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Description of key information

In a study performed in a similar manner to the OECD TG 408 groups of rats were dosed by gavage at up to 750 mg/kg bw/day for 90 days. No systemic effects were noted and the NOAEL was set at 750 mg/kw bw/day.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Referenceopen allclose all

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
1985-09-09 to 1986-05-01
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: GLP study, no guideline specified
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)
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, Margate England.
- Age at study initiation: Not specified
- Weight at study initiation: males: 114 - 153 g, females: 106 - 135 g
- Fasting period before study:
- Housing: polypropylene cages with stainless steel wire mesh tops and bottoms 56 x 38 x 18 cm (Five rats of one sex per cage)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 16 - 25°C
- Humidity (%): 40 - 90%
- Air changes (per hr): Air conditioned
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 7.5, 25 and 75 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analyses were performed by comparison of absorbances of the solutions at 207 nm with those of standard solutions.
Dose concentrations were 99% of theoretical concentration
Duration of treatment / exposure:
91-days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
75 mg/kg bw/day (actual dose received)
Dose / conc.:
250 mg/kg bw/day (actual dose received)
Dose / conc.:
750 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
20 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: 28-day range finding study.
- Rationale for animal assignment (if not random): during acclimatisation period all cages of animals were weighed and a mean weight for each calculated. Animals were then moved to different cages until each cage weighed ± 5 g of mean.
- Rationale for selecting satellite groups: a satelite group of 10 male and 10 female rats were used to provide pre-exposure clinical pathology. These animals were discarded without dosing or necropsy.
Positive control:
No
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: daily
- Mortality: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION
Per cage - weekly mean intake calculated and exressed as g/rat/week

WATER CONSUMPTION
Visual inspection

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: before commencement of treatment and after 4 and 12 weeks of treatment for all animals in the high dose group and control group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Pre-exposure blood and urine samples taken from the 10 male and 10 females of the satallite group. During the treatment period blood and urine samples were obtained from the first surviving 10 male and 10 female rats in each group after 4 and 12 weeks of treatment.
- Anaesthetic used for blood collection: Yes (identity) ether
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined:
Haematocrit, heamoglobin, erythrocyte count, mean cell volume, mean cell haemoglobin concentration, total laucocyte count, leucocyte differential count (neutrophils, lymphocytes, monocyctes and eosinophils), Coagulation tests (prothrombin time and partial thromboplastin time)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Pre-exposure blood and urine samples taken from the 10 male and 10 females of the satallite group. During the treatment period blood and urine samples were obtained from the first surviving 10 male and 10 female rats in each group after 4 and 12 weeks of treatment.
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined:
Blood urea nitrogen, glucose, alkaline phosphatase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, total protein, protein electrophoresis, sodium, potassium

URINALYSIS: Yes
- Time schedule for collection of urine: Pre-exposure blood and urine samples taken from the 10 male and 10 females of the satallite group. During the treatment period blood and urine samples were obtained from the first surviving 10 male and 10 female rats in each group after 4 and 12 weeks of treatment.
- Metabolism cages used for collection of urine: No data
- Animals fasted: Yes
- Parameters examined:
Volume, abnormalities of appearance and colour, specific gravity, pH, glucose, protein, ketones, bilirubin, blood pigments, deposit (erthrocytes, leucocytes, crystals, debris, casts)

Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Other examinations:
Organ weights:
Brain, kidneys, liver, adrenals, heart, lungs, ovaries, pituitary, spleen, testes, thyroids, uterus.

Tissue preservation.
Only comments made on tissues found to be abnormal.

Degree of absorption of test material:
excess plasma samples from the terminal clinical pathology bleed were stored deep frozen to enable an assessment of the degree of absorption to be made if required.
Statistics:
Analysis of variance and an "f" test calculation. These were followed by a "t" test if valid.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
post and pre-dose salivation.
Mortality:
mortality observed, treatment-related
Description (incidence):
post and pre-dose salivation.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in males in mid and high dose groups. High dose females had increase in body weight gain
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in males in high dose group and increase for females in high dose group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Increased volume in high dose females.
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Increased absolute and bodyweight related liver weights in high dose females
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Raised black foci on non-glandular mucosa of stomach in one male in high dose group.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Description (incidence and severity):
See details on results
Histopathological findings: neoplastic:
not specified
Details on results:
CLINICAL SIGNS AND MORTALITY
Post dose salivation was noted in all animals from the high dose group from week 3 of the study onwards, this increased gradually from a minimal amount to marked by week 7, from week 7 onwards pre-dose salivation was also seen in this dose group. Both sexes from the mid dose group showed post dose salivation from week 8 onwards.
Mortality: One female from the control group died at blood sample during week 13. There were no other deaths during the treatment period.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain was reduced in males from the high dose group and the mid dose group by 7% over the 13 week treatment period. Females from the high dose group showed a 7% increase in bodyweight gain over the same time period. All other treatment groups showed bodyweight gains similar to controls throughout treatment.

FOOD CONSUMPTION
Total food consumption over the 13 week treatment period was marginally (5%) lower than the controls for males from the high dose group. Females from this group showed a marginal increase (5%) in total food consumption. These changes existed throughout the treatment period. All other treatment groups maintained food consumption values similar to controls throughout the study period.

WATER CONSUMPTION
A visual inspection of all water bottles throughout the treatment period revealed no changes in water intake thought to be related to the administration of the test compound.

OPHTHALMOSCOPIC EXAMINATION
Ophthalmic examination of control and high dose animals revealed no changes thought to be related to treatment.

HAEMATOLOGY
Haematological examination of blood samples showed decreased haemoglobin in all treated male groups after 4 weeks of treatment, this change was not apparent after 12 weeks. White blood cell count was reduced in males from the high dose group after 4 and 12 weeks of treatment. All these haematological changes were within the historical laboratory ranges and were not considered to be due to test material administration.

CLINICAL CHEMISTRY
Blood biochemical examination after 4 and 12 weeks of treatment showed increased blood urea nitrogen in all female treated groups. Glutamate pyruvate transaminase was reduced in all treated male groups after 4 and 12 weeks of treatment. Both these parameters were within our normal laboratory ranges. There were no other changes on blood biochemistry parameters which might be ascribed to treatment.

URINALYSIS
Urinalysis showed increased volume in females from the high dose group after 4 and 12 weeks of treatment this was associated with reduced specific gravity after 12 weeks of treatment. Males from this group showed marginal changes in volume and specific gravity. There were no other possible treatment related changes in urinalyses.

ORGAN WEIGHTS
Increased absolute and bodyweight related liver weights were seen in females from the high dose group. There were no other changes in organ weight analysis which might be ascribed to treatment.

GROSS PATHOLOGY
There were no abnormalities noted at necropsy which might be attributable to administration of the test material. Macroscopic examination of the stomach and gastro-intestinal tract did not reveal any abnormalities of the mucosa.

HISTOPATHOLOGY
Stomach:
Hyperplasia of the non-glandular squamous epithelium characterised by acanthosis, hyperkeratosis and an increase in the number of mitoses, was seen in 15 out of 20 high dose group males and 17 out of 20 high dose group females and in 5 out of 20 mid dose males and 3 out of 20 mid dose females. In many of these rats focal erosion of the non-glandular squamous epithelium and varying degrees of gastritis were seen in association with the epithelial hyperplasia. These changes were indicative of either irritation to the non-glandular epithelium by the direct action of the gavage-administered test compund on the epitehlial surface, or stress-related gastritis and epithelial erosion with reparative epithelial hyperplasia. Information on the physico-chemical properties and/or pharmacological action of the test compund should help to elucidate the aetiology of these lesions. These treatment and dose-related changes were considered to be of toxicological significance in rats receiving the high and mid level doses.
Hyperplasis of the non-glandular squamous epthelium was seen in 1 out of 20 control females only and in 1 out of 20 low dose males only. In these animals n associated epithelial erosion or gastrtis was seen and this very low incidence of epithelial hyperplasia was not considered to be significant in control rats or rats receiveing the low dose level.
Other minor changes seen in the stomachs of occasional animals and not considered to be significant included distension of gastric glands, single mucosal cysts and a keratin inclusion cyct.
Apart from changes in the stomach, all other morphological entities were recognised as changes which commonly occur in laboratory rats of this age. They were not considered to be significant.
Dose descriptor:
NOAEL
Effect level:
750 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: histopathology
Critical effects observed:
not specified
Conclusions:
The Study Director did not assign a NOAEL value in the study report. The NOAEL value was based on microscopic changes in stomach tissue; there were no other significant effects. It is considered that the stomach changes were consistent with irritation and it may be argued that this kind of effect is better defined by an irritant classification, rather than as though it was a systemic effect. It may also be noted that the non-glandular epithelium (forestomach) does not have a counterpart in humans (except perhaps for oesophagus) so any changes here are not directly relevant to human hazard; the glandular epithelium was clearly resistant to these irritant effects, as is often the case. The substance is classified as an eye irritant, therefore it is reasonable to assign 750 mg/kg/day as the systemic NOAEL.
Executive summary:

In a study performed in a similar manner to the OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) groups of 20 male and 20 female Sprague-Dawley rats were dosed by oral gavage at dose levels of 0, 75, 250 and 750 mg/kg bw/day in distilled water for 91 consecutive days. A further group received distilled water alone and acted as controls.

Animals were observed daily, food intake was recorded weekly and bodyweight group means were calculated weekly during the study. Ophthalmoscopy examinations were made on all animals before treatment and on animals from the high dose and control groups after 4 and 12 weeks of treatment. Clinical pathology examinations were made on satellite animals before commencement of treatment and the first 10 surviving male and female rats of each group after 4 and 12 weeks of treatment.

After 13 weeks of treatment all animals were subjected to a detailed necropsy during which the weights of 10 male and 11 female organs were recorded and a wide range of tissues preserved. A range of tissues from all animals of the high dose and control groups were sectioned and examined microscopically. Due to a possible treatment related effect seen in high dose animals, stomachs from all groups were examined microscopically.

The Study Director did not assign a NOAEL value in the study report. The NOAEL value was based on microscopic changes in stomach tissue; there were no other significant effects. It is considered that the stomach changes were consistent with irritation and it may be argued that this kind of effect is better defined by an irritant classification, rather than as though it was a systemic effect. It may also be noted that the non-glandular epithelium (forestomach) does not have a counterpart in humans (except perhaps for oesophagus) so any changes here are not directly relevant to human hazard; the glandular epithelium was clearly resistant to these irritant effects, as is often the case. The substance is classified as an eye irritant, therefore it is reasonable to assign 750 mg/kg/day as the systemic NOAEL.

Endpoint:
short-term repeated dose toxicity: oral
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
1985-06-21 to 1985-09-17
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
other: Range finding study for 90-day repeat oral dose study, not all parameters of a full guideline study followed.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 407 (Repeated Dose 28-Day Oral Toxicity Study in Rodents)
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, Margate England.
- Age at study initiation: Not specified
- Weight at study initiation: 60 - 75 g
- Fasting period before study: No data
- Housing: polypropylene cages with stainless steel wire mesh tops and bottoms (Five rats of one sex per cage)
- Diet (e.g. ad libitum): ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 10 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3°C
- Humidity (%): 50 ± 20%
- Air changes (per hr): Air conditioned
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS:

VEHICLE
- Concentration in vehicle: 0, 5, 20 and 80 mg/mL
- Amount of vehicle (if gavage): 10 mL/kg/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
No information on analytical method.
Dose concentrations were 98 - 108% of theoretical concentration
Duration of treatment / exposure:
28-days
Frequency of treatment:
Daily
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Dose / conc.:
50 mg/kg bw/day (actual dose received)
Dose / conc.:
200 mg/kg bw/day (actual dose received)
Dose / conc.:
800 mg/kg bw/day (actual dose received)
No. of animals per sex per dose:
5 per sex per dose
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: sponsor and testing facility discussions
Positive control:
No
Observations and examinations performed and frequency:
DETAILED CLINICAL OBSERVATIONS: Yes
Mortality - at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: Daily

FOOD CONSUMPTION
Per cage - weekly mean intake calculated and exressed as g/rat/week

WATER CONSUMPTION
Visual inspection

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At termination
- Anaesthetic used for blood collection: Yes (identity) ether
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined:
Haematocrit, heamoglobin, erythrocyte count, mean cell volume, mean cell haemoglobin concentration, total laucocyte count.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At termination
- Animals fasted: No data
- How many animals: All surviving animals
- Parameters examined:
Blood urea nitrogen, glucose, alkaline phosphatase, glutamate pyruvate transaminase, glutamate oxaloacetate transaminase, total protein.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: No
Other examinations:
Organ weights:
Brain, kidneys and liver

Degree of absorption of test material:
excess plasma samples from the terminal clinical pathology bleed were stored deep frozen to enable an assessment of the degree of absorption to be made if required.
Statistics:
Student's t test for statistical significance compared to controls.
Clinical signs:
effects observed, treatment-related
Description (incidence and severity):
Poor coat condition and post dose salivation. No mortalities.
Mortality:
mortality observed, treatment-related
Description (incidence):
Poor coat condition and post dose salivation. No mortalities.
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduction in females in mid and high dose groups
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduction in females in mid and high dose groups
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
no effects observed
Clinical biochemistry findings:
no effects observed
Urinalysis findings:
not examined
Behaviour (functional findings):
not examined
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
bodyweight related brain and kidney weights increased in high dose females
Gross pathological findings:
effects observed, treatment-related
Description (incidence and severity):
Raised black foci on non-glandular mucosa of stomach in one male in high dose group.
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
CLINICAL SIGNS AND MORTALITY
Poor coat condition was seen in the majority of animals for several days before treatment and at commencement of the study, this improved gradually, and by Day 11 all animals appeared normal, except for females of the high dose group which stayed in poor condition, for the whole study. Poor condition was again noted in females of the mid dose group from day 15 and males of the high dose group from day 22.
Post dose salivation was noted in all animals from the high dose group from day 18.
There were no deaths during the study.

BODY WEIGHT AND WEIGHT GAIN
Bodyweight gain of females from the mid dose group was similar to controls after 2 weeks of treatment but showed a reduction of 8% after 3 weeks and a reduction of 9% after 4 weeks of treatment.
Females from the high dose group showed a reduction in bodyweight gain of 10-12% throughout the treatment period, when compared with controls.
All other treated groups remained similar to controls.

FOOD CONSUMPTION
Females from the high dose group showed a decrease of 7% in food consumption overall when compared to controls. Food consumption was reduced by 5% during week 1 and decreased to a 9% reduction by week 4. Similarly females from the mid dose group showed an overall decrease of 6% in food consumption. The decrease being 4% for week 1 and reducing to 9% by week 4.
The other treated groups remained similar to controls throughout the treatment period.
Food conversion ratios did not indicate any clear pattern of change.

HAEMATOLOGY
No changes of toxicological significance

CLINICAL CHEMISTRY
No changes of toxicological significance

ORGAN WEIGHTS
Bodyweight related brain and kidney weights were statistically increased in females from the high dose group.
Absolute and bodyweight related liver weights were increased in males from the high dose group but were not statistically significant.

GROSS PATHOLOGY
Macroscopic examination at necropsy revealed raised black foci on the non glandular mucosa of the stomach in one male from the high dose group.
Other findings, enlarged cervical lymph nodes (3 control animals) purple foci on thymus (2 control animals) and dilation of a kidney pelvis ( 1 high dose animal), were not considered to be related to treatment with the test material.
Dose descriptor:
NOAEL
Effect level:
800 mg/kg bw/day (actual dose received)
Based on:
test mat.
Sex:
male/female
Critical effects observed:
not specified
Conclusions:
The highest dose level tested is likely to produce some toxicity in the proposed 90-day repeat oral dose study in the rat but without causing mortality
Executive summary:

In a 28-day repeat oral dose range-finding study, male and female rats (5/sex/dose) were dosed with 0, 50, 200 and 800 mg/kg bw/day of the test material in water by oral gavage.

Toxicologically significant and dose-related effects were noted in the clinical observations, on food consumption, on body and organ weights and at gross necropsy.

The highest dose level tested (800 mg/kg bw/day) is likely to produce some toxicity in the proposed 90-day repeat oral dose study in the rat but without causing mortality

Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
750 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Two reliable studies are available (Klimisch =2) covering sub acute and sub chronic exposure.

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

In a study performed in a similar manner to the OECD TG 408 (Repeated Dose 90-Day Oral Toxicity in Rodents) groups of 20 male and 20 female Sprague-Dawley rats were dosed by oral gavage at dose levels of 0, 75, 250 and 750 mg/kg bw/day in distilled water for 91 consecutive days [Hill R (1986)]. A further group received distilled water alone and acted as controls.

Animals were observed daily, food intake was recorded weekly and bodyweight group means were calculated weekly during the study. Ophthalmoscopy examinations were made on all animals before treatment and on animals from the high dose and control groups after 4 and 12 weeks of treatment. Clinical pathology examinations were made on satellite animals before commencement of treatment and the first 10 surviving male and female rats of each group after 4 and 12 weeks of treatment.

After 13 weeks of treatment all animals were subjected to a detailed necropsy during which the weights of 10 male and 11 female organs were recorded and a wide range of tissues preserved. A range of tissues from all animals of the high dose and control groups were sectioned and examined microscopically. Due to a possible treatment related effect seen in high dose animals, stomachs from all groups were examined microscopically.

The Study Director did not assign a NOAEL value in the study report. The NOAEL value was based on microscopic changes in stomach tissue; there were no other significant effects. It is considered that the stomach changes were consistent with irritation and it may be argued that this kind of effect is better defined by an irritant classification, rather than as though it was a systemic effect. It may also be noted that the non-glandular epithelium (forestomach) does not have a counterpart in humans (except perhaps for oesophagus) so any changes here are not directly relevant to human hazard; the glandular epithelium was clearly resistant to these irritant effects, as is often the case. Therefore it is reasonable to assign 750 mg/kg/day as the NOAEL.

Justification for classification or non-classification

According to the Guidance on the Application of Regulation (EC) No 1272/2008 Table 3.9.3, classification in Category 2 is applicable when significant toxic effects observed in a 90 -day repeated dose study conducted in experimental animals are seen to occur within the guidance value range of 10><=100 mg/kg bw/day. The NOAEL from the available 90 -day study is 750 mg/kg bw/day, hence classification is not warranted.