2-benzyl-2-dimethylamino-4'-morpholinobutyrophenone

Endpoint summary

Description of key information

Study conducted to generally recognised scientific standards with GLP.

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

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Reason / purpose for cross-reference:

reference to same study

Qualifier:

equivalent or similar to guideline

Guideline:

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

Version / remarks:

(2008)

Deviations:

yes

Remarks:

No recovery was considered, only two doses used instead of three as recommended by guideline OECD 407.

Principles of method if other than guideline:

The study was based around the OECD guideline 407, which however, was not fully applicable as this study was intended for dose selection for a follow up 90-day study in rats. No recovery was considered; only two doses were used instead of three as recommended by guideline. The dose level of 250 mg/kg bw/day was established as LOAEL. Animals of this group were initially treated with 500 mg/kg bw/day. However, since severe toxicity was seen, treatment was stopped after 9 days, followed by a 5 day recovery period. Subsequently, dosing of these animals was decreased to 250 mg/kg bw/d. Therefore, it could not be excluded that the initial dosing with 500 mg/kg bw/day had, to some degree, contributed to the overall toxicity on which the LOAEL was based.

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Wistar

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Deutschland, Sulzfeld, Germany
- Age at study initiation: 10 weeks (males), 9 weeks (females)
- Weight at study initiation: 288 - 293 (males), 174 - 180 (females)
- Housing: in groups of 5 per sex in Macrolon cages (MIV type)
- Diet ad libitum: pelleted rodent diet (SM R/M-Z from SSNIFF Spezialdiäten GmbH)
- Water ad libitum: tap water
- Acclimatisation: at least 5 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.7 - 21.7
- Humidity (%): 37 - 72 %
- Air changes (per hr): 15
- Photoperiod (hrs dark / hrs light): 12 / 12

Route of administration:

oral: gavage

Vehicle:

propylene glycol

Details on oral exposure:

The choice of vehicle was based on trial formulations performed at NOTOX and on information from the sponsor.
Formulations (w/w) were prepared daily within 6 hours prior to dosing and were homogenised to a visually acceptable level. Formulations were kept away from light as much as possible using amber coloured glassware and/or tin foil. Correction was made for specific gravity of the vehicle.

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

Analysis of stability, homogeneity and concentration of the test article under test conditions was not performed as part of the present study. According to the author, such analyses were to be done within a one-generation reproduction toxicity study performed later.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

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

Remarks:

Reduced to 250 mg/kg bw/day after 9 treatment days a 5 recovery days.

No. of animals per sex per dose:

5

Control animals:

yes, concurrent no treatment

Details on study design:

The test substance, formulated in vehicle, was administered daily for 28 days by oral gavage to the rats. After 9 days and because of severe toxicity, treatment with the highest dose level of 500 mg/kg bw/day (group 3) was stopped for a period of five days (recovery); Subsequently, the same animals were dosed with 250 mg/kg bw/day for 28 days.

Observations and examinations performed and frequency:

MORTALITY AND CLINICAL OBSERVATIONS
The animals were checked for mortality at least twice daily and were examined at least daily for clinical signs.

BODY WEIGHT
Weekly, and additionally on day 11; terminal body weight was also assessed.

FOOD CONSUMPTION
Weekly

HAEMATOLOGY AND CLINICAL CHEMISTRY
Blood samples were collected under iso-flurane anaesthesia on the day of sacrifice after a over night fasting. For details on parameters see table 1.

Sacrifice and pathology:

GROSS PATHOLOGY
All animals were anaesthetized at test ending using iso-flurane and subsequently exsanguinated. After gross pathology the following tissues and organs were collected from all animals and fixed: Lung, adrenal glands, spleen, epididymides, sternum with bone marrow, kidneys, testes, liver and all gross lesions.
From all males sperm samples were taken from the proximal part of the vas deferens to assess sperm motility.

Organ weights: Adrenal glands, liver, kidneys, spleen

HISTOPATHOLOGY
Organ and tissue samples were processed, embedded, cut and stained for histopathology.
All tissues and organs collected at the scheduled sacrifice from all animals of the control and the highest dose group, the samples of the testes of groups 1 and 3 (examination of spermatogenesis) and all gross lesions were examined.
Based on treatment-related morphologic changes in the liver, kidneys, adrenal glands, and sternum with bone marrow of group 3 males and/or females, these organs were also examined from all animals of Group 2 (males and females; kidneys were examined for males only).

Statistics:

The following statistical methods were used to analyse the data:
- If the variables could be assumed to follow a normal distribution, the Dunnett-test (many-to-one t-test) based on a pooled variance estimate was applied for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many-to-one rank test) was applied when the data could not be assumed to follow a normal distribution.
- The exact Fisher-test was applied to frequency data.
- The percentage of motile spermatozoa and progressive motile spermatozoa were subjected to the Kruskal-Wallis nonparametric ANOVA test to determine intergroup difference.
If the results of the ANOVA were significant (p < 0.05), the Wilcoxon test was applied to the data to compare the treated groups to the control group.
All tests were two-sided and in all cases p < 0.05 was accepted as the lowest level of significance. Group means were calculated for continuous data and medians were calculated for discrete data (scores) in the summary tables.

Clinical signs:

effects observed, non-treatment-related

Description (incidence and severity):

Clinical signs consisted of hunched posture, piloerection, a lean appearance, salivation, retching and/or gasping among animals at 500 mg/kg bw/day. These symptoms resolved during the wash-out period of 5 days. During the subsequent treatment at 250 mg/kg bw/day, no toxicologically relevant clinical signs were observed.
One case of rales was seen for 1 female at 250 mg/kg bw/day. This is commonly noted in rats of this age and strain under the conditions in this study.
Salivation was noted intermittently among most animals at 250 mg/kg bw/day as well as among most females at 100 mg/kg bw/day. This effect was considered to be of non-toxicological relevance. No clinical signs were noted among control animals and males at 100 mg/kg bw/day.

Mortality:

no mortality observed

Description (incidence):

No mortality occurred during the study period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight loss (up to 14% of the weight recorded on Day 1) or reduced weight gain was noted among all animals at 500 mg/kg bw/day (see figure). At commencement of treatment at 250 mg/kg/day, body weights of females were similar to control values, whilst body weights of males were still slightly lower than control levels (achieving level of statistical significance). During the first two weeks of treatment at 250 mg/kg bw/day, body weight gain of this group was similar to control levels (taking Day 15 as reference point). Although body weight gain in the last two weeks of treatment could not be compared to concurrent control levels (control animals were sacrificed after 28 days), body weight gain was in the range to be expected for rats of this age and strain.
A statistically significant higher body weight gain of females at 100 mg/kg bw/day on day 15 remained within the range considered normal for rats of this age and strain.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

A lower (relative) food consumption was noted for males and females at 500 mg/kg bw/day, which recovered to control levels after the recovery period.
For females at 250 mg/kg bw/day, relative food consumption appeared slightly higher than control levels. This was due to slightly higher food consumption, as was also observed for females at 100 mg/kg bw/day. Since food consumption levels for these animals remained within the range considered normal, this was considered to be of no toxicological relevance.

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):

The following changes in hematology parameters were of statistical significance in comparison to control animals:
Higher prothrombin time (PT) in males at 100 and 250 mg/kg bw/day, lower reticulocyte and platelet counts in males at 250 mg/kg bw/day, lower hemoglobin concentration and activated partial thromboplastin time (APTT) in females at 250 mg/kg bw/day. However, the both latter effects were within the range considered normal for rats of this age and strain.
Other hematological parameters of animals at 100 and 250 mg/kg bw/day were similar to control values.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following in clinical biochemistry parameters were of statistical significance in comparison to control animals:
Higher alanine aminotransferase activity in males at 250 mg/kg bw/day, and in females at 100 and 250 mg/kg bw/day (not statistically significant at 250 mg/kg bw/day), higher inorganic phosphate levels in females at 250 mg/kg bw/d, lower total protein levels in males at 250 mg/kg bw/day, lower urea levels in males at 250 mg/kg/day, lower sodium levels in males at 250 mg/kg bw/day, lower chloride levels in males and females at 250 mg/kg bw/day. However, the four latter effects were within the range considered normal for rats of this age and strain.
Other clinical biochemistry parameters of animals at 100 and 250 mg/kg/day were similar to control values.

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

The liver weight and liver to body weight ratio was higher in males and females at 100 and 250 mg/kg bw/day. A lower spleen weight was recorded for females at 250 mg/kg bw/day, while spleen to body weight ratio was lower for females at 100 and 250 mg/kg bw/day. Kidney and adrenal weights of animals at 100 and 250 mg/kg bw/day were similar to control levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

At macroscopic examination greenish discolouration of the kidneys was observed among all animals at 250 mg/kg bw/day. Redbrown discolouration of the liver was noted among most females at 250 mg/kg bw/day.
Other necropsy findings included dark-red foci on the pancreas, enlarged adrenal glands, tan discolouration of adrenal glands, and pelvic dilation of the kidneys. These changes were not accompanied by treatment-related histopathological correlates, and hence these were considered not to be of toxicological relevance.
No abnormalities were noted at necropsy of control animals and females at 100 mg/kg bw/day.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

effects observed, treatment-related

Description (incidence and severity):

The following histopathological changes were considered to be related to treatment with the test substance:
Liver: Hypertrophy of the hepatocytes in 4/5 males and 5/5 females of group 3.
Kidneys (males): increased incidence and severity of cortical hyaline droplets in Groups 2 (5/5 males) and 3 (4/5 males). In Group 1 this was seen in 2/5 males at a minimal degree. In this study the occurrence of cortical hyaline droplets was accompanied by a slightly increased incidence and severity of corticomedullary tubular basophilia in Groups 2 (3/5 animals) and 3 (4/5 animals). No tubular basophilia was recorded in males of Group 1 (control).
Bone marrow-sternal: Myeloid atrophy in the sternal bone-marrow in Group 3 in 3/5 males and 3/5 females.

The staging of spermatogenesis of the animals of Groups 1 and 3 suggested normal spermatogenesis; all stages were present.

All other microscopic findings were within the range of background pathology encountered in Wistar rats of this age and strain and occurred at similar incidences and severity in both, control and treated rats.

Other effects:

no effects observed

Description (incidence and severity):

Upon investigation of sperm motility no abnormalities up to 250 mg/kg bw/day were seen.

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

gross pathology

haematology

organ weights and organ / body weight ratios

Dose descriptor:

LOAEL

Effect level:

250 - 500 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

clinical biochemistry

food consumption and compound intake

gross pathology

haematology

histopathology: non-neoplastic

organ weights and organ / body weight ratios

Critical effects observed:

not specified

Table 1: Selected clinical chemistry parameters for females

	control	100 mg/kg bw	250 (500) mg/kg bw
ALAT (U/L)	41.4	63.5 *	59.9
ASAT (U/L)	69	75.9	67.6
ALP (U/L)	67	93	78
Urea (mmol/L)	9.8	8.5	9.5

Table 2: Selected clinical chemistry parameter for males

	control	100 mg/kg bw	250 (500) mg/kg bw
ALAT (U/L)	43.2	47.7	53.4
ASAT (U/L)	77	71.3	83.4
ALP (U/L)	173	154	150
Urea (mmol/L)	10.6	8.6	7.2*

Table 3a and b: Absolute body and liver weights for males and females

males	control	100 mg/kg bw	250 (500) mg/kg bw
body weights (g)	338	331	320
liver weights (g)	8.63	9.78 **	10.76 **

females	control	100 mg/kg bw	250 (500) mg/kg bw
body weights (g)	190	197	197
liver weights (g)	5.23	6.28 *	7.63 **

Table 4a and b: Selected haematology parameters for males and females

males	control	100 mg/kg bw	250 (500) mg/kg bw
Haemoglobin, mmol/L	9.8	9.6	9.4
APTT, s	16.9	18.2	16.2

Females	control	100 mg/kg bw	250 (500) mg/kg bw
Haemoglobin, mmol/L	9.5	9.2	9.0*
APTT, s	16.9	18.7	13.9*

Table 5: Sperm Motility Summary

	control	100 mg/kg bw	500 mg/kg bw
Motility
Mean	55	61	45
St. Dev.	22	23	26
N	5	5	5
Progressive Motility
Mean	32	29	24
St. Dev.	12	10	16
N	5	5	5

Conclusions:

Clear toxicological effects were observed in the 500 mg/kg bw/day dose group at 9 days, this group was subsequently placed on a 5 day recovery period and resumed dosing at 250 mg/kg bw/day for the remainer of the test duration. None adverse effects (haematology, clinical biochemistry, gross necropsy, and organ weight) were observed in the 100 mg/kg bw/day group. The 28 day repeat dose NOAEL for systemic toxicity was therefore set at 100 mg/kg bw/day.

Executive summary:

In this study conducted to generally accepted scientific standards with GLP, the test material (EC 404-360-3) was administered daily via gavage (using propylene glycol as a vehicle) to rats of both sexes in doses of 0, 100 amd 500 mg/kg bw/day. Due to severe toxicity after 9 days, the 500 mg/kg bw/day was stopped for 5 days, before the same animals were re-dosed with a lower amount of 250 mg/kg bw/day for 28 days. Toxic effects were still observed at this level but not at the 100 mg/kg bw/day dose. Thus, the test material was determined to have a NOAEL of 100 mg/kg bw/day and a LOAEL of 250-500 mg/kg bw/day, however, it is not possible to completely exclude that the toxic effects observed at the 250 mg/kg bw/day was due to the previous dosing of 500 mg/kg bw/day.

Reference 2

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

1988-12-07, 1989-10-23

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:

(12.05.1981)

Deviations:

yes

Remarks:

Temperature between 16 - 19 °C for several hours during two days (OECD 22 +/-3 °C). Relative humidity fell between 16 - 35 % for several hours during four days (OECD 30 - 70 %). No reticulocytes were counted and only liimited tissues examined.

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

19.9.84

Qualifier:

according to guideline

Guideline:

other: MITI notification no 61-1014

Qualifier:

according to guideline

Guideline:

other: MOHW notification no 1039

Qualifier:

according to guideline

Guideline:

other: EPA notification no 700 (5.12.86)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Strain as stated in the report: Sprague Dawley rats : Ico : OFA.SD (IOPS Caw)
- Source: IFFA CREDO, Les Oncins, 69210 L'Arbresle, France
- Age at study initiation: 6 weeks
- Weight at study initiation: 179 - 212 g (males); 151 - 177 g (females)
- Fasting period before study: No data
- Housing: Stainless steel mesh cages (555 x 350 x 200 mm), 5 animals of the same group/sex in a cage
- Diet: Ad libitum. Commercial pelleted diet, (A04, Usine d'Alimentation Rationnelle, Villemoisson, France). Sterilised by irradiation and analysed for the absence of chemical and bacteriological contaminants.
- Water: Ad libitum, filtered (0.6 µm) mains water, analysed twice a year (Laboratoire Municipal d'Hygiène de la ville de Lyon, FRANCE) for chemicals and bacterial contamination.
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19 - 25
- Humidity (%): 35 - 75
- Air changes (per hr): minimum 8
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Remarks:

5 mg/l

Details on oral exposure:

PREPARATION OF DOSING SOLUTIONS: The test substance was formulated in suspension in the vehicle. The suspensions were prepared weekly and stored at +4°C. The different concentrations prepared were 1, 5, and 50 mg/ml for groups 2, 3, and 4 respectively.

VEHICLE
- Justification for use and choice of vehicle (if other than water): The test substance is insoluble in water
- Concentration in vehicle: 1, 5 and 50 mg/ml for groups 2, 3 and 4, respectively.
- Amount of vehicle: 10 ml/kg bw

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analytical monitoring was based on high performance liquid chromatography (HPLC). Homogeneity and stability of the test article was assessed. Five samples were used to assess homogeneity and 2 samples were assessed for stability (after 1 and 7 days) for each concentration. The test article was found to be stable (1 and 7 days) and homogenous under testing conditions.

Duration of treatment / exposure:

4 weeks followed by a two week recovery.

Frequency of treatment:

Once daily

Dose / conc.:

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

Dose / conc.:

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

Dose / conc.:

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

No. of animals per sex per dose:

10 animals per sex per dose. 5 animals were sacrificed at ending of treatment period, the 5 remaining animals were sacrificed after 14 day recovery.

Control animals:

yes, concurrent no treatment

Details on study design:

A dose range-finding study in rats was conducted (HAZLETON, 1989). Groups of 5 male and 5 female Sprague Dawley rats were treated at dose levels of 100, 300, and 3000 mg/kg bw/day for 14 days. At 100 mg/kg bw/day, no effects were seen. At the higher doses, treatment-related effects were observed with a dose effect relationship: reduction in body weight gain and food consumption during the first week, slight decrease in haemoglobin, erythrocyte count and packed cell volume, increase in cholesterol and alkaline phosphatase, increase in liver and adrenals weights and decrease in testes weight (3000 mg/kg bw/day). Mortality was seen in the 1000 and the 3000 mg/kg bw/day groups (1 female and 3 females, respectively). The doses selected for the main study were based on these results.

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked: Morbidity, signs of ill-health or adverse reaction to treatment, and mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE: Weekly

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: At weeks 4 and 6
- Anaesthetic used for blood collection: Yes (ether)
- Animals fasted: Yes
- How many animals: 5 animals/sex/group
- Parameters checked in table [1] were examined.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: At weeks 4 and 6
- Animals fasted: Yes (overnight)
- How many animals: 5 animals/sex/group
- Parameters checked in table [1] were examined.

URINALYSIS: Yes
- Time schedule for collection of urine: At weeks 4 and 6
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked in table [1] were examined.

NEUROBEHAVIOURAL EXAMINATION: No

Sacrifice and pathology:

GROSS PATHOLOGY
Five animals/sex/group were killed after 4 weeks of treatment and five animals/sex/group at the end of the treatment-free period. The necropsy included examination of the external surface, all orifices, the thoracic, abdominal and pelvic cavities and viscera, the cervical tissue and organs, the carcass.
The following organs were weighed for all animals: adrenals, gonads, liver, kidneys, and brain
The following organs/tissues were sampled and preserved for all animals: Adrenals, brain (cortex, pons + cerebellum, stem), eyes + optic nerves, femur (bone marrow), heart, kidneys, liver, lungs, ovaries , pituitary, spleen, stomach, testes, thyroid + parathyroid, urinary bladder and all gross lesions.

HISTOPATHOLOGY
Heart, liver, spleen, kidneys, and adrenal glands were examined in group 1 and 4 animals for animals killed at week 4. Additionally, all macroscopic lesions, except those for which the diagnosis was judged unecessary for the outcome of the study by the pathologist.

Statistics:

All parameters (except food consumption) were submitted to an analysis of variance (ANOVAR) which was performed to determine the intergroup variation and when it was statistically significant, the Dunnett's test was applied to determine which group was affected.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Alopecia was observed in both sexes of group 4 at week 4 and in one group 4 female at week 6.

Mortality:

no mortality observed

Description (incidence):

No mortality was observed.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

Body weights and food consumption of treated males and females was comparable to the control values.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Body weights and food consumption of treated males and females was comparable to the control values.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A slight decrease in haemoglobin in group 4 males and females, packed cell volume in group 4 males, and red blood cell count not statistically significant in group 4 males was observed at week 4; these changes were not observed at week 6. The changes observed at week 4 were associated with a slight decrease in mean corpuscular haemoglobin concentration for the group 4 female.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

In comparison with group 1 (control) and after analysis of variance and Dunnett's test the following variations were statistically significant or at the limit of the statistical significance.
- Potassium: slight decrease in group 4 females at week 4.
- Cholesterol: slight increase in group 4 females at week 4.
- Proteins: slight increase in group 4 females at week 4.
- Albumin: slight increase in group 4 males at week 4.
- Creatinine: slight increase in group 4 males at week 4.
- Triglycerides: moderate increase in group 4 females at week 4.
- Alkaline phosphatase: slight increase in group 3 females at week 4.
- Aspartate aminotransferase: slight increase in one group 2 female and in group 3 males at week 4.
- Gamma glutamyltransferase: slight increase in group 4 females at week 4.
At week 4, the individual values of alkaline phosphatase and aspartate aminotransferase (except for the one female in group 2) were within the normal limits and were not dose related; these variations were not treatment-related. For the following parameters, some individual values were :
- Potassium, creatinine, proteins, albumin, triglycerides: at the limits or just without the normal limits
- Cholesterol: moderately higher than the normal limits
- Gamma glutamyltransferase: higher than the control values.
These variations were considered to be treatment-related. They were not present at the end of the 2 week treatment-free period.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Comparison of individual values of control with treated groups showed the following variations:
- Slight increase in reducing substances in group 4 more marked in males than in females at week 4.
- Slight increase in incidence and severity of epithelial cells in group 4 males and in incidence only in group 4 females at week 4.
- Slight increase in incidence and severity in the presence of red blood cells more marked in males than in females group 2 at week 6.
- Slight increase in the presence of white blood cells in group 2 males at week 6.
The treatment effects observed at week 4 were reversible. The presence of blood cells at week 6 was not dose related and was not present at week 4. These changes could not be attributed to treatment.

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

In comparison with group 1 and after analysis of variance and Dunnett's test the following variations were observed :
- Increase in absolute and relative liver weights in group 4 males and females at week 4.
- Increase in absolute and relative adrenal weights in group 3 females and in group 4 males and females at week 4.
- Increase in relative kidney weights in group 4 females at week 4.
The treatment effects observed at week 4 on these organ weights were not present at the end of the treatment-free period.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Two high dose group males killed after the treatment period showed green and mottled kidneys; each one additional male and female had a greenish discoloured kidney. One high dose group male and two high dose group females showed mottled livers. These changes were not observed at the end of the treatment-free period. Alopecia was observed in both sexes of group 4 at week 4 and in group 4 females at week 6.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

No treatment-related histological abnormalities were observed on organs examined after 4 weeks of treatment.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Highest dose level with no observed effects.

Dose descriptor:

LOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

clinical biochemistry

clinical signs

haematology

organ weights and organ / body weight ratios

urinalysis

Critical effects observed:

not specified

Suspensions prepared for the first week and the fourth week of the study were assayed for achieved concentrations by means of HPLC.

Table 2. test article achieved concentrations

Dose Group	Theoretical concentrations (mg/L)	Week 1			Week 4
		(mg/L)	(mg/L)	x̄	(mg/L)	(mg/L)	x̄
1	0	0	0	-	0	0	-
2	1000	911.1	910.4	910.8	948.6	942.8	945.7
3	5000	4632	4670	4651	4830	4829	4830
4	50000	49266	49767	49517	49341	49172	49257

Table 3: Absolute and relative weights of affected organs in males (mean of five animals)

	control	10 mg/kg bw	100 mg/kg bw	500 mg/kg bw
adrenal glands (mg), right	27	28	25	34*
SD	4	4	3	4
adrenal glands (mg), left	25	29	26	38*
SD	7	3	2	5
liver (g)	9.68	8.78	9.62	14.18*
SD	0.95	0.88	0.59	1.15
kidney (g), right	1.29	1.19	1.22	1.42
SD	0.07	0.1	0.11	0.08
kidney (g), left	1.29	1.18	1.2	1.41
SD	0.06	0.13	0.12	0.13
relative adrenal weight, right	0.0083	0.0094	0.079	0.103
relative adrenal weight, left	0.0079	0.0097	0.0082	0.0116
relative liver weight	3.03	2.902	3.007	4.272*
relative kidney weight, right	0.406	0.393	0.382	0.428
relative kidney weight, left	0.403	0.389	0.376	0.426
* p < 0.05

Table 4: Absolute and relative weights of affected organs in females (mean of five animals)

	control	10 mg/kg bw	100 mg/kg bw	500 mg/kg bw
adrenal glands (mg), right	34	34	41*	50*
SD	4	1	5	6
adrenal glands (mg), left	33	36	42*	51*
SD	3	3	2	7
liver (g)	6.08	6.13	6.4	10.85*
SD	0.59	0.58	0.71	1.12
kidney (g), right	0.78	0.81	0.87	0.9
SD	0.07	0.03	0.12	0.05
kidney (g), left	0.77	0.79	0.84	0.89
SD	0.07	0.04	0.11	0.08
relative adrenal weight, right	0.0164	0.0161	0.0189	0.0256*
relative adrenal weight, left	0.016	0.017	0.0196*	0.0265*
relative liver weight	2.978	2.881	2.959	5.567*
relative kidney weight, right	0.38	0.381	0.4	0.463*
relative kidney weight, left	0.375	0.372	0.387	0.458*
* p < 0.05

Conclusions:

The highest dose level with no observed effects (NOEL) was 10 mg/kg bw/day. Clear treatment related toxicological effects were noted in the 50 mg/kg bw/day and 500 mg/kg bw/day groups.

Executive summary:

In this guideline (OECD 407) study conducted with GLP certification, the test material (EC 404-360-3) was prepared in a vehicle of carboxymethycellulose (CMC) to various dosages (0, 10, 50 and 500 mg/kg bw/day) and given to groups of 10 rats of both male and female sex daily via oral gavage for four weeks with a two week recovery period. No mortality was observed although toxic effects were, thus the test material was determined to have a NOEL of 10 mg/kg bw/day and a LOAEL of 500 mg/kg bw/day.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

100 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

Valid

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

A subacute oral toxicity study following OECD 407 and GLP and its dose-range finding study were performed in 1989. In preparation for a one-generation study, a 28-day extended GLP-compliant dose-range-finding study was performed in 2009. The result of the one-generation study was reported in 2011. The studies in 1989 were performed in Sprague-Dawley rats using 0.5 % CMC as vehicle. The later studies were performed with Wistar rats using propylene glycol as vehicle.

In the subacute toxicity study of 1989, doses of 0, 10, 50 or 500 mg/kg bw/day of test article were administered daily via gavage to rats of both sexes for 28 days (Hazleton 1989). Satellite groups with a recovery period of 14 days were included. At the dose level of 500 mg/kg bw/day strong increases in liver weights weights were seen. Kidneys appeard green and mottles and their weights were also increased. Histopathology showed that liver, kidneys and sternal bone marry (myeloid atrophy) were affected. Slight changes in blood and urine parameters were seen. All effects were considered as treatment related but were fully reversible during the recovery period. The NOEL was set on 10 mg/kg bw/day, based on a slight and reversible increase in adrenals at 50 mg/kg bw.

The corresponding dose-range-finder study used doses of 100, 300, 1000 and 3000 mg/kg bw for 14 days. Mortalities were observed in group 4 (1000 mg/kg bw) and 5 (3000 mg/kg bw) animals: One group 4 female and three group 5 females were killed between the third and the fifth day of treatment after a marked body weight loss. At the clinical observations, the group 4 animals showed slight subdued behaviour from day 3 to day 9 of treatment. In group 5, subdued behaviour was observed before the third administration in both sexes with ataxia in females. After treatment, ventral decubitus and tremors were observed in both sexes with hypersalivation in females. These signs were reduced with the time. Alopecia or stained fur were observed in some group 3, 4 and 5 females. At terminal sacrifice, no abnormalities were observed at macroscopic examination except alopecia in some group 3 and 4 animals, stained fur in group 5 females and enlarged liver more frequently in treated animals. Absolute and relative adrenal weights were increased in group 4 and 5 males and in group 3, 4 and 5 females. Liver weights were increased in all treated groups with a dose related effect.

 

In the newer GLP conform 28 -day study in rats, performed as a range-finding test for the one-generation study without the full range of observations of a guideline study, a NOAEL of 100 mg/kg bw/day was established (NOTOX 2009). The study was started with doses of 0, 100 and 500 mg/kg bw/day. Due to severe signs of toxicity (strong body weight loss and clinical signs), the treatment for the high dosed animals was stopped after 9 days for 5 days and then they subsequently received 250 mg/kg bw/day for 28 days. The recovery of effects was not studied in this experiment.

At the dose level of 250 mg/kg bw/day effects in hematology (increased prothrombin time (PT), lower reticulocyte and platelet counts) and clinical chemistry (higher alanine aminotransferase activity, higher inorganic phosphate levels) were seen. Additionally, a greenish discolouration of the kidneys among all animals, along with red-brown discolouration of the liver among most females and changes in histopathology like hypertrophy of hepatocytes was evident. Some of these effects had also been seen in the first subacute study, and found to be reversible. As worst case assumption, the observed effects were considered as adverse. Furthermore, it is not clear whether the observed effects were caused by the previous 500 mg/kg bw/ day treatment or by the final 250 mg/kg bw/ day. Regarding this equivocality, the LOAEL was considered to be 250 -500 mg/kg bw/day in this study, and the NOAEL was established at the dose of 100 mg/kg bw/day.

In a GLP conform one-generation study following OECD guideline 415, rats were treated by gavage with doses of 30, 100 and 300 mg/kg bw/d for 110 to 126 days (BASF 2011). This study is included since it is the only study with subchronic exposure and because organ weights and histopathology in all animals were determined for organs affected upon subacute exposure.

All F0 parental animals were assessed by gross pathology (including weight determinations of several organs) and subjected to histopathological examination, special attention being paid to the organs of the reproductive system. Histopthological evaluation was performed for all animals on liver, kidneys, adrenal glands and thyroids.

Clinically, toxicity was noted in the F0 females at 300 mg/kg bw/d. Food consumption was reduced during lactation, body weights /body weight gain were consistently reduced during gestation and lactation.

All high-dose as well as most mid-dose and many low-dose animals of both sexes showed transient salivation for a few minutes immediately after each treatment. This was likely to be induced by the unpleasant taste of the test substance and/or by local irritation of the upper digestive tract. It is neither considered to be a sign of systemic toxicity nor as adverse.

Pathology (see below) confirmed adverse clinical observations were likely to be subsequent to local irritant effects of the test substance in the fore- and glandular stomach. Also, reduced food consumption and weights/body weight gain in females may have been secondary to these local effects.

Regarding pathology, no histopathological findings were observed in reproductive organs of male and female rats in test group 3 (300 mg/kg bw/d). No histopathological correlate was found for the testes weight increase as well as for the weight decrease in prostate and seminal vesicles of male rats in test group 3 (300 mg/kg bw/d). Therefore, weight changes in male reproductive organs were not considered to be treatment-related. In females, no treatment-related effects were found in reproductive organs however, a minimal decrease in terminal body weight occurred in test group 3 (300 mg/kg bw/d) which was attributed to treatment.

The liver of males and females of test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d) was affected by a significant weight increase which was regarded as treatment-related. In test group 3 (300 mg/kg bw/d) the weight increase correlated with hepatocellular midzonal hypertrophy. Although minimal, hepatocellular pigment storage predominantly affected females of test group 3 (300 mg/kg bw/d). The pigment most probably accounted for the gross “green/brown” liver discoloration. All of these findings were related to treatment but only the strong liver enlargement of the high dose group is considered as adverse. No histopathological correlate was found for the liver weight increase in test group 2 (100 mg/kg bw/d), which was regarded as adaptive.

In the kidneys of male animals, although control weights were minimally above the maximal historical control values, the relative weight increase of 109% and 113% observed respectively in test groups 02 and 03 (100 and 300 mg/kg bw/d) were regarded as treatment-related and were associated with histopathological findings. These showed a dose-dependent increase of eosinophilic droplets in the proximal convoluted tubules. The eosinophilic droplets displayed at the Chromotrope-Aniline Blue stain (CAB) a histomorphologic pattern and tubular distribution characteristic of α2u-globulin (Hard, 2008). α2u-globulin is a male and rat specific poor soluble protein synthesized in the male rat liver, filtered by the glomerulus and reabsorbed in the S2 segment of the proximal tubules where it is slowly hydrolyzed by lysosomal digestion. Reversible binding of the test-substance or their metabolites to α2u-globulin decreases the effectiveness of its lysosomal digestion, resulting in strong accumulation of this protein that can be visible in form of characteristic eosinophilic droplets.The histopathologic changes in the kidneys were considered to be treatment-related but not adverse. No correlate was found for the gross “green brown discoloration” in the kidneys of males and females of test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d).

In the glandular stomach of males and females of test groups 2 (100 mg/kg bw/d) and 3 (300 mg/kg bw/d), minimal to slight mucosal hyperemia correlated with “red focal discolorations”. Males were more affected than females and showed a clear dose relationship in test group 3 (300 mg/kg bw/d). Mucosal hyperemia was attributed to a local effect due to treatment but was regarded as not adverse. In the subacute studies, no macroscopic findings for stomach were reported. Histopathology was only performed in the older subacute study (Hazleton 1989) and no adverse findings on stomach were reported. It is possible that the irritating effects manifest only after prolongued dosing. This would fit with the delayed onset of post-dosing salivation; this started after the fifth week of dosing.

In the thyroid gland, a dose-dependent hypertrophy and / or hyperplasia of the follicular epithelium started to be observed from test group 02 (100 mg/kg bw/d) in males (minimal) and females (minimal to slight). These changes might be associated with an increased thyroxin catabolism occurring in the liver due to hepatic enzyme induction. This well-known phenomenon is characteristic for rodents (Curran et al., 1991), and there is no convincing evidence that humans exposed to chemicals that induce hepatic microsomal enzymes have increased risk for any thyroid gland disorder (Capen, 1997). In males, organ weights including controls were minimally above the maximal historical control values (abs.: 29.000 mg / rel.: 0.008%); however, histopathology was not associated to statistically increased weights. In females, although a significant absolute and relative thyroid weight increase was seen at the high dose (300 mg/kg bw/d), the values were within the historical control range (abs. / rel.: 21.957 g / 0.01%). Thus, the thyroid gland findings in test group 2 and 3 (100 and 300 mg/kg bw/d) were regarded as treatment-related and not adverse. No treatment-related findings were seen in the thyroid gland of males and females of test group 01 (30 mg/kg bw/d). In the subacute studies, no macroscopic findings regarding the thyroid were noted and no further investigations on thyroid were performed.

The adrenal gland of females showed a dose-dependent weight increase starting from test group 02 (100 mg/kg bw/d). In test group 03 (300 mg/kg bw/d), cortical cells with condensed eosinophilic cytoplasm devoid of lipid vacuoles in the zona fasciculata correlated with significantly increased organ weights (absolute/ relative: 122% / 127%). These (abs. / rel.: 94.9 mg / 0.042%) were above the maximal historical control values (abs.: 85.7 mg; rel.: 0.035%). These findings may represent ACTH-induced depletion related to stress (Hamlin II and Banas, 1990). In test group 02 (100 mg/kg bw/d), the weight of the adrenal glands was statistically increased (abs. / rel.: 115% / 113 %) and above the maximal historical control values (abs. / rel.: 89.4 mg / 0.038%) but without any histopathological correlate. Nevertheless, all these findings in test group 02 and 03 females were interpreted as treatment-related, but secondary adaptive. No treatment-related findings were seen in females of test group 01 (30 mg/kg bw/d).

No histopathological correlate was found in the adrenal glands of male animals of test group 03 (300 mg/kg bw/d), which had a statistically significant relative glandular weight increase (112%) but below the maximal historical control values (0.019%). Therefore, the adrenal gland weight increase in males of the high dose group was regarded as incidental and not related to treatment. In the subacute studies, no histopathological findings were observed for adrenals. Absolute and relative weights were increased in the older , but not in the newer subacute study.

Overall, the DNEL is derived from a NOAEL of 100 mg/kg bw for subacute oral exposure.

Justification for selection of repeated dose toxicity via oral route - systemic effects endpoint:
All available repeated-dose studies contribute to the NOAEL definition.

Repeated dose toxicity: via oral route - systemic effects (target organ) digestive: liver

Justification for classification or non-classification

Information is compiled from two subacute oral toxicity studies, a 14-day range-finder study and a one-generation study (subchronic exposure). Studies were performed according to GLP. The substance causes no adverse effects at doses of up to 100 mg/kg bw. A steep dose-response relationship starts at doses exceeding 300 mg/kg bw depeding on rat strain and/or vehicle. In one study, extreme liver enlargement, but no body weight effects were observed at 500 mg/kg bw whereas in the other study, 500 mg/kg bw were not tolerated and the dose had to be discontinued after 9 days of of dosing. The sum of the information is adequate for the hazard assessment. The substances causes an adaptive liver enlargement at lower doses and and adverse findings on liver with secondary effects at high subchronic doses.

From data of two reliable subacute oral toxicity studies, the most sensitive values for NOAEL and LOAEL were 100 mg/kg bw/day and 250 - 500 mg/kg bw/d, respectively. Additionally, no adverse effects of general toxicity were observed in male and female rats in doses up to 100 mg/kg bw/d in a one-generation study, during which animals received the test substance for 16 to18 weeks per gavage and which falls under the definition of subchronic exposure.

Dangerous Substance Directive (67/548/EEC)

The available studies are considered reliable and suitable for classification purposes under 67/548/EEC. No serious irreversible effects were observed at dose levels of less than 150 mg/kg bw upon subacute exposure or at less than 50 mg/kg bw upon subchronic exposure. As a result the substance is not considered to be classified for repeated dose toxicity under Directive 67/548/EEC, as amended for the 31st time in Directive 2009/2/EG.

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

The available experimental test data are reliable and suitable for classification purposes under Regulation 1272/2008. There were no significant toxic effects at doses of less than 300 mg/kg bw upon subacute oral exposure in rats and at less than 100 mg/kg bw upon subchronic exposure. As a result the substance is not classified for repeated dose toxicity under Regulation (EC) No. 1272/2008, as amended for the fifth time in Directive EC 944/2013.