N,N-bis(2-hydroxypropyl)benzamide

Endpoint summary

Description of key information

In a 28-day subacute toxicity study via oral route of administration the NOAEL of the substance was found to be 1000 mg/kg bw.

Test item-related findings were generally restricted to salivation during daily observation and elevated liver weights and slight centrilobular hepatocellular hypertrophy after 4 weeks, in both sexes at the high dose level of 1000 mg/kg bw/day (no treatment related findings at 50 and 200 mg/kg bw/day). All these findings were reversible during the recovery period and histopathological findings in males and females treated with 1000 mg/kg/day, in the absence of commensurate changes in clinical biochemistry parameters, were considered to be metabolic adaptation. Therefore, 200 mg/kg bw/day was considered to be the NOEL and 1000 mg/kg bw/day was considered to be the NOAEL in this study.

No studies are available on dermal and inhalation routes.

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

From June 8th to August 3rd, 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:

July 27th 1995

Deviations:

no

Qualifier:

according to guideline

Guideline:

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

Version / remarks:

September 30, 1996

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Specific details on test material used for the study:

SOURCE OF TEST MATERIAL
- Source and lot/batch No.of test material: HSH 0015.1
- Expiration date of the lot/batch: 31.03.2003
- Purity: 96%

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: At room temperature (17 - 23 °C) in the original container away from direct sunlight
- Stability under test conditions: Stable under storage conditions
- Solubility and stability of the test substance in the solvent/vehicle: Was proven in analytical report
- Reactivity of the test substance with the solvent/vehicle of the cell culture medium: None, see analytical report as appendix to study

TREATMENT OF TEST MATERIAL PRIOR TO TESTING
- Treatment of test material prior to testing: None, material was dissolved in water
- Preliminary purification step (if any): None
- Final dilution of a dissolved solid, stock liquid or gel:
- Final preparation of a solid: Dissolved in bidistilled water

FORM AS APPLIED IN THE TEST (if different from that of starting material): Dissolved in water by gavage

OTHER SPECIFICS:

Species:

rat

Strain:

Wistar

Details on species / strain selection:

HanBrl:WIST(SPF)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: RCC Ltd Biotechnology and Animal Breeding Division CH-4414 Fullinsdorf/Switzerland
- Age at study initiation: 6 weeks
- Weight at study initiation: Males: 135 - 142 g Females: 110 - 124 g
- Housing: In groups of five in Makrolon type-4 cages with wire mesh tops and standardized softwood bedding
- Acllimation period: 7 days

DETAILS OF FOOD AND WATER QUALITY: Pelleted standard Provimi Kliba 3433 rat maintenance diet ad libitum and community tap-water available ad libitum in water bottles. Feed and drinking water quality was analysed and documented in an appendix to the final report.

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 3 °C
- Humidity (%): 30 - 70
- Air changes (per hr): 10 - 15
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Concentration, homogeneity and stability (after 2 hours and 7 days) of the dose formulations were determined in samples taken after experimental start. Concentration and homogeneity of the dose formulations were determined in samples taken during week 3 of the treatment. The analyses were performed using HPLC method.

Duration of treatment / exposure:

28 days

Frequency of treatment:

Daily

Dose / conc.:

0 mg/kg bw/day (nominal)

Remarks:

Group 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Group 2

Dose / conc.:

200 mg/kg bw/day (nominal)

Remarks:

Group 3

Dose / conc.:

1 000 mg/kg bw/day (nominal)

Remarks:

Group 4

No. of animals per sex per dose:

10 for Groups 1 and 4
5 for Groups 2 and 3

Control animals:

yes, concurrent vehicle

Details on study design:

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment. Additional 5 rats per sex and group were used at 0 and 1000 mg/kg.
These animals were treated for 28 days and then allowed a 14-day treatment-free recovery period after which they were sacrificed.

Positive control:

No

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations are included in documents attached.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Weekly

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Not specified

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: Not specified

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Not specified

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes
- Time schedule for collection of blood: After 4 weeks and after 6 weeks (during recovery period)
- Anaesthetic used for blood collection: light isofluorane
- Animals fasted: Yes
- How many animals: all
- Parameters checked are included in documents attached.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: After 4 weeks and after 6 weeks (during recovery period)
- Animals fasted: Yes
- How many animals: all
- Parameters checked are included in documents attached.

URINALYSIS: Yes
- Time schedule for collection of urine: After 4 weeks and after 6 weeks (during recovery period)
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- Parameters checked are included in documents attached.

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations: During week 4.
- Dose groups that were examined: all
- Battery of functions tested: grip strength / motor activity

IMMUNOLOGY: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes (see documents attached)

HISTOPATHOLOGY: Yes (see documents attached)

Statistics:

Statistical methods used to analyze the grip strenght, locomotor activity, body weight, organ weights and ratios, as well as clinical laboratory data:
- The Dunnett-test (many to one t-test) based ona pooled variance estimate was applied if the variables could be assumed to follow a normal distribution for the comparison of the treated groups and the control groups for each sex.
- The Steel-test (many to one rank test) was applied instead of Dunnett-test when the data could not be assumed to follow a normal distribution
- Fisher's exact-test was applied to the macroscopic findings

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

At 1000 mg/kg/day, salivation was noted in one male and two females on day 22 and 23 of treatment, in two males and one female on day 24 of treatment, in two females on day 27 of treatment and in one female on day 28 of treatment. This finding was considered to be a slight test item-related effect.
No clinical signs in rats treated at doses of 50 and 200 mg/kg/day

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, non-treatment-related

Description (incidence and severity):

Statistically higher body weights were noted on days 22 and 28 in males treated with 200 mg/kg/day, but were not evident in males treated with 1000 mg/kg/day and were therefore considered to be incidental.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 4 weeks' treatment, a significant increase in the ratio of middle fluorescent reticulocytes was noted in males treated with 50 mg/kg/day or 200 mg/kg/day, and a significant reduction in the number of low fluorescent reticulocytes was increased in the males treated with 1000 mg/kg/day. The latter finding persisted after the two week recovery period, and the number of high fluorescent reticulocytes was increased in the males previously treated with 1000 mg/kg/day, although this was considered to be due to a low control value.

After four weeks' treatment, the mean levels of methemoglobin were significantly elevated in test item-treated males when compared with the controls. This finding persisted after the two week recovery period for the males treated with 1000 mg/kg/day. This finding was not seen in females, remained within the 95% tolerance limits of the historical control data and was considered to be caused by a low mean value in the control males (0.8 in historical control data and 0.4 in control males). All values remained within the 95% tolerance limits of the historical control data and a clear dose-response relationship was not present.

Slight but statistically significant elevations of the absolute and relative reticulocytes counts were noted in the females treated with 1000 mg/kg/day after 4 weeks' treatment, and the ratio of high fluorescent reticulocytes was increased when compared with the controls. Although statistically significant, these changes remained within the 95% tolerance limits of the historical control data, were not seen in males, were not dose-dependent and were not accompanied by indications of anemia, and therefore considered to be incidental.

After the recovery period, the ratio of high fluorescent reticulocytes remained significantly higher in males and females previously treated with 1000 mg/kg/day when compared with the controls. These differences remained within the 95% tolerance limits of the historical control data and were considered to be incidental.

Clinical biochemistry findings:

effects observed, non-treatment-related

Description (incidence and severity):

After 4 weeks' treatment, males treated with 1000 mg/kg/day had significantly lower glucose levels. Although the glucose levels exceeded the 95% tolerance limits of historical dontrol data, a dose-response relationship was absent and therefore this difference was considered to be incidental.
Significantly higher phospholipid and sodium levels were also noted in males treated with 1000 mg/kg/day, but these differences remained within the 95% tolerance limits of historical control data and therefore were considered to be fortuitous.

Male rats also showed significant differences n the lactate dehydrogenase activity at all dose levels, in creatine kinase levels at 50 and 1000 mg/kg/day, in the phosphorous levels at 50 mg/kg/day and in the potassium levels at 50 mg/kg/day after 4 weeks' treatment. In the absence of dose-response relationship, these differences were considered to be incidental.

Differences noted after the two week recovery period in males previously treated with test item included higher sodium levels and lower potassium levels. All the differences, however, compared favorably with the historical control data and therefore were considered to be due to incidental differences in the control values.

All differences noted after four weeks' treatment in females (increased bilirubin at 50 and 1000 mg/kg/day; increased total cholesterol at 1000 mg/kg/day, increased phospholipids at 1000 mg/kg/day; reduced activity of aspartate aminotransferase at 1000 mg/kg/day; decreased lactate dehydrogenase at 50 mg/kg/day; increased calcium at 200 and 1000 mg/kg/day) were not supported by a dose-response relationship or remained within the 95% tolerance limits of historical control data and therefore were considered to be incidental.
Likewise, the few differences noted after two weeks' recovery in females at 1000 mg/kg/day (increased phosphorus, increased globulin) remined within the 95% tolerance limits of historical control data and therefore considered to be incidental.

Urinalysis findings:

effects observed, non-treatment-related

Description (incidence and severity):

In males and females treated with 1000 mg/kg/day for four weeks, the urinary output (18h) was increased when compared with the controls. Differences were significant in males.
Significant increases in the specific gravity of males an females at 1000 mg/kg/day were noted, and urinary pH of males at 200 and 1000 mg/kg/day was decreased significantly. All values remained within the 95% tolerance limits of the historical control data and were considered to be fortuitous.

Behaviour (functional findings):

effects observed, non-treatment-related

Description (incidence and severity):

The mean forelimb grip strenght of the females treated with 50 mg/kg/day was significantly decreased when compared with the controls. The mean hindlimb grip strenght of the males treated with 1000 mg/kg/day and the females treated with 200 mg/kg/day were significantly increased when compared with the controls. In the absence of similar findings in the opposite sex, or simultaneous changes in the grip strenght of the remaining for/hind paws, these differences were considered to be incidental.

The mean locomotor activity of the test item-treated males compared favorably with those of the control males.
In females treated with 200 mg/kg/day, significantly higher locomotor acticity was noted during the measurement intervals of 0 - 15 minutes, 15 - 30 minutes and 0 - 60 minutes. Females treated with 1000 mg/kg/day had significantly reduced locomotor activity during 0 - 15 minutes and 45 - 60 minutes. In the absence of similar changes in the males (or consistent changes in females), these differences were considered to be incidental.

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

After 4 weeks:
Elevated absolute and relative liver weights were noted in males and females treated with 1000 mg/kg/day. In the same groups, absolute kidney weights were slightly higher. These changes were considered to be test item related.

All other differences in organ weights (increased absolute adrenal weights in males at 200 mg/kg/day, increased adrenal-to-body weight ratio in males at 50 mg/kg/day , decreased testes-to-body weight ratio in males at 200 mg/kg/day) were, in the absence of a dose-response relationship, considered to be incidental.

After 6 weeks:
The absolute and relative organ weights of male and female rats previously treated with 1000 mg/kg/day were considered to be similar to those of the control after the recovery period. Therefore, the test item-related effect was temporary.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of macroscopic findings were observed in rats of all groups. No findings were observed that were considered to be related to the administration of test item at the end of the 28-day period and at the end of the 14-day recovery period. All findings diagnosed either did not distinguish test item-treated rats from controls or were considered to be of no toxicological relevance.

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Minimal to slight centrilobular hepatocellular hypertrophy was diagnosed in all males and females treated with 1000 mg/kg/day. In males, the mean severity grade for this change was 1.4, while in females the mean severity grade was 1.0

Histopathological findings: neoplastic:

no effects observed

Other effects:

effects observed, non-treatment-related

Description (incidence and severity):

A number of microscopic findings were observed in rats of all groups. No findings were observed that were considered to be related to the administration of test item at the end of the 28-day period and at the end of the 14-day recovery period. All findings diagnosed either did not distinguish test item-treated rats from controls or were considered to be of no toxicological significance.

Dose descriptor:

NOEL

Effect level:

200 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Dose descriptor:

NOAEL

Effect level:

1 000 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

behaviour (functional findings)

body weight and weight gain

clinical biochemistry

clinical signs

food consumption and compound intake

gross pathology

haematology

histopathology: neoplastic

histopathology: non-neoplastic

mortality

organ weights and organ / body weight ratios

urinalysis

Key result

Critical effects observed:

no

Lowest effective dose / conc.:

1 000 mg/kg bw/day (nominal)

Organ:

liver

Treatment related:

yes

Dose response relationship:

no

Relevant for humans:

no

Conclusions:

Test item-related findings were generally restricted to salivation during daily observation and elevated liver weights and slight centrilobular hepatocellular hypertrophy after 4 weeks, in both sexes at dose level of 1000 mg/kg bw/day.
All these findings were reversible during the recovery period.
Reversible changes in liver weights and histopathological findings in males and females treated with 1000 mg/kg/day, in the absence of commensurate changes in clinical biochemistry parameters, were considered to be metabolic adaptation.
Based on this considerations, 200 mg/kg bw/day was considered to be the NOEL and 1000 mg/kg bw/day was considered to be the NOAEL.

Executive summary:

In this subacute toxicity study, the substance was administered daily by oral gavage to SPF-bred Wistar rats of both sexes at dose levels of 50, 200 and 1000 mg/kg bw/day for a period of 28 days. A control group was treated similarly with the vehicle, bidistilled water, only.

The groups comprised 5 animals per sex which were sacrificed after 28 days of treatment, and 5 additional rats per sex for control and 1000 mg/kg bw/day dose groups, which were sacrificed after a 14-day recovery period.

Clinical signs, outside cage observation, food consumption and body weights were recorded periodically during pretest, treatment and recovery periods. Functional observational battery, grip strenght and locomotor activity were performed during week 4.

Blood and urine samples were collected at the end of the treatment and at the end of the recovery period for analyses.

All animals were killed, necropsied and examined post mortem. Histological examinations were performed on organs and tissues from all dose groups. From animals of the low and middle dose groups, livers were examined to establish a no-effect level.

Oral administration of the substance resulted in no mortality, no effects upon the weekly behavioural observations (weeks 1 - 3), no changes in grip strenght or locomotor activity, no effects upon food consumption and body weight, and no changes in hematology, clinical biochemistry or urinalysis parameters.

Test item-related findings were generally restricted to salivation during daily observation and elevated liver weights and slight centrilobular hepatocellular hypertrophy after 4 weeks, in both sexes at dose levele of 1000 mg/kg bw/day. All these findings were reversible during the recovery period. Reversible changes in liver weights and histopathological findings in males and females treated with 1000 mg/kg bw/day, in the absence of commensurate changes in clinical biochemistry parameters, were considered to be metabolic adaptation. Based on this considerations, 200 mg/kg bw/day was considered to be the NOEL and 1000 mg/kg bw/day was considered to be the NOAEL.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

1 000 mg/kg bw/day

Study duration:

subacute

Species:

rat

Repeated dose toxicity: inhalation - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: inhalation - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Justification for classification or non-classification

A subacute toxicity study (gavage) on rats revealed no signs of systemic toxicity or negative effects in a dose relevant for classification. Hence, it can be concluded that the substance does not meet the criteria for classification and labeling for repeated dose toxicity (STOT), as set out in Regulation (EC) No. 1272/2008. As a result the substance is not subject to classification for STOT repeat exposure.