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Please be aware that this old REACH registration data factsheet is no longer maintained; it remains frozen as of 19th May 2023.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

Administrative data

Description of key information

Repeated dose toxicity (oral, WoE, 28d/90d), rat: NOAEL ≥1000 mg/kg bw/day (m/f) (RA from CAS 131459-39-7, CAS 68424-31-7 and CAS 146289-36-3)

Repeated dose toxicity (inhalation, 90d), rat: NOAEC = 0.5 mg/L air (m/f) (RA from CAS 67762-53-2)

Repeated dose toxicity (dermal, 90d), rat: NOAEL ≥2000 mg/kg bw/day (m/f) (RA from CAS 67762-53-2)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
17 Nov 1997 - 16 Mar 1998
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
according to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Remarks:
ToxLabs Prüflabor GmbH, Greppin, Germany
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 32 - 38 d
- Weight at study initiation: 148.5 g (mean value males), 136.7 g (mean value females)
- Housing: one or two animals in cages (Makrolon Type 3)
- Diet: Altromin 1326, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 - 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 30 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: distilled water containing 1% Tween 80
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no.: S23350 739
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 2 mL samples of each formulation were taken and stored in the frozen state until measurement
Duration of treatment / exposure:
90 d
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 (control, test and satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: 10 animals each from the high dose and the vehicle group were used to investigate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behavior
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling; occurrence of secretions and excretions
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from the start to the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: All (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes, over night
- How many animals: All animals
- Parameters examined: erythrocyte count, hemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombin time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study (including the satellite groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli) / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: cranial, thoracic and abdominal cavities were opened and examined macroscopically

HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, esophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal cord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus
Other examinations:
Organ weights of adrenals, brain, epididymides, testes, heart, kidneys, liver, ovaries, spleen, testes and thymus
Statistics:
Body weights, food consumption: Welch t-test; Haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test; Differential leukocyte count: Mean, range and standard deviation
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high-dose group (female): fibrinogen concentration of plasma and creatinine content increased, high dose group (male/female): alkaline phosphatase increased, middle and high dose groups (male/female): serum urea nitrogen increased, all non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights for high-dose males and females, non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
intracellular fat and low-grade fatty degenerations of hepatocytes in all male animals, female control, middle dose and high dose groups, non-adverse
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood. None of the animals showed any alterations of their general state of well-being and behaviour at any observation period (few observations were made substance independent and for a short period of time).

BODY WEIGHT AND WEIGHT GAIN
Not affected by the test compound.

FOOD CONSUMPTION AND COMPOUND INTAKE
Not dose-dependently influenced.

OPHTHALMOSCOPIC EXAMINATION
No alterations.

HAEMATOLOGY
Not influenced.

CLINICAL CHEMISTRY
The fibrinogen concentration of the plasma was increased in the female animals of the high dose group, this was no longer apparent at the end of the treatment-free period. The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and females. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period. The serum urea nitrogen was significantly increased in the middle and high dose group of the males and in the high dose group of female animals. The creatinine content was significantly increased in all male and in the high dose group of the female animals. The phosphorus content was significantly increased dose-dependently in all female animals and the sodium content was dose-dependently decreased in the male animals, significantly in the animals of the middle and high dose groups. These effects were no longer apparent at the end of the treatment-free period.

NEUROBEHAVIOUR
No changes in grip strength, motor activity and sensory response.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental.

GROSS PATHOLOGY
No substance-dependent changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Intracellular lipid droplets in hepatocytes of the female animals in the high and mid dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals. Stomach: esophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach; Lungs: Atelectactic and emphysemic areas; Thymus: Partial substitution of the parenchyma by fibrinogenesis; Skin: Benign fibrous proliferation; Sciatic nerve: Thickening of the perineurium and thickening of the adventitia of the vessels
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: non-adverse findings
Critical effects observed:
not specified
Conclusions:
Repeated oral administration of the test material to rats, up to and including a dose level of 1000 mg/kg bw/day for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEL is considered to be ≥1000 mg/kg bw/day for male and female rats.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
Nov - Dec 1992
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: see "remarks"
Remarks:
GLP-Guideline study. According to the ECHA guidance document “Practical guide 6: How to report read-across and categories (Dec 2012)”, the reliability was changed from RL1 to RL2 to reflect the fact that this study was conducted on a read-across substance.
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
no
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderly Park, Macclesfield, Cheshire, UK
- Age at study initiation: 28 d
- Weight at study initiation: 148.45 g (males); 122.6 g (females)
- Housing: sexes separately, five per cage, cages had measurements of 26.5 x 50.0 x 20.0 cm and were constructed of stainless steel mesh with one solid side.
- Diet: CT1 diet; Special Diets Services Limited, Witham, Essex, UK, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65 (71 at one occasion)
- Air changes (per hr): 25 - 30
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: November 1992 To: December 1992
Route of administration:
oral: feed
Vehicle:
other: in diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All diet preparations were based on CT1 diet (Special Diets Services Limited, Witham, Essex, UK). They were prepared by grinding the appropriate amount of test substance with 1 kg of milled CT1 diet. This premix was then added to 14 kg of diet and mixed thoroughly with a Pharma Blender Model PMA 100S (T K Filder).

DIET PREPARATION
- Rate of preparation of diet: 15 kg batches
- Mixing appropriate amounts with (Type of food): CT1 diet (Special Diets Services Limited, Witham, Essex, UK)
- Storage temperature of food: - 20°C, stored at room temperature for usage up to 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical stability was determined for diet preparations over a period of 5 weeks following storage at room temperature or at -20°C. Samples were extracted by chemical shaking with ethyl acetate. The supernatant was diluted with ethyl acetate to give solutions containing appropriate concentrations of the test substance. Extracts were analysed by gas chromatography using flame ionisation detection. The extract concentration was calculated by reference to data from a standard containing a known concentration.
Duration of treatment / exposure:
daily
Frequency of treatment:
28 d
Remarks:
Doses / Concentrations:
1000 ppm, 5000 ppm, 12500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
112, 562, 1450 mg/kg bw/d
Basis:
other: actual ingested for males
Remarks:
Doses / Concentrations:
119, 586, 1613 mg/kg bw/d
Basis:
other: actual ingested for females
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on results of preliminary feeding studies

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: changes in clinical condition and behaviour and significant changes were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Days 8, 15, 22, 29
- observations included, but were not limited to the assessment of autonomic function (e.g. lacrimation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis); description, incidence and severity of any convulsions, tremors, abnormal motor function, alteration in respiration, reactivity to stimuli, changes in the level of arousal, sensorimotor responses

BODY WEIGHT: Yes, measurement in replicate order immediately before feeding and at the same day once a week until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight: Yes, on a weekly basis
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Hemoglobin, red cell count, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, prothrombin time and kaolin-cephalin time

CLINICAL CHEMISTRY: Yes
At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Albumin, total protein, cholesterol, triglycerides, urea, creatinine, glucose, total bilirubin and alkaline phosphatase, plasma gamma-glutamyl transferase, plasma alanine aminotransferase, plasma aspartate aminotransferase, plasma creatine kinase, plasma sodium, plasma potassium, plasma chloride, plasma calcium and plasma phosphorus

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Days 8, 15, 22, 29
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (adrenals, aorta, bladder, bone and bone marrow (femur), brain, caecum, colon, cervical lymph node, cervix, colon, duodenum, epididymis, eye and harderian gland, heart, ileum, jejunum, kidney, liver, lungs, mammary gland, mesenteric lymph node, nasal passages, esophagus, oral cavity, ovaries, pancreas, parathyroid glad, pituitary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, uterus, voluntary muscle)
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (adrenals, aorta, bladder, bone and bone marrow (femur), brain, caecum, colon, cervical lymph node, cervix, colon, duodenum, epididymis, eye and harderian gland, heart, ileum, jejunum, kidney, liver, lungs, mammary gland, mesenteric lymph node, nasal passages, oesophagus, oral cavity, ovaries, pancreas, parathyroid glad, pituary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal chord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, uterus, voluntary muscle)
Statistics:
Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females. Time to tail flick and fore and hindlimb grip strength at weeks 2, 3, 4 and 5 were considered by analysis of variance, separately for both sexes. Haematological and clinical blood parameters were considered by analysis of variance. Organ weights were considered by analysis of variance and covariance on final body weight separately for both sexes.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
reduction in haemoglobin and haematocrit (males), reductions in haemoglobin, haematocrit and in white blood cell count (females).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
minor reductions in plasma cholesterol, triglyceride, total protein levels and plasma alanine transferase activities in males
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights (males), slightly increased kidney weights (females), increased liver weights (males/females) in 5000 and 12500 ppm groups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
kidney: increased tubular hyaline droplet formation, tubular basophilia, granular cast formation (males). liver: minimal hepatocyte hypertrophy in 4/5 male rats
Histopathological findings: neoplastic:
no effects observed
Details on results:
DIET ANALYSIS:
All diets prepared were found to be within 4% of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material, assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use. Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

MORTALITY
There were no mortalities.

FUNCTIONAL OBSERVATION BATTERY
A slightly reduced splay reflex was observed in one female of the 1000 ppm group (on days 29 and 30), in one male of the 5000 ppm group (on day 29) and in one male of the 12.500 ppm group (on day 29). As isolated observations, these were considered to be incidental. There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels. There was no evidence of any treatment related effects on forelimb or hindlimb grip strength. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

FOOD CONSUMPTION
Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

HAEMATOLOGY
There were statistically significant reductions in haemoglobin and haematocrit at 12.500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

CLINICAL CHEMISTRY
There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.
ORGAN WEIGHTS
Kidney weights adjusted for body weight were statistically significantly increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship. Liver weights adjusted for body weight were statistically significantly increased in both sexes at 12500 ppm and in males at 5000 ppm. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

PATHOLOGY:
Macroscopic findings:
No treatment-related macroscopic findings were apparent at the end of the study.

HISTOPATHOLOGY:
Microscopic findings:
Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation. In the liver, there was minimal hepatocyte hypertrophy in 4/5 male rats in the 12500 ppm group. The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 12.500 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).
Dose descriptor:
NOAEL
Effect level:
1 613 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects observed in female rats
Dose descriptor:
NOAEL
Effect level:
1 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: corresponding to 12500 ppm
Critical effects observed:
not specified
Conclusions:
Repeated dietary administration of the test material to rats, up to and including a dose level of 1450 mg/kg bw/day for male rats and 1613 mg/kg bw/day for female rats, did not produce any evidence of overt toxicity. There were no clinical signs indicative of neurological dysfunction or neuropathological changes in the brain related to treatment with the test material at any dose level. In male rats, an increased incidence of renal hyaline droplet formation and tubular basophilia was present at all dose levels, with granular formation and increased kidney weights also present at 562 mg/kg/day and 1450 mg/kg bw/day. This phenomenon is believed to be specific to male rats as such and is not considered to be of relevance to man.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
weight of evidence
Study period:
19 Feb 1998 - 30 Mar 1999
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no analytic of the test compound documented).
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Deviations:
yes
Remarks:
(No analytic of the test compound documented)
Qualifier:
equivalent or similar to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
Deviations:
yes
Remarks:
(No analytic of the test compound documented)
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, UK
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 175 - 208 g (males) and 136 - 181 g (females)
- Fasting period before study: no
- Housing: sexes separately, five per cage in polypropylene grid-floor cages
- Diet: (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, UK), ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the formulations were determined. Formulations were prepared weekly and stored at approx. 4 °C in the dark.

VEHICLE
- Justification for use and choice of vehicle: Stability and homogeneity of the preparation is guaranteed for up to 14 d
- Concentration in vehicle: 7.5 mg/mL; 75 mg/mL; 500 mg/mL
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted with methanol to give a final, theoretical test material concentration of approx. 0.1 mg/mL. Extracts were analysed by gas chromatography. The extract concentration was calculated by reference to data from a standard containing a known concentration (0.1 mg/mL).
Duration of treatment / exposure:
daily
Frequency of treatment:
28 d
Remarks:
Doses / Concentrations:
15, 150 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: based on results of preliminary feeding studies (14 d repeated dose)
- Rationale for animal assignment: sexes separately
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, piloerection, exophthalmia

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing and one and five hours after dosing (immediately before dosing and one hour after dosing at the weekends)

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the start of treatment and on days 7, 14, 21, 28 and at terminal kill

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded for each cage group at weekly intervals.

WATER CONSUMPTION: Yes
- Time schedule: daily visual inspections of the water bottles

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices, total leucocyte count, differential leucocytes count, platelet count, reticulocyte count, prothrombin time, activated thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Animals fasted: No data
- How many animals: all
- Parameters checked: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and on Days 1, 8, 15 and 22
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. (Organ weights: adrenals, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testes, thymus)

HISTOPATHOLOGY: Yes (adrenals, aorta, bone and bone marrow (femur), bone and bone marrow (sternum), brain, caecum, colon, duodenum, muscle, esophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, eyes, gross lesions, heart, ileum, jejunum, kidney, liver, lungs, lymph nodes, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus)
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological and blood chemical parameters, organ weights, weekly bodyweight gain, quantitative functional performance and sensory reactivity data were assessed for dose-response relationships by linear regression analysis followed by one way analysis of variance incorporating Levene´s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett´s test. Where Levene´s test showed unequal variances the data were analyzed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U-test. Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased salivation, diuresis, red/brown staining of the ano-genital region, wet fur
Mortality:
no mortality observed
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):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: slight increase in water consumption in both sexes during the final week of the study
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: males: increase in relative kidney weight; females: increase in relative liver weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: all males: speckled kidneys; 1 female: pallor of the liver with accentuated lobular pattern
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: both sexes: hepatocyte enlargement; 1000 mg/kg/day and 150 mg/kg/day: males: increased incidence and severity of globular accumulations of eosinophilic material in renal tubules
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed. An incidence of increased salivation was detected around the time of dosing and up to five hours after dosing in 1000 mg/kg bw/day animals from Day 14 onwards. Sporadic incidents of diuresis, red/brown staining of the ano-genital region and wet fur were also detected at this dose level.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on body weight development was detected.

FOOD CONSUMPTION AND WATER CONSUMPTION
No adverse effect on dietary intake was detected but a slight increase in water consumption for animals of either sex treated with 1000 mg/kg bw/day during the final week of the study was detected.

NEUROBEHAVIOUR
No intergroup differences detected.

ORGAN WEIGHTS
Males treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weight, relative to terminal body weight, with females from this treatment group showing an increase in relative liver weight when compared with controls. No treatment-related adverse effects were detected among animals of either sex treated with 150 or 15 mg/kg bw/day.

GROSS PATHOLOGY
All males treated with 1000 mg/kg bw/day showed speckled kidneys at terminal kill whilst one female from this treatment group showed pallor of the liver with accentuated lobular pattern. No treatment-related macroscopic abnormalities at necropsy were observed in the other groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of kidney and liver sections revealed treatment-related changes. Hepatocyte enlargement was observed among animals of either sex treated with 1000 mg/kg bw/day. An increased incidence and severity of globular accumulations of eosinophilic material was also observed in the renal tubular epithelium of males only at this dose level and at 150 mg/kg bw/day. Hepatocellular enlargement is considered to be an adaptive change whilst the globular accumulation of eosinophilic material in renal tubules is consistent with male rat specific hydrocarbon nephropathy.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: clinical observations, kidney and liver changes
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: minor kidney and liver changes and clinical observations
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: non-adverse findings
Critical effects observed:
not specified
Conclusions:
The liver changes identified during the study are generally considered to be adaptive in nature whilst the kidney changes are consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. These effects are, therefore, not indicative of a hazard to human health and, for purposes of hazard evaluation, the "No Observed Adverse Effect Level" (NOAEL) should be regarded as 1000 mg/kg bw/day for both sexes.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, UK
- Age at study initiation: 5 - 8 weeks
- Weight at study initiation: 175 - 208 g (males) and 136 - 181 g (females)
- Fasting period before study: no
- Housing: sexes separately, five per cage in polypropylene grid-floor cages
- Diet: (Rat and Mouse SQC Expanded Diet No. 1, Special Diets Services Limited, UK), ad libitum
- Water: ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 40 - 70
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
arachis oil
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: The test material was prepared at the appropriate concentrations as a solution in Arachis oil. The stability and homogeneity of the formulations were determined. Formulations were prepared weekly and stored at approx. 4 °C in the dark.

VEHICLE
- Justification for use and choice of vehicle: Stability and homogeneity of the preparation is guaranteed for up to 14 d
- Concentration in vehicle: 7.5 mg/mL; 75 mg/mL; 500 mg/mL
- Amount of vehicle: 2 mL/kg
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Samples were extracted with methanol to give a final, theoretical test material concentration of approx. 0.1 mg/mL. Extracts were analysed by gas chromatography. The extract concentration was calculated by reference to data from a standard containing a known concentration (0.1 mg/mL).
Duration of treatment / exposure:
daily
Frequency of treatment:
28 d
Remarks:
Doses / Concentrations:
15, 150 and 1000 mg/kg bw/day
Basis:
actual ingested
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: based on results of preliminary feeding studies (14 d repeated dose)
- Rationale for animal assignment: sexes separately
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked: gait, tremors, twitches, convulsions, bizarre/abnormal/stereotypic behaviour, salivation, piloerection, exophthalmia

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: immediately before dosing and one and five hours after dosing (immediately before dosing and one hour after dosing at the weekends)

BODY WEIGHT: Yes
- Time schedule for examinations: prior to the start of treatment and on days 7, 14, 21, 28 and at terminal kill

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food consumption was recorded for each cage group at weekly intervals.

WATER CONSUMPTION: Yes
- Time schedule: daily visual inspections of the water bottles

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Anaesthetic used for blood collection: No data
- Animals fasted: No data
- How many animals: all
- Parameters checked: haemoglobin, erythrocyte count, haematocrit, erythrocyte indices, total leucocyte count, differential leucocytes count, platelet count, reticulocyte count, prothrombin time, activated thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the study (day 28)
- Animals fasted: No data
- How many animals: all
- Parameters checked: urea, glucose, total protein, albumin, albumin/globulin ratio, sodium, potassium, chloride, calcium, inorganic phosphorus, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, creatinine, total cholesterol, total bilirubin

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: prior to the start of treatment and on Days 1, 8, 15 and 22
- Dose groups that were examined: all
- Battery of functions tested: sensory activity / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: Yes, animals were subjected to a full external and internal examination and any macroscopic abnormalities were recorded. (Organ weights: adrenals, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testes, thymus)

HISTOPATHOLOGY: Yes (adrenals, aorta, bone and bone marrow (femur), bone and bone marrow (sternum), brain, caecum, colon, duodenum, muscle, esophagus, ovaries, pancreas, pituitary, prostate, rectum, salivary glands, sciatic nerve, seminal vesicles, eyes, gross lesions, heart, ileum, jejunum, kidney, liver, lungs, lymph nodes, skin, spinal cord, spleen, stomach, testes, thymus, thyroid/parathyroid, trachea, urinary bladder, uterus)
Statistics:
Data were processed to give group mean values and standard deviations where appropriate. Haematological and blood chemical parameters, organ weights, weekly bodyweight gain, quantitative functional performance and sensory reactivity data were assessed for dose-response relationships by linear regression analysis followed by one way analysis of variance incorporating Levene´s test for homogeneity of variance. Where variances were shown to be homogenous, pairwise comparisons were conducted using Dunnett´s test. Where Levene´s test showed unequal variances the data were analyzed using non-parametric methods: Kruskal-Wallis ANOVA and Mann-Whitney U-test. Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: increased salivation, diuresis, red/brown staining of the ano-genital region, wet fur
Mortality:
no mortality observed
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):
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: slight increase in water consumption in both sexes during the final week of the study
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: males: increase in relative kidney weight; females: increase in relative liver weight.
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: all males: speckled kidneys; 1 female: pallor of the liver with accentuated lobular pattern
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
1000 mg/kg bw/day: both sexes: hepatocyte enlargement; 1000 mg/kg/day and 150 mg/kg/day: males: increased incidence and severity of globular accumulations of eosinophilic material in renal tubules
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No mortalities were observed. An incidence of increased salivation was detected around the time of dosing and up to five hours after dosing in 1000 mg/kg bw/day animals from Day 14 onwards. Sporadic incidents of diuresis, red/brown staining of the ano-genital region and wet fur were also detected at this dose level.

BODY WEIGHT AND WEIGHT GAIN
No adverse effect on body weight development was detected.

FOOD CONSUMPTION AND WATER CONSUMPTION
No adverse effect on dietary intake was detected but a slight increase in water consumption for animals of either sex treated with 1000 mg/kg bw/day during the final week of the study was detected.

NEUROBEHAVIOUR
No intergroup differences detected.

ORGAN WEIGHTS
Males treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weight, relative to terminal body weight, with females from this treatment group showing an increase in relative liver weight when compared with controls. No treatment-related adverse effects were detected among animals of either sex treated with 150 or 15 mg/kg bw/day.

GROSS PATHOLOGY
All males treated with 1000 mg/kg bw/day showed speckled kidneys at terminal kill whilst one female from this treatment group showed pallor of the liver with accentuated lobular pattern. No treatment-related macroscopic abnormalities at necropsy were observed in the other groups.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of kidney and liver sections revealed treatment-related changes. Hepatocyte enlargement was observed among animals of either sex treated with 1000 mg/kg bw/day. An increased incidence and severity of globular accumulations of eosinophilic material was also observed in the renal tubular epithelium of males only at this dose level and at 150 mg/kg bw/day. Hepatocellular enlargement is considered to be an adaptive change whilst the globular accumulation of eosinophilic material in renal tubules is consistent with male rat specific hydrocarbon nephropathy.
Dose descriptor:
NOEL
Effect level:
15 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: clinical observations, kidney and liver changes
Dose descriptor:
NOEL
Effect level:
150 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: minor kidney and liver changes and clinical observations
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: non-adverse findings
Critical effects observed:
not specified
Conclusions:
The liver changes identified during the study are generally considered to be adaptive in nature whilst the kidney changes are consistent with well documented changes that are peculiar to the male rat in response to treatment with some hydrocarbons. These effects are, therefore, not indicative of a hazard to human health and, for purposes of hazard evaluation, the "No Observed Adverse Effect Level" (NOAEL) should be regarded as 1000 mg/kg bw/day for both sexes.
Endpoint:
sub-chronic toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Wiga GmbH
- Age at study initiation: 32 - 38 d
- Weight at study initiation: 148.5 g (mean value males), 136.7 g (mean value females)
- Housing: one or two animals in cages (Makrolon Type 3)
- Diet: Altromin 1326, ad libitum
- Water: tap water, ad libitum
- Acclimation period: 6 - 8 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 23
- Humidity (%): 30 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: gavage
Vehicle:
other: distilled water containing 1% Tween 80
Details on oral exposure:
VEHICLE
- Concentration in vehicle: 1%
- Lot/batch no.: S23350 739
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Duplicate 2 mL samples of each formulation were taken and stored in the frozen state until measurement
Duration of treatment / exposure:
90 d
Frequency of treatment:
once daily, 7 days/week
Remarks:
Doses / Concentrations:
100, 300, 1000 mg/kg bw/d
Basis:
actual ingested
No. of animals per sex per dose:
10 (control, test and satellite groups)
Control animals:
yes, concurrent vehicle
Details on study design:
- Rationale for selecting satellite groups: 10 animals each from the high dose and the vehicle group were used to investigate reversibility of possible effects
- Post-exposure recovery period in satellite groups: 28 d
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes, autonomic activity, presence of clonic or tonic movements, stereotypies, bizarre behavior
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes, changes in skin, fur, eyes, mucous membranes, gait, posture: response to handling; occurrence of secretions and excretions
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly from the start to the end of the study

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: prior to the administration and at the end of the study
- Dose groups that were examined: All (surviving) animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study
- Anesthetic used for blood collection: Yes (Ether)
- Animals fasted: Yes, over night
- How many animals: All animals
- Parameters examined: erythrocyte count, hemoglobin concentration, packed cell volume, platelet count, total leukocyte count, leukocyte differential count, prothrombin time, fibrinogen concentration

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Just prior to killing at the end of the study (including the satellite groups)
- Animals fasted: Yes
- How many animals: all animals
- Parameters examined: alkaline aminotransferase, alkaline phosphatase, aspartate aminotransferase, bilirubin, blood urea nitrogen, calcium, creatinine, fasting glucose, phosphorus, total cholesterol, total protein, albumin, chloride, potassium, sodium

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: Prior to administration, at monthly intervals and in the last week of dosing and in the last week of the recovery period.
- Dose groups that were examined: all
- Battery of functions tested: sensory activity (auditory, visual and proprioceptive stimuli) / grip strength / motor activity
Sacrifice and pathology:
GROSS PATHOLOGY: cranial, thoracic and abdominal cavities were opened and examined macroscopically

HISTOPATHOLOGY: adrenals, aorta, brain (3 sections), epididymides, eye, femur, heart, kidney, liver, lungs (incl. mainstem bronchi), mesenteric lymph node, muscle incl. sciatic nerve, esophagus, ovaries, pancreas, pituitary, prostate, seminal vesicle, skin incl. mammary glands, small and large intestine (including peyer´s patches), spinal cord (3 levels), spleen, sternum with bone marrow, stomach, submandibular lymph node, testes, thymus, thyroid (incl. parathyroids), trachea, urinary bladder, uterus
Other examinations:
Organ weights of adrenals, brain, epididymides, testes, heart, kidneys, liver, ovaries, spleen, testes and thymus
Statistics:
Body weights, food consumption: Welch t-test; Haematology, coagulation, clinical biochemistry and absolute and relative organ weights: Dunnett´s test; Differential leukocyte count: Mean, range and standard deviation
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Food efficiency:
no effects observed
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
high-dose group (female): fibrinogen concentration of plasma and creatinine content increased, high dose group (male/female): alkaline phosphatase increased, middle and high dose groups (male/female): serum urea nitrogen increased, all non-adverse
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights for high-dose males and females, non-adverse
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
intracellular fat and low-grade fatty degenerations of hepatocytes in all male animals, female control, middle dose and high-dose groups, non-adverse
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
Two animals died shortly after administration due to incorrect gavage shown by lungs filled with blood. None of the animals showed any alterations of their general state of well-being and behaviour at any observation period (few observations were made substance independent and for a short period of time).

BODY WEIGHT AND WEIGHT GAIN
Not affected by the test compound.

FOOD CONSUMPTION AND COMPOUND INTAKE
Not dose-dependently influenced.

OPHTHALMOSCOPIC EXAMINATION
No alterations.

HAEMATOLOGY
Not influenced.

CLINICAL CHEMISTRY
The fibrinogen concentration of the plasma was increased in the female animals of the high dose group, this was no longer apparent at the end of the treatment-free period. The activity of alkaline phosphatase of the serum significantly increased in the high dose group, males and females. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period. The serum urea nitrogen was significantly increased in the middle and high dose group of the males and in the high dose group of female animals. The creatinine content was significantly increased in all male and in the high dose group of the female animals. The phosphorus content was significantly increased dose-dependently in all female animals and the sodium content was dose-dependently decreased in the male animals, significantly in the animals of the middle and high dose groups. These effects were no longer apparent at the end of the treatment-free period.

NEUROBEHAVIOUR
No changes in grip strength, motor activity and sensory response.

ORGAN WEIGHTS
Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. Other significant differences seem to be incidental.

GROSS PATHOLOGY
No substance-dependent changes.

HISTOPATHOLOGY: NON-NEOPLASTIC
Intracellular lipid droplets in hepatocytes of the female animals in the high and mid dose group (5-90% of the observed area) with cell lesions were clearly caused dose-dependently by the test article. There was no special localization of the changes of hepatocytes in the liver lobules. In most cases only low grade intracellular lipopexia occurred in the male animals. Stomach: esophagal part and cardia with multilayered squamous epithelium, leukocyte infiltration in the submucosa and fibrous repair, fibrotic regions in the submucosa of the glandular stomach; Lungs: Atelectactic and emphysemic areas; Thymus: Partial substitution of the parenchyma by fibrinogenesis; Skin: Benign fibrous proliferation; Sciatic nerve: Thickening of the perineurium and thickening of the adventitia of the vessels
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: non-adverse findings
Critical effects observed:
not specified
Conclusions:
Repeated oral administration of the test material to rats, up to and including a dose level of 1000 mg/kg bw/day for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEL is considered to be ≥1000 mg/kg bw/day for male and female rats.
Endpoint:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
weight of evidence
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
other: Alpk:APfSD
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Zeneca Pharmaceuticals, Alderly Park, Macclesfield, Cheshire, UK
- Age at study initiation: 28 d
- Weight at study initiation: 148.45 g (males); 122.6 g (females)
- Housing: sexes separately, five per cage, cages had measurements of 26.5 x 50.0 x 20.0 cm and were constructed of stainless steel mesh with one solid side.
- Diet: CT1 diet; Special Diets Services Limited, Witham, Essex, UK, ad libitum
- Water: tap water, ad libitum
- Acclimation period: approx. 1 week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 23
- Humidity (%): 45 - 65 (71 at one occasion)
- Air changes (per hr): 25 - 30
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: November 1992 To: December 1992
Route of administration:
oral: feed
Vehicle:
other: in diet
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: All diet preparations were based on CT1 diet (Special Diets Services Limited, Witham, Essex, UK). They were prepared by grinding the appropriate amount of test substance with 1 kg of milled CT1 diet. This premix was then added to 14 kg of diet and mixed thoroughly with a Pharma Blender Model PMA 100S (T K Filder).

DIET PREPARATION
- Rate of preparation of diet: 15 kg batches
- Mixing appropriate amounts with (Type of food): CT1 diet (Special Diets Services Limited, Witham, Essex, UK)
- Storage temperature of food: - 20°C, stored at room temperature for usage up to 14 days
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Chemical stability was determined for diet preparations over a period of 5 weeks following storage at room temperature or at -20°C. Samples were extracted by chemical shaking with ethyl acetate. The supernatant was diluted with ethyl acetate to give solutions containing appropriate concentrations of the test substance. Extracts were analysed by gas chromatography using flame ionisation detection. The extract concentration was calculated by reference to data from a standard containing a known concentration.
Duration of treatment / exposure:
daily
Frequency of treatment:
28 d
Remarks:
Doses / Concentrations:
1000 ppm, 5000 ppm, 12500 ppm
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
112, 562, 1450 mg/kg bw/d
Basis:
other: actual ingested for males
Remarks:
Doses / Concentrations:
119, 586, 1613 mg/kg bw/d
Basis:
other: actual ingested for females
No. of animals per sex per dose:
5
Control animals:
yes
Details on study design:
- Dose selection rationale: Based on results of preliminary feeding studies

Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: changes in clinical condition and behaviour and significant changes were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: on Days 8, 15, 22, 29
- observations included, but were not limited to the assessment of autonomic function (e.g. lacrimation, salivation, piloerection, exophthalmus, urination, defecation, pupillary function, ptosis); description, incidence and severity of any convulsions, tremors, abnormal motor function, alteration in respiration, reactivity to stimuli, changes in the level of arousal, sensorimotor responses

BODY WEIGHT: Yes, measurement in replicate order immediately before feeding and at the same day once a week until termination.

FOOD CONSUMPTION AND COMPOUND INTAKE:
- Food consumption for each animal determined and mean daily diet consumption calculated as mg food/kg body weight: Yes, on a weekly basis
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

HAEMATOLOGY: Yes
At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Hemoglobin, red cell count, haematocrit, mean cell volume, mean cell haemoglobin, mean cell haemoglobin concentration, platelet count, white blood cell count, neutrophil count, lymphocyte count, monocyte count, eosinophil count, prothrombin time and kaolin-cephalin time

CLINICAL CHEMISTRY: Yes
At termination, all rats were bled by cardiac puncture and samples were collected. Parameters determined: Albumin, total protein, cholesterol, triglycerides, urea, creatinine, glucose, total bilirubin and alkaline phosphatase, plasma gamma-glutamyl transferase, plasma alanine aminotransferase, plasma aspartate aminotransferase, plasma creatine kinase, plasma sodium, plasma potassium, plasma chloride, plasma calcium and plasma phosphorus

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: on Days 8, 15, 22, 29
- Dose groups that were examined: All
- Battery of functions tested: sensory activity / grip strength
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes (adrenals, aorta, bladder, bone and bone marrow (femur), brain, caecum, colon, cervical lymph node, cervix, colon, duodenum, epididymis, eye and harderian gland, heart, ileum, jejunum, kidney, liver, lungs, mammary gland, mesenteric lymph node, nasal passages, esophagus, oral cavity, ovaries, pancreas, parathyroid glad, pituitary gland, prostate gland, rectum, salivary glands, sciatic nerve, seminal vesicles, skin, spinal cord, spleen, sternum, stomach, testes, thymus, thyroid gland, trachea, uterus, voluntary muscle)
Statistics:
Bodyweights were considered by analysis of covariance on initial body weight, separately for males and females. Time to tail flick and fore and hindlimb grip strength at weeks 2, 3, 4 and 5 were considered by analysis of variance, separately for both sexes. Haematological and clinical blood parameters were considered by analysis of variance. Organ weights were considered by analysis of variance and covariance on final body weight separately for both sexes.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
reduction in haemoglobin and haematocrit (males), reductions in haemoglobin, haematocrit and in white blood cell count (females).
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
minor reductions in plasma cholesterol, triglyceride, total protein levels and plasma alanine transferase activities in males
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased kidney weights (males), slightly increased kidney weights (females), increased liver weights (males/females) in 5000 and 12500 ppm groups
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
kidney: increased tubular hyaline droplet formation, tubular basophilia, granular cast formation (males). liver: minimal hepatocyte hypertrophy in 4/5 male rats
Histopathological findings: neoplastic:
no effects observed
Details on results:
DIET ANALYSIS:
All diets prepared were found to be within 4% of the target concentration. The homogeneity of the test material in the diet, determined at 1000 and 12500 ppm inclusion levels was within 2 % of the overall mean concentration for both levels. Chemical stability of the test material, assessed at the 1000 and 12500 ppm inclusion levels stored at room temperature or at -20 °C was satisfactory over the period of use. Dose rates (based on nominal dietary levels) were highest at the start of the study and declined rapidly during the period of rapid growth to week 4.

MORTALITY
There were no mortalities.

FUNCTIONAL OBSERVATION BATTERY
A slightly reduced splay reflex was observed in one female of the 1000 ppm group (on days 29 and 30), in one male of the 5000 ppm group (on day 29) and in one male of the 12.500 ppm group (on day 29). As isolated observations, these were considered to be incidental. There were no differences in time to tail flick in either sex which could be attributed to treatment. The statistically significant increase in time to response observed on day 22 for males (5000 ppm) and day 8 for females (1000 ppm) were considered to be incidental to treatment in the absence of similar changes at higher dose levels. There was no evidence of any treatment related effects on forelimb or hindlimb grip strength. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

BODY WEIGHT AND WEIGHT GAIN
There were no statistically significant effects on body weight and all final bodyweights were within 3% of the respective controls, after adjusting for initial weight differences.

FOOD CONSUMPTION
Food consumption in all treated groups remained similar to, or exceeded that, of the respective control group throughout the study.

HAEMATOLOGY
There were statistically significant reductions in haemoglobin and haematocrit at 12.500 ppm in male rats. Statistically significant reductions in haemoglobin and haematocrit were seen in females at 1000 and 5000 ppm and in white blood cell count at 1000 ppm. In the absence of a coherent dose-response relationship, these differences were considered incidental to treatment. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

CLINICAL CHEMISTRY
There were minor reductions in plasma cholesterol, triglyceride and total protein levels and plasma alanine transferase activities in males at 12500 ppm compared to controls. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.
ORGAN WEIGHTS
Kidney weights adjusted for body weight were statistically significantly increased in males at 5000 and 12500 ppm. All the females in the treatment groups had slightly raised kidney weights compared to control, but none achieved statistical significance, and there was no evidence of a coherent dose response relationship. Liver weights adjusted for body weight were statistically significantly increased in both sexes at 12500 ppm and in males at 5000 ppm. Any other statistically significant changes were considered spurious and unrelated to treatment with the test material.

PATHOLOGY:
Macroscopic findings:
No treatment-related macroscopic findings were apparent at the end of the study.

HISTOPATHOLOGY:
Microscopic findings:
Treatment related findings were present in the kidney of male rats from all dose groups. In the 5000 and 12500 ppm dose group these comprised increased tubular hyaline droplet formation and tubular basophilia in all animals, and granular cast formation in four of the 5000 ppm animals and all of the 12500 ppm animals; the latter occurring at the cortico-medullary injection. In the 1000 ppm group, increased renal hyaline droplet formation and/or tubular basophilia were seen, but not granular cast formation. In the liver, there was minimal hepatocyte hypertrophy in 4/5 male rats in the 12500 ppm group. The increased kidney weights and microscopic findings of renal tubular basophilia, granular cast formation and increased hyaline droplet formation present in male rats at 5000 and 12.500 ppm are clearly treatment related. These findings are consistent with the well characterized light hydrocarbon nephropathy described for male rats, following to a variety of chemicals including light hydrocarbons such as unleaded gasoline and trimethyl pentane. The characteristics include an increased accumulation of hyaline droplets in male rat kidneys, the main constituent of which is alpha 2µ-globulin (Alden et al. Adv. Modern Environ Toxicol 7: 107-120 (1984); Stonard et al. Renal Heterogeneity and Target Cell Toxicity. Bach PH and Lock EA Eds, John Wiley and Sons (1985)). It is widely accepted that this phenomenon is specific to male rat and as such appears to have no relevance for man (Swenberg et al. Toxicol and App. Pharmacol. 97: 35-46 (1989)).
Dose descriptor:
NOAEL
Effect level:
1 613 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
female
Basis for effect level:
other: No effects observed in female rats
Dose descriptor:
NOAEL
Effect level:
1 450 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male
Basis for effect level:
other: corresponding to 12500 ppm
Critical effects observed:
not specified
Conclusions:
Repeated dietary administration of the test material to rats, up to and including a dose level of 1450 mg/kg bw/day for male rats and 1613 mg/kg bw/day for female rats, did not produce any evidence of overt toxicity. There were no clinical signs indicative of neurological dysfunction or neuropathological changes in the brain related to treatment with the test material at any dose level. In male rats, an increased incidence of renal hyaline droplet formation and tubular basophilia was present at all dose levels, with granular formation and increased kidney weights also present at 562 mg/kg/day and 1450 mg/kg bw/day. This phenomenon is believed to be specific to male rats as such and is not considered to be of relevance to man.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises adequate and reliable studies (Klimisch score 2) from reference substances with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected studies are thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: inhalation
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (limited parameters examined, no daily observation, no data on test substance purity)
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 413 (Subchronic Inhalation Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(limited parameters examined, no daily observation)
GLP compliance:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic, Germantown, NY, USA
- Weight at study initiation: males: 379 - 388 g; females: 234 - 239 g
- Housing: animals were housed in the exposure chambers (feed and water was removed during exposure)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: 1.0 µm/ approx. 1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L stainless steel and glass exposure chambers; chambers contained catch pans between each of three leveles of cages.
- System of generating particulates/aerosols: The test material was aerosolized directly from the liquid by a modified Lakin nebulizer on each chamber. The test material was in a straight-walled glass flask and the barrels of the nebulizer were immersed under the level of the liquid in order to maximize the amount of material generated. The distance from the nebulizer to the walls of the flask was approx. 3 cm. After exiting the flask, the aerosol passed through a glass impactor to remove most of the larger particles. The remaining aerosol was mixed with the main air stream for each chamber before entering the chamber.
- Temperature and humidity in air chamber (by a Taylor wet/dry bulb hydrometer approx. every 30 min during each exposure): approx. 23 °C, 56 - 64%
- Air flow rate: approx. 300 L/min (mean chamber flow per group: 297, 308, 342, and 243 L/min, respectively)

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric sampling on glass fiber filters (3 times during each exposure); some filters were additionally analyzed by GC/MS
- Samples taken from breathing zone: yes
Nominal concentrations were determined as the loss of weight of fluid from the generator divided by total air flow through the chamber.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day
5 days/week
Remarks:
Doses / Concentrations:
0.00 ± 0.00, 0.05 ± 0.01, 0.17 ± 0.01, and 0.56 ± 0.02 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.05, 0.15, and 0.5 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
15
(Additional 10 male rats per group were included for examination of pulmonary function tests and analysis of pulmonary hydroxyproline following exposure.)
Control animals:
yes, concurrent no treatment
yes, sham-exposed
Details on study design:
- Dose selection rationale: The highest dose was expected to result in abnormal accumulation of test material in the lung and possible impairment of normal clearance mechanisms. The low dose is a factor of 10 above the TLV (threshold limit value) for mineral oil mistes, no untoward effects were expected at this level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (except weekends)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all core animals
- Parameters examined: complete blood count (CBC) (white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelets) and differential count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all core animals
- Parameters examined: glucose, urea nitrogen, total protein, albumin (A), globulin (G), A/G, sorbitol dehydrogenase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, creatinine cholesterol, triglycerides, uric acid, Cl, Ca, Na, K, and P

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Lung function: The animals were anaesthetized and pulmonary function tests were performed (deflation quasistatic pressure-volume curved, functional residual capacity, and maximal forced exhalation maneuver). After the pulmonary function tests, the lungs were removed and all lobes were weighed. Lobes were frozen for analysis of hydroxyproline content and analysis of test material remaining in the lung.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; organ weights (adrenals, kidney, spleen, brain, liver, testes, epididymides, ovaries, thymus, heart, prostate, uterus, right middle lung lobe
HISTOPATHOLOGY: Yes (untreated and high-dose): adrenals, ovaries, sternum, pancreas, brain, salivary gland, eye, spleen heart, stomach, colon, testes, duodenum, thymus, kidneys, thyroid, liver, tracheobronchial lymph nodes, lung, nasal turbinates, thigh muscle, urinary bladder, sciatic nerve, and any gross lesions. Only the lungs and tracheobronchial lymph nodes of the untreated controls were processed. 10 males of group 1, 2 and 5 (untreated, sham-exposed, and high-dose) were evaluated for morphology, number of sperm and number of testicular spermatids.
Statistics:
ANOVA and Tukey´s multiple range test: body weighs, male reproductive endpoints, haematology, and serum chemistry
ANOVA and Duncan´s multiple range test: organ weights, pulmonary function, and pulmonary hydroxyproline
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased total weight of the lung lobes (high-dose)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
mild macrophage accumulation in the lung (high-dose). Non adverse.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
mild macrophage accumulation in the lung (high-dose): Non adverse.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs were and no mortalities observed.

BODY WEIGHT AND WEIGHT GAIN
Increased body weights were observed in treated males. The difference compared to control was statistically significant, but as no clear dose-response was seen and the difference was lower than 7%, it was not considered to be of toxicological relevance.

HAEMATOLOGY
No treatment-related changes were observed.

CLINICAL CHEMISTRY
No treatment-related changes were observed.

ORGAN WEIGHTS
The lungs had a minimal increase in weight after exposure to 0.50 mg/L. Other organ weights were not affected by exposure to the test substance.

GROSS PATHOLOGY
The number of macrophages in the pulmonary alveoli increased slightly. This increase was small considering the high (500 mg/nr) aerosol concentration.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the lungs of animals in the high-dose group revealed one to two plump macrophages with sparse cytoplasmic vacuoles in less than 1.0% of the aveoli (in controls less than 0.1% would be expected).

OTHER FINDINGS
- Sperm morphology: No treatment-related effects were noted in sperm morophology or in sperm and spermatid counts.
- Lung function: There were no significant differences between any groups for any of the pulmonary function parameters. The only parameter affected by exposure was the total weight of the five lung lobes. Weight for the high-dose group was significantly greater than the other groups.
Dose descriptor:
NOAEC
Effect level:
0.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Critical effects observed:
not specified
Conclusions:
Repeated aerosol exposure via the inhalation route of the test material to rats, up to and including a dose level of 0.5 mg/L for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEC is considered to be 0.5 mg/L air for male and female rats.
Endpoint:
sub-chronic toxicity: inhalation
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Taconic, Germantown, NY, USA
- Weight at study initiation: males: 379 - 388 g; females: 234 - 239 g
- Housing: animals were housed in the exposure chambers (feed and water was removed during exposure)
Route of administration:
inhalation: aerosol
Type of inhalation exposure:
whole body
Vehicle:
air
Remarks on MMAD:
MMAD / GSD: 1.0 µm/ approx. 1.8
Details on inhalation exposure:
GENERATION OF TEST ATMOSPHERE / CHAMBER DESCRIPTION
- Exposure apparatus: 1000 L stainless steel and glass exposure chambers; chambers contained catch pans between each of three leveles of cages.
- System of generating particulates/aerosols: The test material was aerosolized directly from the liquid by a modified Lakin nebulizer on each chamber. The test material was in a straight-walled glass flask and the barrels of the nebulizer were immersed under the level of the liquid in order to maximize the amount of material generated. The distance from the nebulizer to the walls of the flask was approx. 3 cm. After exiting the flask, the aerosol passed through a glass impactor to remove most of the larger particles. The remaining aerosol was mixed with the main air stream for each chamber before entering the chamber.
- Temperature and humidity in air chamber (by a Taylor wet/dry bulb hydrometer approx. every 30 min during each exposure): approx. 23 °C, 56 - 64%
- Air flow rate: approx. 300 L/min (mean chamber flow per group: 297, 308, 342, and 243 L/min, respectively)

TEST ATMOSPHERE
- Brief description of analytical method used: gravimetric sampling on glass fiber filters (3 times during each exposure); some filters were additionally analyzed by GC/MS
- Samples taken from breathing zone: yes
Nominal concentrations were determined as the loss of weight of fluid from the generator divided by total air flow through the chamber.
Analytical verification of doses or concentrations:
yes
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
6 hours/day
5 days/week
Remarks:
Doses / Concentrations:
0.00 ± 0.00, 0.05 ± 0.01, 0.17 ± 0.01, and 0.56 ± 0.02 mg/L
Basis:
analytical conc.
Remarks:
Doses / Concentrations:
0.05, 0.15, and 0.5 mg/L
Basis:
nominal conc.
No. of animals per sex per dose:
15
(Additional 10 male rats per group were included for examination of pulmonary function tests and analysis of pulmonary hydroxyproline following exposure.)
Control animals:
yes, concurrent no treatment
yes, sham-exposed
Details on study design:
- Dose selection rationale: The highest dose was expected to result in abnormal accumulation of test material in the lung and possible impairment of normal clearance mechanisms. The low dose is a factor of 10 above the TLV (treshold limit value) for mineral oil mistes, no untoward effects were expected at this level.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (except weekends)

DETAILED CLINICAL OBSERVATIONS: No

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all core animals
- Parameters examined: complete blood count (CBC) (white blood cell count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), and platelets) and differential count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: Yes
- How many animals: all core animals
- Parameters examined: glucose, urea nitrogen, total protein, albumin (A), globulin (G), A/G, sorbitol dehydrogenase, aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase, total bilirubin, creatinine cholesterol, triglycerides, uric acid, Cl, Ca, Na, K, and P

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: No

OTHER:
Lung function: The animals were anaesthetized and pulmonary function tests were performed (deflation quasistatic pressure-volume curved, functional residual capacity, and maximal forced exhalation maneuver). After the pulmonary function tests, the lungs were removed and all lobes were weighed. Lobes were frozen for analysis of hydroxyproline content and analysis of test material remaining in the lung.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; organ weights (adrenals, kidney, spleen, brain, liver, testes, epididymides, ovaries, thymus, heart, prostate, uterus, right middle lung lobe
HISTOPATHOLOGY: Yes (untreated and high-dose): adrenals, ovaries, sternum, pancreas, brain, salivary gland, eye, spleen heart, stomach, colon, testes, duodenum, thymus, kidneys, thyroid, liver, tracheobronchial lymph nodes, lung, nasal turbinates, thigh muscle, urinary bladder, sciatic nerve, and any gross lesions. Only the lungs and tracheobronchial lymph nodes of the untreated controls were processed. 10 males of group 1, 2 and 5 (untreated, sham-exposed, and high-dose) were evaluated for morphology, number of sperm and number of testicular spermatids.
Statistics:
ANOVA and Tukey´s multiple range test: body weighs, male reproductive endpoints, haematology, and serum chemistry
ANOVA and Duncan´s multiple range test: organ weights, pulmonary function, and pulmonary hydroxyproline
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
no effects observed
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:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
increased total weight of the lung lobes (high-dose)
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
mild macrophage accumulation in the lung (high-dose). Non adverse.
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
mild macrophage accumulation in the lung (high-dose): Non adverse.
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related clinical signs were and no mortalities observed.

BODY WEIGHT AND WEIGHT GAIN
Increased body weights were observed in treated males. The difference compared to control was statistically significant, but as no clear dose-response was seen and the difference was lower than 7%, it was not considered to be of toxicological relevance.

HAEMATOLOGY
No treatment-related changes were observed.

CLINICAL CHEMISTRY
No treatment-related changes were observed.

ORGAN WEIGHTS
The lungs had a minimal increase in weight after exposure to 0.50 mg/L. Other organ weights were not affected by exposure to the test substance.

GROSS PATHOLOGY
The number of macrophages in the pulmonary alveoli increased slightly. This increase was small considering the high (500 mg/nr) aerosol concentration.

HISTOPATHOLOGY: NON-NEOPLASTIC
Microscopic examination of the lungs of animals in the high-dose group revealed one to two plump macrophages with sparse cytoplasmic vacuoles in less than 1.0% of the aveoli (in controls less than 0.1% would be expected).

OTHER FINDINGS
- Sperm morphology: No treatment-related effects were noted in sperm morophology or in sperm and spermatid counts.
- Lung function: There were no significant differences between any groups for any of the pulmonary function parameters. The only parameter affected by exposure was the total weight of the five lung lobes. Weight for the high-dose group was significantly greater than the other groups.
Dose descriptor:
NOAEC
Effect level:
0.5 mg/L air (analytical)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: no adverse effects
Critical effects observed:
not specified
Conclusions:
Repeated aerosol exposure via the inhalation route of the test material to rats, up to and including a dose level of 0.5 mg/L for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEC is considered to be 0.5 mg/L air for male and female rats.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEC
500 mg/m³
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: dermal
Type of information:
experimental study
Adequacy of study:
key study
Study period:
09 Jul - 10 Oct 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Comparable to guideline study with acceptable restrictions (no data on test substance purity; only 2 dose groups, open application, limited parameters examined)
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 411 (Subchronic Dermal Toxicity: 90-Day Study)
Deviations:
yes
Remarks:
(no data on test substance purity, only 2 dose groups, open application, limited parameters examined)
GLP compliance:
no
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, Lakeview, NJ, USA
- Age at study initiation: approx. 7 weeks
- Housing: individually in hanging, stainless steel cages with wire bottoms and fronts
- Diet: Purina Certified Lab Chow ' 5002 in pellet form; ad libitum
- Water: tap water; ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 40 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: no data
- Type of wrap if used: no wrap used, open
- Time intervals for shavings or clippings: 24 h before the first treatment; at least weekly afterwards
- Application site: back (shaved)

REMOVAL OF TEST SUBSTANCE
- Washing: no washing; wiping off with a gauze pads every saturday (applications on working days)

TEST MATERIAL
- Amount(s) applied: no data
- Concentration: undiluted
- Constant volume or concentration used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (collars), removal during the weekend
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week (65 exposures), 24 hours/day, removal of substance on saturdays (once a 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
(5 additional animals of the control and the high dose group were included for dermal bioavailability experiments only.)
Control animals:
yes, concurrent no treatment
Details on study design:
The test substance was dispensed by volume from a syringe and left uncovered on the shaved skin. The rats were fitted with cardboard Elizabethan collars to minimize ingestion of the test material. The controls were treated in the same manner except that no material was applied to their skin.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: appearance, behaviour, secretory function and discharges

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: weekly
- Parameters evaluated: erythema and edema according to Draize, chronic deterioration: flaking, thickening, stiffening, cracking and slouthing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: red blood cells, white blood cells, and platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: glucose, urea nitrogen, alanine aminotransferase, albumin, phosphorus; only females: lactate dehydrogenase, aspartate aminotransferase

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

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sperm morphology: at termination
- Parameters: percentage normal sperm, abnormal heads, headless, tailless, and curled tail
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; organ weights: kidneys, liver; only males: brain, spleen; only females: thyroids

HISTOPATHOLOGY: Yes (no further information available)
Statistics:
The level of statistical significance was P <0.05.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
slight erythema and flaking; slight epidermal hyperplasia and chronic inflammation (both treatment groups)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain in males (800 mg/kg: 7% and 2000 mg/kg: 10%)
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related effects were observed.

BODY WEIGHT AND WEIGHT GAIN
Treated males gained slightly less weight than the controls (800 mg/kg bw: 7%, 2000 mg/kg bw: 10%). As the difference is low, the decrease in body weight was not interpreted as a sign for systemic toxicity.

HAEMATOLOGY
No adverse effects on any haematologic parameters measured were observed.

CLINICAL CHEMISTRY
A few of the serum paramters of the high-dose animals were statistically different from the controls, but the differences were small, not consistent between males and females, and did not present any pattern suggestive of effects on any specific organ (no corresponding histological findings). Thus, the effects were considered not to be of toxicological relevance.
- high-dose males (compared to controls): glucose: -14%, albumin: -3%, and phosphorus: +16%
- high-dose females (compared to controls): lactate dehydrogenase: +45% (low-dose: +22%), and aspartate aminotransferase: +22%

URINALYSIS
No additional data given on Urinalysis in study report.

ORGAN WEIGHTS
Increased thyroid weight in the low-dose (+ 25%) females and decreased spleen weight (- 10%) in the low-dose males were not considered to be toxicologically relevant, as these effects were not observed in the high-dose groups.

GROSS PATHOLOGY
No abnormalities were detected.

HISTOPATHOLOGY:
No abnormalities were detected.

OTHER FINDINGS
- Sperm morphology: No effects on sperm morphology were detected.
- Local effects: Slight erythema and flaking of the skin were observed in the treated groups during the dosing phase. Microscopic examination of the skin indicated trace to slight epidermal hyperplasia and chronic inflammation of the superficial dermis.

SKIN PENETRATION
Skin penetration values of 2 - 6% were obtained.
The results of the in vivo skin penetration study indicate that the 13-week treatment with the test substance does not increase the skin penetration of the test substance (only the value for females was statistically different from the penetration in untreated animals). The skin penetration of untreated rats was less than 2% and the mean value for treated rats was approx. 6%. The recovery of radioactivity was measured in the urine and feces as well as the remaining radioactivity in tissue samples.
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
Conclusions:
Repeated dermal application of the test material to rats, up to and including a dose level of 2000 mg/kg bw for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEL is considered to be ≥2000 mg/kg bw/day for male and female rats.
Endpoint:
sub-chronic toxicity: dermal
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
refer to analogue justification provided in IUCLID section 13
Reason / purpose for cross-reference:
read-across source
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River, Lakeview, NJ, USA
- Age at study initiation: approx. 7 weeks
- Housing: individually in hanging, stainless steel cages with wire bottoms and fronts
- Diet: Purina Certified Lab Chow ' 5002 in pellet form; ad libitum
- Water: tap water; ad libitum
- Acclimation period: at least 14 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 22
- Humidity (%): 40 - 60
- Photoperiod (hrs dark / hrs light): 12/12
Type of coverage:
open
Vehicle:
unchanged (no vehicle)
Details on exposure:
TEST SITE
- Area of exposure: no data
- Type of wrap if used: no wrap used, open
- Time intervals for shavings or clippings: 24 h before the first treatment; at least weekly afterwards
- Application site: back (shaved)

REMOVAL OF TEST SUBSTANCE
- Washing: no washing; wiping off with a gauze pads every saturday (applications on working days)

TEST MATERIAL
- Amount(s) applied: no data
- Concentration: undiluted
- Constant volume or concentration used: no

USE OF RESTRAINERS FOR PREVENTING INGESTION: yes (collars), removal during the weekend
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
5 days per week (65 exposures), 24 hours/day, removal of substance on saturdays (once a 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
(5 additional animals of the control and the high dose group were included for dermal bioavailability experiments only.)
Control animals:
yes, concurrent no treatment
Details on study design:
The test substance was dispensed by volume from a syringe and left uncovered on the shaved skin. The rats were fitted with cardboard Elizabethan collars to minimize ingestion of the test material. The controls were treated in the same manner except that no material was applied to their skin.
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations included: appearance, behaviour, secretory function and discharges

DETAILED CLINICAL OBSERVATIONS: No

DERMAL IRRITATION: Yes
- Time schedule for examinations: weekly
- Parameters evaluated: erythema and edema according to Draize, chronic deterioration: flaking, thickening, stiffening, cracking and slouthing

BODY WEIGHT: Yes
- Time schedule for examinations: weekly

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: No

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

WATER CONSUMPTION: No

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: red blood cells, white blood cells, and platelets

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at study termination
- Animals fasted: No data
- How many animals: all animals
- Parameters examined: glucose, urea nitrogen, alanine aminotransferase, albumin, phosphorus; only females: lactate dehydrogenase, aspartate aminotransferase

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

NEUROBEHAVIOURAL EXAMINATION: No

OTHER: sperm morphology: at termination
- Parameters: percentage normal sperm, abnormal heads, headless, tailless, and curled tail
Sacrifice and pathology:
GROSS PATHOLOGY: Yes; organ weights: kidneys, liver; only males: brain, spleen; only females: thyroids

HISTOPATHOLOGY: Yes (no further information available)
Statistics:
The level of statistical significance was P <0.05.
Clinical signs:
no effects observed
Dermal irritation:
effects observed, treatment-related
Description (incidence and severity):
slight erythema and flaking; slight epidermal hyperplasia and chronic inflammation (both treatment groups)
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
reduced body weight gain in males (800 mg/kg: 7% and 2000 mg/kg: 10%)
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:
no effects observed
Urinalysis findings:
no effects observed
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
CLINICAL SIGNS AND MORTALITY
No treatment-related effects were observed.

BODY WEIGHT AND WEIGHT GAIN
Treated males gained slightly less weight than the controls (800 mg/kg bw: 7%, 2000 mg/kg bw: 10%). As the difference is low, the decrease in body weight was not interpreted as a sign for systemic toxicity.

HAEMATOLOGY
No adverse effects on any haematologic parameters measured were observed.

CLINICAL CHEMISTRY
A few of the serum paramters of the high-dose animals were statistically different from the controls, but the differences were small, not consistent between males and females, and did not present any pattern suggestive of effects on any specific organ (no corresponding histological findings). Thus, the effects were considered not to be of toxicological relevance.
- high-dose males (compared to controls): glucose: -14%, albumin: -3%, and phosphorus: +16%
- high-dose females (compared to controls): lactate dehydrogenase: +45% (low-dose: +22%), and aspartate aminotransferase: +22%

URINALYSIS
No additional data given on Urinalysis in study report.

ORGAN WEIGHTS
Increased thyroid weight in the low-dose (+ 25%) females and decreased spleen weight (- 10%) in the low-dose males were not considered to be toxicologically relevant, as these effects were not observed in the high-dose groups.

GROSS PATHOLOGY
No abnormalities were detected.

HISTOPATHOLOGY:
No abnormalities were detected.

OTHER FINDINGS
- Sperm morphology: No effects on sperm morphology were detected.
- Local effects: Slight erythema and flaking of the skin were observed in the treated groups during the dosing phase. Microscopic examination of the skin indicated trace to slight epidermal hyperplasia and chronic inflammation of the superficial dermis.

SKIN PENETRATION
Skin penetration values of 2 - 6% were obtained.
The results of the in vivo skin penetration study indicate that the 13-week treatment with the test substance does not increase the skin penetration of the test substance (only the value for females was statistically different from the penetration in untreated animals). The skin penetration of untreated rats was less than 2% and the mean value for treated rats was approx. 6%. The recovery of radioactivity was measured in the urine and feces as well as the remaining radioactivity in tissue samples.
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
Conclusions:
Repeated dermal application of the test material to rats, up to and including a dose level of 2000 mg/kg bw for male and female rats did not produce any evidence of overt toxicity. Therefore, the NOAEL is considered to be ≥2000 mg/kg bw/day for male and female rats.
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:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
11.24 mg/cm²
Study duration:
subchronic
Species:
rat
Quality of whole database:
The available information comprises an adequate and reliable study (Klimisch score 2) from a reference substance with similar structure and intrinsic properties. Read-across is justified based on common functional group(s), common precursors/breakdown products, similarities in PC/ECO/TOX properties (refer to endpoint discussion for further details). The selected study is thus sufficient to fulfil the standard information requirements set out in Annex VIII-IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006.

Additional information

Justification for read-across

There are no data available on repeated dose toxicity of Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid. In order to fulfil the standard information requirements set out in Annex VIII - IX, 8.6, in accordance with Annex XI, 1.5, of Regulation (EC) No 1907/2006, read-across from structurally related substances was conducted. In accordance with Article 13 (1) of Regulation (EC) No 1907/2006, "information on intrinsic properties of substances may be generated by means other than tests, provided that the conditions set out in Annex XI are met.” In particular for human toxicity, information shall be generated whenever possible by means other than vertebrate animal tests, which includes the use of information from structurally related substances (grouping or read-across) “to avoid the need to test every substance for every endpoint”. For each specific endpoint the source substance(s) structurally closest to the target substance is/are chosen for read-across, with due regard to the requirements of adequacy and reliability of the available data. Structural similarities and similarities in properties and/or activities of the source and target substance are the basis of read-across. A detailed justification for the analogue read-across approach is provided in the technical dossier (see IUCLID Section 13) and within Chapter 5.1 of the CSR.

 

Repeated dose toxicity: oral

CAS 146289-36-3

A reliable oral repeated dose toxicity study (90-day) with Isononanoic acid, mixed esters with 2-methylbutanoic acid, 3-methylbutanoic acid, pentaerythritol and valeric acid (CAS 146289-36-3) is available and was performed according to OECD TG 408 (Müller, 1998) and in compliance with GLP. Groups of 10 male and 10 female Wistar rats were exposed to the test substance at dose levels of 100, 300 and 1000 mg/kg bw/day by oral gavage once daily for 7 days/week for 90 consecutive days. A satellite control and high dose group containing 10 male and female animals each were included in the study and observed for additional 28 days. Control animals (10 per sex and dose) received the concurrent vehicle, distilled water containing 1% Tween 80 only. Observations and examinations of the animals included clinical signs, body weight, food consumption, haematology, clinical chemistry, organ weights, neurobehaviour, gross necropsy and histopathology. The daily oral administration of the test substance was tolerated without any adverse effects up to and including the highest dose tested (1000 mg/kg bw/day). No mortality was observed except for two animals that died shortly after administration due to incorrect gavage. Absolute and relative kidney weights were increased in all male animals in the high dose group which was still present after the recovery period. However, histopathology revealed no adverse effects in the kidney. Absolute and relative liver weights were increased in both sexes but this was no longer apparent after the recovery period in females. The activity of serum alkaline phosphatase was significantly increased in the high dose group in both sexes. This indicates damage to liver cells and/or an increased function rate. This finding was no longer apparent at the end of the treatment-free period. Except for the increased kidney and liver weights in the males, all changes (e.g. clinical chemical changes) were no longer apparent at the end of the treatment-free period. The increase in kidney weights in all male animals could be correlated to the formation of hyaline droplets a phenomenon widely accepted to be specific to male rats and as such considered to have no relevance to man. Thus, a 90-day oral NOAEL of 1000 mg/kg bw/day was derived for Isononanoic acid, mixed esters with 2-methylbutanoic acid, 3-methylbutanoic acid, pentaerythritol and valeric acid (CAS 146289-36-3) in male and female rats.

 

CAS 131459-39-7

A 28-day oral repeated dose toxicity study with 3,5,5-trimethylhexanoic acid mixed tetraesters with PE and valeric acid (CAS 131459-39-7) was conducted equivalent or similar to OECD TG 407 and under GLP conditions (Jones, 1999). Groups of 5 male and 5 female Sprague-Dawley rats were administered the test substance at dose levels of 15, 150 and 1000 mg/kg bw/day by oral gavage once daily for 7 days/week for 28 consecutive days. Control animals (5 per sex and dose) received the vehicle (arachis oil) only. No toxicologically significant effects on body weight, food consumption, mortality, neurobehaviour, haematology and clinical chemistry up to and including the highest dose level were observed. Clinical signs such as increased salivation, diuresis, red/brown staining of the ano-genital region and wet fur were observed in animals of the high dose group (1000 mg/kg bw/day). Males treated with 1000 mg/kg bw/day showed a statistically significant increase in kidney weight, relative to terminal body weight, with females from this treatment group showing an increase in relative liver weight when compared with controls. All males treated with 1000 mg/kg bw/day showed speckled kidneys at terminal kill whilst one female from this treatment group showed pallor of the liver with accentuated lobular pattern. No treatment-related macroscopic abnormalities at necropsy were observed in the other groups. Microscopic examination of kidney and liver sections revealed treatment-related changes. Hepatocyte enlargement was observed among animals of either sex treated with 1000 mg/kg bw/day. An increased incidence and severity of globular accumulations of eosinophilic material was also observed in the renal tubular epithelium of males only at this dose level and at 150 mg/kg bw/day. Hepatocellular enlargement is considered to be an adaptive change whilst the globular accumulation of eosinophilic material in renal tubules is consistent with male rat specific hydrocarbon nephropathy. This phenomenon is widely accepted to be specific to the male rat and as such is considered to have no relevance to man. In conclusion, 28-day NOEL values of 15/150 (m/f) mg/kg bw/day and a NOAEL value of 1000 mg/kg bw/day were derived for male and female rats, respectively.

 

CAS 68424-31-7

A further 28-day oral repeated dose toxicity study with Pentaerythritol tetraesters of n-decanoic, n-heptanoic, n-octanoic and n-valeric acids (CAS 68424-31-7) was conducted equivalent or similar to OECD TG 407 and under GLP conditions (Brammer, 1993). The test substance was administered in concentrations of 1000, 5000 and 12500 ppm (corresponding to 112, 562 and 1450 mg/kg bw/day for male and 119, 586 and 1613 mg/kg bw/day for female rats) to 5 Alpk:APfSD rats per sex and dose for 28 consecutive days. Control animals (5 per sex and dose) received the plain diet only. There were no toxicologically significant effects on body weight, food consumption and clinical condition and mortality up to and including the highest dose level. Changes in clinical chemistry and red cell-related parameters were observed in male rats at 12500 ppm, but these were minor and considered not to be of toxicological significance. A minimal hepatocyte hypertrophy present in males of the 12500 ppm group was observed and considered to be evidence of an adaptive response. Microscopic examination of the kidneys from male animals from all dose groups revealed an increase in hyaline droplet formation (the main constituent of which is alpha-2µ-globulin) and tubular basophilia. This phenomenon is widely accepted to be specific to the male rat and as such is considered to have no relevance to man. In conclusion, a 28-day NOAEL of 1450 and 1613 mg/kg/day was derived for male and female rats, respectively.

 

Repeated dose toxicity: inhalation

CAS 67762-53-2

A reliable inhalation repeated dose toxicity study (90-day) with Carboxylic acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) is available and was performed equivalent or similar to OECD TG 413 (Dulbey, 1992). Groups of 15 Sprague-Dawley rats of each sex were exposed via whole body to the test substance aerosol for 6 hours/day, 5 days/week at concentrations of 0.05, 0.15 and 0.5 mg/L air. Additionally, 10 male rats per dose group were included for examination of pulmonary function tests and analysis of pulmonary hydroxyproline following exposure to the test substance aerosol. The respective controls (15 animals per sex and dose) inhaled clean air under the same conditions. Animals were observed for clinical sings, body weight, haematology, clinical chemistry, organ weights, gross necropsy and histopathological examinations. Briefly, no substance-related adverse effects were observed for body weight, body weight gain, mortality, clinical biochemistry and haematological parameters. The lungs of the high dose animals revealed a minimal increase in weight which correlated with slightly increased numbers of macrophages in the pulmonary alveoli. Based on the results of the study and the absence of any toxicological relevant findings the subchronic NOAEC is considered to be 0.5 mg/L air for male and female rats.

 

Repeated dose toxicity: dermal

CAS 67762-53-2

A reliable dermal repeated dose toxicity study (90-day) with Carboxylic acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2) is available and was performed equivalent or similar to OECD TG 411 (Cruzan, 1988). Groups of 10 Sprague-Dawley rats of each sex were exposed to the test substance open without coverage once daily (5 days/week, 24 hours/day) at 800 and 2000 mg/kg bw for 90 days (65 applications in total). Animals were observed for clinical signs, body weight, dermal irritation, haematology, clinical chemistry, urinalysis, organ weights, gross necropsy and histopathology. Briefly, no adverse effects were found after dermal application of the test substance for 90 days on the parameters investigated. Males gained less body weight compared to control animals but this effect was low and no dose-dependency was observed. Therefore, it was not considered as a sign of systemic toxicity. Some serum parameters of the high dose group animals were significantly different compared to the control, but since the differences were small and they did not present any pattern suggestive of effects on a specific organ, they were considered not to be of toxicological relevance. Both test groups exhibited minimal erythema and flaking of the skin during the dosing phase. At microscopic examination it was identified as very minor epidermal hyperplasia and chronic inflammation of the superficial dermis. Since no effects of systemic toxicity were identified up to the highest dose tested, the 90-day dermal NOAEL was found to be 2000 mg/kg bw/day in male and female rats for Carboxylic acids, C5-9, tetraesters with pentaerythritol (CAS 67762-53-2).

Overall conclusion for repeated dose toxicity

Taken together, the available data on subacute and subchronic repeated dose toxicity from the analogue substances do not indicate adverse effects up to and including the recommended limit values. Therefore, as the available data did not identify any hazard for repeated dose toxicity, Tetraesters of pentaerythritol with heptanoic acid and 3,5,5-trimethylhexanoic acid is not considered to be hazardous following repeated exposure.

Justification for classification or non-classification

Based on the analogue read-across approach, the available data on repeated dose toxicity do not meet the classification criteria according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.