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Administrative data

Description of key information

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
Remarks:
combined repeated dose and reproduction / developmental screening
Type of information:
experimental study
Adequacy of study:
supporting study
Study period:
28 February 2014 to 3 September 2014
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
guideline study
Qualifier:
according to guideline
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Deviations:
yes
Remarks:
(Microscopic analysis of urine observation). No effect of the study integrity or validity.
GLP compliance:
yes (incl. QA statement)
Limit test:
yes
Species:
rat
Strain:
Wistar
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Animal Breeding Facility, Jai Research Foundation.
- Age at study initiation: At acclimatization rats were 10 to 11 weeks old.
- Weight at study initiation: Body weight variations among the animals were within ± 20% of the mean body weight for each sex.
- Housing: Animals were housed individually in solid floor polypropylene cages (size: 41.0 cm x 28.2 cm x 18.0 cm) except during the mating period where rats were housed 2 rats/cage (one male and one female). Mated female rats caged individually and nesting material was provided at gestation day 14.
- Diet (e.g. ad libitum): ad libitum - Teklad Certified Global 16% Protein Rodent diet manufactured by Harlan, USA.
- Water (e.g. ad libitum): ad libitum - unlimited supply of clean and filtered drinking water.
- Acclimation period: 6 days prior to randomization (observed once daily for clinical signs and twice for mortality and morbidity).

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 21 - 24
- Humidity (%): 66 - 68
- Air changes (per hr): 19 - 20
- Photoperiod (hrs dark / hrs light): 12/12 (fluorescence lighting from approximately 06.00 - 18.00 hours).

IN-LIFE DATES: From: 15 April 2014 to 11 June 2014
Route of administration:
oral: gavage
Vehicle:
other: Reverse osmosis water
Details on oral exposure:
PREPARATION OF DOSING SOLUTIONS: Test formulations were freshly prepared (daily) in reverse osmosis water and dosed within 2 hours of preparation. Stability of test item in vehicle was not performed.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
Analytical Method: Dry empty petridish weighed using calibrated balance (x g). A known quantity of dose formulation added and the petridish along with the test item (dose formulation) were reweighed (y g). Petridish along with sample were kept in an oven (105 ± 5°C for 2 hours). The petridish along with samples were removed and cooled in desiccators. The petridish along with the dried sample were weighed (z g). Petridish along with the dried sample were kept in oven until constant weight achieved.
Duration of treatment / exposure:
Treatment was administered in graduated doses to each dose group. Males were dosed for 6 weeks (i.e. 2 weeks prior to mating, during mating and post-mating) until ca. 80% of the females had delivered. Females were dosed for 2 weeks prior to mating, the variable time to conception, the duration of the pregnancy and at least four days after delivery up to and inclusive of the day before scheduled sacrifice.
Frequency of treatment:
Daily for seven days a week (male and female).
Dose / conc.:
0 mg/kg bw/day (actual dose received)
Remarks:
Group 1 - Control
Dose / conc.:
100 mg/kg bw/day (actual dose received)
Remarks:
Group 2 - low dose
Dose / conc.:
300 mg/kg bw/day (actual dose received)
Remarks:
Group 3 - mid dose
Dose / conc.:
1 000 mg/kg bw/day (actual dose received)
Remarks:
Group 4 high dose
No. of animals per sex per dose:
8
Control animals:
yes
Details on study design:
- Dose selection rationale: Dose formulations were administered daily by oral administration (an exaggerated model of foreseeable human exposure) at a dose volume of 10 mL/kg body weight. The control group animals received the vehicle only. The first day of dosing was designated as day 1.
- Rationale for animal assignment (if not random): Healthy rats were randomly allocated to different groups using an in-house developed validated computer software program (Gad and Weil, 1994).
Positive control:
No
Observations and examinations performed and frequency:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: All animals were observed twice daily for mortality, and twice daily throughout the experimental period for clinical observations.
- Cage side observations: Home Cage Observation: Posture and the absence/presence of convulsions

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical signs were recorded twice daily throughout the experimental period, these includes changes in skin, fur, eye, mucous membranes as well as behavioural pattern and abortion.

BODY WEIGHT: Yes
- Time schedule for examinations: Recorded for males on the first day of dosing and subsequently at weekly intervals. Recorded for females on first day of dosing and at weekly intervals during pre-mating and mating period. During the gestation period, females were weighed on gestation days 0, 7, 14 and 20 and within 24 hours of parturition (day 0 post-partum), and post-partum day 4 (i.e., lactation day 4). Bodyweight of all rats were recorded at the time of sacrifice. Parturition day 0 is defined as the day on which the female littered.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
In males, feed consumption was recorded weekly during pre-mating and post-mating period. In females this was recorded during the pre-mating, gestation and lactation periods at the same day when body weight was recorded.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: Clinical signs were recorded twice daily throughout the experimental period, these includes changes in eye.
- Dose groups that were examined: All dose groups

HAEMATOLOGY: Yes
- Time schedule for collection of blood: All animals at terminal sacrifice
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes (overnight; water allowed)
- How many animals: 64 (32 males; 32 females)
- Parameters examined: Haematology Parameters: Haematocrit, Platelets Count, Haemoglobin, Erythrocyte count (Red Blood Cells), Mean Corpuscular Haemoglobin, Differential leukocyte Count, Mean Corpuscular Haemoglobin, Concentration Reticulocyte Count, Mean Corpuscular Volume, Prothrombin Time, Total leukocyte count (White Blood Cells), Activated Partial Thromboplastine Time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: All the animals at terminal sacrifice
- Animals fasted: Yes (overnight; water allowed)
- How many animals: 64 (32 males; 32 females)
- Parameters examined (Serum used): Alanine Aminotransferase, Total Cholesterol, Albumin, Total Protein, Alkaline Phosphatase, Total Bilirubin, Aspartate Aminotransferase, Triglycerides, Creatinine, Blood Urea Nitrogen, Creatine kinase, Calcium, Gamma Glutamyl Transpeptidase, Albumin:Globulin ratio, Glucose, Potassium, Globulin, Sodium, Inorganic Phosphorous, Chloride, Lactate dehydrogenase, Urea, Bile acids.

URINALYSIS: Yes
- Time schedule for collection of urine: Overnight
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No data
- Parameters examined: Appearance, protein, colour, glucose, volume, ketone, specific gravity or osmolality, blood, pH, bilirubin, urobilinogen.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: In all animals prior to initiation of treatment and once weekly thereafter. Following Functional Observational Battery parameters including Neurobehavioral Observations were performed the day before terminal sacrifice in five animals from G1 to G4 groups.
- Dose groups that were examined: 0 (G1 - Control), 100 (G2 - Low Dose), 300 (G3 - Mid Dose) and 1000 (G4 - High Dose) mg/kg bw/day.
- Battery of functions tested:
Neurobehavioral Observations: Home cage obs: Posture, Convulsions (Clonic/Tonic movement), Handling obs: Handling reactivity, Ease of removing rat from cage, Palpebral closure, Lacrimation, Eye examination, Piloerection, Skin examination, Salivation, Open field obs: Gait, Mobility, Arousal, Vocalisation, Rearing, Respiration, Clonic/Tonic movement, Urination, Defecation, Stereotypy, Bizarre behaviour.
- Functional observation battery: Approach, Touch, Click, Pupil and Tail pinch response, Air righting reflex, Hindlimb foot splay, motor activity and grip strength.

OTHER:
- Pups: After delivery, each litter was examined to determine the number/sex/dead/undersized (runts) pups, stillbirths and the presence of gross anomalies. Pups which died prior to scheduled necropsy were weighed and subjected to post-mortem examination. Individual pup body weight was recorded on lactation days 0 and 4.
Sacrifice and pathology:
GROSS PATHOLOGY: Yes (Necropsy)
- All surviving rats were euthanized by CO2 overdose. Males were sacrificed after ca. 80% of females had delivered. Five females/group were sacrificed on lactation day 5. Females and pups were sacrificed on post-partum day 4. Females which had not delivered by day 25 post-coitum were sacrificed. Pups were sacrificed by intraperitoneal administration of thipentol sodium. All pups were examined macroscopically for structural or pathological changes. Special attention was paid to reproductive organs. The number of corpora lutea and implantation sites was recorded in each dam; Organ Weight and Preservation of Organ/Tissues. At sacrifice, adrenals, brain (cerebrum, cerebellum, and medulla/pons), heart, kidneys, liver, spleen and thymus were excised and weighed from 5 animals/sex/group. Testes and epididymes of all male rats were excised, weighed and preserved).

HISTOPATHOLOGY: No (organs showed no macroscopic lesion)
Statistics:
Statistical analysis was performed using validated software developed at JRF (non-pregnant animals were excluded from analysis). Data such as body weight, body weight change, feed consumption, uterine data and litter parameters were analyzed using Bartlett‟s test of homogeneity of variance. If not significant then analysis of variance (ANOVA) was performed. If ANOVA was significant then Dunnett‟s multiple comparison tests was performed. If Bartlett‟s test of homogeneity was significant that data was subjected to Student ‘t’ test. Data such as mortality rate, gestation index, parturition index, pregnancy rate, pups survival index, live birth index and fertility index were analyzed using Chi-Square test.
Clinical signs:
no effects observed
Mortality:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Decrease in bw change during treatment in male high dose group. Decrease in bw change in male treatment group (treatment days 1-43) & female treatment group (treatment days 1-8 & 1-15). Treatment related decrease in terminal bw in male high dose group.
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Statistically significant decrease in food consumption in high dose group males (treatment days 22-29, 29-36 & 36-43, compared to control group.
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Haematological findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant treatment related increases in MCV, MCH, MCHC, retic values and decrease in RBC values in high dose female animals, compared to control group.
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant treatment related decrease in glucose in mid and high dose male animals and an increase in albumin in high dose group male animals, compared to control group.
Urinalysis findings:
effects observed, treatment-related
Description (incidence and severity):
Statistically significant treatment related pH increase in all group males, and an increase in urine volumes in high dose group females, compared to control group.
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Description (incidence and severity):
Decrease absolute weight of thymus in male mid & high dose.Increase relative weight of liver, heart, spleen & kidneys in male high dose,spleen in male mid dose & liver, spleen & kidneys in female high dose.Increase absolute weight of liver in female high dose.
Gross pathological findings:
no effects observed
Histopathological findings: non-neoplastic:
not examined
Histopathological findings: neoplastic:
not examined
Details on results:
BODY WEIGHT AND WEIGHT GAIN:
Males: No relevant change in body weight in test item treated groups, compared with the control group. Statistically significant decrease in body weight change (%) throughout treatment days (except days 15-22) in high dose group, compared to control group. Statistically significant decrease in body weight change (%) throughout treatment days 36-43 in mid dose group, compared to control group. Statistically significantly decrease in overall body weight change (%) during treatment days 1-43 in all groups treated with test item, compared to control group.

Females: Statistically significant decrease in body weight change (%) during pre-mating period (days 1-8 & 1-15) in all groups treated, compared to control group. During gestation and lactation period, no difference in mean body weight were observed in all groups treated, compared with control group.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Males: No test item related effect was observed in feed consumption in low dose group animals, compared with the control group. A statistically significant decrease in feed consumption during treatment (days 29-36) in the mid dose group was observed, compared to control group. In the high dose group, a statistically significant decrease in feed consumption during treatment (days 22-29, 29-36 & 36-43) was observed, compared to control group. The observed decrease in feed consumption in high dose males is considered treatment related due to parallel decrease in body weight change (%) across treatment days.

Females: Feed consumption were comparable throughout the treatment period in all groups, compared with control group.

HAEMATOLOGY:
Males: All haematology parameters were comparable among test item treated and concurrent control group.

Females: Treatment related statistically significant increase in MCV, MCH, MCHC, retic values and decrease in RBC values were observed in high dose animals, compared to control group. All remaining parameters were comparable between test item treated groups and the control group.

CLINICAL CHEMISTRY: Male: Treatment related statistically significant decrease in glucose in mid and high dose animals and an increase in albumin in high dose group animals, compared to control group. Female: All clinical chemistry parameters were comparable among test item treated animals and the concurrent control group.

URINALYSIS: Male: All group animals showed statistically significant treatment related pH value increase, compared to control group. Females: High dose group animals had statistically significant increase in urine volume, compared to control group. All remaining parameters were comparable between test item treated groups and control group.

NEUROBEHAVIOUR:
- Home Cage Observation: Normal posture was observed in all rats from treated and vehicle control groups. All rats revealed normal postures. Clonic and tonic movements were not observed in home cage during weekly NBO.
- Observation during Removal and Handling: Did not reveal any treatment related abnormalities. All rats were of normal behaviour during removal and handling and none showed lacrimation, salivation or piloerection. The eyelids were wide open in all rats, and eye and skin examination did not reveal any abnormality.
- Open Field Observation: All vehicle control and treated group rats showed normal gait and mobility. Respiration examination did not reveal any abnormality. Clonic and tonic movements, vocalization, stereotypy and bizarre behaviour were absent in all groups. No alterations were observed in rearing, urination and defecation count in male or female rats of treated groups, compared with vehicle control.
- Sensory Reactivity Observations: Sensory reactivity parameters in all treated groups were comparable to vehicle control.
- Hindlimb Foot Splay: Mean hindlimb foot splay values of male and female treated groups were comparable to the vehicle control.
- Motor Activity: Motor activity data of all treated groups were comparable with vehicle control.
- Grip Strength: Mean grip strength values of treatment groups were comparable with vehicle control group.

ORGAN WEIGHTS:
Males: Statistically significantly decrease in absolute weight of thymus in mid and high dose group, compared to control group.
- Statistically significant increase in relative weight of liver, heart, spleen and kidneys in high dose, compared to control group.
- Statistically significant increase in relative weight of spleen in mid dose, compared to concurrent control group.
Females: Statistically significant increase in absolute weight of liver animals of high dose group, compared to control group.
- Statistically significant increase in relative weight of liver, spleen and kidneys in high dose, compared to concurrent control group.

GROSS PATHOLOGY
Parental animals, no macroscopic findings were observed following external and internal examination of terminally sacrificed animals. In all group animals, no manifestation of lesions of pathological significance was observed.

HISTOPATHOLOGY: Not examined as no organs showed macroscopic lesion.

OTHER FINDINGS:
Fertility Data: No relevant test item-related effects were observed on male and female fertility index, or female gestation index, compared with the control group. Pregnancy rate and parturition index were unaffected by treatment. The duration of the gestation period and pre-coital interval in test item treated group were comparable to the control group. No treatment related difference was observed with respect to pre and post-implantation loss and post-natal loss in test item treated groups, compared to the control group.

Pup Data
- Mortality: No relevant test item related difference in pup mortality was observed during the lactation period in pups in test item treated groups, compared to the control group.
- Clinical Sign: No relevant test item related clinical signs were observed in pups of test item treated dose group, compared to the control group (exception: pup No. 6 of dam No. 29, low dose group, had weakness on lactation day 3).
- Body Weight: No treatment related difference in pup body weight was observed on lactation days 0 and 4 in test item treated groups, compared to the control group.
- Live Birth and Survival Index: No treatment related difference was observed in mean number of live pups, live birth index and postnatal index in test item treated groups, compared to the control group.
- Litter Size and Sex Ratio: No treatment related differences were observed in mean male, female and total (male + female) litter size and sex ratio in test item treated groups, compared with the control.
Macroscopic Findings:
- Pups: External and internal examination of terminally sacrificed pups and found dead pups did not reveal any lesions of pathological significance. However, some internal non-treatment related lesions (minimal congestion in liver) were observed in found dead pups: Dam No. 28 (Pup No.12), Dam No. 58 (Pups No. 1 and 2), Dam No. 62 (Pup No. 11).
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:
body weight and weight gain
clinical signs
mortality
organ weights and organ / body weight ratios
Key result
Critical effects observed:
yes
Lowest effective dose / conc.:
1 000 mg/kg bw/day (actual dose received)
System:
hepatobiliary
Organ:
kidney
liver
spleen
Treatment related:
yes
Dose response relationship:
yes
Relevant for humans:
yes
Conclusions:
On the basis of results of the present study following dose levels are proposed for main study:

Option I
Low Dose: 100
Mid dose: 300
High Dose: 1000

Option II
Low Dose: 100
Mid dose: 300
High Dose: 900

Option III
Low Dose: 212.5
Mid dose: 425
High Dose: 850
Endpoint conclusion
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
1 000 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Quality of whole database:
Klimisch 1

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

Justification for classification or non-classification

The results from the studies indicate that this substance will not be classified for repeat does toxicity.