Registration Dossier

Diss Factsheets

Administrative data

Description of key information

NOAEL (28d) (male and female): 200 mg/kg bw

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:
short-term repeated dose toxicity: oral
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Study period:
From May 21st to June 25th, 1986
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
test procedure in accordance with generally accepted scientific standards and described in sufficient detail
Justification for type of information:
Justification for Read Across is detailed in the IUCLID section 13.
Reason / purpose for cross-reference:
read-across source
Qualifier:
according to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
Version / remarks:
May 12, 1981
Qualifier:
according to guideline
Guideline:
EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))
GLP compliance:
yes
Limit test:
no
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: KFM KIeintierfarm Madoerin AG, CH 4414 Fuellinsdorf/Switzerland.
- Age at study initiation: 9 - 7 weeks
- Weight at study initiation: males 156 - 185 g, females 165 - 187 g.
- Housing: individually in Makrolon type-3 cages with standard softwood bedding.
- Diet: peIIeted standand Kliba no. 343 rat Batch 43/86 and 44/86 maintenance diet ('Kliba', Klingentalmuehle 46, 4303 Kaisenaugst, Switzerland), ab Iibitum.
- Water: community tap water from Itingen, ad libitum.
- Acclimation period: 7 days under laboratory conditions, after veterinary examination.

DETAILS OF FOOD AND WATER QUALITY: results of analysis for diet abd water contaminants were included in the study report.

ENVIRONMENTAL CONDITIONS
- Temperature: 22 ± 3 °C
- Humidit: 40 - 70 %
- Air changes: 10 - 15 air changes per hour.
- Photoperiod: 12 hours artificial fluorescent light / 12 hours dark.
- Other: music / light period.
Route of administration:
oral: gavage
Vehicle:
CMC (carboxymethyl cellulose)
Remarks:
4 % solution of carboxymethyl cellulose in distilled water
Details on oral exposure:
- Dose volume: 10 mI/kg body weight

DOSE PREPARATION
For doses 50 and 200 mg/kg bw/day the test article was weighed into a gIass beaker on a tared Mettler PK 300 balance and the vehicle, carboxymethyl ceIIuIose 4 % in distilled water was added. The mixture was prepared daily prior to administration using a homogenizer and kept stable during application with a magnetic stirrer.
For group treated with 1000 mg/kg bw/day the test article was prepared as described above. In addition 5 mI of a 1N hydrochloric acid was added and after dispersion of the dilution the pH-value was adjusted to neutral with 5 ml of a 1N sodium hydroxide solution.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The stability, homogeneity and concentration of the test article/vehicle mixtures were determined once during the study.
The concentrations of the test item found in the test medium were in the range of 86.7 - 95.2 % of the nominal concentrations.
The homogeneity varied in the range of -8.2 to +4.9 % of the mean concentration.
The test article/vehicle mixture is stable for at least 90 minutes.
Duration of treatment / exposure:
28 days
Frequency of treatment:
Once daily, 7 days per week
Dose / conc.:
50 mg/kg bw/day (nominal)
Dose / conc.:
200 mg/kg bw/day (nominal)
Dose / conc.:
1 000 mg/kg bw/day (nominal)
No. of animals per sex per dose:
five males and five females
Control animals:
yes, concurrent vehicle
Observations and examinations performed and frequency:
VIABILITY / MORTALITY
Each animal was observed twice daily for viability. At the last treatment day animals were observed only once.

SIGNS AND SYMPTOMS
Each animal was observed twice daily for toxicity or clinical symptoms. A description of any abnormality was recorded.

FOOD CONSUMPTION
The food consumption was recorded once during the acclimation period and weekly thereafter using an on-Iine electronic recording system consisting of a Metter PK 4800 balance.

BODY WEIGHTS
The body weight of each animal was recorded weekIy during the acclimation and treatment periods using an on-Iine electronic recording system consisting of a Mettler PK 4800 balance.

OPHTHALMOSCOPIC EXAMINATIONS
The animals were examined for ocular abnormalities at 4 weeks, using a Heine-BifocaI ophthalmoscope (miraflex type). A description of any abnormality was recorded.

HEMATOLOGY
The following anticoagulants were EDTA-K2 (hematology): Sodium Citrate, 3.8 % (coagulation)
Parameters: erythrocyte count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, pIatelet count, reticulocyte count, nucleated erythrocytes - normoblasts, totaI Ieukocyte count, differential Ieukocyte count, red ceII morphology.
Coagulation: thromboplastin time, partial thromboplastin time, thrombin time.

CLINICAL CHEMISTRY
Blood samples for hematology and clinical biochemistry were collected from all animals under light ether anesthesia. The animals were fasted for 18 hours before blood sampling but water was provided. BIood samples were collected from each animal between the hours of 7.00 and 9.00 a.m. to reduce biologic variation caused by circadian rhythm. BIood samples were drawn from the retro-orbital plexus.
Blood sampling: after 4 hours.
The assays of blood parameters were performed under quality control conditions to assure reliable test results.
Parameters: glucose, urea, creatinine, bilirubin total, choIesterol total, aspartate aminotransferase, alanine aminotransferase, lactate dehydrogenase, alkaline phosphatase, calcium, phosphorus, sodium, potassium, chloride, albumin, protein total.

URINALYSIS
Urine was collected over an 18-hour period into a specimen vial using a metabolism cage.
Urine sampling: after 4 hours.
The assays of urine parameters were performed under quality control conditions to assure reliable test results.
Parameters: voIume, specific gravity, pH, protein, glucose, ketone, bilirubin, blood, nitrite, urobiIinogen, urine sediment.
Sacrifice and pathology:
ORGAN WEIGHTS
The following organ weights were recorded on the scheduled date of necropsy: adnenal glands, heart, kidneys, liver and testes.

NECROPSY AND HISTOPATHOLOGY
AII anirnals were necropsied and descriptions of all macroscopic abnormalities were recorded. Necropsies were performed by experienced prosectors supervised by a board certified pathologist. AII animals surviving to the end of the observation period were anesthetized by intraperitioneal injection of sodium pentobarbitone and killed by exsanguination.
Samples of the following tissues and orgfan were collected from aIl animals at necropsy and fixed in 10 % phosphate buffered neutral formalin: adrenal glands, heart, kidneys, liver, gross lesions.
AII organ and tissue samples were processed, embedded and cut at a thickness of 2-4 micrometers and stained with hematoxylin and eosin. Special stains were used at the discretion of the pathologist.
SIides of all tissues collected at terminal sacrifice from the animals of the contnol and high-dose groups were examined by a pathologist. Treatment-related morphologic changes in the organs of any high-dose animal required histological evaluation of the same organs in Iower dose groups until a no-effect IeveI was determined.
All abnormalities were described and included in the report.
Statistics:
The following statistlcal methods were used to analyze the body weights, food consumption, organ weights and clinical Iaboratory data:
- univariate one-way analysis ot variance was used to assess the significance of intergroup differences.
- if the variables could be assumed to follow a normal distribution, the Dunnett-test (many to one t-test) based on a pooled variance estimate was applied for the comparison between treated groups and control group.
- the SteeI-test (many-one rank test) was applied when the data could not be assumed to foIIow a normal distribution.
For the overall spontaneous mortality data, the Fisher's exact test for 2×2 tables was applied.
Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.
Individual values, means, standard deviations and statistics were rounded off before printing.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
With the exception of a slight diarrhea noted in the rats of the group dosed at 1000 mg/kg bw during the test period, no symptoms which could be related to the test article treatment was observed.
Mortality:
no mortality observed
Description (incidence):
No death occurred
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
There were no significant differences between the animals of the test article-treated groups and control group.
The initial significant differences of the female rats of group treated with 1000 mg/kg bw/day were random and considered unrelated to test article treatment.
Food consumption and compound intake (if feeding study):
no effects observed
Description (incidence and severity):
No differences in food consumption or body weight gain were observed between the animals of the test article treated and control groups.
Ophthalmological findings:
no effects observed
Description (incidence and severity):
No ophthalmoscopic differences were observed between the animals of the test article-treated and control groups at termination of treatment.
Haematological findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of hematology data indicated no changes or toxicological significance at termination of treatment.
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
The assessment of clinical biochemistry data indicated no changes or toxicological significance at termination of treatment.
However, a slight increase in the total cholesterol Ievel was noted for male and female rats of group treated with 1000 mg/kg bw/day. This finding is considered to be of an adaptive nature related to the treatment.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
The assessment of urinalysis data indicated no changes or toxicological significance at termination of treatment.
An abnormally strong yellow color contributed by the test-article was noted in the urine for rats of groups treated with 200 and 1000 mg/kg bw/day.
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increases were noted in the Iiver weight-, kidney weight- and adrenal weight- to body weight ratios of the males treated at 1000 mg/kg bw/day.
The absolute kidney weights of the groups treated at 50 and 200 mg/kg bw/day were slightly increased.
Increases in the Iiver weight- to body weight- ratios of the females treated at 50, 200 and 1000 mg/kg bw/day were noted.
Other increased relative organ weights included the kidney weight to body weight ratios of females treated at 200 and 1000 mg/kg bw/day, and the adrenal weight- to body weight ratio of the females at 1000 mg/kg bw/day.
The slight increases in organ weights and/or ratios noted for the animals of groups dosed at 50 and 200 mg/kg bw/day were considered to be rather be rather incidental and in control ranges than treatment related.
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
All pathology findings recorded were of a spontaneous nature common to rats of this age and strain; there was no evidence of abnormal histopathological findings resulting from treatment with test item.
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
No treatment-related macroscopic or microscopic organ changes were noted.
Dose descriptor:
NOAEL
Effect level:
200 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
organ weights and organ / body weight ratios
Critical effects observed:
no
Conclusions:
NOAEL (28d) (male and female): 200 mg/kg bw
Executive summary:

In the subacute 28-day study, test item was administered daily by gavage to SPF-bred Wistar rats. The study included four groups, each containing five male and five female rats. The following nominal test article concentrations were administered: 0, 50, 200 and 1000 mg/kg bw/day.

No death occurred. In all animals treated with 1000 mg/kg bw/day slight diarrhea was observed during the treatment period. No differences in food consumption or body weight gain were observed between the animals of the test article treated and control groups.

No ophthalmoscopic differences were observed between the animals of the test article-treated and control groups at termination of treatment.

The assessment of hematology, clinical biochemistry and urinalysis data indicated no changes or toxicological significance at termination of treatment.

All pathology findings recorded were of a spontaneous nature common to rats of this age and strain; there was no evidence of abnormal histopathological findings resulting from treatment with test item.

Organ enlargement of kidneys, liver and adrenals was noted in the absence of any histopathological changes. The slight increases in organ weights or ratios noted for the animals treated at 50 and 200 mg/kg bw/day were considered to be rather incidental and in control ranges than treatment related.

Conclusion

Based upon the results obtained in the study, NOAEL of test item was determined to be 200 mg/kg bw/day for male and females rats when administered orally by gavage.

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
200 mg/kg bw/day
Study duration:
subacute
Species:
rat

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

Available information on the structural analogue Similar Substance 02 have been used to assess repeated dose toxicity; the read across approach can be considered as appropriate and suitable to assess the property under investigation (details about the approach are reported into the IUCLID section 13).

In the subacute 28-day study, Similar Substance 02 was administered daily by gavage to Wistar rats. The study was conducted according to the OECD guideline 407. The following nominal test article concentrations were administered: 0, 50, 200 and 1000 mg/kg bw/day. The stability, homogeneity and concentration of the test article/vehicle mixtures were determined once during the study: the concentrations of the test item found in the test medium were in the range of 86.7 - 95.2 % of the nominal concentrations, the homogeneity varied in the range of -8.2 to +4.9 % of the mean concentration and the test article/vehicle mixture resulted to be stable for at least 90 minutes.

No death occurred during the experiment. In all animals treated with 1000 mg/kg bw/day slight diarrhea was observed during the treatment period. No differences in food consumption or body weight gain were observed between the animals of the test article treated and control groups.

No ophthalmoscopic differences were observed between the animals of the test article-treated and control groups at termination of treatment.

The assessment of hematology, clinical biochemistry and urinalysis data indicated no changes or toxicological significance at termination of treatment.

All pathology findings recorded were of a spontaneous nature common to rats of this age and strain; there was no evidence of abnormal histopathological findings resulting from treatment with test item.

Organ enlargement of kidneys, liver and adrenals was noted in the absence of any histopathological changes. The slight increases in organ weights noted for the animals treated at 50 and 200 mg/kg bw/day were considered to be rather incidental and in control ranges than treatment related.

Based upon the results obtained in the study, NOAEL of test item was determined to be 200 mg/kg bw/day for male and females rats when administered orally by gavage.

Data on Basic Orange 064 derived from a screening test of reproductive toxicity (OECD 421) was reported for completeness sake. Dosing was done for 48 days in male rats and for 51 -55 days in female rats at doses of 0, 100, 300 and 1000 mg/kg bw/day. A NOAEL for systemic toxicity was identified at 100 mg/kg bw/day; effects in terms of soft stool, piloerection, decreased activity or decreased body tone and changes in the body weight were observed in the male and female animals at 300 mg/kg bw/day; at the highest dose of 1000 mg/kg bw/day, further effects in terms of macroscopic and microscopic local changes in the stomach (thickened and erythematous mucous membrane, focal squamous cell hyperplasia in the mucous membrane of non-glandular stomach) were seen in male rats.

Overall, despite inherent differences in repeated dose and reproductive toxicity studies, e.g. in terms of duration and type of observations, experimental findings for systemic toxicity and effects levels were found to be comparable and consistent.

Justification for classification or non-classification

According to the CLP Regulation (EC 1272/2008), 3.9 Specific target organ toxicity - repeated exposure section, substances are classified as specific target organ toxicants following repeated exposure by the use of expert judgment, on the basis of the weight of all evidence available, including the use of recommended guidance values, which take into account the duration of exposure and the dose/concentration, which produced the effect(s), and are placed in one of two categories, depending upon the nature and severity of the effect(s) observed.

In order to decide whether a substance shall be classified in categroy 1 or 2 or not classified, dose/concentration ‘guidance values’ are provided for consideration of the dose/concentration which has been shown to produce significant health effects. The guidance values refer to effects seen in a standard 90-day toxicity study conducted in rats. However, they can be used as a basis to extrapolate equivalent guidance values for toxicity studies of greater or lesser duration (the assessment shall be done on a case-by- case basis). For example, for a 28-day study, the guidance values are increased by a factor of three, thus the limit for sub-acute studies should be 300 mg/kg bw/day.

 

In the specific case, a NOAEL was established at 200 mg/kg bw/day, on the basis of the results from the subacute study on rat. Such findings were consistent with systemic effects seen in a reproductive toxicity study.

Overall, considering the effects and the severity of the impact of the findings recorded at the higher doses, it can be concluded that the substance does not meet the criteria to be classified for repeated dose toxicity, according to the CLP Regulation (EC 1272/2008).