H112323

Endpoint summary

Description of key information

Based on the 28 d subacute oral gavage study in rats, the NOAEL of the test substance was determined to be 1000 mg/kg bw/day. 

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

May-July, 1991

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: Study was conducted equivalent or similar to EU Method B.7. and OECD Guideline 407 in compliance with GLP

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

Qualifier:

equivalent or similar to guideline

Guideline:

EU Method B.7 (Repeated Dose (28 Days) Toxicity (Oral))

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River UK Limited, Manston Road, Margate, Kent, UK
- Age at study initiation: 6-7 wk
- Weight at study initiation: 147-167 g Males; 125-151 g females
- Housing: Housed individually in suspended cages (26.5cm x 50cm x 20.7cm)
- Diet (e.g. ad libitum): CT1 Diet, Special Diet Services Ltd, ad libitum
- Water (e.g. ad libitum): water, ad libitum
- Acclimation period: at least 6 d

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21±2 °C
- Humidity (%): 55±15 %
- Air changes (per hr): 25-30
- Photoperiod (hrs dark / hrs light): 12 h dark/12 h

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
The preparations were made as suspensions in deionised water. For each dose level, an appropriate amount of water was added to a weighed amount of the undiluted test sample. Each preparation was thoroughly mixed and subdivided into aliquots. Further preparations were made at appropriate intervals throughout the study.
The doses used in the study were adjusted-for water content of the test substance(i.e., the daily dose levels administered were approximately equal
to 50, 200 and 1000 mg/kg body weight), but were not otherwise corrected for purity.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

A sample of each preparation was analysed prior to dosing to verify the achieved concentration of test substance in water. Trial dilutions of 5.6 and 11.7 mg/mL of the suspension were made prior to the study for the determination of chemical stability (over a 21-d period) and homogeneity of the test sample in deionised water.

Duration of treatment / exposure:

28 d

Frequency of treatment:

Once a day for 28 d

Remarks:

Doses / Concentrations:
50, 200 and 1000 mg/kg bw/d
Basis:
other: Nominal in vehicle

No. of animals per sex per dose:

5/sex/dose in main groups
5/sex/dose in recovery groups

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results obtained from a pilot oral study (CIL study number KR1130). In that study, groups of two male rats were given 14 consecutive daily doses of 250 or 1000 mg/kg/day. No signs of toxicity were seen in any of the animals and there were no macroscopic abnormalities at post mortem examination.
- Rationale for selecting satellite groups: To check the reversibility of the effects, if any
- Post-exposure recovery period in satellite groups: yes, 14 d

Positive control:

Not used in the study

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily in all groups

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily throughout the study, immediately before dosing, when appropriate. In addition, all the animals were checked between 1 and 3 h after dosing

BODY WEIGHT: Yes
- Time schedule for examinations: weighed daily on days 1-29. Recovery group animals were additionally weighed on Day 36 and prior to termination on Day 43.

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

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period (Day 29 or 43)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the termination of the study and after the recovery period (Day 29 or 43)

URINALYSIS: Yes
- Time schedule for collection of urine: Few days before termination (Day 22-23) of the study as well as before (Day 37-38) the end of the recovery period
- Animals fasted: Yes, 16-18 h

OTHER: Bone marrow samples were taken and stored, but were not examined

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals were necropsied and each animal was subjected to a macroscopic examination of major abdominal and thoracic viscera.

HISTOPATHOLOGY: Yes, Following tissues/organs were preserved in a suitable fixative and processed for histopathological investigations: adrenal gland, bone marrow (femur), brain, heart, colon, jejunum, kidneys, liver, lungs, lymph nodes (mandibular and iliac), ovaries, uterus, pituitary gland, thyroid and parathyroid glands, prostate gland, spleen, stomach, testis, epididymides, thymus, trachea, urinary bladder, Harderian gland and abnormal tissue.

Other examinations:

No data

Statistics:

- ANCOVA for body weights
- ANOVA for food consumption, haematology, blood and urine clinical chemistry
- Organ weights were considered by ANOVA and ANCOVA on final bodyweight, separately for males and females
- All analyses were carried out using the GLM procedure in SAS
- Differences from control were tested statistically by comparing each treatment group LSM with the control group least square mean using a two-sided Student's t-test, based on the error mean square in the analysis

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

effects observed, treatment-related

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

not examined

Details on results:

CLINICAL SIGNS AND MORTALITY: No treatment related toxicity was observed in any group. All the animals dosed with the test substance had black faeces throughout most of the dosing phase of the study; this was not evident from Day 30 onwards in the animals retained until Day 43.

BODY WEIGHT AND WEIGHT GAIN: Statistically significantly lower body weights in males at 1000 mg/kg bw than the corresponding control group

FOOD CONSUMPTION: Males dosed with 1000 mg/kg killed on Day 29 was statistically significantly lower than their corresponding control group

HAEMATOLOGY: Slight decrease in haemoglobin on Day 29 in males and on Day 43 in both sexes dosed with 1000mg/kg,

CLINICAL CHEMISTRY: Increase in plasma total bilirubin in both sexes dosed with 1000 mg/kg. All the plasma samples from animals dosed with
1000 mg/kg and killed on Day 29 were coloured blue/grey or brown/grey.

URINALYSIS: Renal epithelial cells were present on Day 22-23 in the urine of females dosed with 1000mg/kg and killed on Day 29 and on Days 37-38 in both sexes dosed with 1000 mg/kg and killed on Day 43. At 1000 mg/kg, the urine samples were various shades of grey, brown, black or blue on Day 22-23, and light grey or grey on Day 37-38. As a consequence, none of the qualitative tests (ketones, glucose, urobilinogen and blood) could be done on Day 22-23 on the urine of animals dosed with 1000 mg/kg, the test strips being unreadable. Those at Day 37-38 were read normally.

ORGAN WEIGHTS: No significant change in organ weights

GROSS PATHOLOGY: Males and females dosed with 1000 mg/kg and killed on Day 29 or 43 showed blue discolouration of a wide range of organs. Similar, but reduced discolouration was present in rats dosed with 200 mg/kg, where it was confined to the stomach and kidney. Discolouration was not present in the animals dosed with 50 mg/kg.

HISTOPATHOLOGY: NON-NEOPLASTIC: Treatment-related findings were confined to the kidney and lung of females dosed at 1000 mg/kg. Minimal and subtle increase in neutral staining granularity of the cytoplasm of the proximal convoluted tubules. In the lung of two females there was a moderate pneumonitis characterised by thickening of the alveolar septae and mononuclear cell accumulation

Dose descriptor:

NOAEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No significant biological effects were observed at any dose levels

Critical effects observed:

not specified

Results-Analysis of Dosing Preparations:

The concentrations of test substance in deionised water were within 11% of the nominal concentrations.

The homogeneity of test substance in deionised water was determined at 5.6 and 111.7 mg/mL and was shown to be satisfactory.

The chemical stability of test substance in deionised water was determined at 5.6 and 111.7mg/ml and was shown to be satisfactory over a 21-d period.

Conclusions:

Under the test conditions, the NOAEL is 1000 mg/kg body weight/day.

Executive summary:

A study was conducted to assess the sub acute repeated dose toxicity of the test substance in rats equivalent or similar to EU Method B.7. and OECD guideline 407 in compliance with GLP

Groups of five male and five female rats were given 28 consecutive daily doses of 50, 200 or 1000 mg/kg of the test substance and were then subjected to a full post mortem examination. A similar group of animals was dosed with deionised water only and used as a control. Two further groups of five male and five female rats were dosed with either 1000 mg/kg of the test substance or deionised water and these animals were retained for 14 d without further treatment to allow a two week recovery period following the final dose of the test substance.

There were no clinical abnormalities of toxicological significance. The only clinical signs were black faeces during the dosing period and blue skin, both of which were due to colouration by the test substance.

Males dosed with 1000 mg/kg and killed on Day 29 had lower bodyweights and lower food consumption than the control group, but this was not evident when compared with the recovery control group and is considered to be unrelated to treatment.

There were no toxicologically significant effects on clinical chemistry, other than staining of the plasma and urine by the test substance. There were no toxicologically significant effects on haematology or organ weights.

The only pathological effects were in the kidneys and lungs of females dosed with 1000 mg/kg and killed on Day 29 and in the kidneys of females dosed with 1000mg/kg and killed on Day 43. In the kidney there was increased granularity in the proximal convoluted tubules which is considered to be due to, an accumulation of test substance within the kidney. Renal epithelial cells, seen in the urine of both groups of females dosed with 1000mg/kg and in the males killed on day 43, show that some exfoliation was occurring, but as there was no clear evidence of kidney damage this is considered not to be of toxicological significance. In the lung, two females showed pneumonitis. Leading to a NOAEL of 1000 mg/kg/day to male rats and 200 mg/kg/day to female rats. However, this NOAEL for female rats is only true for rats dosed by gavage. The test item administration at a high concentration and volume can lead to reflux of the administered formulation in the oesophagus and aspiration into the lung, leading to pneumonitis. This is not relevant for humans.

Hence, the NOAEL relevant for risk assessment is 1000 mg/kg body weight /day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 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

A study was conducted to assess the sub acute repeated dose toxicity of the test substance in rats equivalent or similar to EU Method B.7. and OECD guideline 407 in compliance with GLP

Groups of five male and five female rats were given 28 consecutive daily doses of 50, 200 or 1000 mg/kg of the test substance and were then subjected to a full post mortem examination. A similar group of animals was dosed with deionised water only and used as a control. Two further groups of five male and five female rats were dosed with either 1000 mg/kg of the test substance or deionised water and these animals were retained for 14 d without further treatment to allow a two week recovery period following the final dose of the test substance.

There were no clinical abnormalities of toxicological significance. The only clinical signs were black faeces during the dosing period and blue skin, both of which were due to colouration by the test substance.

Males dosed with 1000 mg/kg and killed on Day 29 had lower bodyweights and lower food consumption than the control group, but this was not evident when compared with the recovery control group and is considered to be unrelated to treatment.

There were no toxicologically significant effects on clinical chemistry, other than staining of the plasma and urine by the test substance. There were no toxicologically significant effects on haematology or organ weights.

The only pathological effects were in the kidneys and lungs of females dosed with 1000 mg/kg and killed on Day 29 and in the kidneys of females dosed with 1000mg/kg and killed on Day 43. In the kidney there was increased granularity in the proximal convoluted tubules which is considered to be due to, an accumulation of test substance within the kidney. Renal epithelial cells, seen in the urine of both groups of females dosed with 1000 mg/kg and in the males killed on day 43, show that some exfoliation was occurring, but as there was no clear evidence of kidney damage this is considered not to be of toxicological significance. In the lung, two females showed pneumonitis, leading to a NOAEL of 1000 mg/kg/day to male rats and 200 mg/kg/day to female rats. However, this NOAEL for female rats is only true for rats dosed by gavage. The test item administration at a high concentration and volume can lead to reflux of the administered formulation in the oesophagus and aspiration into the lung, leading to pneumonitis. This is not relevant for humans.

Hence, the NOAEL relevant for risk assessment is 1000 mg/kg body weight /day (Parr-Dobrzanski R J & Leah AM, 1991).

Justification for classification or non-classification

Based on the 28 d oral gavage study in rats, the NOAEL of the test substance was determined to be 1000 mg/kg bw/day. Based on these results, the test substance does not meet the requirement for classification according to EC criteria (67/548/EEC) and according to CLP criteria (EC 1272/2008).