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Description of key information

NOAEL (13 weeks, rat, oral) > 10000 ppm (subchronic) (OECD 408; GLP compliant)

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records
Reference
Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1996-08-06 to 1996-12-02
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: see 'Remark'
Remarks:
GLP study reliable without restrictions Deviations from the guidelines OECD 408 (1998) and EU method B.26 (2008): - according to the guideline, consideration should be given to include an additional satellite group of ten animals (five per sex) in the control and in the top dose group for observation, after the treatment period, of reversibility or persistence of any toxic effect. In this study 10 animals per sex were used for the top dose group and 5 animals per sex for the control group. - according to the guideline, the morbidity and mortality of the test animals should be inspected twice daily. In this study this was not done on weekends and public holidays. - according to the guideline, towards the end of exposure period and any case not earlier than in week 11, sensory reactivity to stimuli of different types, assessment of grip strength and motor activity assessment should be conducted. These investigations were not conducted in this study. - according to the guideline, the epididymis, thymus and uterus were not weighed during gross pathology.
Reference:
Composition 0
Qualifier:
equivalent or similar to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
2001
Deviations:
yes
Remarks:
please refer to the "Rationale for reliability incl. deficiencies" above
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
1981-05-12
Deviations:
yes
Remarks:
please refer to the "Rationale for reliability incl. deficiencies" above
GLP compliance:
yes
Limit test:
no
Test material information:
Composition 1
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS - SPF-bred Wistar rats (strain Hsd Cpb:WU)
- Source: Harlan-Winkelmann, formerly Winkelmann, Borchen
- Age at study initiation: 5 - 6 weeks
- Weight at study initiation (mean): males: 138 (121 -155) g; females: 119 (104 - 132) g
- Housing: from their arrival until randomization the animals were kept in groups of 10 in Makrolon® cages type III. During experimental period animals were kept individually, under conventional conditions in Makrolon® cages type Ila (slightly modified as described by Spiegel and Gönnert, 1961 and Meister 1965) on Iow-dust wood granules (supplier: Ssniff Spezialdiäten Inc. Soest/Westfalen; manufacturer: J. Rettenmeier, Ellwangen-Holzmühle). Cages and bedding were changed weekly. The cages with study animals were kept by groups on shelves.
- Diet (ad libitum, except during the urine collection period): a fixed-formula standard diet (supplied by Altromin® GmbH, Lage)(Altromin® 1321 meal)
- Water (ad libitum): tap water
- Acclimation period: 8 days; during which time the health status was monitored. Only healthy animals, free of clinical signs were used for the study. The animals were not vaccinated or treated with anti-infectives either before delivery, during the adaptation or study period. Females were nulliparae and not pregnant.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 2°C
- Air humidity: 55% ± 5%
- Air pressure: 20 Pa above the normal pressure (in animal rooms)
- Air exchange: approx. 15 - 20 passages per hour
- Photoperiod (hrs dark / hrs light): 12/12
Route of administration:
oral: feed
Vehicle:
not specified
Details on oral exposure:
DIET PREPARATION
The test substance was blended (using a mixing granulator manufactured by Loedige, Paderborn) with Altromin® 1321 meal. The amount of test substance per concentration was not corrected according to the actual content. The diet was prepared weekly.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Data on homogeneity and stability of the test substance in the feed covering the concentration range used were obtained before the start of this study (method: high-performance liquid chromatography).
The test substance content of the diet mixtures was checked three times during the study (at start of study and end of study, and once in between) (method: high-performance liquid chromatography). This was done by analysing samples of food mixes used which were deep frozen (at temperatures of -15° C) until examination.
Reserve samples from each mixture were stored at least for 8 weeks at -15° C.

The analytical investigations showed the test substance to be homogeneously distributed and stable in the concentration range used beyond the period of use(formulation(s) stable over a period of at least 8 days).
The content of the test item in the diet was checked three times during the study. The analytical data verified that the test compound content agreed with the target concentrations within the defined limits.
Duration of treatment / exposure:
13 weeks
Frequency of treatment:
daily (ad libitum, except during the urine collection)
Remarks:
Doses / Concentrations:
0, 500, 1500, 5000 and 10000 ppm (m/f; main group)
Basis:
nominal in diet
Remarks:
Doses / Concentrations:
0 and 10000 ppm (m/f; recovery group)
Basis:
nominal in diet
No. of animals per sex per dose:
Main group: 10 males / 10 females
Recovery group: 5 males / 5 females (0 ppm); 10 males / 10 females (10000 ppm)
Control animals:
yes, plain diet
Details on study design:
- Dose selection rationale: The dosage scheme was based on a previous 14-day range-finder in which groups of 3 male and female Wistar rats were treated with concentrations of 0 - 5000 - 10000 ppm. No test-substance related effects on clinical signs, mortality, body weights, as well as food and water consumption were seen (Bomhard, 1996 unpublished data of BAYER AG).
On the basis of these results the following dose scheme was selected for the present subchronic study: 0 - 500 - 1500 - 5000 -10000 ppm.

- Post-exposure recovery period in satellite groups: 4 weeks
Positive control:
no data
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily, once daily on weekend and public holidays
- Cage side observations: any clinical signs (findings) and abnormalities were recorded.

DETAILED CLINICAL OBSERVATIONS: Yes
- A detailed weekly report on the conditions of the individual animals assessed the following: body surfaces and orifices, posture, general behaviour, breathing and excretory products.

BODY WEIGHT: Yes
- Time schedule for examinations: before initial application and thereafter weekly; furthermore, body weights were recorded immediately before scheduled necropsies, for calculation of relative organ weight.

FOOD CONSUMPTION AND COMPOUND INTAKE:
Food intake was calculated for all animals per group individually once a week from the difference of food supplied and not consumed. From these primary data the following was calculated, if appropriate:
For each interval:-
a) daily food intake per animal
b) mean daily food intake per animal
For the total feeding period (for recovery groups calculations were done separatedly for treatment and recovery period):-
c) mean food intake per animal and day
d) mean food intake per kg body weight and day
The calculation of the cumulative data:
e) cumulative food intake per animal
f) cumulative food intake per kg body weight and day
Furthermore, from the primary data the following was calculated:
g) cumulative test substance intake per animal and per kg body weight
h) mean test substance intake per animal/day and per kg body weight/day for the entire duration of the study

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 AND COMPOUND INTAKE: Yes
- Time schedule for examinations:weekly; comparable calculations as for food consumption were done for water intake of all animals.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before start of treatment (week 0) and at the end of the treatment period (week 12)
- Dose groups that were examined: all animals for the control group and the highest dose group.
- The pupillary reflex of both eyes was first tested in a darkened room. After dilating the pupils with Mydriaticum-Stulln® drops, the refractive elements of the eye and the fundus were examined using an indirect ophthalmoscope. The eyes were also examined using a photo-slit lamp.

NOTE: clinical laboratory tests on blood and urine samples were performed on 10 animals (5 recovery control) per sex and group. Occasionally, sample quantity may have been insufficient to permit determination of all intended parameters, or no determination was possible due to technical faults.Therefore, 10 (5) determinations per group are not necessarily available in all cases.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: blood samples used for determining the parameters in peripheral blood were collected in the morning (weeks 4 and 13 (main groups) or week 17 (recovery group)) from the retro-orbital venous plexus of the animals.
- Blood obtained was treated as follows: the samples for the haematological determinations were collected in tubes coated with EDTA (anticoagulant); samples for the determination of the thromboplastin time (HQuick) were collected in tubes with sodium-citrate.
- Anaesthetic used for blood collection: Yes; diethyl ether (Nöller, H.G. (1955). Die Blutentnahme aus dem retroorbitalen Venenplexus. Klin. Wschr. 33, 770 - 771)
- Animals fasted: No
- How many animals: 10 animals (5 recovery control) per sex and group
- Parameters examined: differential blood count, erythrocyte morphology, erythrocyte count, haemoglobin concentration in the blood, haematocrit, leucocyte count, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, mean corpuscular cell volume, reticulocyte count, thrombocyte count and thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: blood samples for determination of glucose concentration in deproteinized whole blood were taken from one of the caudal veins of non-anesthetized animals weeks 4 and 13 (main groups) or week 17 (recovery group). Blood samples used for determining the other parameters in peripheral blood were also collected in the morning (weeks 4 and 13 (main groups) or week 17 (recovery group)) from the retro-orbital venous plexus of animals anesthetized with diethyl ether (Nöller, H.G. (1955). Die Blutentnahme aus dem retroorbitalen Venenplexus. Klin. Wschr. 33, 770 - 771).
- Blood obtained was treated as follows: the samples for glucose determinations were mixed with perchloric acid (1:10) to precipitate proteins; samples for other biochemical tests were heparinized. The samples for the determinations of electrolyte concentrations remained untreated.
- Animals fasted: No
- How many animals: 10 animals (5 recovery control) per sex and group
- Parameters examined:
Enzyme activities in plasma: alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase;
Substrates in plasma: albumin, bilirubin, cholesterol, creatinine, total protein, triglycerides (after 4 weeks only) and urea;
Substrate in deproteinized whole blood: glucose;
Electrolytes in serum: chloride, calcium, inorganic phosphate, potassium and sodium.

URINALYSIS: Yes
- Time schedule for collection of urine: urine samples were collected at room temperature during a period of 16 hours (overnight)(main groups: weeks 4 and 12/13; recovery groups: week 17)
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes; during the urine collection periods water was offered ad libitum but feed was not supplied.
- Parameters examined:
Semiquantitative: blood, bilirubin, glucose, ketone bodies, pH, protein, sediment and urobilinogen;
Quantitative: density and volume.

NEUROBEHAVIOURAL EXAMINATION: No
Sacrifice and pathology:
GROSS PATHOLOGY: Yes
All animals scheduled for necropsies were killed by exsanguination under diethyl ether anesthesia, necropsied and their organs and tissues subjected to thorough gross pathological examination. Changes were described in terms of localization, size, color and consistency whenever appropriate.

Necropsy of intercurrent deaths: animals that died spontaneously or were killed in a moribund state during the study were dissected at the earliest opportunity. From these animals the organs and tissues were handled as described below ("Scheduled necropsies"). Tissues modified by autolysis were fixed only if they were still usable for further histological examination.

Scheduled necropsies: at the end of the treatment period (main groups) or at the end of the recovery period (recovery groups) all surviving animals were necropsied. The following organs and tissues, in whole or in part, as well as all tissues with macroscopic findings were fixed in 10% buffered formaldehyde solution: adrenals, aorta, brain (cerebrum, cerebellum, pons/medulla), cecum, colon, duodenum, epididymides, esophagus, eyes (with eyelids), exorbital lacilmal glands, femur (inc.t. bone marrow and knee Joint), harderian glands, head-nose-pharynx area, heart, ileum, jejunum, kidneys, larynx, liver, lungs (prefixation by instillation with 4% formaldehyde), lymph nodes (mandibular and mesenteric), mammary glands, optic nerves, ovaries (incl. oviduct), pancreas, physical identifier (tattooed ears), pituitary, prostate, rectum (with remaining intestine), salivary glands, sciatic nerve, seminal vesicies (with coagulating glands), skeletal muscle, skin (mammary region), spinal cord (cervical, thoracal, lumbar), spleen, stemum (with bone marrow), stomach (forestomach and glandular Stomach), testes, thymus (if present), thyroid (with parathyroids), tongue, trachea, ureter, urethra, urinary bladder, uterus, vagina, zymbal glands and all tissues showing abnormalities.

ORGAN WEIGHTS:
The following organs of the animals killed at the end of the treatment and the recovery period were weighed before fixation: brain, heart, liver, spleen, kidneys (both), adrenal glands (both), ovaries and testes (both).

HISTOPATHOLOGY: Yes
The organs and tissues Iisted above ("Scheduled necropsies"), were handled as follows: osseous tissues (femur, head, sternum, and vertebrae with spinal cord) were first decalcified by EDTA (ethylenediaminetetraacetic acid tetrasodium salt) and then, like all other organs (except eyelids, head, larynx, remaining intestinal tissue, ureters, urethra, optic nerve, zymbal glands and physical identifier) embedded in Paraplast. Sections approximately 5 micrometer thick were prepared from the organs assigned for histopathological examination (see below) and stained with hematoxylin and eosin (H&E). Cryocuts obtained from the Formalin-fixed livers were stained with Oil Red 0 (ORO).
The histopathological examination was performed on all mentioned organs of controls and 10000 ppm main group animals. In addition kidneys, liver, lungs and macroscopic findings were investigated in all animals of the 1500 and 5000 ppm groups and all recovery animals (except lungs).
Statistics:
The statistical evaluation of data related to clinical chemistry, hematology, body and organ weights as well as feed and water intake is performed using SAS® routines.
Statistical evaluations on body weight and organ weight data were done using the Dunnet-test in connection with a variance analysis. A Kruskal-Wallis Test with Steel test was erformed when data of feed and water intake were analyzed. The statistical test used to evaluate the remaining parameters were as follows: Dunnet test, adjust Welch test and Kruskal-Wallis test followed by adjusted Mann-Whitney-Wilcoxon tests (U test)
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:
not examined
Water consumption and compound intake (if drinking water study):
no effects observed
Ophthalmological findings:
no effects observed
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:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
There were no clinical findings attributable to the treatment.
Death of one control female in week 4 was causally related to blood collection.
These data produced no evidence of a test substance-related effect on mortality.

BODY WEIGHT AND WEIGHT GAIN
There was no significant effect on the body weights in the whole dose-range investigated.

FOOD CONSUMPTION AND COMPOUND INTAKE
No toxicologically relevant differences in mean food per animal/day or per kg body weight/day were detected in both sexes of all treatment groups.
Average test compound intake of main groups was 34.1, 103.6, 350.6, 695.1 mg/kg bw/d in males and 43.6, 129.5, 433.3, 824.5 mg/kg bw/d in females (in ascending order of concentrations).

WATER CONSUMPTION AND COMPOUND INTAKE
No toxicologically relevant differences in mean water consumption per animal/day or per kg body weight/day were detected in both sexes of all treatment groups.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmological examinations performed on all surviving animals from control and high dose groups at the end of the treatment period did not reveal any test substance-related effect.

HAEMATOLOGY
A few values which were significantly different from control values are considered to be of no toxicological relevance since either the differences to the respective control group were negligibly small or they did not show a correlation with the dose administered and with the time of treatment.
The determination of differential blood count did not indicate treatment-related effects.

CLINICAL CHEMISTRY
There were no relevant and/or dose-dependent changes at both dates during treatment, which could be attributed to the test substance administration.
At the end of the recovery period enzyme activity means of 10000 ppm males were slightly above controls due to a few individual values. Since they did not exceed the physiological range and gross and histopathology in these animals did not reveal any abnormal findings no toxicological relevance is attributed to them.
Occasionally, examinations revealed some parameters which were slightly but significantly different from controls. These values are considered to be of no toxicological relevance since either the differences from control were neglectably low and/or they did not show a correlation with the dose administered and with the time of treatment.
The determination of blood electrolyte concentrations did not indicate treatment related effects. Occasionally, a few means of electrolyte concentrations were identified as being significantly different from controls. However, these differences are of no toxicological relevance since these were not distributed dose dependently and/or the corresponding individual data were within the normal biological scatter of the parameter in question.

URINALYSIS
The excreted urine volume and the density were not affected by the test substance administration.
The semi-quantitatively determined blood, bilirubin, glucose, protein, urobilinogen and ketone body concentrations as well as the pH values were unremarkable in all animal groups. The sediments showed no abnormalities in the males or females.
Urinalyses, thus did not indicate treatment-related effects.

ORGAN WEIGHTS
Kidney weights seemed to be slightly increased in 10000 ppm females at the end of the recovery but were inconspicuous at the end of the treatment.
There were no other toxicologically relevant effects on the organ weights. The few statistically significant deviations are distributed dose independently and are within the bounds of values usually determined in rats of this strain and age.

GROSS PATHOLOGY
Necropsy of intercurrent deaths: the female rat of the 0 ppm main group, which died after blood sampling, showed dark-red discolouration of parts of the lungs.
None of the animals treated with the test substance died intercurrently.
Necropsy of main group: at the end of the treatment period gross pathological examinations did not indicate evidence for treatment-specific organ changes in males and females in all dose groups.
Necropsy of recovery group: at the end of the recovery period gross pathological examinations did not indicate evidence for treatment-specific organ changes in males and females.

HISTOPATHOLOGY: NON-NEOPLASTIC
Histopathological examinations revealed no organ and tissue changes attributable to the administration of the test substance.
Dose descriptor:
NOAEL
Effect level:
>= 10 000 ppm
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Treatment was very well tolerated without any signs of systemic toxicity. Average test compound intake was 695 and 825 mg/kg bw/d in male and female animals, respectively.
Critical effects observed:
not specified
Conclusions:
Under the conditions described the administration of the test item to male and female rats was tolerated without adverse effects up to and including 10000 ppm. The average test compound intake per day was about 695 and 825 mg/kg bw/d in males and females of the main study groups, respectively.
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
695 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
CLP compliant key study (Klimisch score=1)

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion
Endpoint conclusion:
no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion
Endpoint conclusion:
no study available

Mode of Action Analysis / Human Relevance Framework

Additional information

Repeated dose toxicity: oral

In a 13 -week oral toxicity study in rats (0, 500, 1500, 5000 and 10000 ppm in diet) described in Bomhard & Geiß (1997), treatment of the animals with the test item was very well tolerated without any signs of systemic toxicity. Therefore, the NOAEL can be expected above 10000 ppm. In conclusion, the administration of Neo Heliopan® AP to male and female rats was tolerated without adverse effects up to and including 10000 ppm which is equivalent to an average test compound intake of 695 mg/kg bw/d for male and 824 mg/kg bw/d for female rats.

Repeated dose toxicity: inhalation

According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute inhalation toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -week oral toxicity study in rats with Neo Heliopan® AP.

Repeated dose toxicity: dermal

According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute dermal toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -weeks oral toxicity study in rats with Neo Heliopan® AP.


Justification for selection of repeated dose toxicity inhalation - systemic effects endpoint:
According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute inhalation toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -week oral toxicity study in rats with Neo Heliopan® AP.

Justification for selection of repeated dose toxicity inhalation - local effects endpoint:
According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute inhalation toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -week oral toxicity study in rats with Neo Heliopan® AP.

Justification for selection of repeated dose toxicity dermal - systemic effects endpoint:
According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute dermal toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -weeks oral toxicity study in rats with Neo Heliopan® AP.

Justification for selection of repeated dose toxicity dermal - local effects endpoint:
According to regulation (EC) 1907/2006 Annex XI (weight of evidence), testing for subacute dermal toxicity is not considered to be required, for the following reason: - In accordance with ECHA guidance on information requirements and chemical safety assessment-chapter R.8: characterisation of dose [concentration]-response for human health, December 2010, a DNEL for systemic effects could be derived by route-to-route extrapolation from a 13 -weeks oral toxicity study in rats with Neo Heliopan® AP.

Justification for classification or non-classification

Repeated dose toxicity, oral

Reference Bomhard & Geiß (1997) is considered as the key study for repeated dose oral toxicity and will be used for classification. The NOAEL was determined to be above 10000 ppm (equivalent to 695 (M) or 825 (F) mg/kg bw/d ) in a 13 -week repeated dose oral toxicity study.

Specific target organ toxicant (STOT)- repeated exposure: oral

No classification and labelling of Neo Heliopan ® AP according to regulation (EC) 1272/2008 as specific target organ toxicant (STOT) – repeated exposure, oral is necessary, since the guidance value for a Category 1 classification of C<10 mg test substance/kg bw/day (based on sub-chronic toxicity study), and the guidance value for a Category 2 classification of 10<C<100 mg test substance/kg bw/day (based on sub-chronic toxicity study) are not met. In the 13 -week oral toxicity study in rats, no signs of systemic toxicity and therefore no specific organ toxicity was observed, and the NOAEL is determined to be above the highest dose tested (10000 ppm equivalent to 695 (M) or 825 (F) mg/kg bw/d).