2,2'-[(4-methylphenyl)imino]bisethanol

Repeated dose toxicity: oral

Administrative data

Endpoint:

short-term repeated dose toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Study period:

14 Nov 2012 - 18 Jan 2013

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2013

Report date:

2013

Materials and methods

Test guideline

Test guideline 1

Qualifier:

according to guideline

Guideline:

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

Test guideline 2

Qualifier:

according to guideline

Guideline:

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

Test guideline 3

Qualifier:

according to guideline

Guideline:

other: United States Environmental Protection Agency (EPA). Health Effects Test Guidelines, OPPTS 870.3050, Repeated dose 28-day oral toxicity study in rodents. Office of Prevention, Pesticides and Toxic Substances (7101), EPA 712-C-00-366, July 2000

GLP compliance:

yes

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

2,2'-[(4-methylphenyl)imino]bisethanol

EC Number:

221-359-1

EC Name:

2,2'-[(4-methylphenyl)imino]bisethanol

Cas Number:

3077-12-1

Molecular formula:

C11H17NO2

IUPAC Name:

2,2'-[(4-methylphenyl)imino]diethanol

Constituent 2

Reference substance name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Cas Number:

878391-30-1

Molecular formula:

C13H21NO3

IUPAC Name:

2-{[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino}ethan-1-ol

Test material form:

liquid: viscous

Details on test material:

- Appearance: clear, slightly yellowish to brown, viscous liquid
- Storage condition of test material: at room temperature in the dark

Test animals

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: 8 weeks
- Weight at study initiation: males 223 - 282 g; females 147 - 192 g
- Fasting period before study: no
- Housing: Group houses (5 animals per sex) in Macrolon cages
- Diet: Free access to pelleted rodent diet (SM R/M-Z from SSNIFF® Spezialdiäten GmbH, Soest, Germany); During motor activity measurements, animals had no access to food; Animals were deprived of food overnight (with a maximum of 24 hours) prior to scheduled necropsy
- Water: ad libitum, tap water
- Acclimation period: at least 7 days

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24
- Humidity (%): 40 - 70
- Air changes (per hr): approximately 15
- Photoperiod (hrs dark / hrs light): 12/12

IN-LIFE DATES: From: 14 Nov 2012 To: 18 Jan 2013

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

polyethylene glycol

Details on oral exposure:

VEHICLE
- Justification for use and choice of vehicle: Based on trial formulations performed at WIL Research Europe and on information from the sponsor and on the results of the dose range finding study.
- Concentration in vehicle: 20, 60, 120, 200 mg/mL
- Amount of vehicle: 5 mL/kg

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The accuracy of preparation was considered acceptable if the mean measured concentrations were 90 - 110% of the target concentration for solutions. Homogeneity was demonstrated if the coefficient of variation is ≤ 10%. Formulations were considered stable if the relative difference before and after storage was maximally 10%.

Duration of treatment / exposure:

At least 28 days

Frequency of treatment:

Once daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

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

Doses / concentrations 2

Dose / conc.:

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

Doses / concentrations 3

Dose / conc.:

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

Doses / concentrations 4

Dose / conc.:

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

No. of animals per sex per dose:

5

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: Based on the results from the dose range finding study.

Positive control:

No.

Examinations

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: At least twice daily.

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations were conducted after dosing at no specific time point, but within a similar time period after dosing for the respective animals, based on the range finding study.

BODY WEIGHT: Yes
- Time schedule for examinations: Weekly

FOOD CONSUMPTION: Yes
- Time schedule for examinations: Weekly

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

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: Subjective appraisal was maintained during the study, but no quantitative investigation introduced as no effect was suspected.

OPHTHALMOSCOPIC EXAMINATION: No
- Time schedule for examinations:
- Dose groups that were examined:

HAEMATOLOGY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Anaesthetic used for blood collection: Yes (isoflurane)
- Animals fasted: Yes
- How many animals: all
- Parameters following OECD guideline were checked

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: at the end of the treatment
- Animals fasted: Yes
- How many animals: all
- Parameters following OECD guideline were checked

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Motor activity testing was performed

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all animals
HISTOPATHOLOGY: Yes
The following slides were examined by a pathologist:
- all tissues collected at the scheduled sacrifice from all control animals and animals exposed to 600 mg/kg bw/day.
- all tissues from all animals of control group and animals exposed to 100, 300 and 600 mg/kg bw/day, which died spontaneously or were terminated in extremis,
- all gross lesions.
No histopathology was performed on animals exposed to 1000 mg/kg bw/day that died spontaneously or that were sacrificed.

On detection of treatment-related morphological changes in the urinary bladder of the females in the high dose group (600 mg/kg bw/day) the histological examination was extended to that particular organ of all females exposed to 100 and 300 mg/kg bw/day. All abnormalities were described and included in the report. An attempt was made to correlate gross observations with microscopic findings.

Statistics:

The following statistical methods were used to analyze 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 if the data could not be assumed to follow a normal distribution.
- The Fisher Exact-test was applied to frequency data.
- The Kruskal-Wallis nonparametric ANOVA test (Ref. 4) was applied to motor activity data to determine intergroup differences. In case intergroup differences were seen, the Wilcoxon test was applied 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. Test statistics were calculated on the basis of exact values for means and pooled variances. Individual values, means and standard deviations may have been rounded off before printing. Therefore, two groups may display the same printed means for a given parameter, yet display different test statistics values.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

The animals treated at 1000 mg/kg bw/day for one day showed slight lethargy, severe clonic spasms, tremor, flat posture, and/or hypersensitivity to touch on Day 1 prior to death. Hunched posture or piloerection was noted in the surviving animals at 1000 mg/kg bw/day on Days 2 and/or 3.

The animals at 600 mg/kg bw/day showed during the study period: lethargy, clonic spasms, tremors, flat and/or hunched posture, quick breathing, rales, laboured and/or shallow respiration, piloerection, salivation and/or chromodacryorrhoea.

The males at 300 mg/kg bw/day showed incidentally over the study period flat posture, quick breathing, rales and/or chromodacryorrhoea. The females showed lethargy, tremor, hunched posture, uncoordinated movements, laboured or shallow respiration, rales, piloerection, salivation and/or ptosis during the study period predominantly in the first three days.

No toxicologically relevant clinical signs were noted in control animals and animals treated at 100 mg/kg bw/day. Scabs were noted in one male at 100 mg/kg bw/day in a short period. This finding occurred within the range of background findings to be expected for rats of this age and strain which are housed and treated under the conditions in this study. At the incidence observed, this was considered a sign of no toxicological significance.

Mortality:

mortality observed, treatment-related

Description (incidence):

At the highest concentration (1000 mg/kg bw/day) two males and three females died on Day 1. The highest dose was lowered to 600 mg/kg bw/day and animals were replaced. At 600 mg/kg bw/day one male and one female died after first exposure. No other mortality occurred in the animals that survived the treatment with 1000 mg/kg bw/day, or in the animals that replaced the animals that died on the first day.
No mortality occurred in control animals and animals at 100 and 300 mg/kg bw/day.

Body weight and weight changes:

no effects observed

Description (incidence and severity):

No toxicologically significant changes in body weights and body weight gain were noted.
The body weight gain in the first week appeared to be lower at 600 mg/kg bw/day compared to controls but recovered thereafter.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

No toxicologically significant changes in food consumption before or after correction for body weight were noted.

Food efficiency:

no effects observed

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

No toxicologically relevant changes occurred in haematological parameters of treated rats.
In animals at 600 mg/kg bw/day increased red blood cell counts and hematocrit level was noted. These changes were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain.

Minor statistically significant differences (platelet count and red blood cell distribution width) arising between controls and animals receiving 100 and 300 mg/kg bw/day were considered not to represent a change of biological significance.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

The following (statistically significant) changes in clinical biochemistry parameters distinguished treated animals at 600 mg/kg bw/day from control animals and were outside the range considered normal for rats of this age and strain:
- higher total bilirubin in males and females
- higher cholesterol in females (not statistically significant in males)

Statistically significant changes (at 600 mg/kg bw/day) remaining within the range considered normal for rats of this age and strain included:
- higher alanine aminotransferase level in females (ALAT); increased in all exposed groups, dose-related increase
- higher bile acid, calcium and inorganic phosphate level in females (higher inorganic phosphate also noted at 300 mg/kg bw/day)

Any statistically significant changes at 100, 300 or 600 mg/kg bw/day were considered to be of no toxicological significance as they occurred in the absence of a treatment-related distribution and remained within the range considered normal for rats of this age and strain. These findings included lower glucose level, higher sodium and chloride level.

Urinalysis findings:

not examined

Behaviour (functional findings):

no effects observed

Description (incidence and severity):

Hearing ability, pupillary reflex, static righting reflex and grip strength were normal in all animals.
Motor activity in males was similar between treated and control groups. The females showed lower total movements and lower ambulation counts at 600 mg/kg bw/day.
All groups showed a similar motor activity habituation profile with high activity in the first interval that decreased over the duration of the test period.

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Higher liver weight (absolute and relative) were noted at 300 (females; >10% larger than control) and 600 mg/kg bw/day (male and female). Higher thyroid weight (absolute and relative) were found at 600 mg/kg bw/day (males and females)
Other organ weights and organ to body weight ratios among the dose groups were similar to control levels.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

The animals treated at 1000 mg/kg bw/day, which were found dead on Day 1, showed discoloration of the stomach and/or foci in the thymus. The animals at 600 mg/kg bw/day, which were sacrificed on Day 1 showed foci in the thymus and/or lungs and thickened thymus.

In the other animals necropsy revealed the following findings in single animals: nodules in stomach (control animal), discoloration of clitoral gland (100 mg/kg bw/day), fluid in the uterus (100 and 300 mg/kg bw/day), foci in stomach (300 mg/kg bw/day), and enlarged liver, pelvic dilation in the kidney, reduced size of testes and epididymides, foci in clitorial gland and/or adrenal glands and enlarged clitorial gland (600 mg/kg bw/day). These necropsy findings were considered to be of no toxicological relevance the incidence was very low and within the expected range of findings that are encountered among rats of this age and strain.

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

In 2/5 female rats at 600 mg/kg bw/day a minimal degree of diffuse hyperplasia of the urothelium of the urinary bladder was recorded. Based on the low grade and the absence of additional histopathologic findings, this was considered to be a non-adverse treatment-related effect.
All remaining microscopic findings recorded were considered to be within the normal range of background pathology encountered in rats of this age and strain.

Effect levels

Key result

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: Increased liver weights (female) at 300 mg/kg bw/day coinciding with elevated levels of ALAT, total bilirubin, and cholesterol (levels increased in exposed groups in a dose-dependent way).

Target system / organ toxicity

Key result

Critical effects observed:

yes

Lowest effective dose / conc.:

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

System:

hepatobiliary

Organ:

liver

Treatment related:

yes

Dose response relationship:

yes

Relevant for humans:

yes

Applicant's summary and conclusion

Conclusions:

A sub-acute 28 days repeated dose study was performed according to OECD 407 and GLP principles. Based on effects seen at 300 mg/kg bw/day (enlarged liver with dose-related changes in biochemical parameters), the NOAEL was found to be 100 mg/kg bw/day.

Executive summary:

In a sub-acute repeated dose study performed according to OECD guideline and GLP principles, rats were treated with 100, 300 or 1000 mg/kg bw/day Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]-. Due to mortality in the highest dose group on day 1 (2/5 males and 3/5 females), the highest dose was lowered to 600 mg/kg bw/day and the animals were replaced. One male and one female died shortly after the first dose of 600 mg/kg bw/day. At macroscopic examination these animals showed foci in the thymus and/or lungs and thickened thymus. No further mortality occurred. Clinical signs were noted during the study period at 600 mg/kg bw/day (lethargy, clonic spasms, tremors, flat and/or hunched posture, quick breathing, rales, laboured and/or shallow respiration, piloerection, salivation and/or chromodacryorrhoea). During the first week the body weight gain was slightly lower but recovered from week 2 onwards. No changes were noted in food consumption, or functional behaviour were noted compared to control animals. At the highest dose level of 600 mg/kg bw/day higher liver weights and thyroid gland weights were noted in males and females, coinciding with changes in total bilirubin and cholesterol levels. Liver weights were also increased in females at 300 mg/kg bw/day. In females ALAT-values, total bilirubin, and cholesterol were elevated in all groups and increased in a dose-dependent way (significant at 600 mg/kg bw/day).

Microscopic examination revealed in some females diffuse hyperplasia of the urothelium of the urinary bladder (2/5 females at 600 mg/kg bw/day). Based on the low grade and the absence of additional histopathologic findings, this was considered to be a non-adverse treatment-related effect. Based on the effects on liver at 300 mg/kg bw/day with dose-related changes in biochemical parameters (ALAT, cholesterol, total bilirubin and bile acids), a No Observed Adverse Effect Level (NOAEL) for Reaction mass of 2,2'-[(4-methylphenyl)imino]bisethanol and Ethanol 2-[[2-(2-hydroxyethoxy)ethyl](4-methylphenyl)amino]- of 100 mg/kg bw/day was established.