Diisobutyl hexahydrophthalate

Endpoint summary

Description of key information

Sub-acute toxicity: NOAEL - Males and females: 1000 mg/kg/day

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: GLP compliant study conducted according to internationally recognised test methods.

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Deviations:

no

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
- Source: Charles River Italy SpA
- Age at study initiation: 8-9 weeks
- Weight at study initiation: Males: 388-420 g; Females: 242-277 g
- Fasting period before study: No
- Housing: Polysulphone solid bottom cages, Group caged except for in pairs (1 male/1 female) for mating and females individually caged during gestation
- Diet (e.g. ad libitum): Mucedola 4RF21 pelleted diet available ad libitum
- Water (e.g. ad libitum): Municipal supply tap water available ad libitum
- Acclimation period: 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20-24
- Humidity (%): 40-70
- Air changes (per hr): 15-20
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 2012-08-16 To: 2012-10-21

Route of administration:

oral: gavage

Vehicle:

corn oil

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: Prepared daily by dissolution/suspension in corn oil.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Proposed formulation procedure checked for concentration and homogeneity in the range from 20 to 200 mg/mL to confirm that the method was suitable. All levels were within 90-110% of nominal for concentration and the CV for homogeneity was < 10%. Stability after 24 hours at room temperature was verified in the range from 20 to 200 mg/mL and determined to be within acceptable limits (90-110% of nominal of concentration and CV for homogeneity < 10%).
Samples of the formulations, prepared on Weeks 1 and 6, were analysed to check for homogeneity and concentration. Results were within acceptable limits (90-110% of nominal for concentration and the CV for homogeneity was < 10%).

Duration of treatment / exposure:

Males: from 14 days before pairing for a total of approximetaly 6 weeks
Females: from 14 days before pairing to day 3 post partum (total of approximately 6 weeks)
Satellite (recovery) animals (male and female) dosed daily for at least 4 weeks

Frequency of treatment:

Daily

Remarks:

Doses / Concentrations:
0, 100, 300 and 1000 mg/kg/day
Basis:
other: nominal in corn oil

No. of animals per sex per dose:

10 males & 10 females/group
Additional satellite groups of 5 males/5 females in control (0 mg/kg) and high dose (1000 mg/kg) to assess recovery following treatment

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on data findings from structural analogues of the substance
- Rationale for animal assignment (if not random): Random, stratified body weight
- Rationale for selecting satellite groups: As above
- Post-exposure recovery period in satellite groups: 2 weeks

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily for signs of ill health and moribund condition
- Cage side observations were included.: Yes - Daily, 2 or 3 times following dose administration

DETAILED CLINICAL OBSERVATIONS: Yes (functional observation battery)
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Males: Weekly; Females: days 0, 7, 14, 20, gestation days 0, 7, 14 and 20, post partum Days 1 and 4.

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 - Weekly
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable as animals dosed by gavage

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No - Not applicable - Not a dietary study

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No - Not applicable as animals dosed by gavage:

OPHTHALMOSCOPIC EXAMINATION: No


HAEMATOLOGY: Yes
- Time schedule for collection of blood: Males - after 42 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes - overnight
- How many animals: 5 males/5 females/group
- Parameters examined: Haematocrit, Haemoglobin, Red blood cell count, Reticulocyte count, Mean red blood cell volume, Mean corpuscular haemoglobin, Mean corpuscular haemoglobin concentration, White blood cell count, Differential leucocyte count (Neutrophils, Lymphocytes, Eosinophils, Basophils, Monocytes, Large unstained cells), Platelets, Prothrombin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Males - after 42 days of treatment; Females - after day 4 of lactation. Terminal blood samples taken from the abdominal vena cava
- Anaesthetic used for blood collection: Yes (isofluorane)
- Animals fasted: Yes - overnight
- How many animals: 5 males/5 females/group
- Parameters examined: Alkaline phosphatase, Alanine aminotransferase, Aspartate aminotransferase, Gamma-glutamyltransferase, Urea, Creatinine, Glucose, Triglycerides, Phosphorus, Total bilirubin, Total cholesterol, Total protein, Albumin, Globulin, A/G Ratio, Sodium, Potassium, Calcium, Chloride

URINALYSIS: Yes (males only)
- Time schedule for collection of urine: overnight collection from 5 males/group after 42 days of treatment
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes - ovenight
- Parameters examined: Appearance, Volume, Specific gravity, pH, Protein, Glucose, Total reducing substances (only if Glucose is higher than 100 mg/dL), Ketones, Bilirubin, Urobilinogen, Blood.
The sediment, obtained from centrifugation at approximately 3000 rpm for 10 minutes, examined microscopically for: Epithelial cells, Leucocytes, Erythrocytes, Crystals, Spermatozoa and precursors, Other abnormal components

Sacrifice and pathology:

GROSS PATHOLOGY: Yes - Macroscopic examination of organs and tissues including organ weights: adrenal glands, brain, epididymides, heart, kidney, liver, ovaries (with oviduct), spleen, testes, thymus.
- Preservation: adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), nasal cavity, oesophagus, ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal column, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina. All tissues fixed and preserved in 10% neutral buffered formalin with the exception of testes and epididymides which were fixed in modified Davidson's fluid and preserved in 70% ethyl alcohol.

HISTOPATHOLOGY: Yes (5 males/5 females examined from high dose and control groups) - adrenal glands, bone marrow, brain, caecum, colon, duodenum, epididymides, heart, ileum, jejunum (including Peyer’s patches, kidneys, liver, Lungs (including mainstem bronchi), lymph nodes (cervical and mesenteric), ovaries with oviducts, pituitary gland, prostate gland, rectum, sciatic nerve, seminal vesicles with coagulating gland, spinal cord (cervical, thoracic and lumbar), spleen, stomach, testes, thymus, thyroid, trachea, urinary bladder, uterus and cervix, vagina.

Other examinations:

No

Statistics:

For continuous variables the significance of the differences amongst group means was assessed by Dunnett’s test or a modified t test, depending on the homogeneity of data. Statistical analysis of histopathological findings was carried out by means of the non-parametric Kolmogorov-Smirnov test if n was more than 5. The non-parametric Kruskal-Wallis analysis of variance was used for the other parameters. Intergroup differences between the control and treated groups was assessed by the non-parametric version of the Williams test. The criterion for statistical significance was p<0.05

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Salivation noted in animals treated at 1000 mg/kg following dosing

Mortality:

mortality observed, treatment-related

Description (incidence):

Salivation noted in animals treated at 1000 mg/kg following dosing

Body weight and weight changes:

no effects observed

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:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Reduced thymus weight in females treated at 1000 mg/kg/day

Gross pathological findings:

no effects observed

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY – No mortality occurred in the study. Clinical observations for neurotoxicity assessment (removal of animals from the home cage and open arena) did not reveal changes attributable to the tested substance. Salivation was the most relevant clinical sign, observed approximately 1 hour after dosing in high dose (1000 mg/kg b.w./day) main and recovery animals during the treatment phase of the study. This finding disappeared during the 2 week recovery period.

BODY WEIGHT AND WEIGHT GAIN - Body weight and body weight gain were unaffected by treatment.

FOOD CONSUMPTION AND COMPOUND INTAKE - Food consumption was unaffected by treatment.

HAEMATOLOGY - No changes were noted in the haematology investigation performed.

CLINICAL CHEMISTRY - A number of treated males showed increases of triglycerides, albumin and calcium, and decrease of potassium. Changes were within 16% with the exception of triglycerides, which were increased by approximately 90%. In addition, increases of transaminases enzymes were noted in 2 individuals, one treated at 300 mg/kg/day and one at 1000 mg/kg/day. Treated females showed increased phosphorus (15% to 22%). Potassium was increased in those receiving 1000 mg/kg/day (11%) and creatinine showed a statistically significant decrease in females treated with 1000 mg/kg/day (approx 20%) and 300 mg/kg/day (approx 24%). The above findings were of low severity/incidence, not dose-related and/or inconsistent between sexes, therefore considered of no toxicological significance. No relevant changes were noted in males of the recovery groups at the end of the 2 week recovery period. Those statistically significant differences between control and treated animals that were observed (gamma-glutamyltransferase, protein and phosphorus) were of minimal severity and not observed during the dosing phase in the main groups, therefore considered unrelated to treatment.

URINALYSIS - Slight reduction of pH and increase of glycosuria and ketonuria were noted in some treated males, with no dose-relationship apparent. In the absence of any other changes, these differences were not considered as adverse. No differences between control and treated males were apparent after 2 weeks of recovery from treatment.

GROSS PATHOLOGY - No treatment-related changes were noted in treated animals, both in main and recovery groups. Those changes that were observed were regarded as being incidental, having a comparable incidence in control and treated groups, and/or characteristically seen in untreated Sprague Dawley rats of the same age.

ORGAN WEIGHTS – A statistically significant decrease was noted in the absolute thymus weight of females treated at 1000 mg/kg/day (approximately 28%). A slight, statistically significant, increase was also noted in the relative kidney weight of males treated at 300 and 1000 mg/kg/day (approximately 10%) and in the relative kidney and liver weights of females of the recovery group treated at 1000 mg/kg/day (approximately 7%).

HISTOPATHOLOGY: NON-NEOPLASTIC –No treatment-related changes were noted in randomly selected animals from the control group and high dose (1000 mg/kg/day) group. Those changes that were observed were regarded as being incidental, having a comparable incidence in control and treated groups, and/or characteristically seen in untreated Sprague Dawley rats of the same age. Examples of such minimal, spontaneous, age-related changes included hepatic and heart inflammatory cell foci. In particular, two female rats treated at 1000 mg/kg/day exhibited mild atrophy of the thymic cortex. As this kind of change is sporadically seen in untreated female rats it was not considered as related to treatment.
A detailed qualitative evaluation of testes was performed on randomly selected control and high dose (1000 mg/kg/day) males. The evaluation took into account the tubular stages of the spermatogenic cycle, in order to identify potential treatment-related effects, such as: missing germ cell layers or types, retained spermatids, multinucleated or apoptotic germ cells and sloughing of spermatogenic cells into the lumen. PAS-H stained sections were used to identify the spermatogenic stages. Seminiferous tubules were evaluated with respect to their stage in the spermatogenic cycle and to the integrity of the various cell types within the different stages. Regular layering in the germinal epithelium was noted

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall lack of effects on: clinical signs; mortality; body weight; food consumption; haematology; clinical chemistry; gross pathology; organ weights; histopathology

Critical effects observed:

not specified

Conclusions:

A combined repeat-dose toxicity reproduction/developmental toxicity screen has been undertaken according to OECD TG 422 methods. Dose levels of 0, 30, 125 and 500 mg/kg/day were investigated. No treatment-related findings were noted during the in-vivo phase. Changes in clinical pathological investigations were considered of no toxicological significance. No treatment-related changes were observed both in male and female animals of main and recovery groups at post mortem examinations. Changes in organ weights were considered of no toxicological significance and were not correlated with histological changes. On the basis of these findings, the NOAEL (No Observed Adverse Effect Level) for general toxicity is considered to be 1000 mg/kg/day for both males and females.

Executive summary:

A combined repeat-dose toxicity reproduction/developmental toxicity screen has been undertaken according to OECD TG 422 methods. Dose levels of 0, 30, 125 and 500 mg/kg/day were investigated.No treatment-related findings were noted during thein-vivophase. Changes in clinical pathological investigations were considered of no toxicological significance. No treatment-related changes were observed both in male and female animals of main and recovery groups at post mortem examinations. Changes in organ weights were considered of no toxicological significance and were not correlated with histological changes. On the basis of these findings, the NOAEL (No Observed Adverse Effect Level) for general toxicity is considered to be 1000 mg/kg/day for both males and females.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Single study available for evaluation

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

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A combined repeat-dose toxicity reproduction/developmental toxicity screen has been undertaken according to OECD TG 422 methods. Dose levels of 0, 30, 125 and 500 mg/kg/day were investigated.No treatment-related findings were noted during thein-vivophase. Changes in clinical pathological investigations were considered of no toxicological significance. No treatment-related changes were observed both in male and female animals of main and recovery groups at post mortem examinations. Changes in organ weights were considered of no toxicological significance and were not correlated with histological changes. On the basis of these findings, the NOAEL (No Observed Adverse Effect Level) for general toxicity is considered to be 1000 mg/kg/day for both males and females.

Justification for classification or non-classification

No significant toxicity was observed at a concentration of 1000 mg/kg/day in a repeated-dose subacute study of at least 28 days duration.

 

According to EU Directive 67/548/EEC and EU Classification, Labelling and Packaging of Substances and Mixtures (CLP) Regulation (EC) No. 1272/2008, classification and labelling is not needed for repeated dose toxicity, as the effects seen in the repeated dose toxicity test do not indicate significant functional change or organ disfunction occuring at levels below indicated cut-off values. Those effects that were observed are regarded as being of minimal toxicological significance insufficient to warrant classification.