Phosphonic acid, monoethyl ester, sodium salt (1:1)

Endpoint summary

Description of key information

Subacute (28 days, rat): NOAEL oral (female, male) > 1000 mg/kg bw/day
Subchronic (90 days, rat): NOAEL oral (female, male) > 1000 mg/kg bw/day
No data are available for repeated dose toxicity after dermal exposure and inhalation, respectively.

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:

30 Jan - 01 Jun 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:

(adopted 1995)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2008

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

(adopted 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Republique Francaise, Premier Ministre, Groupe Interministeriel Des Produits Chimiques, Paris Cedex, France

Limit test:

no

Species:

rat

Strain:

other: Wistar (AF) RJ: WI (IOPS AF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 7 weeks
- Weight at study initiation: 223 - 281 g (males), 161 - 206 g (females)
- Fasting period before study: animals were fasted overnight for diet only before blood sampling and for diet and water before urine sampling
- Housing: individual in suspended stainless steel, wire mesh cages
- Diet: certified and irradiated rodent pellet diet "A04C" (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 13 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15 (average, not monitored)
- Photoperiod (hrs dark / hrs light): (12 / 12)

IN-LIFE DATES: From: 30 Jan To: 01 Mar 2001

Route of administration:

oral: gavage

Details on route of administration:

not specified

Vehicle:

water

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was sufficiently soluble in water.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dosing solutions were sent for analysis to RCC Ltd, Environmental Chemistry and Pharmanalytics Division, Switzerland. Homogeinity of the test substance was verified at the lowest and highest doses. Concentrations of the test item in the vehicle were verified for each dose tested on the three first preparations. Stability of the test substance was demonstrated during the study at the lowest and highest dose.

Results of analysis
The results obtained for homogeneity, stability and concentration were within the expected ranges except the lowest concentration of the first preparation which was 111% of the nominal concentration (e.g. test 1: homogeneity: 91 - 98 and 103 - 105% of nominal concentration for 0.2 and 200 g/L, respectively; concentration: 95 and 104% of nominal concentration for 0.2 and 200 g/L, respectively; stability: 90 -95 and 92 - 104% of nominal concentration for 0.2 and 200 g/L, respectively; test 2: homogeneity: 111.2 - 112.5 and 90 - 96.2% of nominal concentration for 20 and 200 g/L, respectively; concentration: 90.4 - 111 and 93 - 102% of nominal concentration for 20 and 200 g/L, respectively).

Duration of treatment / exposure:

28 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Remarks:

(adjusted for purity)

Dose / conc.:

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

Remarks:

(adjusted for purity)

Dose / conc.:

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

Remarks:

(adjusted for purity)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: The dose levels were selected on the basis of an acute oral study
- Rationale for animal assignment (if not random): random
- Fasting period before blood sampling for clinical biochemistry: animals were fasted overnight
- Rationale for selecting satellite groups: no satellite groups included

Positive control:

not included

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations checked for ill-health including blood or loose feces were included. Detailed physical examinations were performed at least weekly during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice during the acclimatisation period, on the first day of dosing and at weekly intervals thereafter and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation period and during week 4
- Dose groups that were examined: control and high-dose group

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once during the study on Day 24 or 25
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all survivng animals
- Parameters checked: red blood cell count (RBC), haemoglobin, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC) and differential count evaluation and platelet count (PLT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once during the study on Day 24 or 25
- Animals fasted: Yes
- How many animals: all suriving animals
- Parameters checked: total bilirubin (TBIL), glucose (GLUC), urea, creatinine (CREA), total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, and aspartate aminotransferase, alanine aminotransferase (ALAT), alkaline phosphatase (AP) and gamma-glutamyltransferase (GGT) activities were assayed on plasma samples, total protein and albumin concentrations

URINALYSIS: Yes
- Time schedule for collection of urine: on Days 29, 30 or 31, prior to sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: volume, pH, urinary refractive index (RI), glucose (GLU), bilirubin (BIL), ketone bodies (KET), occult blood (OCBL), protein (PRO) and urobilinogen (UOR), microscopic examination of the urinary sediment ( presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation period and during week 4
- Functions tested: Grasping reflex, righting reflex, corneal reflex, pupillary reflex, auditory reflex and head shaking reflex

Sacrifice and pathology:

SACRIFICE: All surviving animals from all dose groups were sacrificed on study Days 29, 30 or 31. An approx. equal number of animals was randomly distributed amongst all groups.

GROSS PATHOLOGY: Yes
- Organs weighed: Adrenal glands, brain, epididymides, heart, kidneys, liver, ovaries, pituitary gland, prostate, spleen, testes, thymus, thyroid (with parathyroid) and uterus were weighed fresh at scheduled sacrifice only. Paired organs were weighed together.

HISTOPATHOLOGY: Yes
- Dose groups: all animals of the control and high-dose group, all decedents in the low- and mid-dose groups (lung, liver, thyroid gland,kidney and all macroscopic findings were also examined in the low- and mid-dose groups)

The following organs were sampled for pathological examinations:
Adrenal gland, Aorta, Articular surface (femoro-tibial), Bone (sternum), Bone marrow (sternum), Brain, Epididymis, Esophagus, Eye and optic nerve, Exorbital (lachrymal) gland, Harderian gland, Heart, Intestine (duodenum, jejunum, ileum,cecum, colon, rectum), Kidney, Larynx, Liver, Lung, Lymph nodes (submaxillary, mesenteric), Mammary gland, Ovary, Pancreas, Pituitary gland,Prostate, Sciatic nerve, Seminal vesicle, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Submaxillary (salivary) gland, Testis,Thymus,Thyroid (with parathyroid), Tongue, Trachea, Urinary bladder, Uterus (including cervix), Vagina

Statistics:

Means and standard deviations were calculated for the analysed parameters for each sex separately for each group at each time period. All tests were performed at 1 and 5% significance levels. Statistical analysis were performed using ANOVA. Homogeinity of variance assumption was examined by Levene´s Test. If Levene's test indicated lack of homogeneity of variance (p<0.05), robust linear regression methods were used to test all treatment effects. The robust regression methods use variance estimators that make no assumptions regarding homogeneity of variance or normality of the data. They were used to test for overall treatment group differences (Wald chi-square tests), followed by individual Mests for exposed vs. control group comparisons when the overall treatment effect was significant. If Levene's test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS® 6.12 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to control via Dunnett's test.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

At 100 and 300 mg/kg bw/day, two and one animals, respectively, showed ocular discharge. At 300 mg/kg bw/day, two and one animals showed nasal discharge and abnormal dentition, respectively. However, since there was no dose-response relationship, the effects were considered incidental.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1000 mg/kg bw/day: one female died
300 mg/kg bw/day: one female died

The two females from the 300 and 1000 mg/kg bw/day group died during anesthesia for blood sampling on Day 24 and 25, respectively.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No effects on body weight were observed and the weight gain was comparable among the groups (mean weight gain weeks 0 -4: males: 148.9 ± 20.5, 142.0 ± 21.9, 147.5 ± 18.9 and 137.6 ± 14.0; females: 54.3 ± 10.9, 56.3 ± 8.2, 55.6 ± 13.5 and 58.7 ± 5.8 for control, low-, mid- and high-dose animals, respectively).

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Food consumption was unaffected by the test substance (mean food consumption weeks 0 - 4: males: 27.7 ± 1.7, 27.4 ± 1.3, 27.8 ± 1.8 and 27.1 ± 1.4; females: 19.6 ± 1.6, 19.1 ± 1.5, 19.5 ± 1.5 and 19.3 ± 0.7 for control, low-, mid- and high-dose animals, respectively).

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related abnormalities at the ophthalmological examination.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

100 and 300 mg/kg bw/day: decreased RBC in males

When compared with the control group, there were no treatment-related changes in hematological parameters in either sex. The lower RBC observed in males at 300 and 100 mg/kg/day (8.697 ± 0.302, 8.293* ± 0.329, 8.318* ± 0.257 and 8.469 ± 0.345 for control, low-, mid- and high-dose group, respectively) were considered to be of no toxicological significance because this change was slight and not seen at 1000 mg/kg bw/day and the RBC level was comparable in females (8.301 ± 0.318, 8.313 ± 0.569, 8.031 ± 0.527 and 8.226 ± 0.381 for control, low-, mid- and high-dose group, respectively). The other examined parameter did not reveal treatment-related changes and were comparable among the groups.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: decreased Cl (males)
300 mg/kg bw/day: decreased ASAT (males) and decreased Na (females)

When compared with the control group, there were no treatment-related changes in clinical chemistry parameters in either sex. Any statistical significant differences were considered to be of no toxicological significance because they were either not dose-related or the values were within the normal range of historical control (please refer to table 1).

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

300 and 1000 mg/kg bw/day: males showed decreased urinary pH values and a lower number of crystals (non adverse)

Males of the mid- and high-dose group revealed a statistically reduced mean urinary pH (males: 6.72 ± 0.36, 6.67 ± 0.25, 6.33 ± 0.25* and 5.83 ± 0.35***; females: 5.8 ± 0.42, 5.83 ± 0.35, 5.44 ± 0.39 and 5.38 ± 0.35 for control, low-, mid- and high-dose group, respectively). Furthermore, a lower number of crystals was determined in mid- and high-dose males (please refer to Table 2). As these changes were only slight and not linked to alterations in clinical or histopathological parameter, they are not considered as adverse.

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

The neurotoxicity assessment did not reveal any effects on neurobehavior.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

100 mg/kg bw/day: increased relative liver weight in males, increased absolute brain weight in females (non adverse)

Low-dose males revealed a statistically significant higher relative liver weight whereas the absolute liver weight and the liver-brain/weight ratio remained unaffected. Furthermore, the absolute brain weight of low-dose females was statistically significant increased without altering the relative brain weight (please refer to Table 3). In the absence of a dose-relationship, the effects on organ weights in low-dose animals were considered as fortuitous and not considered as adverse. The weights of the remaining organs (heart, pituitary gland, spleen, kidneys, adrenal glands, thymus, thyroid gland, epididymis, prostate, testes, uterus and ovaries) were comparable among the dose groups in both genders.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

all groups: abnormalities in harderian gland, kidneys, liver, lung, spleen or uterus (non adverse)

Scheduled sacrifices: In general, most animals from control and test groups did not show abnormalities in gross pathology (males: 6, 7, 5 and 6; females: 9, 6, 2 and 8 animals without abnormalities in control, low-, mid- and high-dose groups). Changes observed on harderian gland, kidney, liver, lung, spleen, eyes or uterus were observed in only few animals without a dose-relationship and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 4).

Unscheduled death: The deceased from the high-dose group showed red foci in the thymus and adrenal gland. Moreover, the liver appeared dark. All organs from the deceased of the mid-dose group appeared normal.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

High-dose males revealed a higher incidence of slight vacuolation in the zona fasciculata of the adrenal gland (2/10 vs 5/10 in control and high-dose males). As the effect was not observed in any female, toxicological significance of this effect is questionable. Moreover, 2/10 high-dose males showed signs of slight, unilateral, focal atrophy of the retina, which was also determined in 1/3 mid-dose females. In the kidney, increased incidences of focal basophilic tubulus and mineralisation of the inner medulla was observed in males (1/10, 1/10, 3/10 and 5/9; 0/10, 0/10, 0/10 and 3/9 of the control, low-, mid-and high-dose group, respectively). Furthermore, male animals showed increasing incidences for the following abnormalities of the lung: mononuclear inflammation (5/10, 9/10, 9/10 and 7/10 for control, low-, mid- and high-dose animals) and chronic interstitial inflammation (3/10 for control and 6/10 for each dose group). As the effects observed are only present in one sex and no clear dose-response was present, biological relevance remains questionable. Further abnormalities observed in histopathology were comparable among the groups and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 5).

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

Male animals showed increasing incidences for the following abnormalities of the lung: hyperplasia of regenerative type II pneumocytes (2/10, 4/10, 3/10 and 4/10 for control, low-, mid- and high-dose animals) and slight, bronchiolar regenerative hyperplasia (0/10, 1/10, 0/10 and 2/10 for control, low-, mid- and high-dose animals). Female test animals showed a higher incidence of diffuse, epithelial hyperplasia in the high-dose group (1/10 vs 3/9 for control and high-dose group) in the trachea. As the effects observed are only present in one sex and no clear dose-response was present, biological relevance remains questionable. Further abnormalities observed in histopathology were comparable among the groups and are therefore considered as incidental in origin and unrelated to treatment (please refer to Table 5).

Other effects:

not examined

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 were observed on the examined parameters (applied concentrations were adjusted for purity).

Key result

Critical effects observed:

no

Conclusions:

In this 28-day repeated dose toxicity study performed according to OECD 407 and in compliance with GLP, the NOAEL for both sexes was greater than 1000 mg/kg bw/day.

Reference 2

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

23 May - 11 Nov 2001

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:

(adopted 1998)

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

2018

Deviations:

yes

Remarks:

No hormonal analysis (thyroid), no HDL and LDL, no histopathology on cervix, no determination of estrous cycle stage at necropsy

Qualifier:

according to guideline

Guideline:

EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Version / remarks:

(adopted 1992)

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

Republique Francaise, Premier Ministre, Groupe Interministeriel Des Produits Chimiques, Paris Cedex, France

Limit test:

no

Species:

rat

Strain:

other: Wistar (AF) RJ: WI (IOPS HAN)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: R. Janvier, Le Genest St Isle, France
- Age at study initiation: 6 - 7 weeks
- Weight at study initiation: 267 - 297 g (males), 174 - 201 g (females)
- Fasting period before study: animals were fasted overnight for diet only before blood sampling and for diet and water before urine sampling
- Housing: individual in suspended stainless steel, wire mesh cages
- Diet: certified rodent pellet diet "A04C-P1" (Usine d'Alimentation Rationnelle, Villemoisson-sur-Orge, France), ad libitum
- Water: filtered and softened water from the municipal water supply, ad libitum
- Acclimation period: 8 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 20 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): 10 - 15 (average, not monitored)
- Photoperiod (hrs dark / hrs light): 12 / 12

IN-LIFE DATES: From: 23 May To: 11 Nov 2001

Route of administration:

oral: gavage

Vehicle:

water

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS:

All doses were adjusted for purity so that the dose levels correspond to the given concentration of ethyl sodium phosphonate.
Rate of preparation: Dosing solutions were prepared approx. every 2 to 4 weeks.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test item was sufficiently soluble in water.
- Amount of vehicle (if gavage): 5 mL/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of dosing solutions were sent for analysis to RCC Ltd, Environmental Chemistry and Pharmanalytics Division, Switzerland. Homogeneity and concentration of the test substance were verified according to OECD 408. Homogeneity was verified on the first formulation at the highest and lowest concentrations. Concentrations of the test item in the vehicle were verified on all formulations (concentrations: 20, 60 and 100 g/L). Stability of the test substance was demonstrated in the 28-day repeated dose study (# M-203654-01-1).

Results of analysis
The results obtained for homogeinity and concentration were within the expected ranges (90 - 110%) except the lowest concentration of the fifth preparation (20 g/L) which was 88.6% of the nominal concentration.

Duration of treatment / exposure:

90 days

Frequency of treatment:

once daily, 7 days/week

Dose / conc.:

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

Remarks:

(adjusted for purity)

Dose / conc.:

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

Remarks:

(adjusted for purity)

Dose / conc.:

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

Remarks:

(adjusted for purity)

No. of animals per sex per dose:

10

Control animals:

yes, concurrent vehicle

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily (observation of clinical signs) or twice daily (observation for moribundity and mortality)
- Cage side observations checked for ill-health including blood or loose feces were included. Detailed physical examinations were performed at least weekly during the treatment period.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: at least once daily

BODY WEIGHT: Yes
- Time schedule for examinations: twice during the acclimatisation period, on the first day of dosing and at weekly intervals thereafter and before necropsy

FOOD CONSUMPTION: Yes
- Time schedule: weekly

OPHTHALMOSCOPIC EXAMINATION: Yes
- Time schedule for examinations: once during the acclimatisation period (all animals) and during week 13 (control and high-dose group)

HAEMATOLOGY: Yes
- Time schedule for collection of blood: once during the study on Day 85 or 86
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all survivng animals
- Parameters checked: red blood cell count (RBC), haemoglobin, haematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), white blood cell count (WBC) and differential count evaluation and platelet count (PLT), prothrombine time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: once during the study on Day 85 or 86
- Animals fasted: Yes
- How many animals: all suriving animals
- Parameters checked: total bilirubin (TBIL), glucose (GLUC), urea, creatinine (CREA), total cholesterol, triglycerides, chloride, sodium, potassium, calcium and inorganic phosphorus concentrations, aspartate aminotransferase, alanine aminotransferase (ALAT), alkaline phosphatase (AP) and gamma-glutamyltransferase (GGT) activities were assayed on plasma samples, total protein and albumin concentrations

URINALYSIS: Yes
- Time schedule for collection of urine: on Days 91, 92 or 93, prior to sacrifice
- Metabolism cages used for collection of urine: No
- Animals fasted: Yes
- Parameters checked: volume, pH, urinary refractive index (RI), glucose (GLU), bilirubin (BIL), ketone bodies (KET), occult blood (OCBL), protein (PRO) and urobilinogen (UOR), microscopic examination of the urinary sediment ( presence of red blood cells, white blood cells, epithelial cells, bacteria, casts and crystals)

Sacrifice and pathology:

SACRIFICE: All surviving animals from all groups were sacrificed on study Days 91, 92 or 93. An approx. equal number of animals randomly distributed amongst all groups. Animals were fasted overnight.

GROSS PATHOLOGY: Yes

HISTOPATHOLOGY: Yes
- Dose groups: all animals of the control and high-dose group, all decedents in the low- and mid-dose groups (lung, liver, thyroid gland, kidney and all macroscopic findings were also examined in the low- and mid-dose groups).

The following organs were sampled for pathological examinations:
Adrenal gland, Aorta, Articular surface (femoro-tibial), Bone (sternum), Bone marrow (sternum), Brain, Epididymis, Exorbital (lachrymal) gland, Esophagus, Eye and optic nerve, Harderian gland, Heart, Intestine (duodenum, jejunum, ileum,cecum, colon, rectum), Kidney, Larynx, Liver, Lung, Lymph nodes (submaxillary, mesenteric), Mammary gland, Ovary, Pancreas, Pituitary gland, Prostate, Sciatic nerve, Seminal vesicle, Skeletal muscle, Skin, Spinal cord (cervical, thoracic, lumbar), Spleen, Stomach, Submaxillary (salivary) gland, Testis, Thymus, Thyroid (with parathyroid), Tongue, Trachea, Urinary bladder, Uterus (including cervix), Vagina

Optional endpoint(s):

Optional endpoints: No

Statistics:

Means and standard deviations were calculated for the analysed parameters for each sex separately for each group at each time period. All tests were performed at 1 and 5% significance levels. Statistical analysis was performed by the following tests:
The treatment groups were compared using either parametric ANOVA under the standard assumptions or robust linear regression methods. The homogeneity of variance assumption was examined via Levene's test. If Levene's test indicated lack of homogeneity of variance (p<0.05), robust linear regression methods
were used to test all treatment effects. The robust regression methods use variance estimators that make no assumptions regarding homogeneity of variance or normality of the data. They were used to test for overall treatment group differences (Wald chi-square tests), following by individual t-tests for exposed vs. control group comparisons when the overall treatment effect was significant. If Levene's test did not reject the hypothesis of homogeneous variances, standard ANOVA techniques were applied for comparing the treatment groups. The GLM procedure in SAS® 6.12 was used to evaluate the overall effect of treatment and, when a significant treatment effect was present, to compare each exposed group to control via Dunnett's test.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: most animals showed sporadic salivation after gavage

Salivation was observed on Day 17 (3 out of 20), Day 57 (9 out of 19), Day 83 (all animals) and Day 85 (1 out of 17). A complete recovery was noted 24-hour post-dosing. This observation may have been related to the administration of the test material administration. However, in view of its sporadic appearance, this clinical sign was considered to be not toxicologically significant.
There were no treatment-related clinical signs in either sex at 100 and 300 mg/kg bw/day.

Mortality:

mortality observed, non-treatment-related

Description (incidence):

1000 mg/kg bw/day: 2 males and 1 female died
300 mg/kg bw/day: 1 male and 1 female animal died

At 1000 mg/kg bw/day, one male was found dead on Day 63 following an accidental trauma. Another male was found dead on Day 55 following a significant body weight loss and a concomitant reduction in food consumption between Day 36 to 43.
Macroscopic examination at necropsy revealed no significant findings. Histopathological examination indicated few changes that were considered to be not treatment-related. One female of the same treated group was found dead on Day 81. Ante mortem clinical investigation and macroscopic examination at necropsy showed no significant findings. Histopathological examination revealed few changes that were considered to be not treatment-related. These three deaths were therefore considered not to be treatment related.
At 300 mg/kg bw/day, one male and one female died during anesthesia for blood sampling on Day 86 and 85, respectively.
There were no deaths at 100 mg/kg bw/day.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: reduced body weight in males (non adverse)

At 1000 mg/kg bw/day, mean body weight was consistently lower in males throughout the dosing period when compared to controls, the statistical significance being achieved on Day 64 ((males: 102.94, 101.25 and 93.38*%; females: 99.68, 99.36 and 99.21 for low-, mid- and high-dose groups, respectively) -6.5%, p<0.05) and at terminal sacrifice (males: 103.42, 101.93 and 92.65*%; females: 103.81, 97.92, 97.95 for low-, mid- and high-dose groups, respectively). Mean body weight gain was consistently lower in males throughout the dosing period when compared to controls, the statistical significance was only achieved between Day 29 to 57 ((males: 105.8, 104.9 and 80.7*%; females: 105.3, 84.3 and 85.3% for low-, mid- and high-dose groups, respectively) -19%, p<0.05). However, no statistically significant difference was observed on overall body weight gain and no similar effect was noted in high-dose females (mean body weight change Days 1-90: males: 107.4, 103.9 and 87.7%; females: 111.2, 98.5 and 96.9% for low-, mid- and high-dose groups, respectively). These slight changes were therefore considered not to be adverse.
There were no effects on body weight in either sex at 100 and 300 mg/kg bw/day and in females at 1000 mg/kg bw/day.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: slight reduced food intake in males (non-adverse)

At 1000 mg/kg bw/day, mean food consumption was lower in males throughout the dosing when compared to controls, the statistical significance being achieved between Days 36-64 (Days 36-43: -14%, p<0.01, Days 43-50: -9%, p<0.01, Days 50-57: -8%, p<0.05, Days 57-64: -8%, p<0.05). However, no statistically significant difference was observed on overall mean food consumption. This slight change was therefore considered not to be adverse.
There were no effects on food consumption in either sex at 100 and 300 mg/kg bw/day and in females at 1000 mg/kg bw/day.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

no effects observed

Description (incidence and severity):

There were no treatment-related ophthamological changes.

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

300 mg/kg bw/day: In males, statistically significant decreased number of lymphocytes

There were no treatment-related changes in the hematology except the number of lymphocytes, which was significantly decreased in mid-dose males (males: 14.8 ± 2.76, 13.86 ± 2.36, 11.82 ± 1.61* and 13.04 ± 1.82x 1E+09/L; females: 8.4 ± 1.18, 8.96 ± 2.65, 8.83 ± 1.96 and 8.63 ± 1.72x 1E+09/L for control, low-, mid- and high, respectively). The finding reported in males was considered as indicental because there was no dose-relationship and the effect was not observed in females.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: reduced creatinine concentration in females

A tendency to increased urea concentrations was observed in either sex in a dose-dependent manner without reaching statistical significance. In females at 1000 mg/kg bw/day, a statistically significant difference was noted in creatinine concentration (-15%, p<0.05). This finding was considered to be not toxicologically relevant in the absence of any variation of the individual values between control and treated groups.

Endocrine findings:

not examined

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: decreased urinary pH values and lower number of crystals in either sex
100 mg/kg bw/day: increased urinary volume in males

Lower mean urinary pH was noted at 1000 mg/kg bw/day in males and females ((males: 7.00 ± 0.47, 6.8 ± 0.48, 6.67 ± 0.66 and 5.50 ± 0.38***, -21%, p<0.001; females: 6.00 ± 0.53, 6.00 ± 0.53, 6.06 ± 0.53 and 5.28 ± 0.36**, -12%, p<0.01 for control, low-, mid- and high-dose group, respectively). Furthermore, a decreased number of crystals was determined in test animals of both sexes resulting in an absence of urinary crystals in high-dose males and females. As biological significance of these changes remains unclear, this effect was considered to be doubtful of toxicological significance and therefore, not considered adverse.
The statistically significantly higher mean volume seen in males at 100 mg/kg bw/day (males: 4.12 ± 2.25, 6.79* ± 1.80, 5.97 ± 2.99 and 4.46 ± 1.66; females: 3.03 ± 1.33, 3.52 ± 1.57, 3.26 ± 0.99 and 2.22 ± 1.73 for control, low-, mid- and high-dose group, respectively) was judged to be incidental as no relevant change was seen at 300 and 1000 mg/kg bw/day.
There were no treatment-related changes in the urine parameters assayed at 100 and 300 mg/kg bw/day.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

1000 mg/kg bw/day: increased relative kidney weight in both sexes
100 mg/kg bw/day: increased absolute kidney weight in males

Statistically significant differences in kidney weights, when compared to controls, included a higher mean kidney to body weight ratio in males and females at 1000 mg/kg bw/day (+14%, p<0.01 and +15%, p<0.01, respectively) and a higher mean absolute kidney weight in males at 100 mg/kg bw/day (+10%, p<0.05). These differences were considered to be incidental in origin in view of the lack of corroborative macroscopic or microscopic findings.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

all groups: abnormalities in adrenal gland, caecum, harderian gland, kidneys, liver, lung, submaxillary lymph node, prostate, spleen, stomach, testis or uterus

Scheduled sacrifice: Most animals revealed organs with normal appearance (males: 6/10, 5/10, 4/9 and 3/8; females: 9/10, 5/10, 2/9 and 9/9 for control, low-, mid- and high-dose group, respectively). The changes seen at macroscopic examination were few and considered to be incidental and unrelated to the treatment. In addition, the few changes observed in decedent animals were not considered to be treatment-related and consisted mainly of signs of congestion (please refer to table 6).

Unscheduled death: The deceased male of the mid-dose group revealed abnormal findings in the kidney, liver and lung described as dark colouring. Furthermore, the thymus showed red foci. The female deceased of the mid-dose group showed dark red colouring of the liver and lung. In the high-dose group, 1/2 male decedents showed white foci in the lung and white and black foci in the spleen. Moreover, an empty stomach and nodule masses were determined in 1/2 deceased high-dose male. In addition, soiled fur, liquid content of the thoracic cavity and autolysis and red appearance was observed in 1/2 deceased high-dose males. The high-dose deceased female revealed dark appearance of the kidney, lung, liver and submaxillary lymph node. In addition, the thymus was determined as mottled and red. In general, autolysis was observed.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

all groups: abnormalities in liver, kidney, urinary bladder, lung, thyroid gland, adrenal gland, pituitary gland, spleen, thymus, submaxillary gland, stomach, pancreas, harderian gland, testis, epididymis, prostate, caecum, and uterus

Decedent animals showed changes similar to those seen at terminal sacrifice. At terminal sacrifice, no treatment-related changes were seen. The few changes seen were considered to be incidental in origin and within the range of expected changes for animals of this age kept under laboratory conditions (please refer to table 7). In addition, no dose-response relationship was present in either sex.

Unscheduled death: No abnormal findings were determined in histopathology.

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 were observed on the examined parameters (applied concentrations were adjusted for purity).

Key result

Critical effects observed:

no

Conclusions:

In this 90-day repeated dose toxicity study performed according to OECD 408 and in compliance with GLP, the NOAEL was established to be greater than 1000 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Study duration:

subchronic

Species:

rat

Quality of whole database:

The available information comprises adequate, reliable studies (Klimisch score 1) and consistend studies, and is thus sufficient to fulfil the standard information requirement set out in Annex VIII-IX, 8.6, of Regulation (EC) No 1907/2006.

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

Repeated dose toxicity: oral administration

Data on repeated dose toxicity are available after exposure for 28- and 90-days to characterize the potency of the test substance to induce toxicity after short-term and sub-chronic exposure.

 

Groups of 10 Wistar rats/per sex were exposed daily via gavage to 100, 300 and 1000 mg/kg bw/day for at least 90 days according to OECD Guideline 408 and in compliance to GLP (M-210431-01-1).

Dosing solutions were prepared from an aqueous solution with a purity of 22.1% and were adjusted for purity. A control group consisting of 10 rats/sex was included in the study, which received the respective volume of the vehicle (water). Homogeneity and concentration of dosing solutions were analytically verified by gas chromatography. Stability of the test substance was demonstrated in the 28-day repeated dose study (see below). Clinical signs were recorded daily and body weight and food consumption were measured weekly. Detailed physical examination was performed once during acclimatisation and weekly throughout the study. Ophthalmological examinations were performed during the acclimatisation phase and during Week 13 for control and high-dose animals. Blood samples of fasted animals were collected one week before necropsy for haematology and clinical chemistry. Furthermore, urine samples were collected overnight before necropsy from all animals. All animals were necropsied, and the major organs were weighed and included in macroscopic and microscopic evaluation. Unscheduled mortality was observed in the mid- and high-dose group: one mid-dose male and female died during anesthesia for blood sampling. Moreover, one male of the high-dose group was found dead following accidental trauma. One further male of the same dose group was found which showed reduced food consumption (between Day 36 - 43) and a significant body weight loss. Furthermore, a high-dose female was found dead on Day 81. As gross and histopathology did not reveal significant findings, the unscheduled deaths were not considered as treatment-related. No clinical signs of toxicity were observed in the low- and mid-dose group. In the high-dose group, most animals showed signs of sporadic increased salivation few minutes post-dosing. In view of the sporadic appearance, this effect was considered to be not of toxicological significance. Body weights and weight gain appeared normal in the low- and mid-dose group for either sex compared to the control animals. High-dose males revealed a consistently reduced mean body weight reaching statistical significance only on Day 64 and at terminal sacrifice. As no statistically significant effect on overall body weight gain was noted over the study period and no similar effect was observed in high-dose females, the slight changes on body weight and weight gain were considered as non-adverse. Overall mean food consumption was comparable among the groups. No alterations were determined in haematology in either sex except the number of lymphocytes, which was significantly decreased in mid-dose males. As no dose-response was present, the effect was considered as not treatment-related. Blood samples revealed reduced creatinine concentrations in test animals reaching statistical significance in high-dose females. Due to the absence of any variation in individual values between control and treated groups, the finding was not considered to be of toxicological relevance. The remaining clinical parameters were unremarkable among the groups. Urinalysis determined a statistically reduced mean urinary pH in high-dose animals. Furthermore, a decreased number of crystals was determined in test animals of both sexes resulting in the absence of urinary crystals in high-dose males and females. As the effects were not correlated with clinical signs or pathological alterations, biological significance of these changes remain unclear and the effects were considered as doubtful for toxicological significance and thus not considered as adverse. Furthermore, a higher mean urinary volume was determined for low-dose males. In the absence of a dose-response, the effect was judged as incidental. No further abnormalities were determined in either sex or dose group in the evaluated parameter. Moreover, organ weights were unremarkable among the groups except increased kidney weights around 14 and 15% for high-dose males and females, respectively. Due to the lack of correlated findings in pathology, the effect was interpreted as incidental and not treatment-related. In histopathology, abnormalities in organs like the liver, kidney, urinary bladder, lung, thyroid gland, adrenal gland, pituitary gland, spleen, thymus, submaxillary gland, stomach, pancreas, harderian gland, testis, epididymis, prostate, stomach, caecum, pancreas and uterus were observed in few sacrificed animals including controls. As no dose-response was present in either sex, all abnormalities were considered as incidental and not treatment-related. In conclusion, no treatment-related mortality or effects on the elevated parameters were determined in the low- and mid-dose group. In the high-dose group, males showed reduced body weight and decreased body weight gain. However, as no effect was determined on overall body weight gain and food consumption, observed alterations were not interpreted as adverse. Moreover, changes determined in haematology, clinical chemistry and urinalysis were considered as non-adverse. Macroscopically or microscopically detected abnormalities were detected in control and test animals and were thus interpreted as incidental. Therefore, a NOAEL of > 1000 mg/kg bw/day was defined for male and female rats for the test substance.

  

Further, a subacute toxicity study was performed with the test substance according to OECD Guideline 407 and in compliance with GLP (M-203654-01-1). Ten Wistar rats /sex were exposed to 100, 300 and 1000 mg/kg bw/day prepared from an aqueous solution (purity of 22.1% concentrations adjusted for purity) via gavage for 28 days. The respective control group received the vehicle (water). Homogeneity, concentration and stability of dosing solutions were analytically verified by gas chromatography. Elevated endpoints included clinical observations, body weight and food consumption measurements, ophthalmoscopy, neurobehavioral tests, haematology, clinical biochemistry, urinalysis and pathology. Histopathology was carried out on the control and high-dose animals of all major tissues and organs. Further, organs like lung, liver, thyroid gland, kidney and organs with macroscopic findings were also examined in the low- and mid-dose groups. No treatment-related mortalities occurred during the study and no clinical signs of toxicity were observed. One mid- and high-dose female died during anaesthesia for blood sampling. Body weight and weight gain were comparable among control and test animals. Moreover, food consumption was unaffected by the treatment. Absolute and relative organ weights were comparable among the groups except increased relative liver and absolute brain weight in low-dose males and females, respectively. In the absence of a dose-relationship, these changes are interpreted as fortuitous and not treatment-related. In haematology, only a slight decrease in red blood cell count (RBC) was observed reaching statistical significance only in low- and mid-dose males. As no dose-response was present and the RBC level was unaffected in females, the effect is considered as fortuitous. Statistically significant alterations in aspartate aminotransferase activity, chloride and sodium levels were determined. However, as the effects were neither dose-related nor were the values still within the range of historical data, adversity of these effects is excluded. In urinalysis, males of the mid- and high-dose group revealed a statistically reduced mean urinary pH and a lower number of crystals. As these changes were only slight and not linked to alterations in clinical or histopathological parameters, they are not considered as adverse. Most animals from the control and test groups did not show abnormalities in gross pathology. Changes observed on harderian gland, kidney, liver, lung, spleen, eyes or uterus were observed only in a few animals without a dose-relationship and are therefore considered as incidental in origin and unrelated to treatment. High-dose males revealed a higher incidence of slight vacuolation in the zona fasciculata of the adrenal gland. As the effect was not observed in any female, toxicological significance of this effect remains questionable. Moreover, few high-dose animals showed signs of slight, unilateral, focal atrophy of the retina. In the kidney, increased incidences of focal basophilic tubulus and mineralisation of the inner medulla was observed in males. Furthermore, some abnormalities were determined in the lung of male animals including mononuclear inflammation, chronic interstitial inflammation, hyperplasia of regenerative type II pneumocytes and slight, bronchiolar regenerative hyperplasia. High-dose females showed a higher incidence of diffuse, epithelial hyperplasia in the trachea. As the effects observed are only present in one sex and no clear dose-response was present, biological relevance of histopathological findings remains questionable. Further abnormalities observed in histopathology were comparable among the groups and are therefore considered as incidental in origin and unrelated to treatment. Based on these data, a NOAEL > 1000 mg/kg bw/day was defined for both sexes after exposure to the test substance for 28 days.

 

Taken these data together, short-term and sub-chronic exposure to the test substance for 28 or 90 days did not result in adverse effects. Therefore, the test substance is not considered as hazardous after repeated exposure. 

Justification for classification or non-classification

The available data on repeated dose toxicity do not meet the criteria for classification according to Regulation (EC) 1272/2008, and are therefore conclusive but not sufficient for classification.