Reaction mass of [[(2-hydroxyethyl)imino]dimethylene]bisphosphonic acid, sodium salt and 4-(Phosphonomethyl)-2-hydroxy-2-oxo-1,4,2-oxazaphosphorinane, sodium salt

Repeated dose toxicity: oral

Administrative data

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

7 April 2020 to 21 May 2021

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2021

Report date:

2021

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

25th June 2018

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

EC Number:

939-513-8

Molecular formula:

C4H12NNaO7P2, C4H11NNa2O7P2 and C4H10NNa3O7P2

IUPAC Name:

tetrasodium 4-(hydrogen phosphonatomethyl)-2-oxo-1,4,2λ⁵-oxazaphosphinan-2-olate hydrogen {[(hydrogen phosphonatomethyl)(2-hydroxyethyl)amino]methyl}phosphonate

Test animals

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: SPF breeding, VELAZ s.r.o., Praha – Lysolaje, Czech Republic, RČH CZ 11760500
- Females: nulliparous and non-pregnant: yes
- Age at study initiation: 6-7 weeks on arrival
- Weight at study initiation: Males approximately 370 ± 30g; females approximately 213 ± 10g
- Fasting period before study: No
- Housing: The study was performed in a SPF (Specified Pathogen Free) animal house of CETA in SPF conditions according to internal SOP No. 12. Animals were housed in the animal room, 2 rats of the same sex in one plastic cage (polycarbonate cages for rats TECNIPLAST, type 1291H, Italy)
- Diet (e.g. ad libitum): Ad libitum
- Water (e.g. ad libitum): Ad libitum
- Acclimation period: 19 days

DETAILS OF FOOD AND WATER QUALITY: Complete pelleted diet for rats – Altromin 1321 (Altromin Spezialfutter GmbH & Co. KG, Germany). Water quality corresponded to Regulation No. 252/2004 Czech Coll. of Law, Ministry of Health. The standard pelleted laboratory animal diet is analysed for nutrients (once a year) and bacteriological contaminants every 2 months on a regular basis. Results are retained in the CETA archives. Certificates of drinking water analysis (performed twice a year) are retained in the CETA archives. Analysis of diet and water did not reveal any findings that could affect study integrity.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +3°C
- Humidity (%): 30-70 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 hours light / 12 hours dark

IN-LIFE DATES: From: 22 April 2020 To: 9 September 2020

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

Concentration level 18.3 mL/ 100 mL: 1000 μL water solution was added into a 100 mL volumetric flask, weigh and diluted to volume by UPW. 500 μL water solution was added into a 50 mL volumetric flask and diluted to volume by UPW. Solution was shaked and analysed by ICP-OES.

Concentration level 1.8 mL/ 100 mL: 1000 μL water solution was added into a 100 mL volumetric flask, weigh and diluted to volume by UPW. Solution was shaked and analysed by ICP-OES.

The test item was weighed into a glass beaker and the beaker was replenished by aqua pro iniectione was stirred by magnetic stirrer (250 rpm) for 30 minutes. For each dose level concentration, the solution was prepared separately. The application forms were prepared daily just before administration. The administration of the test item to animals was performed during the two hours after preparation of application form. The stirring of solutions continued during administration.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The stability and the homogeneity of application form were determined in CETA analytical laboratories (Analytical Group III – ICP-OES). Stability and homogeneity were determined by means of measuring of a concentration P (mg/kg) in the test item application form. The determination of elements was performed by ICP OE spectroscopy.

Concentration level 18.3 mL/ 100 mL: The samples were taken after 30 min of mixing by magnetic stirrer (250 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from all places.

Concentration level 1.8 mL/ 100 mL: The samples were taken after 30 min of mixing by magnetic stirrer (250 rpm) from 3 given places - the bottom, the middle and the surface of the beaker content. Two samples were taken from all places.

Duration of treatment / exposure:

90 days

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

No. of animals per sex per dose:

10 males and females per dose, with an additional 6 males and females in the control and high dose group for the recovery phase.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale:
- Rationale for animal assignment (if not random): Random
- Fasting period before blood sampling for clinical biochemistry: 18 hours before blood collection
- Rationale for selecting satellite groups: To investigate recovery in case of suspected adverse findings at the end of the treatment period.
- Post-exposure recovery period in satellite groups: 28 days

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations: This observation was made in order to record possible clinical effects after application and all changes in behaviour of animals. So it was done after application at the same time every day (11.00 – 13.00 pm.). Animals were observed in natural conditions in their cages.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Before first dose and then weekly. Firstly, behaviour of animals in the cage was monitored: piloerection, posture, eyes (pupils), breathing, tonic or clonic movements, stereotypes or bizarre behaviour. Secondly, animals were removed from the cage for further examination of reaction to handling, elasticity of skin, colour of visible mucous membranes, salivation, lacrimation, cleanliness of fur around foramina.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

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

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Yes

WATER CONSUMPTION: Yes
- Time schedule for examinations: The drinking water consumption was recorded. The mean values in groups (water consumption per animal and per day) were calculated for each week of the study: total initial volume – total remainder of water = group consumption → group consumption/number of animals/number of days = mean water consumption per animal and per day.

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: During the first week of application and at the end of the administration and recovery periods. Both eyes were examined by means of an ophthalmoscope without chemical dilatation. The following examinations were made: visual acuity, eyelids, palpebral fissure, eyelash, bulb, lacrimal apparatus, conjunction, sclera, cornea, iris, pupil, lens, eye ground.
- Dose groups that were examined: The ophthalmologic examination was performed in all animals at the beginning of the study. An ophthalmologic examination was also performed at the end of the study in animals of the control group, high dose group and in both satellite groups (control and 1000 mg/kg bw/day) the. No changes related to application of the test item were observed in the 1000 mg/kg bw/day group (highest dose level) group; therefore, animals of lower dose groups were not examined.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: Day 91 (day after completion of 90 days of exposure) and Day 119 (day after completion of the recovery period)
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes, 18 hours
- How many animals: All animals
- Parameters checked in table [No.1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: Day 91 (day after completion of 90 days of exposure) and Day 119 (day after completion of the recovery period)
- Animals fasted: Yes, 18 hours
- How many animals: All animals
- Parameters checked in table [No.2] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: Day 90 (last day of the treatment period) and Day 118 (last day of the recovery period)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: No
- Parameters checked in table [No.3] were examined.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: End of administration and recovery periods
- Dose groups that were examined: All groups
- Battery of functions tested: sensory activity / grip strength / motor activity / other: During functional examination, the sensory reactivity on auditory, visual, proprioceptive stimuli (reaction to approximation, to contact point, to noise and to pain) and pupillary reflex, were evaluated and motor activity assessment (number of upstanding, emiction and defecation within 3 minutes) was conducted. Moreover, the individual observations of grip strength (grip power in Newton) were performed using dynamometer. Measurements were made on: 1) pectoral legs, 2) pelvis legs.


OTHER: Blood samples from males and females were assessed for serum levels of thyroid hormones (thyroxine - T4, triiodothyronine - T3 and rat Thyroid Stimulating Hormone -TSH) by ELISA kit + supplier of reagents. The examination of thyroid hormones was conducted in animals of control groups, all treated groups and recovery groups were analysed.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see table 4), on 91st (main group); 119th (satellite group) day of study. The absolute weights of liver, kidneys, adrenals, testes or ovaries, epididymides or uterus, prostate gland, thymus, spleen, brain, pituitary gland, thyroid gland and heart in control, all treated and satellite groups of animals were recorded. Afterwards the somatic indexes - SI (= relative weight of organ) were computed according to the following formula: SI = weight of organ x 100/ body weight.

HISTOPATHOLOGY: Yes (see table 4). In the study the full histopathology of the preserved organs and tissues was performed for all high dose, control animals, recovery animals and animals with macroscopical changes.

Statistics:

For statistical evaluation the software Statgraphic ® Centurion (version XVII, USA) was used.
Results that were statistically significant were indicated using the probability values as follows: p<0.05 (significant) and p≥0.05 (not significant)

The parametric tests were used for: body weight; haematology parameters: haematocrit, haemoglobin concentration, mean volume of erythrocyte, differential leucocyte count, monocytes, granulocytes, lymphocytes, values of blood coagulation: fibrinogen, partial thromboplastin time, prothrombin time; glucose, cholesterol total, inorganic phosphorus, urea, protein total, aspartate aminotransferase, alanine aminotransferase, albumin, alkaline phosphatase, calcium, creatinine, sodium, potassium, chloride, bile acids, triglycerides, cholinesterase, LDL and HDL; urinalysis (volume, pH); biometry of organs (absolute and relative weight)
As the first step the test for normality (Shapiro-Wilk test) was performed. If data were not normally distributed the transformation of data was done (Box-Cox transformation). If data were normally distributed the variance check was performed (Levene´s test) to verify if standard deviations within each group were equal. One-Way ANOVA (probability level 0.05) was used to detect whether there are any significant differences amongst the means. If significant differences were found, then the post hoc statistical testing was performed (Fisher's least significant difference - LSD test). If the presence of outliers was noted, the appropriate non-parametric test (Kruskal-Wallis Test, Mann-Whitney test) was chosen.

Non-parametric tests were used for: haematology parameters with non-continuous distribution (total erythrocyte count, total leucocyte count, total platelets count)
At first the Kruskal-Wallis test for the comparison of the measured effect in all treatment groups with the vehicle control group was used as global test, and then the two-groups Mann-Whitney test (probability level 0.05) was applied.

Results and discussion

Results of examinations

Clinical signs:

no effects observed

Description (incidence and severity):

There were no clinical signs in female animals.

The only observation was soft excrement in males of the high dose group during weeks 12 and 13 of treatment.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

One single male rat from the mid-dose group died during the 5th week due to an intubation error. No other deaths occurred.

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Males
The statistical analysis was performed for necropsy body weights and body weights of the 13th week. Statistically significant differences were not found.
The body weights of males in all treated groups were similar compared to those of the control group.
The necropsy body weights of males in all treated groups were similar compared to those of the control group.
Body weight increments were similar in males of treated groups compared to those of the control group for the whole application period.

Satellite males
The statistical analysis was performed for necropsy body weights and body weights of the 17th week. Statistically significant differences were not found.
Increased body weights in males of the treated group were recorded from the 9th week to the end of the study compared to the control group. The necropsy body weights of treated males were similar to the control group males.
Body weight increments were similar in males of the treated group compared to the control group for the whole application period and during the recovery period.During the 16th week of the study, males of the control and treated groups lost weight (negative body weight increment). The weight loss was not statistically significant and not considered to be adverse

Females
The statistical analysis was performed for necropsy body weights and body weights of the 13th week. Statistically significant differences were not found.
The body weights and necropsy body weights were similar in females of treated groups compared to the controls for the whole application period.
The body weight increments were variable in females of treated and control groups during the whole application period. During the 9th week of the study, females of the 350 mg/kg bw/day group lost weight (negative body weight increment). The weight loss was not statistically significant or considered to be adverse.

Satellite females
The statistical analysis was performed for necropsy body weights and body weights of the 17th week. Statistically significant differences were not found.
The body weights and necropsy body weights were similar in females of the satellite treated group compared to the satellite control for the whole application period and recovery period.
The body weight increments were similar in females of treated and control groups during the whole application and recovery period. During the 9th and 15th week of the study the satellite control females and during the 16th week of the study the satellite treated females lost weight (negative body weight increment). The weight loss was not statistically significant and not considered to be adverse.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption for males and females was similar for control and treated animals during the treatment and recovery periods.

Food efficiency:

no effects observed

Description (incidence and severity):

Food conversion for males and females was variable but appropriate for the species, sex and age of the animals in the test.

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

no effects observed

Description (incidence and severity):

There were no treatment-related effects on drinking water consumption throughout the treatment and recovery periods.

Ophthalmological findings:

no effects observed

Description (incidence and severity):

No changes related to application of the test item were observed in the 1000 mg/kg bw/day group (highest dose level) group; therefore, animals of lower dose groups were not examined.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
For parameters relating to haemocoagulation statistically significantly prolonged APTT in males at the dose level 1000 mg/kg bw/day HEBMP-H was recorded.
For red blood cell components the percentage of reticulocytes in blood in males at the dose level 100 mg/kg bw/day HEBMP-H was statistically significantly increased and on the contrary in males at the dose levels 350 and 1000 mg/kg bw/day HEBMP-H the percentage of reticulocytes in blood was decreased.
For the white blood cell components, the values were not affected by the administration of the test item.
All values of haematological parameters were within historical control limits.

Satellite males
Statistically significantly shortened PT in satellite treated males was detected (in a range of historical control).
Other haematological parameters of satellite treated males were similar with the satellite control males.

Females
The following statistically significant differences were registered for haemocoagulation parameters: prolonged APTT in females at the dose level 1000 mg/kg bw/day HEBMP-H.
For red blood cell components the percentage of reticulocytes in blood of females of all treated groups was statistically significantly decreased.
For the white blood cell components, the values were not affected by the administration of the test item.
All values of haematological parameters were within historical control limits.

Satellite females
For red blood cell components statistically significantly increased values of haemoglobin and decreased percentage of reticulocytes in blood in satellite treated females were recorded.
For white blood cell components a change in differential leucocyte counts (percentage of basophils statistically significantly increased in satellite treated females) was recorded.
Other haematological parameters of satellite treated females were not statistically significantly different to the satellite control females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

Males
Statistically significantly increased cholinesterase activity in males at all dose levels HEBMP-H was recorded (without dose dependence). In males of the 100 and 1000 mg/kg bw/day HEBMP-H groups glucose concentration was statistically significantly decreased compared to the control group. There was also a statistically significantly increased albumin concentration and AST activity recorded for the 1000 mg/kg bw/day HEBMP-H group. The urea concentration in males of the dose level 1000 mg/kg bw/day HEBMP-H was statistically significantly decreased.
Differences in values of other biochemical parameters did not show any statistically significant changes.
All values of biochemical parameters were within historical control limits.

Satellite males
Statistically significantly increased cholesterol total, triglyceride and LDL cholesterol concentrations were recorded in treated males. A statistically significantly decreased concentration of inorganic phosphorus in satellite treated males was recorded.
Other biochemical parameters of satellite treated males were similar to the satellite control males.
All values of biochemical parameters were within historical control limits.

Females
Statistically significantly increased concentrations of cholesterol total, bilirubin total and LDL cholesterol at the dose levels 100 and 1000 mg/kg bw/day HEBMP-H were recorded. Bilirubin total concentration in females of the 350 mg/kg bw/day HEBMP-H group was increased (without statistical significance). Statistically significantly increased triglyceride and albumin concentrations in the 1000 mg/kg bw/day HEBMP-H group were detected. The activity of ALT in females of the 350 mg/kg bw/day HEBMP-H group was decreased with statistical significance. Statistically significantly decreased activity of AST in females of the 350 and 1000 mg/kg bw/day HEBMP-H groups was recorded. Statistically significantly increased concentration of inorganic phosphorus in females of the 350 and 1000 mg/kg bw/day HEBMP-H groups was detected.
Differences in values of other biochemical parameters did not show any significant changes.
All values of biochemical parameters were within historical control limits.

Satellite females
Statistically significantly increased bilirubin total concentration in satellite treated females was recorded.
Other biochemical parameters of satellite treated females were similar to the satellite control females.
All values of biochemical parameters were within historical control limits.

Endocrine findings:

no effects observed

Description (incidence and severity):

Blood samples from all groups of males and females were assessed for serum levels of thyroid hormones (T4, T3 and TSH).

Males
Statistically significant differences in the concentrations of T4, T3 and TSH hormone were not detected.
Mean concentrations of T3 and TSH hormone were similar for treated and control males. A slightly decreased concentration of T4 hormone in males of the 1000 mg/kg bw/day HEBMP-H group was recorded.

Satellite males
Statistically significant differences in the concentrations of T4, T3 and TSH hormone were not detected.
Mean concentrations of T3 and TSH hormone were similar for satellite treated an satellite control males. A slightly decreased concentration of T4 hormone in satellite treated males compared with satellite control males was recorded.
All values of hormone concentration were within historical control limits.

Females
Statistically significant differences in the concentrations of T4, T3 and TSH hormone were not detected.
Mean concentrations of T4 and T3 hormone were similar for treated and control females. A decreased concentration of TSH hormone in females of the 100 and 1000 mg/kg bw/day HEBMP-H groups was recorded.

Satellite females
Statistically significant differences in the concentrations of T4, T3 and TSH hormone were not detected.
Mean concentrations of T4 and T3 hormone were similar in satellite treated and satellite control females. A decreased concentration of TSH hormone in satellite treated females was recorded.
All values of hormone concentration were within historical control limits.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of animals with findings in urine is expressed in numeric form and arranged in sequence of the dose levels 0-100-350-1000 in the main groups and 0S-1000S in the satellite groups further in the text.
Statistical evaluation was performed for pH and volume of urine.

Males
A statistically significant decrease in volume of urine was detected in males of all treated groups compared with the control group.
A statistically significantly decreased pH of urine was detected in males of the 350 and 1000 mg/kg bw/day HEBMP-H groups compared with the control group.
A change of colour of urine (yellow) was observed in 0-1-1-7 males. Presence of protein in urine was recorded for 0-4-7-4 males. Presence of blood in urine was observed in 1-2-6-4 males. Presence of leukocytes in urine was observed in 1-4-6-9 males.

Satellite males
Statistically significantly decreased pH of urine was detected in satellite treated males.
Presence of protein in urine was recorded in 2-2 males. Presence of leukocytes in urine was observed in 6-4 males.
Presence of protein, blood and leucocytes in urine was normal for the sex and age of animals used in experiments.

Females
A statistically significantly decreased volume of urine was recorded for females of the 350 mg/kg bw/day HEBMP-H group. At 1000 mg/kg bw/day HEBMP-H the decrease in volume of urine was not statistically significant compared with the control group. Statistically significantly decreased pH of urine was detected in females of the 350 mg/kg bw/day HEBMP-H group.
Change of colour of urine (yellow) was observed in 0-0-2-2 females. Presence of blood in urine was recorded in 0-2-1-2 females. Presence of leukocytes in urine was observed in 0-0-0-2 females. The presence of blood and leucocytes in urine was appropriate to the sex and age of the animals used in experiments.

Satellite females
The volume of urine was statistically significantly increased in satellite treated females compared with satellite control females.
The presence of protein in 1-0 female, presence of blood in 0-1 female and presence of leucocytes in 1-0 female were detected (finding appropriate to the age of the animals).

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Reactions to touch, noise, pain, pupillary reflex and upstanding of treated animals were the same as in the control group. Emiction and defecation of treated animals was without important changes. The examination of grip strength of treated males was without important changes. The mild variability was adequate to species, sex and age of animals used in experiments.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

Absolute organ weights

Males
Absolute weights of organs of treated males (main and satellite) were comparable with the control males. No statistically significant changes of absolute weight of organs were recorded.

Females
The absolute weight of brain was statistically significantly decreased in all treated groups compared to the control group. Non-statistically significant changes in the absolute weight of uterus of females in all treated groups were detected. This finding probably related to the oestrus cycle of females.
Absolute weights of other organs of the treated females were comparable with control females.

Satellite females
Absolute weights of organs of the satellite treated females were comparable with satellite control females. No statistically significant changes in the absolute weight of organs were recorded.

Relative organ weights

Males
Statistically significantly decreased relative weight of kidneys was recorded in males of the 100 mg/kg bw/day HEBMP-H group and on the contrary in males of the 350 mg/kg bw/day HEBMP-H group the relative weights of kidneys were statistically significantly increased.
Relative weights of other organs of treated males were comparable with control males.

Satellite males
Statistically significantly decreased relative weight of kidneys was recorded in the satellite treated males compared with the satellite control males.
Relative weights of other organs of treated males were comparable with control males.

Females
The relative weight of brain was statistically significantly decreased for all treated groups compared to the control group. Non-statistically significantly decreased relative weight of uterus in females in all treated groups was detected. This finding probably related to the oestrus cycle of females.
Relative weights of other organs of treated females were comparable with control females.

Satellite females
Statistically significantly decreased relative weight of pituitary gland was recorded in satellite treated females.
Relative weights of other organs of satellite treated females were comparable with satellite control females.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

The incidence of affected animals is expressed in numerical form and according to the dose level sequence 0-100-350-1000 mg/kg bw/day in the main groups and 0S-1000S mg/kg bw/day in the satellite groups.

Males
In 10-7-8-8 males no pathological findings were recorded during necropsy. Testicle enlarged or reduced in 0-2-1-2 males, epididymis reduced in 0-0-0-1 male were observed. Fusion of the left kidney and spleen in 0-1-0-0 male was detected; this finding was incidental and was not related to the test item administration.

Satellite males
In 6-6 males no macroscopic findings were recorded during necropsy.

Females
In 10-10-10-9 females no pathological findings were recorded during necropsy (Table 41). Cyst on left ovary in one female at the dose of 1000 mg/kg bw/dayHEBMP-H was observed.
Non-pathological finding – uterus dilatation in 6-2-5-3 females was detected.

Satellite females
In 6-6 females no macroscopic findings were recorded during necropsy.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

The histopathological examination of organs in all groups showed only sporadic microscopic changes and these changes were not related to the test item treatment.

Histopathological findings: neoplastic:

not examined

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

other: active acid

Sex:

male/female

Remarks on result:

other: No adverse effects at doses up to 1000 mg/kg bw/day

Target system / organ toxicity

Key result

Critical effects observed:

no

Applicant's summary and conclusion

Conclusions:

In the 90-day oral repeated dose toxicity study with 28-day recovery period with HEBMP (1-3Na), conducted according to OECD Test Guideline 408 and in compliance with GLP, the concluded NOAEL for systemic toxicity was at least 1000 mg active acid/kg bw/day based on no treatment-related adverse effects up to the highest dose tested of 1000 mg active acid/kg bw/day.