[No public or meaningful name is available]

Endpoint summary

Description of key information

Repeated dose toxicity oral: NOAEL = 300 mg/kg bw/day (OECD 408, GLP, K, rel.1)

Reproduction/Developmental toxicity screening study oral: parental NOAEL = 1000 mg/kg bw/day (OECD 421, GLP, rel.1)

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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:

20/10/2008-20/07/2009

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Remarks:

According to the OECD Guideline and in compliance with GLP

Reason / purpose for cross-reference:

reference to other study

Remarks:

Preliminary study

Qualifier:

according to guideline

Guideline:

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

Deviations:

no

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

HanRcc: WIST (SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Harlan Laboratories Ltd (Switzerland)

- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standard softwood bedding

Number of Animals:
Group 1: 15 males and 15 females
Group 2: 10 males and 10 females
Group 3: 10 males and 10 females
Group 4: 15 males and 15 females

Reserve animals:
Group 10: 2 males and 2 females

Age (at Delivery): Ca. 7 weeks

Body Weight Range (at Acclimatization):
Males: 176 g to 190 g (mean 184 g)
Females: 136 g to 156 g (mean 147 g)

- Diet (e.g. ad libitum): Pelleted standard Kliba Nafag 3433 (batch no.44/08) rat / mouse maintenance diet (Provimi Kliba SA, 4303 Kaiseraugst / Switzerland),ad libitum
- Water (e.g. ad libitum): Community tap-water, ad libitum

- Acclimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY:
The feed batch was analyzed for contaminants.
Water: Bacteriological assay, chemical and contaminant analyses were made.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22±3ºC
- Humidity (%): 30-70
- Air changes (per hr): 10-15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

unchanged (no vehicle)

Details on oral exposure:

A control group was treated similarly with the vehicle, PEG 300, only
Dose Volume: 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

On treatment day 1, samples of the control group as well as three samples (top, middle and bottom) of about 2 g of each concentration were taken prior to dosing for analysis of homogeneity and concentration were taken during week 4, 8 and 13 after commencement of gosing to confirm homogeneity and concentration. The analysis was performed using HPLC coupled to a UV detector and quantified with area under the peak. The test item was used as analytical standard.

Duration of treatment / exposure:

91/92 days

Frequency of treatment:

Daily

Dose / conc.:

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

Remarks:

actual ingested

Dose / conc.:

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

Remarks:

actual ingested

Dose / conc.:

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

Remarks:

actual ingested

No. of animals per sex per dose:

10 animals/sex/dose for the following groups : 0, 30, 100 and 300 mg/kg body weight/day
Additional satellite group of 5 animals/sex/dose for the following groups: 0 and 300 mg/kg body weight/day

Control animals:

yes

Details on study design:

- Dose selection rationale: The dose levels were selected based on a previous dose range finding toxicity study in Wistar rats (Harlan Laboratories study C02788).
In this dose range-finding toxicity study, AA 15 was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 100, 300 or 1000 mg/kg body weight for a period of 14 days. The study comprised five animals per group and sex which were sacrificed after 14 days of treatment.
No clinical signs of toxicological relevance were noted at any dose level.
Initial, but slight reductions in mean daily food consumption were noted at the highest dose of 1000 mg/kg/day, but these differences were transient and no longer evident during the second week of treatment. Slightly lower mean daily body weight gain values were noted in males at 1000 mg/kg/day, whereas females were considered to be unaffected.
Elevated mean absolute and relative liver weights were noted in males treated with 100 mg/kg/day and in both sexes at 300 mg/kg/day and 1000 mg/kg/day. These differences were considered to be related to the test item. Although such changes are often due to metabolic adaptation, these tissues were not examined for morphological changes and therefore, any associated microscopic alterations were not evaluated.
There were no macroscopical findings of toxicological relevance.
Based on the results of this 14-day dose range-finding study, dose levels of 30, 100 or 300 mg/kg body weight/day would be considered appropriate for a subsequent study with AA 15.


Dose Levels in Main study:
Group 1: 0 mg/kg/day
Group 2: 30 mg/kg/day
Group 3: 100 mg/kg/day
Group 4: 300 mg/kg/day

Dose Volume: 5 mL/kg body weight
Dose Concentrations:
Group 1: 0 mg/mL/day
Group 2: 6 mg/mL/day
Group 3: 20 mg/mL/day
Group 4: 60 mg/mL/day

Duration of Treatment Period: 91/92 days
Duration of Recovery Period: 28 days

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: twice daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before commencement of administration as well as twice daily on days 1 to 3, once daily from on day to the end of the treatment period , and once daily during days 1 to 28 (recovery period).

BODY WEIGHT: Yes
- Time schedule for examinations: weekly during acclimatization, treatment and recovery periods and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once during the acclimatization period and weekly thereafter

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: in all animals (satellite A and B) during acclimatization, during week 13 of treatment in animals of the control and high dose groups, and during week 17 in rats of satellite B.
- Dose groups that were examined: all groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Anaesthetic used for blood collection: Yes : light isoflurane anesthesia
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals:
- Parameters checked in Table 7.5.1/1 were examined.

CLINICAL BIOCHEMISTRY: Yes
- Time schedule for collection of blood: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Animals fasted: Yes but allowed access to water ad libitum
- How many animals:
- Parameters checked in Table 7.5.1/2 were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: After 13 weeks (satellite groups A and B), after 17 weeks (satellite group B)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in Table 7.5.1/3 were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: observations were performed once before commencement of administration and once weekly (weeks 1 to 12) thereafter.
- Dose groups that were examined: 0, 30, 100, 300 mg/kg bw/day
- Battery of functions tested: grip strength / locomotor activity. During week 13, relevant parameters from a modified Irwin screen test were evaluated in all animals.

IMMUNOLOGY: No

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see Table 7.5.1/4)

HISTOPATHOLOGY: Yes : Slides of all organs and tissues (as defined in the table 7.5.1/4) which were collected at scheduled sacrifices from all animals of the control and high-dose groups and all gross lesions from all animals were examined by the study pathologist. Organ and tissue samples taken from animals which died spontaneously were evaluated similarly to those organs taken from animals of the high-dose group.
As test item-related morphologic changes were detected in the organs of high-dose animals, the same organs (hearts (males only), livers and thyroids) from animals of the mid- and low-dose groups were examined.

Statistics:

The following statistical methods were used to analyze body weight, grip strength, locomotor activity, organ weights and ratios as well macroscopic findings: the Dunnett-test, the steel-test and fisher's exact-test

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Test item-related effects seen during daily observations included initial salivation at the highest dose level. This finding was noted more sporadically as the treatment period progressed.
Pale feces was noted in both sexes from day 47 of treatment until the end of the treatment period and for one day of the recovery period. In females treated with 300 mg/kg/day, salivation was noted during weeks 2-6 in some animals, and then intermittently during several of the remaining treatment weeks.
At 100 mg/kg/day and 30 mg/kg/day, a variety of clinical signs (such as breathing noises, salivation, localized scabs) were noted sporadically in some animals. These findings were considered to be typical background clinical signs and considered to be of no toxicological significance, as indicated by the very sporadic occurrence and/or small number of affected animals.

CONCLUSION: No adverse effects

Mortality:

no mortality observed

Description (incidence):

One male (nº41) treated with 300 mg/kg/day was found dead on day 71 of treatment. This was considered to be a possible dosing error. All other animals survived until scheduled necropsy.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

A test item-related reduction in the mean body weights was noted during the treatment period in males treated with 300 mh/kg/day. These differences were largely reversible after the recovery period. In females, the differences to the control group during the treatment period were negligible and considered to be due to biological variation.
No effects were noted in the groups treated with 30 mg/kg/day or 100 mg/kg/day.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

The mean daily food consumption of the test item-treated rats was generally similar to that of the controls.

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No test item-related changes were noted after 13 weeks of treatment.

Haematological findings:

no effects observed

Description (incidence and severity):

No test item-related changes of toxicological relevance were noted at any dose level.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Test item-related changes in the clinical biochemistry parameters were characterized by alterations in the lipid metabolism parameters in males and females treated at 300 mgkg/day, as well as elevated activity of aspartate aminotransferase in males at 300 mg/kg/day.
The latter changes were considered to be related to metabolic adaptation. None of the remaining minor difference to the control values were considered to be of toxicological relevance.

CONCLUSION: No adverse effects

Urinalysis findings:

no effects observed

Description (incidence and severity):

There were no test item-related changes in the urinalysis parameters at any dose level.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Functional Observational Battery
During the Functional Obsevational Battery performed after 13 weeks of treatment, slightly to moderately decreased activity was recorded in males treated with 100 mg/kg/day and 300 mg/kg/day. No other differences were noted.

Grip Strength
At 300 mg/kg/day, the mean hind limb grip strength was significantly reduced (p<0.01) in females when compared with that of the controls. Insofar as the forelimb grip strength of these females was unaffected, this was considered to be incidental. The mean fore- and hindlimb grip strength values of the males and females were considered to be unaffected at all dose levels.
No differences were ascertained at 30 mg/kg/day or 100 mg/kg/day.

Locomotor Activity
Minor differences in the mean locomotor activity were considered to be incidental.

CONCLUSION: No adverse effects

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

At 300 mg/kg/day, elevated mean absolute liver weights were noted in males and females when compared with controls.
In females, the mean absolute heart weight was elevated, along with mean brain-to-body weight, mean heart-to-brain weight, mean kidney-to-body weight and mean uterus-to-body weight ratios.
Females treated with 300 mg/kg/day also showed elevated liver-to-brain weight ratio at the end of the treatment period.

CONCLUSION: No adverse effects

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

No test item-related macroscopical changes were noted at any dose level.
The item-related microscopical inflammatory and degenerative changes were noted in the hearts of males treated with 100 and 300 mg/kg/day and centrilobular hypertrophy of the hepatocytes was recorded in the livers of males and females treated with 300 mg/kg/day. After the 28-day recovery period, the heart and liver changes reverted to normal.

CONCLUSION: No adverse effects

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Dose descriptor:

NOEL

Effect level:

30 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: no effects observed at this dose level

Key result

Dose descriptor:

NOAEL

Effect level:

300 mg/kg bw/day (nominal)

Sex:

male/female

Basis for effect level:

other: Treatment-related effects detected at 300 mg/kg bw/day were not considered to represent an adverse health effect.

Critical effects observed:

not specified

Conclusions:

Based on the results of this study, 30 mg/kg body weight/day of the substance was established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the substance as the no-observed-adverse-effect-level (NOAEL).

Executive summary:

In a repeated dose toxicity study performed according to the OECD test guideline No. 408 and to the EU Method B.26 and in compliance with GLP, test material was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 30, 100 and 300 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after the end of the treatment period. Additional 5 rats per sex and group were used at 0 and 300 mg/kg. These animals were treated for 91/92 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed. Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

Oral administration of test substance to Wistar rats at doses of 30, 100 and 300 mg/kg/day for 91/92 days resulted in no deaths from systemic toxicity, no relevant differences in mean fore- or hindlimb grip strength or locomotor activity, no ophthalmoscopic changes, no differences in mean daily food consumption, no changes of toxicological relevance in hematology or urinalysis and no macroscopical changes indicative of systemic findings. The single male (no 41) treated with 300 mg/kg/day which died on day 71 of treatment was considered to result from a dosing error that caused lung edema, hemorrhage and alveolar necrosis.

Test item-related findings were generally restricted to initial salivation at 100 mg/kg/day and 300 mg/kg/day (considered to be related to a bitter taste of the test item), pale feces at 300 mg/kg/day from day 47 of treatment onwards, decreased activity in both sexes at 300 mg/kg/day and in males at 100 mg/kg/day, reduced mean body weight development in males at 300 mg/kg/day, changes in clinical biochemistry (metabolic adaptation characterized by alterations in the lipid metabolism parameters in males and females treated at 300 mg/kg/day, as well as elevated activity of aspartate aminotransferase in males at 300 mg/kg/day), increased absolute and/or relative liver weights in males and females at 300 mg/kg/day and males at 100 mg/kg/day.

At the end of the treatment period, inflammatory and degenerative changes were seen in the heart of males treated with 100 mg/kg/day and 300 mg/kg/day. These heart changes reversed after a 28-day recovery period. Furthermore, centrilobular hypertrophy of the hepatocytes was seen in males and females treated with 300 mg/kg/day that reversed after a 28-day recovery period. This hypertrophy most likely reflected an induction of the mixed function oxidase (microsomal enzymes) that is responsible for the metabolism of the xenobiotics. The hypertrophy of the follicular epithelium of the thyroid gland was secondary to the hepatocytic hypertrophy. It is well documented, particularly in rats, that an increased turnover of T4 (due to the increased liver metabolism) is associated to an activation of the thyroid gland through the increased secretion of TSH (thyroid stimulating hormone) by the pituitary gland. The exposure to xenobiotics can induce the mixed function oxidase (MFO) in the liver with an increase in the microsome content resulting in an increase of the liver weight and a corresponding hepatocyte hypertrophy (predominantly centrilobular). In general, when the animals undergo a recovery period, the MFO activation disappears with a return to the normal morphology of the liver. The hypertrophy of the follicular epithelium of the thyroid gland of males and females at 300 mg/kg/day was very likely the result of the liver change. It is well documented that the induction of the mixed function oxidase in the liver determines an increased metabolism of the thyroid hormones (T4). As a result, the low level of T4 induces a feedback mechanism that leads to increased synthesis of TSH by the pituitary gland (pituitary-thyroid axis). This pathogenic mechanism is particularly observed in rats, the most sensitive species to the metabolism of thyroxine. Humans are far less sensitive to such mechanism.

Based on the results of this study, 30 mg/kg body weight/day of the substance was established as the no-observed-effect-level (NOEL) and 300 mg/kg body weight/day of the substance as the no-observed-adverse-effect-level (NOAEL).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

300 mg/kg bw/day

Study duration:

subchronic

Species:

rat

Quality of whole database:

The key study is GLP compliant and of high quality (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

Additional information

Repeated dose toxicity: oral

- A repeated dose toxicity study was performed according to the OECD test guideline No. 408 and to the EU Method B.26 and in compliance with GLP.

The dose levels used in the main study were determined in a dose range-finding toxicity study. AA 15 was administered daily by oral gavage to Wistar rats at 100, 300 and 1000 mg/kg bw/day for 14 days. No clinical signs of toxicological relevance were noted at any dose level. Initial, but slight reductions in mean daily food consumption were noted at the highest dose of 1000 mg/kg/day, but these differences were transient and no longer evident during the second week of treatment. Slightly lower mean daily body weight gain values were noted in males at 1000 mg/kg/day, whereas females were considered to be unaffected. Elevated mean absolute and relative liver weights were noted in males treated with 100 mg/kg/day and in both sexes at 300 mg/kg/day and 1000 mg/kg/day. These differences were considered to be related to the test item. Although such changes are often due to metabolic adaptation, these tissues were not examined for morphological changes and therefore, any associated microscopic alterations were not evaluated. There were no macroscopical findings of toxicological relevance.

Based on the results of this 14-day dose range-finding study, in the main study test material was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 30, 100 and 300 mg/kg body weight/day for a period of 91/92 days. A control group was treated similarly with the vehicle, PEG 300, only. The groups comprised 10 animals per sex which were sacrificed after the end of the treatment period. Additional 5 rats per sex and group were used at 0 and 300 mg/kg. These animals were treated for 91/92 days and then allowed a 28-day treatment-free recovery period after which they were sacrificed.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, the treatment and recovery periods. Functional observational battery, locomotor activity and grip strength were performed during week 13. At the end of the dosing and the treatment-free recovery period, blood samples were withdrawn for hematology and plasma chemistry analyses. Urine samples were collected for urinalyses. All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all control and high dose animals, and all gross lesions from all animals.

No deaths from systemic effects were observed. Only one male died on day 71 resulting from a dosing error that caused lung edema, hemorrhage and alveolar necrosis.

No relevant differences in mean fore- or hindlimb grip strength or locomotor activity, no ophthalmoscopic changes, no differences in mean daily food consumption, no changes of toxicological relevance in hematology or urinalysis and no macroscopical changes indicative of systemic findings.

Test item-related findings were generally restricted to initial salivation at 100 mg/kg/day and 300 mg/kg/day (considered to be related to a bitter taste of the test item), pale feces at 300 mg/kg/day from day 47 of treatment onwards, decreased activity in both sexes at 300 mg/kg/day and in males at 100 mg/kg/day, reduced mean body weight development in males at 300 mg/kg/day, changes in clinical biochemistry (metabolic adaptation characterized by alterations in the lipid metabolism parameters in males and females treated at 300 mg/kg/day, as well as elevated activity of aspartate aminotransferase in males at 300 mg/kg/day), increased absolute and/or relative liver weights in males and females at 300 mg/kg/day and males at 100 mg/kg/day.

At the end of the treatment period, inflammatory and degenerative changes were seen in the heart of males treated with 100 mg/kg/day and 300 mg/kg/day. These heart changes reversed after a 28-day recovery period. These effects are particularly orbserved in rats which are the most sensitive species to the metabolism of thyroxine. Humans are far less sensitive to such mechanism.

NOAEL = 300 mg/kg bw/day

- In a reproduction/developmental toxicity screening study performed according to the OECD Guideline 421 and in compliance with GLP, test substance was daily administered at a dose level of 100, 300 and 1000 mg/kg bw/day by gavage to groups of 10 males and 10 females rats over a 14 -pre-mating period and during the mating period for males (approx. 4 weeks) and also during gestation and lactation up to day 4 post partum for dams (approx 7 weeks).

No test item-related deaths occurred during the study.

No clinical signs or body weight changes were observed in the control group and at the dose level of 100 mg/kg bw/day.

No effects on organ weight or macroscopicl findings were observed in the control and test groups.

At the dose levels of 300 and 1000 mg/kg bw/day in both sexes pushing head through the bedding material and salivation after application were observed during the most of the treatment period. These symptoms were considered to be signs of discomfort caused by the treatment with the test item but not adverse effect. A short-lasting diarrhea was observed in males at the dose levels of 300 and 1000 mg/kg bw/day and females at the dose level of 1000 mg/kg bw/day and activity reduced for 1 to 3 days was observed in males at the dose level of 1000 mg/kg bw/day.

Treatment with the test item at the dose level of 300 mg/kg bw/day caused in males a statistically significant reduction of body weight gain at the beginning of the treatment period. Afterwards body weight gain increased and was similar to the respective control values.

Treatment with the test item at the dose level of 1000 mg/kg bw/day caused a statistically significant reduction of body weight gain in males and females at the beginning of the treatment. This resulted in a statistically significant reduction of body weights in both sexes. Afterwards body weight gain and body weights increased in both sexes and were similar to the control values during the remaining treatment period.

None of the effects observed are considered to be adverse.

NOAEL = 1000 mg/kg bw/day in this study

Conclusion: based on both studies, the NOAEL is determined at 300 mg/kg bw/day considering the longest period of exposure

Justification for classification or non-classification

Harmonized classification:

The substance has no harmonized classification according to the Regulation (EC) No 1272/2008.

Self-classification:

Based on the available data, no additional classification is proposed regarding the specific target organ toxicity after oral dose-repeated exposure according to the Regulation (EC) No 1272/2008 and to the GHS.

There were no data regarding the dermal and inhalation routes.