1,3-di-o-tolylguanidine

Endpoint summary

Description of key information

A 28-day repeated oral study is available in rat. A NOAEL of 30 mg/kg bw was determined based on mortality, decrease of food consumption and bodyweight at 60 mg/kg bw. No organ specific toxicity attributable to DOTG exposure was apparent.

Key value for chemical safety assessment

Toxic effect type:

dose-dependent

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

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Qualifier:

according to guideline

Guideline:

other: Guideline for 28-Day Repeated Dose Toxicity Test in Mammalian Species (Chemical Substances Control Law of Japan)

Deviations:

not specified

Qualifier:

equivalent or similar to guideline

Guideline:

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

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Atsugi Breeding center, Charles River Japan Co.
- Age at study initiation: 6 weeks of age
- Weight at study initiation: 197.5-225.2 g (males), 142.5-168.0 g (females)
- Fasting period before study: no data
- Housing: stainless steel barrier system cages
- Diet (e.g. ad libitum): CRF-1 (Oriental yeast Co. Ltd.), ad libitum
- Water (e.g. ad libitum): water to which sodium hypochlorite (about 2 ppm) had been added, ad libitum
- Acclimation period: 7 days (males), 8 days (females)

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24+/-2
- Humidity (%): 55+/-10
- Air changes (per hr): 13-15
- Photoperiod (hrs dark / hrs light): 12/12 (light : 7 am to 7 pm)

Route of administration:

oral: gavage

Vehicle:

other: 0.5 % Sodium carboxymethylcellulose solution mixed with 0.1 % Tween 80

Details on oral exposure:

Administration volume = 10 ml/kg
The administration dose for each animal was calculated based on the most recent weight.
The same dose of the medium was administered in tyhe control group.

PREPARATION OF DOSING SOLUTIONS:
A solution of 0.1% Tween 80 and 0.5% sodium carboxymethyl cellulose CMC-Na was measured using a scale for the amount required in each concentration of test substance to prepare solutions of 0.75, 1.5, 3 and 6mg/mL. Preparation was conducted weekly, and the prepared mixture was stored at room temperature in the sample storage containers in the area where kept. Solutions with 0.1 and 100mg/mL of N,N’ bis (2-methylphenyl) guanidine suspended in a solution of 0.1% Tween 80 and 0.5% CMC-Na were uniform, and verified to be stable for 8 days when stored in a clear glass container either at room temperature or refrigerated.

DIET PREPARATION : no (gavage)

VEHICLE
The medium was sodium carboxymethyl cellulose (Lot No.: M1R6775, Nacalai Tesque K.K.) and Tween 80 (Lot No.: M0A8651, Nacalai Tesque K.K.) dissolved in purified water and then a solution of 0.1% Tween 80 and 0.5% sodium carboxymethyl cellulose (CMC-Na) was used.
- Justification for use and choice of vehicle (if other than water): no data
- Concentration in vehicle: Control (0 mg/ml), low dose group (0.75 mg/ml), medium dose group 1 (1.5 mg/ml), medium dose group 2 (3 mg/ml), high dose group ( 6 mg/ml).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

no data

Duration of treatment / exposure:

28 days

Frequency of treatment:

daily

Remarks:

Doses / Concentrations:
7.5, 15, 30 and 60 mg/kg/day
Basis:
actual ingested

No. of animals per sex per dose:

Control group : 12 animals/sexe/dose
Treated group (with 7.5 and 15 mg/kg) : 6 animals/sex/dose
Treated group (with 30 and 60 mg/kg) : 12 animals/sex/dose

Control animals:

yes, concurrent vehicle

Details on study design:

Post-exposure period: 14 days

Positive control:

no

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes, four times on the day of administration, including before administration, immediately after administration, 30 min-1h after administration and 4 hours after administration. During the recovery : once per day.
DETAILED CLINICAL OBSERVATIONS: Yes, one time before starting administration, once a week during the administration and recovery periods, and 30min-1h after administration.
1) Observations Inside Cages : Posture, twitching, abnormal locomotion, tremors
2) Observations Outside Cages : Ease of handling, abnormal emission of sounds, tremors, muscle spasms, twitching, breathing, salivation, tearing, pupil diameter, bulging eyeballs, eye or nose secretions, skin, raised fur, fur care, visible mucous, urinary incontinence, muscle contractions, temperature.

BODY WEIGHT: Yes, before administration on the first day of administration, and then at a frequency of one week throughout the administration period and the recovery period for both sexes, and finally on the last day of the administration period and the last day of the recovery period.
FOOD CONSUMPTION : Yes, during the administration period and the recovery period at a frequency of weekly for both sexes
WATER CONSUMPTION : No
OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood: during necropsy at the end of the administration period and at the end of the recovery period.
- Anaesthetic used for blood collection: Yes with 30 mg/kg of sodium pentabarbitol
- Animals fasted: Yes, at least 18 hours prior to blood collection
- How many animals: all animals
- Parameters examined : white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin concentration, platelets, reticulocyte ratio, white blood cell shape examination, prothrombin time, activated partial thromboplastin time.

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: during necropsy at the end of the administration period and at the end of the recovery period.
- Animals fasted: Yes, at least 18 hours prior to blood collection
- How many animals: all animals
- Parameters examined : total protein, albumin, A/G ratio, total bilirubin, GOT, GPT, gamma-glutamyl transpeptidase, alkaline phosphatase, total cholesterol, triglycerides, phospholipids, glucose, blood urea nitrogen, creatinine, inorganic phosphorus, calcium, potassium, chloride, sodium.²

URINALYSIS: Yes
- Time schedule for collection of urine: in the morning (on week 4 of administration was during the time before administration). Additionally, 24 hours of continuous fresh urine provided, although drinking water was provided normally.
- Metabolism cages used for collection of urine: Yes
- Animals fasted: Yes
- How many animals: 6 males and 6 females with the lowest animal numbers in each group
- Parameters examined : Urine volume, color, specific gravity, osmotic pressure, pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen.
- Urinary sediment : 470g of the fresh urine collected was subject to 5 minutes of centrifugal separation. the sediment obtained was examined under a microscope. Parameters examined : epothelial cells, red blood cells, white blood cells, cylinders, non-cellular sediment.

NEUROBEHAVIOURAL EXAMINATION: Yes
1) Field Observations : Excited state, abnormal locomotion, abnormal/stereotypy behavior, drooping eyelids, diarrhea, feces, urine
2) Examination of Sensory Functions : on week 4 of administration and week 2 of recovery. Examinations were performed in the field or on a table on auditory and visual senses, touch, pain, frontal reflex and pupillary reflex.
3) Measurements of Grip Strengh : on week 4 of administration and week 2 of recovery.
4) Measurements of spontaneous locomotion : on week 4 of administration and week 2 of recovery.

Sacrifice and pathology:

GROSS PATHOLOGY: Yes, all the organs
HISTOPATHOLOGY: Yes, all the organs

Other examinations:

Measurement of organs weights : brain, pituary gland, thyroid gland, heart, thymus, lungs, liver, spleen, kidney, adrenals, testes, epididymis, ovaries, uterus.

Statistics:

Mean values and standard deviations were determined for body weight, food consumption, observations on feces and urine function, front limb grip, back limb grip, spontaneous locomotion, urine examinations (excluding qualitative reactions), hematological examinations, blood chemistry examinations, organ weights and relative organ weight to body weight for each group using the Barlett method of uniform dispersion. If the dispersion was uniform, Dunnetts weighted comparison assay was employed, while if the dispersion was not uniform, Steel’s weighted comparison assay was used for comparisons with the control group. Assays were performed on both sides.
Wilcoxon rank sum test was performed for function observations (excluding feces and urine) and examination of sensory function. Fisher’s exact test was performed on the necropsy results. Mann Whitney U assay test was performed on histopathological examination results.

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Mydriasis and salivation was observed in both sexes of the groups with doses of 30mg/kg and higher. Tremors, reduction in spontaneous motor activity, bradypnea, reduction in body temperature and soiled lower abdomen was observed in the 60mg/kg group. Prone position, lateral position and gasping for breath was noted in the cases that perished.

Mortality:

mortality observed, treatment-related

Description (incidence):

1 male and 7 females in the 60mg/kg group died during the administration period.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

In the 60mg/kg group, low values or a trend of low values in weight were noted from day 8 to day 42 of administration in males and from day 8 to day 28 in females of administration.

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

Both sexes in the 60mg/kg group exhibited low values for food consumption on day 2, 8, 15 and 28 of administration.

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):

Males in the 30mg/kg and higher groups exhibited a reduction in APTT.

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

Both sexes in the 60mg/kg group exhibited low values for total protein and high values for GPT and potassium. Furthermore, males exhibited low values for albumin and high values for alkaline phosphatase and blood urea nitrogen. Females exhibited low values for GOT and sodium, and high values for total cholesterol, triglycerides and phospholipids. Also, high values for total cholesterol and phospholipids were notes in females in the 30mg/kg group.

Urinalysis findings:

effects observed, treatment-related

Description (incidence and severity):

Males in the 30mg/kg and higher groups exhibited an increasing trend in urine volume, and females in the 15mg/kg and higher groups exhibited an increase in urine volume, and low values for osmotic pressure and specific gravity were confirmed in conjunction with these changes.

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

Furthermore, during examinations on sensory functions, males in the 60mg/kg group exhibited reduced pupillary reflex, and females exhibited reduced pupillary reflex and reduced visual response. During measurements on spontaneous motor activity, both sexes in the 60mg/kg group exhibited low values in the count for spontaneous motor activity, or a trend of low values when measured for one hour.

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Description (incidence and severity):

Females in the 30mg/kg and higher groups exhibited high values for the relative organ weights for livers.

Gross pathological findings:

effects observed, treatment-related

Description (incidence and severity):

Of the mortalities, one female in the 60mg/kg group exhibited rose colored spots on the proventriculus during visual examination, and slight erosion of the proventriculus was confirmed in histological examination.
Of the survivors, one female in the 60mg/kg group exhibited hypertrophy of the renal cells during histopathological examination.

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

Of the mortalities, one female in the 60mg/kg group exhibited rose colored spots on the proventriculus during visual examination, and slight erosion of the proventriculus was confirmed in histological examination.
Of the survivors, one female in the 60mg/kg group exhibited hypertrophy of the renal cells during histopathological examination.

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Key result

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

female

Basis for effect level:

other: Females in the 15mg/kg and higher groups exhibited an increase of urine volume, and low values for osmotic pressure and specific gravity were confirmed in conjunction with these changes.

Dose descriptor:

NOEL

Effect level:

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

Based on:

test mat.

Sex:

male

Basis for effect level:

other: see 'Remark'

Dose descriptor:

NOAEL

Effect level:

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

Sex:

male/female

Basis for effect level:

other: Based on mortality and decreases on bw and food consumption at 60 mg/kg bw. At 60 mg/kg bw, effects on clinical chemistry and neurobehaviour effects were observed also.

Critical effects observed:

no

Conclusions:

The NOELs are considered to be 15 mg/kg/day for males and 7.5 mg/kg/day for females, based on clinical signs, haematological and urinalysis data.

Executive summary:

Group of male and female rats were exposed to DOTG by gavage at 7.5, 15, 30 and 60 mg/kg bw/d during 28 days. Animals were examined each day for mortality, clinical signs, weight and food consumption ; urinalysis, hematological examination, blood chemistry examination, histopathological examinations were analyzed. Animals were observed 14 days after the final administration.

Mortalities were observed only at the higher dose (60 mg/kg bw), 1/12 males and 7/12 females died. Mydriasis and salivation was observed in both sexes of the groups with doses of 30 mg/kg and higher. Tremors, reduction in spontaneous motor activity, bradypnea, reduction in body temperature and soiled lower abdomen was observed in the 60 mg/kg group. Decreases of food consumption and body weight were observed in rats exposed to 60 mg/kg bw only. Females in the 30mg/kg and higher groups exhibited high values for the relative organ weights for livers.

The NOELs are considered to be 15 mg/kg/day for males and 7.5 mg/kg/day for females, based on clinical signs, haematological and urinalysis data. The NOAEL (males/females) is determined at 30 mg/kg bw based on mortality, decrease of food comsumption, decrease of bodyweight and neurobehavioural effects at 60 mg/kg bw.

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEL

30 mg/kg bw/day

Study duration:

subacute

Species:

rat

Quality of whole database:

The study is the reliable study with a klimisch score of 1.

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

28-day repeated oral study on rats (2003):

Groups of male and female rats were exposed to DOTG by gavage at 7.5, 15, 30 and 60 mg/kg bw/d during 28 days. Animals were examined each day for mortality, clinical signs, bodyweight and food consumption ; urinalysis, hematological examination, blood chemistry examination, histopathological examinations were analyzed. Animals were observed 14 days after the final administration.

Mortalities were observed only at the higher dose (60 mg/kg bw), 1/12 males and 7/12 females died. Mydriasis and salivation was observed in both sexes of the groups with doses of 30 mg/kg and higher. Tremors, reduction in spontaneous motor activity, bradypnea, reduction in body temperature and soiled lower abdomen was observed in the 60 mg/kg group. Decreases of food consumption and body weight were observed in rats exposed to 60 mg/kg bw only. Females in the 30 mg/kg and higher groups exhibited high values for the relative organ weights for livers.

The NOELs are considered to be 15 mg/kg/day for males and 7.5 mg/kg/day for females, based on clinical signs, haematological and urinalysis data. The NOAEL is determined at 30 mg/kg bw based on mortality, decrease of food comsumption, decrease of bodyweight and neurobehavioural effects at 60 mg/kg bw.

 

Justification for classification or non-classification

Based on the available data, no classification for repeated toxicity is required for 1,3-di-o-tolylguanidine according to the Regulation EC N°1272/2008.