95% confidence level) and statistically significant increase in clinical chemistry parameters was only seen in serum globulin concentration in females. Relative to body weight, the livers were mildly enlarged (p<0.05) in exposed rats of both sexes at 2000 mg/kg bw/day and in males at 800 mg/kg bw/day. The mean absolute kidney weight was slightly increased in males and females at 2000 mg/kg bw/day and in males at 800 mg/kg bw/day. A statistically significant increase in relative kidney weights was observed in both sexes and at both dose levels. In treated females, relative thymus weights were significantly decreased, whereas the decrease in relative thymus weight was not significant in males. Since no concomitant changes in histopathology were observed in liver, kidney and thymus, the effects on organ weights in the animals were considered to be non-adverse and adaptive responses (kidney, liver) to treatment with the test substance. Microscopic examination revealed mild hyperplasia of sebaceous glands of both sexes at 2000 mg/kg bw/day. No further treatment-related lesions were observed at gross and histopathological examination.

Based on the results of this study, the systemic NOAEL for subchronic dermal toxicity in Bis(tridecyl) adipate was considered to be ≥ 2000 mg/kg bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles (no data on haematology, clinical chemistry, urinalysis and behavioural analysis).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no data on haematology and clinical chemistry

GLP compliance:

not specified

Species:

mouse

Strain:

B6C3F1

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: NCI Frederick Cancer Research Center, Frederick, Maryland
- Age at study initiation: 4 weeks old
- Housing: 5 per cage in solid bottom suspended polycarbonate cages equipped with disposable nonwoven fiber filter sheets and Aspen-bed hardwood chips as bedding
- Diet: powdered Wayne Lab Blox diet, ad libitum
- Water: available via an Edstrom automatic watering system, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-3
- Humidity (%): 10-88
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at least every 14 days
- Mixing appropriate amounts with (Type of food): chemical was mixed with aliquot of powdered Wayne Lab Blox animal feed, placing the mixture in a Petterson-Kelly twin-shell intensifier bar V-blender with the remainder of the feed and mix for 10 minutes
- Storage temperature of food: 4 °C for no longer than 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The amounts of the test substance in 12500 and 25000 ppm samples were determined by vapour phase chromatography. One-gram samples of each of the above mixtures were triturated twice with 50-mL portions of methanol. The supernatant solutions were combined and diluted to a volume of 100 ml and analyzed. The mean of the analytical concentration was usually within 10% of the theoretical.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1600, 3100, 6300, 12500, 25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
200, 387, 787, 1562, and 3125 mg/kg bw
Basis:
other: actual ingested (recalculated based on food consumption). Only the corresponding value to 1600 ppm were given in the study report. The other doses were calculated with the mean food consumption used to calculate this given value.

No. of animals per sex per dose:

10

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: doses used are based on an acute study and a 14 day study
(groups of five mice per sex were treated with five dose levels of the test substance in feed (up to 25000 ppm) for 14 days, followed by 1 day of observation with control diet. 1 group per sex were maintained as untreated controls. All surviving animals were killed after 15 days.

Observations and examinations performed and frequency:

CAGE SIDE & CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- no specifics given on how feed consumption was observed

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1 female died (12500 ppm)

Mortality:

mortality observed, treatment-related

Description (incidence):

1 female died (12500 ppm)

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

body weight depression in males (from 1600 ppm) and females (from 6300 ppm)

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY
One mouse died as a result of an accident.

BODY WEIGHT AND WEIGHT GAIN
Weight gain depression was 10% or more for male mice fed 3100 ppm or more. Weight gain depression was 10% or more for female mice fed 6300 or 25000 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No compound-related reduction in feed consumption was observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No compound-related histopathological effects were observed.

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain at 387 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

1 600 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain at 3100 ppm

Dose descriptor:

LOAEL

Effect level:

387 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain

Dose descriptor:

LOAEL

Effect level:

3 100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain

Dose descriptor:

NOAEL

Effect level:

387 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain at 1562 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

3 100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain at 6300 ppm

Dose descriptor:

LOAEL

Effect level:

787 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain

Dose descriptor:

LOAEL

Effect level:

6 300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain

Critical effects observed:

not specified

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

other: Acceptable, well-documented study report which meets basic scientific principles (no data on haematology, clinical chemistry, urinalysis and behavioural analysis).

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

no data on haematology and clinical chemistry

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: NCI Frederick Cancer Research Center, Frederick, Maryland
- Age at study initiation: 3 weeks old
- Housing: 5 per cage in solid bottom suspended polycarbonate cages equipped with disposable nonwoven fiber filter sheets and Aspen-bed hardwood chips as bedding
- Diet: powdered Wayne Lab Blox diet, ad libitum
- Water: available via an Edstrom automatic watering system, ad libitum
- Acclimation period: 2 weeks

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18-31
- Humidity (%): 10-88
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: feed

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

DIET PREPARATION
- Rate of preparation of diet (frequency): at least every 14 days
- Mixing appropriate amounts with (Type of food): chemical was mixed with aliquot of powdered Wayne Lab Blox animal feed, placing the mixture in a Petterson-Kelly twin-shell intensifier bar V-blender with the remainder of the feed and mix for 10 minutes
- Storage temperature of food: 4 °C for no longer than 14 days

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The amounts of the test substance in 12500 and 25000 ppm samples were determined by vapour phase chromatography. One-gram samples of each of the above mixtures were triturated twice with 50-mL portions of methanol. The supernatant solutions were combined and diluted to a volume of 100 mL and analyzed. The mean of the analytical concentration was usually within 10% of the theoretical.

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
1600, 3100, 6300, 12500, 25000 ppm
Basis:
nominal in diet

Remarks:

Doses / Concentrations:
females: 140, 271, 551, 1094, 2187 mg/kg bw/day; males: 160, 310, 630, 1250, and 2500 mg/kg bw/day
Basis:
other: actual ingested (recalculated based on food consumption). Only the corresponding values to 6300 ppm were given in the study report. The other doses were calculated with the mean food consumption used to calculate these given values.

No. of animals per sex per dose:

10 males and 10 females

Control animals:

yes, plain diet

Details on study design:

- Dose selection rationale: doses used are based on an acute study and a 14 day study
(groups of five rats per sex were treated with five dose levels of the test substance in feed (up to 100000 ppm) for 14 days, followed by 1 day of observation with control diet. 1 group per sex were maintained as untreated controls. All surviving animals were killed after 15 days.

Observations and examinations performed and frequency:

CAGE SIDE & CLINICAL OBSERVATIONS: Yes
- Time schedule: twice daily

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- no specifics given on how feed consumption was observed

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
HISTOPATHOLOGY: Yes

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

one female (1600 ppm) died,

Mortality:

mortality observed, treatment-related

Description (incidence):

one female (1600 ppm) died,

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

dose-related body weight depression up to 18% (males); no effects in females

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not specified

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not specified

Details on results:

CLINICAL SIGNS AND MORTALITY
One female rat recieving 1600 ppm died, but its death was not considered to be compound-related.

BODY WEIGHT AND WEIGHT GAIN
Weight gain depression was 11% or more for male rats fed 12500 ppm or 25000 ppm.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study)
No compound-related reduction in feed consumption were observed.

HISTOPATHOLOGY: NON-NEOPLASTIC
No compound-related histopathological effects were observed.

Dose descriptor:

NOAEL

Effect level:

630 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight at 1250 mg/kg bw/day

Dose descriptor:

NOAEL

Effect level:

6 300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight at 12500 ppm

Dose descriptor:

LOAEL

Effect level:

1 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight

Dose descriptor:

LOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight

Dose descriptor:

NOAEL

Effect level:

2 187 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects observed at the highest dose

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects observed at the highest dose

Critical effects observed:

not specified

Reference 3

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Please see Section 13 below.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain at 387 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

1 600 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain at 3100 ppm

Key result

Dose descriptor:

LOAEL

Effect level:

387 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain

Key result

Dose descriptor:

LOAEL

Effect level:

3 100 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight gain

Key result

Dose descriptor:

NOAEL

Effect level:

387 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain at 1562 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

3 100 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain at 6300 ppm

Key result

Dose descriptor:

LOAEL

Effect level:

787 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain

Key result

Dose descriptor:

LOAEL

Effect level:

6 300 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: decreased body weight gain

Key result

Critical effects observed:

not specified

Reference 4

Endpoint:

sub-chronic toxicity: oral

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Please see Section 13 below.

Reason / purpose for cross-reference:

read-across source

Key result

Dose descriptor:

NOAEL

Effect level:

630 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight at 1250 mg/kg bw/day

Key result

Dose descriptor:

NOAEL

Effect level:

6 300 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight at 12500 ppm

Key result

Dose descriptor:

LOAEL

Effect level:

1 250 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight

Key result

Dose descriptor:

LOAEL

Effect level:

12 500 ppm

Based on:

test mat.

Sex:

male

Basis for effect level:

other: decreased body weight

Key result

Dose descriptor:

NOAEL

Effect level:

2 187 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects observed at the highest dose

Key result

Dose descriptor:

NOAEL

Effect level:

25 000 ppm

Based on:

test mat.

Sex:

female

Basis for effect level:

other: no effects observed at the highest dose

Key result

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

200 mg/kg bw/day

Study duration:

subchronic

Species:

mouse

Quality of whole database:

Klimisch 2 studies

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Reference

Reference 1

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)

Reason / purpose for cross-reference:

reference to same study

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

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

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

Remarks:

Doses / Concentrations:
800 and 2000 mg/kg bw/ day
Basis:
nominal per unit body weight

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.

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).

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.

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)

Critical effects observed:

not specified

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.

Reference 2

Endpoint:

sub-chronic toxicity: dermal

Type of information:

read-across from supporting substance (structural analogue or surrogate)

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Justification for type of information:

Please see Section 13 below.

Reason / purpose for cross-reference:

read-across source

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)

Key result

Critical effects observed:

not specified

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

2 000 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

Klimisch 2

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

Based on the above discussed results for repeated dose toxicity, diisodecyl adipate does not have to be classified and has no obligatory labelling requirement for repeated dose toxicity according to the CLP Regulation (EC) No 1272/2008.