1-imino-1H-isoindol-3-amine

Endpoint summary

Description of key information

Dose range finding 14-day repeated dose toxicity via oral route

A dose range finding study was performed which preceeded the presented key 28-day repeated dose study.

To assess the possible health hazards which could arise from repeated exposure of the test item via oral administration to rats, the test item was administered daily to 3 groups of test animals at 5, 10 and 25 mg/kg bw/day for a treatment period of 14 days. Under the conditions of the present study, no major signs of toxicity or mortality were observed.

There are no regulatory guidelines for this type of dose range-finding study, but the study design was based on the principles specified in OECD TG 407. The study was non-GLP. No NOAEL was determinded for this study as it served as a dose range finder. This study is used as supporting study.

28-day repeated dose toxicity study via oral route

For this endpoint there is one key study available: a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test in Wistar rats administered 5, 10, and 25 mg/kg bw/d.

In this study the NOAEL for general toxicity is considered to be at least 25 mg/kg bw/day for the test item.

Repeated dose toxicity via other routes

There are no studies available in which the repeated dose toxicity via inhalation or dermal route is assessed.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2017-02-08 to 2017-06-14

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

July, 2016

Qualifier:

according to guideline

Guideline:

other: OPPTS 870.3650, Combined Repeated Dose Toxicity Study with the Reproduction/ Develop mental Toxicity Screening Test. EPA 712-C-00-368

Version / remarks:

July, 2000

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Test animals:
Species: Wistar rat, Crl: WI(Han) (Full Barrier)
- Source: Charles River, 97633 Sulzfeld, Germany
- Age at study initiation: approx. 14-15 weeks old
- Weight at study initiation: males: 327 - 487 g (mean: 397.89 g), females: 213 - 277 g (mean: 240.33 g)
- Fasting period before study: not reported
- Housing: Animals were housed in groups of 5 per sex in type IV polysulphone cages
- In each cage Altromin saw fibre was used as bedding
- Diet (ad libitum): free access to Altromin 1324 maintenance diet for rats and mice
- Water (ad libitum): free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- Acclimation period: At least 5 days.

Environmental conditions:
- Temperature (°C): 22 +/- 3°C
- Humidity (%): 55 +/- 10 %
- Air changes (per hr): 10
- Photoperiod (hrs dark / hrs light): 12 hours artificial light / 12 hours dark

Route of administration:

oral: gavage

Details on route of administration:

Prior to the start of the treatment period a detailed clinical observation outside the home cage was
made. None of the animal showed pathological signs before the first administration.
Before dosing all females were screened for two weeks for regular estrous cyclicity and animals (10
females/ group) with regular estorus cycle (4-5 day cycle) were used in the study.
The test item and vehicle were administered at a single dose to the animals by oral gavage. The appli
cation volume for all groups was 5 mL/kg body weight.
Before the first administration all animals to be used for the study were weighed and assigned to the
experimental groups
Each animal was marked with its identification number by individual ear tattoo marking

Vehicle:

water

Details on oral exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females. Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Before the treatment period, formulation samples were prepared and analysed for stability and homogeneity of the test item in the selected vehicle.

Study pre-start stability analysis were included on the samples from high dose and low dose group and the investigation was made for 0 h, 6 h (RT), 10 day (RT), 10 day (2-8 °C) and 10 day -15 to -35 °C (10 samples). Pre-start homogeneity investigation was included on the samples collected from various levels (top, middle and bottom) of high dose and low dose groups (6 samples).

The test item was homogenous (after 60 min without stirring), therefore during the study samples were collected for the investigation of homogeneity and only samples were taken for substance concentration in study week 1 (pre-mating period), 3 (first week of mating), 5 (gestation) and in the last week of the study (gestation / lactation) from all dose groups (12 samples).

Each sample taken during the study was retained in duplicate (sample A, sample B, each of at least 5 mL). The A-samples were analysed at Eurofins Munich and until then stored under appropriate conditions based on available stability data. The B-samples were retained at -15 to -35 °C at BSL Munich (test facility) and discarded after completion of the final study report.

Duration of treatment / exposure:

The animals were treated with the test item formulation or vehicle on 7 days per week for a maximum period of 63 days, i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females.

Then in females, treatment was done during the gestation period and up to post-natal day 12. Males were dosed after the mating period until the minimum total dosing period of 28 days was completed.

Frequency of treatment:

7 days per week

Dose / conc.:

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

Remarks:

control (C) group

Dose / conc.:

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

Remarks:

low dose (LD) group

Dose / conc.:

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

Remarks:

medium dose (MD) group

Dose / conc.:

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

Remarks:

high dose (HD) group

No. of animals per sex per dose:

10 animals per sex per group

Control animals:

yes, concurrent vehicle

Details on study design:

100 animals (40 males and 60 females) were included in the study.

Prior to the start of the treatment period a detailed clinical observation outside the home cage was made. None of the animal showed pathological signs before the first administration.

Before the first administration all animals to be used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively (randomisation was performed with IDBS Workbook 10.1.2
software).

Dose selection rationale: According to the results of a previous dose range finding study the doses were selected for the 3 dose groups (LD = low dose, MD = medium dose, HD = high dose).

Body weight and food consumption: The animals were weighed once before the assignment to the experimental groups, on the first day of dosing and weekly thereafter as well as at the end of the study. All animals were weighed directly before termination. Food consumption was measured on the corresponding days of the body weight measurements after the beginning of the dose administration. Food consumption was not measured during the mating period in males and females and the post-mating period in males.

Positive control:

not applicable

Observations and examinations performed and frequency:

Clinical observations:
General clinical observations were made at least once a day, preferably at the same time each day. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once
daily.Once before the first exposure, and at least once a week thereafter, detailed clinical observations were made in all animals outside the home cage in a standard arena. Clinical observations included spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in the skin and fur, eyes and mucous membranes (salivation, discharge),piloerection and pupil size. Changes in gait, posture, response to handling as well as the presence of clonic or tonic movements, stereotypes, difficult or prolonged parturition or bizarre behaviour were recorded.

Functional observations:
Multiple detailed behavioural observations were made in the week before the first treatment and during the last week of the treatment in 5 randomly selected males and during the last week of lactation of the lactation period in 5 randomly selected females (only lactating females were evaluated) of each group outside the home cage using a functional observational battery of tests. Sensory reactivity to different modalities, grip strength and motor activity assessments and other behavioural observations as well as rearing supported and not supported, urination, defecation, startle/ auditory response, equilibrium reflex, positional passivity, visual placing, fore and hind limb grip strength, tail pinch response, toe pinch reflex, extensor thrust/limb tone, hind limb reflex, righting reflex on the ground, air righting reflex, pupil response, body temperature and ophthalmoscopy (anterior chamber of the eye and fundus of eye).

Haematology:
Haematological parameters were examined in 5 randomly selected males and females (only lactating females were evaluated) from each group at the end of the treatment prior to or as part of the sacrifice of the animals.
The following haematological parameters were examined: haematocrit value (Hct), haemoglobin content (Hb), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), reticulocytes (Re), platelet count (PLT), white blood cells (WBC), neutrophils (Neu), lymphocytes (Lym), monocytes (Mono),eosinophils (Eos), basophils (Baso), large unstained cells (Luc).

Blood coagulation:
Coagulation parameters from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. The following coagulation parameters were examined: prothrombin time (PT), activated partial thromboplastin time (aPTT).

Clinical biochemistry:
Parameters of clinical biochemistry from 5 randomly selected males and females (only lactating females were evaluated) of each group were examined at the end of the treatment prior to or as part of the sacrifice of the animals. The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total bile acids (TBA), total bilirubin (TBIL), total cholesterol (Chol), glucose (Gluc), sodium (Na), potassium (K).

Urinalysis:
A urinalysis was performed with samples from 5 randomly selected males and females (only lactating females were evaluated) prior to or as part of the sacrifice of the animals. Additionally, urine colour/appearance were recorded. The following parameters were measured: specific gravity, nitrite, ph-value (pH), protein, glucose, ketone bodies (ketones), urobilinogen (ubg), bilirubin, blood, leukocytes.

Sacrifice and pathology:

The males were sacrificed any time after the completion of the mating period (after a minimum dosing period of 28 days) and females were sacrificed on the respective PND 13 by using anesthesia (ketamine/xylazine). Non-pregnant females were sacrificed on study day 26 using the sperm-positive vaginal
smear or from the last day of mating period.
All animals were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ weights at necropsy:
The wet weight of the organs of 5 randomly selected male and female animals (only lactating females were evaluated) from each group was recorded as soon as possible. Paired organs were weighed together. Organ weights of animals found dead were not recorded. Reproductive organs were
weighed from all animals. Thyroid/parathyroid glands from all adult males and females were preserved. Weight of thyroid/para thyroid glands was measured after fixation.

The following tissues/organs were examined: testes (paired weight), epididymides (paired weight), prostate, seminal vesicles and coagulating glands (complete weight), thyroid/parathyroid glands (from all adult males and females), liver, kidneys (paired weight), heart. Further tissues/organs from the same selected animals were preserved. All animals found dead were subjected to a gross necropsy and the organs preserved for a hist
opathological examination. Thyroid/parathyroid glands from non-selected adult animals were preserved for potential histopathological examination.

Histopathology:
A full histopathology was carried out on the preserved organs and tissues of the selected animals of the control and high dose groups which were sacrificed at the end of the treatment period. Evaluation of thyroid/parathyroid glands from the remaining non-selected adult animals was not deemed necessary as no test item related histopathological findings were observed in thyroid/parathyroid gland of selected animals and there was also no test item related effect observed on T4 hormone level in males sacrificed on PND 13.
A full histopathology was carried out on the preserved organs and tissues of all animals which died during the study. Any gross lesion macroscopically identified was examined microscopically in all animals.

Statistics:

A statistical assessment of the results of body weight, food consumption and litter data was performed for each gender by comparing values of dosed with control animals using a one-way ANOVA and a post-hoc Dunnett Test. Results of absolute and relative organ weights, parameters of haematology, blood coagulation and
clinical biochemistry were statistically analysed by comparing values of dosed with control animals using either a parametric one-way ANOVA and a post-hoc Dunnett Test or a non-parametric Kruskal-Wallis Test and a post-hoc Dunn’s Test, based on the results of homogeneity and normality tests. These statistics were performed with GraphPad Prism V.6.01 software or Ascentos 1.1.3 software (p<0.05 was considered as statistically significant).

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

In terminally sacrificed males, predominant clinical signs observed during the treatment period (premating day 1 to mating/post mating day 14) were nasal discharge in one animal of control group (PMD 12-13), severely increased salivation in one animal of HD (PMD 11) and moving the bedding in 2-3 animals of HD group during very few days of premating, mating and postmating period.
In terminally sacrificed females, major clinical signs observed during the treatment period (Premating day 1 to PND 12) were moving the bedding on one day during gestation period in three females of HD group, moderately increased salivation in two females of HD group on one day during gestation period and abnormal breathing on one day in one female of the HD group on gestation day 8.

The clinical signs salivation and moving the bedding on few days were observed immediately after the dose administration and therefore were considered to be a sign of a local reaction to the test item rather than a systemic adverse effect and has no toxicological relevance.

During the weekly detailed clinical observation, no relevant differences between the groups were found.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

During the treatment period of this study, one mortality was observed on day of terminal sacrifice (PND 13):
- Female no. 49 (Control) was found dead on post natal day (PND) 13.
No specific clinical signs were observed in this animal before death or during entire study period.
Histopathologically, the cause of death was not evident.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In both males and females, there was no test item treatment related effect observed on body weight and body weight gain in the dose groups during the study period. There were no statistically significant differences observed for body weight and body weight gain between the dose groups and the control group except statistically significantly lower body weight gain in HD males during premating day 1-7 when compared with the controls. As this difference was marginal and within the biological variation, it was not considered to be adverse effect due to treatment with test item.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In correlation to the body weight and body weight gain, the food consumption in both males and females tended to increase with the progress of the study in the control, the LD, the MD and the HD group. No test item related or statistically significant effect on food consumption was observed in males and females during the whole study period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males sacrificed at the end of treatment period, no test item related adverse effects were observed for haematological parameters in treatment groups when compared to the control group.

In females sacrificed at the end of treatment period, no test item related effect or statistically significant effect observed on any of the haematology except statistically lower reticulocytes in LD group when compared with the controls. Due to lack of dose dependency, this effect on reticulocytes in LD group was considered as incidental.

No test item related effect was observed on coagulation parameters in males and females when compared with the respective controls.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females sacrificed at the end of treatment period, no test item related or statistically significant effect on any clinical biochemistry parameter in treatment groups was observed when compared with the control except statistically significantly lower TBA group mean value in male HD group. This effect on TBA in male HD group was considered as incidental and toxicologically irrelevant as all individual values were within the historical data range (6.38 - 93.82 µmol/L). All other group mean and most of the individual values for clinical chemistry parameters in male and females were comparable with control group.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The urinalysis performed in selected male and female animals sacrificed at the end of treatment period revealed no test item treatment related effect and all urinary parameters were in the normal range of variation. High protein levels were found in the urine of few male and females of all groups including control group. Therefore, this effect on urine parameters was not considered to be test item related.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males, no relevant effects were observed in any of the parameters of the functional observation battery before and at the end of the treatment period when compared with the controls. There were no biologically relevant differences observed in body temperature between the groups.

In females, statistically significantly higher not supported rearing count in HD group before initiation of treatment and statistically significantly higher urination count in LD group during last week of treatment was observed. As this type of difference was either marginal, before imitation of treatment or without dose dependency/consistency, it has no toxicological relevance and could be considered as biological variation.

There were no biologically relevant differences in body temperature between the groups.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, there were no statistically significant differences in the absolute and relative organ weights of the dose groups when compared to the control group except statistically significantly higher absolute liver weights were observed in HD group females when compared with the controls. In the light of fact that no test item related histopathological findings and effects on liver enzymes were observed, this marginal increase in female liver weight was not considered to be adverse.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Few specific macroscopic changes were recorded for the male and female animals, which based on microscopic examination were not considered to be of test item treatment relevance.
The predominant macroscopic changes observed were brown soft mass observed at the head of the epididymis (male no. 15 of LD group), small right thyroid gland (male no. 12 of LD group), liver- diaphragmal herniation (female no. 79 of HD group), cyst on ovary (Female no. 46 of control group), red mass and red fluid filled uterus and red vaginal discharge (female no. 59 of LD group) and enlarged mandibular lymph nodes (female no. 53 of LD group). Few organs like gastrointestinal tract, lung, kidney and spleen from female no. 49 of control group were autolytic.
Macroscopic findings correlating with histopathological observations were observed in following animals:
- animal no. 15 the brown, soft mass observed at the head of the epididymis histologically correlated with severe diffuse necrosis of the epididymis head
- animal no. 46 the cyst observed at necropsy histologically correlated with an ovarian follicular cyst
The above mentioned findings were deemed incidental. All other observed gross lesions reflected the animal physiology or, in absence of corresponding histopathological findings, were considered of no toxicological relevance.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Ingrain Blue 2.2 did not produce morphological or histological evidence of toxicities in the organs and tissues examined in this study.

In conclusion, a histopathological NOEL (No Observed Effect Level) could be established at 25 mg//kg bw/day.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No test item related effect of toxicological relevance or statistical significance was observed in male thyroxine hormone (T4) in the treatment groups when compared to the controls.

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: general toxicity

Critical effects observed:

no

Conclusions:

The NOAEL for the test item in this study is considered to be at least 25 mg/kg bw/day for general toxicity.

Executive summary:

The aim of this study performed according to OECD TG 422 and in compliance to GLP, was to assess the possible adverse effects of Ingrain Blue 2.2 on male and female Wistar rats after repeated dose administration with dose levels of 5, 10, and 25 mg/kg body weight/day.

The test item was administered daily in graduated doses to 4 groups of test animals (each comprising 10 male and 10 female rats), one dose level per group for a treatment of 63 days i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed. The doses applied were: control, low dose (5 mg/kg bw/day), medium dose (10 mg/kg bw/day) and high dose (25 mg/kg bw/day).

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, all animals were sacrificed and observed macroscopically.

In conclusion:

- There was one mortality observed in the study on day of terminal sacrifice (PND 13). Histopathologically cause of the death was not evident. 

-  No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, thyroid hormone analysis in parental males, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

The NOAEL of the test item in this study for general toxicity is considered to be at least 25 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

supporting study

Study period:

2017-01-10 to 2017-01-25

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)

Version / remarks:

adopted 03 October, 2008

Principles of method if other than guideline:

There are no regulatory guidelines for this type of dose range-finding study, but the design of this study is based on the principles specified in following test guidelines on the toxicity testing of chemicals. OECD Guideline for the Testing of Chemicals (Section 4 Health Effects), Test No. 407, Repeated
Dose 28-Day Oral Toxicity Study in Rodents (October 03, 2008)

GLP compliance:

no

Limit test:

no

Species:

rat

Strain:

Wistar

Remarks:

Crl: WI(Han) (Full Barrier)

Details on species / strain selection:

Crl: WI(Han) (Full Barrier)

Sex:

male/female

Details on test animals or test system and environmental conditions:

Animals:
- female animals were non-pregnant and nulliparous
- Age at the start of the treatment period: 7-8 weeks old
- Body weight at the allocation of the animals to the experimental groups:
-- males: 180 – 210 g (mean: 194.86 g, ± 20% = 155.89 – 233.83 g)
-- females: 155 – 187 g (mean: 168.86 g, ± 20% = 135.09 – 202.63 g)

Housing and Feeding Conditions:
- Full barrier in an air-conditioned room
- Temperature: 22 ± 3 °C
- Relative humidity: 55 ± 10%
- Artificial light, sequence being 12 hours light, 12 hours dark
- Air change: 10 x / hour
- Free access to Altromin 1324 maintenance diet for rats and mice
- Free access to tap water, sulphur acidified to a pH of approximately 2.8 (drinking water, municipal residue control, microbiological controls at regular intervals)
- The animals were group housed (3 animals/sex/cage) in IVC cages (type III, polysulphone cages) on Altromin saw fibre bedding
- Adequate acclimatization period (at least five days)

Route of administration:

oral: gavage

Details on route of administration:

The test item formulation and vehicle were administered at a single dose to the animals by oral gavage. The application volume for all groups was 5 mL/kg body weight. For each animal the individual dosing volume was calculated on the basis of the body weight most recently measured.

Vehicle:

water

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Dose formulation analysis have not been conducted for this dose range-finding study.

Duration of treatment / exposure:

2 weeks

Frequency of treatment:

7 days per week for a period of 14 days.

Dose / conc.:

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

Remarks:

control C)

Dose / conc.:

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

Remarks:

low dose (LD)

Dose / conc.:

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

Remarks:

medium dose (MD)

Dose / conc.:

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

Remarks:

high dose (HD)

No. of animals per sex per dose:

3

Control animals:

yes, concurrent vehicle

Details on study design:

Justification for Dose Level:
The dose level was selected based on a previous acute oral toxicity study in rats where LD50 was 50-300 mg/kg. The dose levels selected for this study will be 5, 10 and 25 mg/kg for low, mid and high dose groups, respectively.
Control group will be administered with vehicle.

Number and Sex of Animals:
- 24 animals (12 males and 12 females) were used for the study (3 male and 3 female animals per group). The control group animals (3 males and 3 females) were used together in another study

Preparation of the Animals:
- Prior to the start of the treatment period a detailed clinical observation outside the home cage was made
- Before the first administration all animals used for the study were weighed and assigned to the experimental groups with achieving a most homogenous variation in body weight throughout the groups of males and females, respectively
- Each animal was marked for individual identification with an ear tattoo and/or tail number.

Evaluation of Results:
The findings of this study were evaluated in terms of the observed effects. Parameters like body weight gain were calculated for each animal and food consumption for each cage as the difference in weight measured from one week to the next. The relative organ weights were calculated in relation to the brain weight and in relation to the body weight (measured at necropsy) and presented as percentage.
All results were reported in tabular form (summarised in mean or summary tables and/or listed in individual data tables).

Deviations:
Macroscopic findings and the weighed organs were not preserved. These deviations did not influence the quality or integrity of the present study .

Positive control:

not applicable

Observations and examinations performed and frequency:

Body Weight and Food Consumption:
The body weight was recorded once before assignment to the experimental groups and on study days 1, 8 and 14 during the treatment period as well as on the day of necropsy.
Food consumption was measured on study days 1, 8 and 14 for each cage.

Clinical Observations:
All Animals were observed for clinical signs during the entire treatment period of 14 days.
General clinical observations were made at least once a day, approximately at the same time each day and considering the peak period of anticipated effects after dosing. The health condition of the animals was recorded. Twice daily all animals were observed for morbidity and mortality except on weekends and public holidays when observations were made once daily.
Detailed cage side observations considering spontaneous activity, lethargy, recumbent position, convulsions, tremors, apnoea, asphyxia, vocalisation, diarrhoea, changes in skin and fur, eyes and mucous membranes (salivation, discharge), piloerection and pupil size were made outside the home cage in a standard arena once before the first administration and at least once a week thereafter.

Sacrifice and pathology:

Haematology:
Haematological parameters were examined at the end of the treatment prior to or as part of the sacrifice of the animals. The following haematological parameters were examined: haematocrit value (HCT), haemoglobin content (Hb), red blood cell count (RBC),platelet count (PLT),
white blood cells (WBC).

Clinical Biochemistry:
Parameters of clinical biochemistry were examined at the end of the treatment prior to or as part of the sacrifice of the animals.
After overnight fasting, blood from the abdominal aorta of the animals was collected in serum separator tubes.
The following parameters of clinical biochemistry were examined: alanine aminotransferase (ALAT), aspartate-aminotransferase (ASAT), alkaline phosphatase (AP), creatinine (Crea), total protein (TP), albumin (Alb), urea, total cholesterol (Chol), sodium (Na), potassium (K).

Pathology:
On study day 15, all animals of the study were sacrificed using anesthesia (ketamine, xylazin) and were subjected to a detailed gross necropsy which included careful examination of the external surface of the body, all orifices and the cranial, thoracic and abdominal cavities and their contents.

Organ Weight:
The wet weight of the following organs was taken from all sacrificed animals as soon as possible. Paired organs were weighed together.
Organ weights at necropsy: liver, kidneys, adrenals, testes, epididymides, prostate, seminal vesicles and coagulating glands, ovaries, uterus with cervix thymus, thyroid/ parathyroid glands, spleen, brain, heart.

Other examinations:

not specified

Statistics:

not specified

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

No test item related clinical signs were observed in any male and female animals during the entire study period. However, Moving the bedding was observed on one day (Day 10) in all animals of HD group. This clinical finding could be considered of toxicological relevance due to test item administration.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred in the control or any of the dose groups during the treatment period of this study and all animals were sacrificed on day 15.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, mean body weight and body weight gain increased with the progress of the study in all treatment groups when compared with the controls and no test item related effect was observed on body weight and body weight change in treatment groups during the entire study period. However, statistically significantly higher body weight change was observed during day 1-8 in MD group when compared with the controls. Due to lack of dose dependency and consistency, this effect on body weight change in MD group male was not considered to be test item related and toxicologically relevant.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

In males and females, no test item related effect on food consumption was observed during the whole study period. In correlation to the body weight and body weight gain, the food consumption was comparable in treatment groups when compared with the control group.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females, no test item related effect on haematological parameters was observed in treated groups when compared with the controls. Although variation in WBC values in male and female LD groups were observed, due to lack of dose dependency, it was not considered to be test item related. All individual and group mean values were within the biological variation and historical control data range.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

In males and females, no test item related effect on clinical biochemistry was observed in treated groups and all individual and group mean values were comparable with controls and within the historical control data range.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In males, statistically significantly higher absolute and relative (to body weight) thymus weight in MD group and liver weights in LD group were observed in males.Absolute kidney weights were also statistically significantly higher in LD group when compared to the control group. Due to lack of dose dependency and consistency, this effect on organ weight in LD group male was not considered to be test item related and toxicologically relevant. However, consistent decrease in prostate and adrenal weight in males and increase in thyroid and parathyroid weight in both male and females (in females no clear dose dependency), this effect could be attributed to treatment with test item although statistical significance was not achieved.
There was increase or decrease in few absolute and relative organs weights (brain, testes, spleen, heart and epididymides in males and brain, spleen, kidneys, adrenals, heart, liver, ovaries, uterus with cervix in females) observed when compared with the controls. However, due to lack of dose dependency, this effect was not considered to be test item related.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

At necropsy of males and females macroscopic examination of the animals revealed no macroscopic findings except in females fluid filled ureters (1/3 in C and MD group) and fluid filled uterus (1/3 in HD) were observed. These gross pathological finding in females were spontaneous in nature and as such not due to a systemic effect due to the test item administration.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Remarks on result:

other: Not determinable because of methodological limitations. This RSS relates to the Dose Range Finder study. The dose descriptor is determined in the main test.

Conclusions:

Under the conditions of the present study, the repeated oral application of the test item to male and female Wistar rats at doses of 5, 10 and 25 mg/kg body weight for 14 days was associated with no major signs of toxicity or mortality.

Executive summary:

The aim of this study was to assess the possible health hazards which could arise from repeated exposure of the test item via oral administration to rats over a period of 14 days. There are no regulatory guidelines for this type of dose range-finding study, but the study design is based on the principles specified in OECD TG 407. The study was not-GLP, since there was not audit by the quality assurance unit .

The test item was administered daily in graduated doses to 3 groups of test animals, one dose level per group for a treatment period of 14 days. Animals of an additional control group were handled identically as the dose groups but received the vehicle only (sterile water). The 4 groups comprised 3 male and 3 femaleWistar rats each at doses of 0 (control), 5 (low dose), 10 (mid dose) and 25 mg/kg bw/day (high dose).

    

The test item formulation was prepared freshly on each day of administration. The test item was dissolved in sterile water and administered daily during a 14-day treatment period to male and female animals. Dose volumes were adjusted individually based on weekly body weight measurement. During the period of administration, the animals were observed precisely each day for signs of toxicity. Body weight and food consumption were measured weekly. Haematological and clinical biochemistry evaluations were performed on blood samples collected at terminal sacrifice. At the conclusion of the treatment period, all animals were sacrificed and subjected to necropsy. The wet weight of a subset of tissues was taken.

No mortality occurred in the control or any of the dose groups during the treatment period of this study and all animals were sacrificed on day 15. Except moving the bedding observed on one day (Day 10) in all animals of the high dose group, no test item related clinical signs were observed in any male and female animals during the entire study period.

In males and females, mean body weight and body weight gain increased with the progress of the study in all treatment groups when compared with the controls and no test item related effect was observed on body weight and body weight change in treatment groups during the entire study period.

In males and females, no test item related effect on food consumption was observed during the whole study period. In correlation to the body weight and body weight gain, the food consumption was comparable in treatment groups when compared with the control group.

In males and females, no test item related effect on haematological and clinical biochemistry parameters was observed in treated groups when compared with the controls.

At necropsy of males and females, macroscopic examination of the animals revealed no macroscopic findings except in females fluid filled ureters (1/3 in the control and medium dose group) and fluid filled uterus (1/3 in the high dose group) were observed. These findings were considered to be spontaneous in nature. In males and females, no test item related organ weight effects were observed in all treatment groups when compared with the controls.

In conclusion, under the conditions of the present study, the repeated oral application of the test item to male and female Wistar rats at doses of 5, 10 and 25 mg/kg body weight for 14 days was associated with no major signs of toxicity or mortality.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

25 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

In accordance to OECD guideline 422 and in compliance with GLP regulations.

System:

other: general systemic toxicity

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

28-day repeated dose toxicity study

The aim of this study was to assess the possible adverse effects of the test item on male and female Wistar rats after repeated dose administration with dose levels of 5, 10, and 25 mg/kg bw/day.

The test item was administered daily in graduated doses to 4 groups of test animals (each comprising 10 male and 10 female rats), one dose level per group for a treatment of 63 days i.e. during 14 days of pre-mating and maximum 14 days of mating in both males and females, during the gestation period and up to post-natal day 12 in females. Males were dosed after the mating period until the minimum total dosing period of 28 days were completed.

During the period of administration, the animals were observed each day for signs of toxicity. At the conclusion of the test, all animals were sacrificed and observed macroscopically.

In conclusion:

- There was one mortality observed in the study on day of terminal sacrifice (PND 13). Histopathologically cause of the death was not evident. 

-  No adverse effects of test item were found on male and female clinical observations, functional observations, body weight development, food consumption, thyroid hormone analysis in parental males, haematology and coagulation, clinical biochemistry, urinalysis, gross macroscopic findings at necropsy, organ weights and histopathology in all treatment groups. 

The NOAEL of the test item in this study for general toxicity is considered to be at least 25 mg/kg bw/day.

Justification for classification or non-classification

A 28 -day repeated dose toxicity study was conducted. The NOAEL of the test item in this study for general toxicity is considered to be at least 25 mg/kg bw/day. The substance did not induce any adverse effects after 28 days of repeated dosing.

According to the criteria set out in the CLP Regulation No 1272/2008 in section 3.9, the substance does not meet the criteria for classification according to CLP regulation. The substance is therefore found to be not toxic after repeated dosing and should hence not be classified.