1-o-tolylbiguanide

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:

Sub-acute, with treatment up to 48 days for reproduction toxicity screening animals

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Justification for type of information:

Guideline study
The pH was adjusted with citric acid in view of alkaline nature of the substance
The highest initial treatment levels were reduced to minimise clinical signs to allow tolerance for the treatment period.

Data source

Reference

Reference Type:

study report

Title:

Unnamed

Year:

2017

Report date:

2017

Materials and methods

Test guideline

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

GLP compliance:

yes (incl. QA statement)

Limit test:

no

Test material

Test material information

Constituent 1

Reference substance name:

1-o-tolylbiguanide

EC Number:

202-268-6

EC Name:

1-o-tolylbiguanide

Cas Number:

93-69-6

Molecular formula:

C9H13N5

IUPAC Name:

1-o-tolylbiguanide

Test material form:

solid: particulate/powder

Remarks:

migrated information: powder

Specific details on test material used for the study:

Manufactured Date : October 2016
Retest Date : October 2017
Purity as per Certificate of Analysis : 97.4 %
Physical Appearance : White, dusty powder
Storage Conditions : Ambient (+15 to +25°C)

Test animals

Species:

rat

Strain:

Wistar

Details on species / strain selection:

Wistar rats – HanTac: WH
Source : Vivo Bio Tech Ltd.
Sy. #349/A, Pregnapur-502311,
Gajwel Mandal, Medak District, Telangana
Justification for selection
of species
: Rat is the standard laboratory rodent species
used for toxicity assessment and also
recommended by various regulatory
authorities.

Sex:

male/female

Details on test animals or test system and environmental conditions:

No. of animals for acclimatization : 60 males and 60 females
No. of rats/group : Main groups : 10 males+10 females per group
Recovery groups : 5 males + 5 females per group
Total = 100 (50 males + 50 females)
Age at the start of treatment : 14 – 16 weeks. nBody weight range : Males: 354.93 to 440.13 g Females: 224.83 to 268.39 g
At the commencement of the treatment, the weight variation of rats used did not exceed ± 20 % of the mean body weight in each sex and group
After detailed clinical examination for good health and the suitability for the study, the rats were acclimatized for a period of five days
before Pre-treatment period. During the acclimatization period, animals were observed once daily for any abnormalities. Only the animals that are determined by the veterinarian to be suitable for use were assigned to this study. Female rats used in this study were nulliparous and non-pregnant.

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on oral exposure:

Based on the solubility test, the test item forms suspension in 0.5% carboxymethyl cellulose sodium salt (medium viscosity), while it could not be dissolved/suspended in Milli-Q® water. The Sponsor requested to check the pH of test item. The pH was checked at 1% suspension in Milli-Q® water. The observed pH was 11.78. The observed pH was informed to sponsor. The sponsor had suggested neutralizing the pH using suitable buffer. Hence, the 0.5% sodium carboxy methyl cellulose was prepared in 0.2M citrate buffer with pH 4

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The dose formulations were analysed for active ingredient (a.i.) concentration on Days 1, 3 and during 2nd (Day 38) month of the treatment period. The results indicated that the analysed concentrations were within ± 15 % of variations from the nominal concentrations.

Duration of treatment / exposure:

The dose formulations were administered once daily to specific group of rats prior to mating, during mating and post-mating periods (for males), during pregnancy and up to Lactation Day (LD) 13 for females. In the control and high dose recovery groups, the treatment period was followed by a 14 day no treatment (recovery) period.

Frequency of treatment:

Daily

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

50 mg/kg bw/day (nominal)

Remarks:

Treatment started at 300 mg/kg/day, then reduced from Day 3 after adverse clinical signs

Doses / concentrations 2

Dose / conc.:

25 mg/kg bw/day (nominal)

Remarks:

Treatment started at 50 mg/kg/day, then reduced from Day 3 after adverse clinical signs

Doses / concentrations 3

Dose / conc.:

10 mg/kg bw/day (nominal)

No. of animals per sex per dose:

Main groups : 10 males+10 females per group
Recovery groups : 5 males + 5 females per
group
Total = 100 (50 males + 50 females)

Control animals:

yes, concurrent vehicle

Examinations

Observations and examinations performed and frequency:

The dose levels of 10 (G2), 50 (G3) and 300 (G4/G4R) mg/kg/day was selected for this study in consultation with the Sponsor.
Based on clinical signs observed, body weight and and food consumption changes, the mid dose of 50 mg/kg/day was decreased to 25 mg/kg Bwt/day and high dose (G4/G4R) of 300 mg/kg Bwt/day was decreased to 50 mg/kg Bwt/day from treatment day 3 in consultation with sponsor. At Low dose, the treatment was continued at 10 mg/kg Bwt/day. In addition to the test doses, vehicle control and vehicle control recovery groups were included. Animals in the vehicle control and recovery were handled in a manner similar to the treatment groups except for test item administration.

Sacrifice and pathology:

Oral administration of Ortho Tolyl Biguanide in Wistar rats did not reveal any treatment related changes in the hematology, coagulation, clinical chemistry, urinalysis, terminal fasting body weights/organ weights and gross pathological changes in both the sexes.

Statistics:

ProvantisTM: Parameters of laboratory Investigations - Haematology (Coagulation tests PT and APTT data was entered retrospectively in ProvantisTM) and Clinical Chemistry data was analysed using Provantis built-in
statistical tests.
The statistical analysis of the experimental data was carried out using the
validated package in Excel and also using licensed copies of SYSTAT
Statistical package ver.12.0. All quantitative variables like neurological
observations (neuromuscular observation/body temperature/body weights),
body weight, net weight gain, food consumption, oestrous cycle length,
hormone levels, ano-genital distance, body weights, ano-genital index, mean
number and weight of pups, organ weights and organ weight ratios were tested
for normality (Shapiro-Wilk test) and homogeneity of variances (Levene’s test)
within the group before performing a one-factor analysis of variance (ANOVA)
modeling by treatment groups. Non-optimal (non-normal or heteroschedastic)
data was transformed, before ANOVA was performed. Comparison of means
between treatment groups and control group was done using Dunnett’s test
when the overall treatment, ‘F’ test is found to be significant.
In case of recovery groups, data was analysed using Two sample t-test.
Comparison of means between treatment recovery group(s) and vehicle control
recovery group was performed.
Post-implantation loss (%), no. of implantations, pre-coital interval, mean litter
size, sex ratio and gestation length (Days) was analysed after suitable
transformation (√ x + ½) of the data. One-way ANOVA was carried out for the
transformed data. Dunnett’s pair-wise comparison of the treated means with the
control mean was done when the group differences are found significant.
Z test was performed for testing the differences in proportions for Day 4 survival
index, mating and fertility indices.
All analyses and comparisons were evaluated at the 5% (p<0.05) level.

Results and discussion

Results of examinations

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

There were no clinical signs observed in both sexes at 10 mg/kg/day dose.
The clinical sign of slight salivation was observed, approximately after 50 minutes after dosing on Day 1 at 50 mg/kg/day dose.
At 300 mg/kg/day, the clinical signs of slight salivation, piloerection, dehydration and emaciation were observed. Based on the severity of the clinical signs, the doses of 50 and 300 mg/kg/day were reduced to 25 and 50 mg/kg/day, respectively from treatment Day 3.
There were no clinical signs observed at the reduced mid dose of 25 mg/kg/day.
At the final high dose of 50 mg/kg/day, all animals become normal from two to three days after reducing the dose and slight salivation was observed 30 minutes after dosing towards the end of treatment period (Day 48 to 51).

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

The mean body weights and net weight gains unaffected by the treatment at low and mid doses in both sexes when compared to vehicle control.
At 300 mg/kg/day dose, the mean body weights were significantly lower, measured on Day 3, with reduction up to 10% in both the sexes. After reducing the dose to 50 mg/kg Bwt/day, the mean body weights were apparently (-2.6% to -6.7%) lower and net body weight gains were significantly lower during treatment period in males, when compared to concurrent vehicle control. In females, the mean body weights and net body weight gains were comparable to concurrent vehicle control group. The net body weight gains were apparently higher during the recovery period in males indicating the reversibility of effects observed during the treatment period.

Food consumption and compound intake (if feeding study):

no effects observed

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

no effects observed

Urinalysis findings:

no effects observed

Behaviour (functional findings):

no effects observed

Immunological findings:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

not specified

Effect levels

Dose descriptor:

NOAEL

Effect level:

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

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Target system / organ toxicity

Critical effects observed:

no

Any other information on results incl. tables

No adverse effecte at maximum tolerated treatment levels.

Applicant's summary and conclusion

Conclusions:

Daily oral (gavage) administration at the dose levels 10, 25 and 50 mg/kg/day for 2 weeks prior to mating, during mating, and approximately 4 weeks post mating (males) or 2 weeks prior to mating, during mating, and during pregnancy until 13 days after delivery (females) had no effects on general health, pre-coital time, gestation length, mating and fertility parameters.
Initial treatment levels of 300 mg/kg/day resulted in clinical signs that were judged to have been unlikely to be tolerated over longer periods, leading to the decision to reduce treatment levels in the first week of administration
A dose range finding study was not performed to reduce the number of experimental animals.