Tetrahydro-1,3,4,6-tetrakis(hydroxymethyl)imidazo[4,5-d]imidazole-2,5(1H,3H)-dione

Endpoint summary

Description of key information

The test substance revealed a NOAEL in male and female rats of 1.000 mg/kg body weight in a subacute and a subchronic oral repeated dose toxicity study. Both studies were GLP and OECD guideline studies.

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:

2001

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1995

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: BASF AG; Batch No. 0767
- Expiration date of the lot/batch: March 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Stored at < 25°C, in die dark
- Stability of the test substance in the solvent/vehicle: The analysis of the formaldehyde content in the test substance revealed negligible loss for a period of 4 hours.

FORM AS APPLIED IN THE TEST: dissolved in distilled water; dose solution was prepared daily prior to dosing

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as a test system because it is a readily available laboratory rodent. species. lt has been historically shown to be a suitable model for repeated dose toxicity assessment and is recommended by the OECD and other regulatory authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jai Research Foundation, Valvada - 3 96 108, Dist. Valsad, Gujarat, India
- Females nulliparous and non-pregnant: yes
- Age at study initiation: about 7 weeks
- Fasting period before study: not specified
- Weight at start of treatment: males: 118 - 158 g; females: 95 - 130 g
- Housing: individually; during acclimation period 5/cage
- Diet: ad libitum
- Water: ad libitum
- Acclimation period: 5 days

DETAILS OF FOOD AND WATER QUALITY:
Every new batch of feed used was analysed or microbial contaminants. Rat pellet feed and drinking water were analysed for potential contaminants once in six months.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24°C
- Humidity (%): 67% (mean)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: June 16, 2001 To: July 20 / August 03, 2001

Route of administration:

oral: gavage

Details on route of administration:

The oral administration represents a possible route of human exposure.

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:
Required quantities of test substance were dissolved in distilled water to get the desired dose solutions with appropriate concentrations for various treatment groups. Dose solution was prepared daily just prior to dosing.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Purity of formaldehyde content in the test substance in dose solution was detected by the folowing procedure:

A volume of 1 ml samnple was pipetted out and diluted to 100 ml with distilled water. A volume of 100 ml diluted sample was placed in different distillation apparatus with the addition of 85% phosphoric acid (15 ml)
and distilled out. A series of standard solutions was pipetted out from 10 ml standard solutions, i. e., 0.25, 0.5, 1.0, 1.5, 2.0 ml in to 25 ml of volumetric flask with addition of10 ml of reagent solution and volume was made with distilled water (concentration of standard solutions were 1 ppm , 2 ppm, 4 ppm, 6 ppm, 8 ppm). A volume of 1.0 ml of the above distilled solution was pipetted out into a 25 ml volumetric flask with the addition of 10 ml of antona reagent and volume was adjusted with distilled water. Then standard and sample flasks were kept for 3.0 minutes in a water bath at 40°C and was cooled down at 20°C. The Blank solution was prepared with distilled water using the same procedure. Above prepared standard and sample solution was again diluted to 5 times with distilled water. The absorbance of the diluted standard and sample was measured against die blank at 412 nm.

Duration of treatment / exposure:

28 days; recovery group animals were kept under observation for further 14 days

Frequency of treatment:

once daily

Dose / conc.:

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

Remarks:

Low dose

Dose / conc.:

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

Remarks:

Mid dose

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

Remarks:

High dose

No. of animals per sex per dose:

5; 3 males and females in one additonal control and high dose group

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In order to find out die irritancy, if any, of the test substance at the dose level of 1000 mg/kg body weight two additional groups of animals i. e., one control and one high dose, each consisting of 3 males and 3 females, were included in die 28 day study and dosed
for a period of 14 days. On the 15th day these animals were subjected to a complete gross pathology examination which showed no treatment related irritation in the gastrointestinal tract. Hence, the 28 day repeated dose oral toxicity study was performed at the same highest dose.

- Post-exposure recovery period in satellite groups: yes, 14 days

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: once a day; twice daily observation for morbidity and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once a day

BODY WEIGHT: Yes
- Time schedule for examinations: on the day of initiation of treatment and at weekly intervals thereafter

FOOD CONSUMPTION: Yes
- Time schedule for examinations: once a week

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once before commencement of the treatment and prior to terminal sacrifice
- Dose groups that were examined: all animals

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment and revovery period
- Anaesthetic used for blood collection: Yes; light ether anesthesia
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked in table [No. 1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment and revovery period
- Anaesthetic used for blood collection: Yes; light ether anesthesia
- Animals fasted: Yes; overnight
- How many animals: all animals
- Parameters checked in table [No. 1] were examined.

NEUROBEHAVIOURAL OBSERVATIONS: yes
- Time schedule: once before commencement of the treatment and at weekly intervals thereafter
- Battery of functions tested: posture, convulsions, ease of removal from the home cage, handling reactivity of the animal, autonomic signs such as palpebral closure, lacrimation, salivation and piloerection
- Open field observations (gait, monilty score, arousal level, vocalisations, rearing, respiration, clonic or tonic movements, urination and defecation, stereotypy, bizarre behaviour)

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table no. 2)

HISTOPATHOLOGY: Yes; All the preserved organs (see table no. 2) from control and high dose groups as well as liver, kidneys, heart, spleen and lungs of all the animals

Statistics:

Raw data were processed by the Department of Biostatistics and Systems Information, Jai Research Foundation to give group means and standard deviations with significance between the control and treated groups, using in-house developed statistical software. All the parameters characterised by continuous data such as rearing count, urination count, defecation, body weight, feed consumption, organ weight, relative organ weight, haematological and clinical chemnistry data were subjected to Bartlett's test to meet the homogeneity of variance before conducting Analysis of Variance (ANOVA) and Dunnett' s t-test. Where the data did not meet die homogeneity of variance, Student's t-test was performed to calculate significance.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

In high and mid dose males, the monocyte count was significantly enhanced. This effect was not observed at the end of the recovery phase.
Females of all dose groups showed an increase in mean corpuscular volume and an according decrease in measn corpuscular haemoglobin concentration. In the absence of any other alterations in the red blood corpuscle, these changes are considered biologically insignificant.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males: decreased sodium and potassion values mainly in the low dose animals were considered incidental
Females: Marginally, though statistically significantly increased phophorus values in mid and high dose animals without deviations in calcium levels and/or histopathological alterations in the kidney were considered biologically irrelevant. A statistical increase in total protein, though marginal and hardly dose related, in all dose groups was not considered adverse. All other changes occured with no relation to dose and were considered incidental.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute prostate weights were increased in high dose recovery males, but not at the end ot the treatment period.
In females of the recovery group, the absolute spleen weight was decreased, and the relative liver weight was increased.
As for males, in the absence of histopathological correlates and since the changes were not observed at the end of the treatment period, they were considered as irrelevant and incidental.

Gross pathological findings:

no effects observed

Neuropathological findings:

no effects observed

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Degenerative or inflammatory changes were observed at similar incidence and severity in control and treated animals.

Histopathological findings: neoplastic:

no effects observed

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no adverse effects observed

Critical effects observed:

no

Conclusions:

Based on the results of this study, it is concluded that the No Observed Adverse Effect Level (NOAEL) of the test substance in Wistar rats exposed over a period of 28 days is 1000 mg/kg body weight.

Executive summary:

This study was conducted to assess the toxic effects of the test substance to rats, when administered orally by gavage for 28 consecutive days according to OECD 407. Three groups of Wistar rats comprising 5 males and 5 females per group with the test substance at 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg b.wt. A concurrent control group comprising die same number of animals was also maintained in die study. In addition, a high dose recovery (1000 mg/kg b. wt.) and a control recovery group were included in the study to investigate the persistence, recovery or delayed effect of the test substance for an other period of 14 days.

Each rat was observed for visible signs of reactions to the treatment once a day and for rnortality and morbidity twice a day. Individual body weight and feed consumption were monitored weekly. Detailed clinical examination/neurobehavioural observations were conducted for each animal once prior to initiation of treatment and at weely intervals thereafter. Ophthalmological exanmination was conducted on all animals prior to treatment and prior to sacrifice. Haematological observations and biochemical analysis of blood samples were performed on all animals at the end of treatment and recovery periods. All the rats were sacrificed and subjected to a gross pathological examination at the end of treatment and recovery period. Absolute organ weights were recorded and relative organ weights were calculated for die organs viz., adrenal, testes/ovaries, epididymis/uterus, posate, kidneys, brain, heart, spleen, liver and thymus in all rats. Histopathological examination was made on all preserved organs from control and high dose group animals as well as liver, kidneys, spleen, heart and lungs of all animals.

No treatment related mortality or visible clinical symptoms were observed in any of the treatment group aimals. Body weight, feed consumption and opthalmological examination did not reveal treatmnent related alterations.

The test substance did not alter the neurobehavioural pattern of animals.

Haematology parameters studied in treated males were comparable to control group except in monocyte count; significantly increased monocyte count was observed in mid and high dose groups.

The clinical chemistry parameters did not reveal any major alterations in treated males. In female increased total protein in all the dose groups (low, mid and high dose) and phosphorus in mid and high dose groups were observed. All other parameters studied were comparable to control group.

No treatment related changes were observed in the absolute organ weight and relative organ weight of treated groups as compared to control groups.

Gross pathology examination did not reveal any treatment related lesions.

The histopathological examination of various organs /tissues from control and high dose (1000 mg/kg b. wt.) group animals did not reveal any treatment related effect.

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) of the test substance in Wistar rats exposed for a period of 28 days is 1000 mg/kg b. wt.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Jul 2001 - May 2002

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

1998

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source; batch No.of test material: BASF AG Ludwigshafen, Germany; 0767
- Expiration date of the batch: Mar 2002

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: Kept container tightly closed at room temperature in the Substance Control Office (TSCO)
- Stability in the dose solution: Was performed for preliminary 28-day oral toxicity study and a separate analysis was not performed
- Homogeneity: Homogeneity and active ingredient tests were performed once during start of the study and at monthly intervals thereafter
- Stability under test conditions: The chemical analysis results indicated that the test substance was stable for 4 hours and homogeneous in the gavage solution

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
Treatment of test material: required qunatity of the test sustance was obtained from the test substance control office and dissolved in distilled water to attain the required concentrations

OTHER:
The test substance was analyzed in the dose solution via the method of a calibration curve.

Species:

rat

Strain:

Wistar

Details on species / strain selection:

The rat was selected as a test system because it is a readily available laboratory rodent species. lt has been historically shown to be a suitable model for subchronic toxicitiy assessment and is recommended by the OECD and other regulatory authorities. The oral administration represents a possible route of human exposure and was agreed by the study sponsor. The results of the studie are believed to be of value in predicting the toxicity of the test substance to human.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Jai Research Foundation, Valvada - 396 108, Dist. Valsad, Gujarat, India
- Females nulliparous and non-pregnant: yes
- Age at start of treatment: approximately six to seven weeks
- Fasting period before study: no (only overnight before blood sampling)
- Housing: During acclimatization period rats were housed in groups of five per sex per cage. After acclimatization period rats were housed in groups of two of same sex..
- Diet: rat pellet feed, ad libitum
- Water: ad libitum
- Acclimation period: Five days
- Body weight variation at the start of the treatment: within the 20'% of mean body weight for each sex; males: 108 g - 155 g, females: 94 g - 130 g

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 - 24
- Humidity (%): 67 (mean)
- Photoperiod (hrs dark / hrs light): 12/12
- Air changes: A minimum of 21 air changes per hour were maintained

Route of administration:

oral: gavage

Details on route of administration:

All animals were dosed once daily for a minimum of 90 consecutive days using a metal cannula attached to a Borosilicate hard glass syringe. The rats of the control group were administered distilled water only. Dose volume was adjusted weekly based on the body weight of individual rat.

Vehicle:

water

Remarks:

distilled

Details on oral exposure:

TREATMENT
Animals were treated for a period of 90 consecutive days. Recovery group animals were kept under observation further for a period of 14 days.

PREPARATION OF DOSING SOLUTIONS:
- Rationale for the selection of the starting dose: Doses were selected based on the recommendations of the sponsor
- Dose solution was prepared daily just prior to dosing
- Rate of preparation of solution(frequency): The required quantity of the test substance was dissolved in distilled water to attain the desired concentration freshly every day prior to dosing.
- Dose volume: was calculated on the basis of a constant factor of 10 ml/kg bw throughout the study


VEHICLE
- distilled water
- amount of vehicle: 10 ml/kg bw

OTHER
In order to find out irritancy at the dose level of 1,000 mg/kg bw two additional groups of animals, i.e. one control and one high dose, each consisting of three males and three females, were included and dosed for a period of 14 days. On the 15th day these animals were subjected to a complete gross pathology examination.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Yes, by UV spectrophotometry at 412 nm.

A volume of 1 ml sample was pipetted out and diluted to 100 ml with distlled water. A volume of 100 ml diluted sample was placed in different distillation apparatus with the addition of 85% phosphoric acid (15 ml) and distilled out for 12 minutes.
A volume of 1.0 ml of the above distilled solution was pipetted out into a 25 ml volumetric flask with the addition of 10 ml of antona reagent and volume was adjusted with distilled water. Flasks were kept for 30 minutes in a water bath at 40°C and was cooled down at 20'C.
A series of standard solutions was pipetted out from 100 ppm standard solutions, i. e., 0. 25, 0.5, 1.0, 1.5, 2.0 ml in to 25 ml of volumetric flask and 10 ml reagent solution and volume was reached with distilled water (concentration of standard solutins were 1 ppm , 2 ppm, 4 ppm, 6 ppm, 8 ppm).
The Blank solution was prepared with distilled water using the same procedure. Above prepared standard and sample solution was again diluted to5 times with distilled water. The absorbance of the diluted standard and sample was measured against the blank at 412 nm.

Duration of treatment / exposure:

90 days. Recovery animals were kept under observation for further 28 days to find out reversibility, persistence or delayed occurrence of toxic effects.

Frequency of treatment:

once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

1 000 mg/kg bw/day (actual dose received)

No. of animals per sex per dose:

Ten animals pe sex per group and five males and five females each in one additional control and high dose group (satellite groups)

Control animals:

yes, concurrent vehicle

Details on study design:

- Duration of observation period following administration: 28 days (recovery group)

- Rationale for selecting satellite groups: In order to observe reversibility, persistence, or delayed occurrence of toxic effects, a satellite group for the control (three animals per sex) and the top dose group (three animals per sex) was used

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, for mortality and morbidity twice daily and visible signs were recorded once a day

BODY WEIGHT: Yes
- Time schedule for examinations: At the start of treatment and at the terminal sacrifice

FOOD CONSUMPTION:
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/rat/week: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: No

OPHTHALMOSCOPIC EXAMINATION: Yes, see table 1.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period
- Anaesthetic used for blood collection: Yes, light ether anestesia
- Animals fasted: Yes, overnight (water allowed)
- How many animals: all animals
- Parameters checked in table 2 were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At the end of the treatment and recovery period
- Animals fasted: Yes, overnight (water allowed)
- How many animals: all animals
- Parameters checked in table 2 were examined.

NEUROBEHAVIOURAL EXAMINATION (detailed clinical examination): Yes
- Time schedule for examinations: all animals once before commencement of the treatment and at weekly intervals thereafter.
- Dose groups that were examined: all animals
- Battery of functions tested: sensory activity (tail-pinch, click, touch, pupil, response and air righting refelx), grip strength, motor activity

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, see table 3

HISTOPATHOLOGY: Yes, see table 3. All preserved organs from control and high dose groups and from animals which died during the study as well as liver, kidneys, heart, spleen, bone marrow, smear, gastro intestine and lungs of all the animals.

Other examinations:

Spermatology count (Sperm motility, epididymal sperm, homogenisation resistant testicular sperm head count, epididymal sperm morphology) was performed for all surviving males from all animals at the end of the treatment and recovery period to find out reversibility, persistence, recovery or delayed effect of the test substance.

Statistics:

Bartlett's test, ANOVA, Dunnett's test, Student's t-test

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

A few symptoms, although considered not treatment-related, were observed including snuffles (1/10 males control, low dose and mid dose group each), wry-neck (1/10 males high dose recovery group) and conjunctivitis (1/10 males in high dose) in male rats.
Female rats showed wry-neck (one from control group and low dose) and one rat from low dose group revealed piloerection and aggression (1/10 females in low dose group) during the experimental period.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

one male rat from mid dose group was found dead on day 84 of experimental period and it was considered to be incidental.

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

effects observed, non-treatment-related

Description (incidence and severity):

Decrease in feed consumption during weeks 9 and 10 (male high dose group). Decrease in feed consumption during week 10 (male low dose group).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male:
Significantly increased mean corpuscular volume (MCV) and mean corpuscular haemoglobin (MCH) values in high dose group. However, no changes in the values of erythrocyte count (RBC) and haemoglobin (HGB), thus findings are considered to be biologically irrelevant. Low dose group showed incidental increase in platelet count.
Significantly increased monocyte count in high dose group. However, increased values were within the range of historical control data. On completion of recovery period, high dose recovery group animals revealed significantly increased clotting time. However, no changes were observed at the end of the treatmetn period.

Female:
Significantly increased red blood corpuscle (RBC), haemoglobin (HGB) and haematotocrit (HCT) values were observed in low, mid, and high dose groups. However, changes were not dose dependent and values are within the historical control data. Slight elevation in these haematological parameters is not an adverse effect. On completion of recovery period, high dose recovery group animals revealed significantly decreased clotting time. However, no changes were observed at the end of the treatmetn period and values were within the histotrical control data.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males, significantly decreased gamma glutamyltranspeptidase values were observed in high dose recovery group which are considered to be biologically insignificant.

In females, a significant increase in total bilirubin in mid and high doses, increased phosphorus in high dose groups and increased chloride in low and high dose groups were observed. However, values of total bilirubin and chloride are within the range of historical control data. Alterations in phosphorus are marginal and are not accompanied by any other corresponding deviations in calcium, which is usually the case.

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

In males, an incidental decrease in rearing count during week 1 and 9 in high dose recovery group was observed.
In females, transient variations were observed in urination and defecation counts. Incidental decrease in mean urination count was observed in low dose group animals during week 7 and high dose recovery group animals during pre-exposure revealed incidental increase in mean urination count. Significanfly decreased mean defecation counts were observed in low dose group during pre-exposure and week 3 and in high dose group during pre-exposure, as compared to control group animals.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Males:
High dose recovery group animals revealed increased mean absolute and relative organ weight of brain as compared to control recovery group. However, no changes were observed at the end of 90-day treatment period in the treatment groups sacrificed at day 90 and relative weight values are within the range of historical control data.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Male: The internal examination of the carcasses from different groups showed varying degree of lesions in organs like lungs (consolidation/pneumonic foci/hepatization/suppuration, congestion); kidneys (congestion, whitish foci, contraction, blotched), liver (congestion/mottling, parasitic cyst), spleen (whitish deposition), urinary bladder (suppuration/abscess), small intestine (mucus exudation) and mesenteric lymph node (enlarged/cystic, haemorrhagic).

Females: Lesions were comparable to those observed in males. Additional findings included changes in lungs (haemorrhages and white nodule containing pus in left lobe), spleen (atrophy), small intestine (large cyst), ovary (cystic ovarian bursa, mesovarian cyst) and uterus (hydrometra and thickening of uterine wall).

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

see attached background material.

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Description (incidence and severity):

No effects in both sexes in motor activity, hind limb foot splay and grip strength.

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: no treatment related or adverse effetcs observed up to the limit dose

Critical effects observed:

no

Conclusions:

Based on the results of this study, it is concluded that the no observed adverse effect level (NOAEL) of the test substance in Wistar rats exposed over a period of 90 days is 1,000 mg/kg bw.

Executive summary:

This study was conducted to assess the toxic effects of the test substance to rats, when administered orally by gavage for 90 consecutive days according to OECD guideline 408.

Three groups of Wistar rats comprising 10 males and 10 females per group were treated with the test substance at 63 (low dose), 250 (mid dose) and 1000 (high dose) mg/kg bw. A concurrent control group comprising the same number of animal was also maintained in the study. In addition, a high dose recovery (1000 mg/kg b. wt.) and a control recovery groups were included in the study and observed further for a period of 28 days to investigate the persistence, recovery or delayed effect of the test substance. The rats from the control groups were administered distilled water only. Required quantity of the test substance was dissolved in distilled water to attain the required concentrations, i.e. 6.3, 25, 100 mg/ml for the low, mid and high dose groups, respectively. The chemical analysis results indicated that the test substance was stable for 4 hours and homogenous in the gavage solution. Fresh dosing solution of the test substance in distilled water was prepared every day prior to dosing.

Each rat was observed for visible signs of reaction to the treatment once a day and for rnortality and morbidity twice a day. Individual body weight and feed consumption were monitored weekly. Detaile clinical examiation/neurobehavioural observations were conducted for each animal once prior to initiation of treatment and at weely intervals thereafter. Ophthalmologiacal examination was conducted on all animalsprior treatment and prior to sacifie. Functional observational battery tests were performed on all surviving animalstowards the end of treatment and recovery period. Haematological observations and biochemical analysis of blood samples were performed on all animals at the end of treatment and recovery periods. All surviving rats were sacrificed and subjected to a gross pathological examination at the end of treatment and recovery period. Spermatology was performed for males of all the groups during sacrifice. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., adrenal, testes/ovaries, epididymis/uterus, prostate, kidneys, brain, heart, spleen, liver and thymus in all rats surviving the study period. Histopathological examination was made on all preserved organs from control and high dose group animals and from animals that died during the study as well as liver, kidneys, bone marrow smear, gastro intestine, spleen, heart and lungs of all animals.

No treatment related mortality or visible signs of toxicity were observed in any of the treatment group aimals. Body weight, feed consumption and ophthalmological examination did not reveal treatment related alterations.

The test substance did not alter the neurobehavioural pattern of animals. Sensory reactivity, hind limb foot splay measurements, grip strength and motor activity of treatmetn group animals were comparable to control group animals.

Haematology parameters analysed did not reveal treatment related alterations in both sexes.

No treatment related alterations were observed in the analysed clinical chemistry parameters.

The observed Spermatological parameters of treatment groups were comparable to control groups.

No treatment related changes were observed in absolute organ weights and relative organ weights of animals treated with the test substance.

Gross pathology and histopathological examination of various organs/tissues from control and high dose (1000 mg/kg b. wt.) group animals did not reveal any treatment related effect.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subchronic

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

28 -day oral repeated dose toxicity study:

This study was conducted to assess the toxic effects of the test substance to rats, when administered orally by gavage for 28 consecutive days according to OECD 407. Three groups of Wistar rats comprising 5 males and 5 females per group with the test substance at 100 (low dose), 300 (mid dose) and 1000 (high dose) mg/kg b.wt. A concurrent control group comprising die same number of animals was also maintained in die study. In addition, a high dose recovery (1000 mg/kg b. wt.) and a control recovery group were included in the study to investigate the persistence, recovery or delayed effect of the test substance for an other period of 14 days.

Each rat was observed for visible signs of reactions to the treatment once a day and for rnortality and morbidity twice a day. Individual body weight and feed consumption were monitored weekly. Detailed clinical examination/neurobehavioural observations were conducted for each animal once prior to initiation of treatment and at weely intervals thereafter. Ophthalmological exanmination was conducted on all animals prior to treatment and prior to sacrifice. Haematological observations and biochemical analysis of blood samples were performed on all animals at the end of treatment and recovery periods. All the rats were sacrificed and subjected to a gross pathological examination at the end of treatment and recovery period. Absolute organ weights were recorded and relative organ weights were calculated for die organs viz., adrenal, testes/ovaries, epididymis/uterus, posate, kidneys, brain, heart, spleen, liver and thymus in all rats. Histopathological examination was made on all preserved organs from control and high dose group animals as well as liver, kidneys, spleen, heart and lungs of all animals.

No treatment related mortality or visible clinical symptoms were observed in any of the treatment group aimals. Body weight, feed consumption and opthalmological examination did not reveal treatmnent related alterations.

The test substance did not alter the neurobehavioural pattern of animals.

Haematology parameters studied in treated males were comparable to control group except in monocyte count; significantly increased monocyte count was observed in mid and high dose groups.

The clinical chemistry parameters did not reveal any major alterations in treated males. In female increased total protein in all the dose groups (low, mid and high dose) and phosphorus in mid and high dose groups were observed. All other parameters studied were comparable to control group.

No treatment related changes were observed in the absolute organ weight and relative organ weight of treated groups as compared to control groups.

Gross pathology examination did not reveal any treatment related lesions.

The histopathological examination of various organs /tissues from control and high dose (1000 mg/kg b. wt.) group animals did not reveal any treatment related effect.

Based on the results of this study, the No Observed Adverse Effect Level (NOAEL) of the test substance in Wistar rats exposed for a period of 28 days is 1000 mg/kg b. wt.

90 -day oral repeated dose toxicity study:

This study was conducted to assess the toxic potential of the test substance to rats, when administered orally by gavage for a minimum period of 90 consecutive days. The methods followed were as per the guideline of OECD No 408 (1998).

Three groups of Wistar rats comprising 10 males and 10 females per group were treated with the test substance at dose levels of 63 (G2 -low dose), 250 (G3 -mid dose) and 1000 (G4 -high dose) mg/kg body weight for a period of 90 consecutive days. A concurrent control group (G1) comprising the same number of animals was also maintained in the study. In addition, a high dose recovery (G6 -1000 mg/kg body weight) and a control recovery group (G5 -0 mg/kg body weight) were included in the study and observed further for a period of 28 days to investigate the persistence, recovery or delayed toxic effect of the test substance, if any. The rats from the control groups were administered distilled water only. Required quantity of the test substance was dissolved in distilled water to attain the required concentrations i.e., 100, 25 and 6.3 mg/mL for the high, mid and low dose groups, respectively. The chemical analysis results indicated that the test substance was stable for 4 hours and homogenous in the gavage solution. Fresh dosing solution of the test substance in distilled water was prepared every day prior to dosing.

Each rat was observed for visible signs of reaction to the treatment once a day and for mortality and morbidity twice a day. Body weight and feed consumption were monitored weekly. Detailed clinical examination / neurobehavioural observations were conducted for each animal once prior to initiation of treatment and at weekly intervals thereafter. Ophthalmological examination was conducted on all animals prior to treatment and prior to sacrifice. Functional observational battery (FOB) tests were performed on all surviving animals towards the end of treatment and recovery period. Haematological observations and biochemical analysis of blood samples were performed on all surviving animals at the end of treatment and recovery periods. All surviving rats were sacrificed and subjected to a gross pathological examination at the end of treatment and recovery period. Spermatology was performed for males of all the groups during sacrifice. Absolute organ weights were recorded and relative organ weights were calculated for the organs viz., adreanls, testes/ovaries, epididymis/uterus, prostate, kidneys, brain, heart, liver, spleen and thymus from all rats that survived till the end of the study. Histopathological examination was made on all preserved organs from control and high dose group and from animals that died during the study as well as liver, kidneys, lungs, heart, bone marrow smear, gastro intestine and spleen of all animals.

No treatment related mortality or visible signs of toxicity was observed in animals treated with the test substance. Body weight and feed consumption of treatment group animals were comparable to control group animals in both the sexes. Ophthalmologic observations did not reveal any treatment related alterations.

Neurobehavioural observations performed during 13 weeks of treatment period and 4 weeks of recovery period did not reveal consistent alterations in the neurobehavioural status of the animals pertaining to the treatment. Sensory reactivity, hind limb foot splay measurements, grip strength and motor activity of treatment group animals were comparable to control group animals.

Haematological parameters analysed did not reveal treatment related alterations in both the sexes.

No treatment related alterations were observed in the analysed clinical chemistry parameters.

The observed Spermatological parameters of treatment groups were comparable to control groups.

No treatment related changes were observed in absolute organ weights and relative organ weights of animals treated with the test substance.

Gross pathology and histopathological examination of various organs/tissues from control and high dose (1000 mg/kg b.wt.) group animals did not reveal any treatment related effect.

The NOAEL was set to 1000mg/kg b.w. for both sexes.

Justification for classification or non-classification

Classification, Labelling, and Packaging Regulation (EC) No. 1272/2008

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses up to 1.000 mg/kg body weight upon subacute and subchronic oral exposure in rats. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the seventh time in Regulation (EC) No. 2015/1221.