1-o-tolylbiguanide

Toxicity to reproduction

Administrative data

Endpoint:

screening for reproductive / developmental toxicity

Type of information:

experimental study

Adequacy of study:

key study

Study period:

Dosing up to 48 days

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.

Cross-reference

Reason / purpose for cross-reference:

reference to same study

Reference

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.

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

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)

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.

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

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

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

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

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.

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

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

Critical effects observed:

no

No adverse effecte at maximum tolerated treatment levels.

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.

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

Justification for study design:

The highest initial treatment levels were reduced to minimise clinical signs to allow tolerance for the treatment period.

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

Sex:

male/female

Details on test animals or test system and environmental conditions:

The main groups i.e., G1 to G4 consisted of 10 male and 10 female rats per group and recovery groups consisted of 5 male and 5 female rats per group. 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. The recovery period of the study was started from the first scheduled kill of dams

Administration / exposure

Route of administration:

oral: gavage

Vehicle:

CMC (carboxymethyl cellulose)

Details on exposure:

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.

Details on mating procedure:

Pre-mating:
Two rats of same sex were housed per cage in sterilized standard polysulfone cages (Size: L 425 x B 266 x H 185 mm), with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles with stainless steel sipper tubes except for last animal in the recovery group wherein one animal was housed.


Mating and post-mating:
During mating, two rats (one male and one female) were housed in standard polysulfone cages with stainless steel top grill having facilities for pelleted food and drinking water in polycarbonate bottles. After confirming the presence of sperm in the vaginal smear or vaginal plugs (Day ‘0’ pregnancy), the mated pairs were separated. Males were housed with their former cage mates while females were housed individually in polysulfone cages. The sterilised nesting material (paper shreds) was provided near-term.
Polycarbonate Rat huts was provided to the animals as environmental enrichment objects during pre-mating period and post-mating period for males and pre-mating period for females. For recovery groups, enrichment was provided throughout the experimental period.
During the study period, animals were housed in a single experimental room of T4 Barrier Area (Room No.: SC-22).

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For homogeneity and active ingredient (a.i.) concentration analysis, prepared formulation samples were sampled in duplicate sets on Day 1 of the treatment period. In addition, for active ingredient (a.i) concentration analysis, samples of dose formulation were taken from reduced mid (25 mg/kg Bwt/day) and high (50 mg/kg Bwt/day) dose groups prepared on Day 3 of the treatment period including control. During 2nd month (day 38) of treatment, prepared formulation samples were sampled in duplicate sets form 10, 25 and 50 (mg/kg Bwt/day) doses. The collected samples were analysed in-house.
For each set, duplicate samples were drawn from top, middle and bottom layers of each preparation and in case of control duplicate samples from the middle layer were drawn.
The analysis was done as per the method validated under Advinus Study No. G12743. One set of samples were analysed for concentration (a.i) analysis.
Formulations were considered acceptable as mean results are within ± 15 % of the theoretical concentration and the relative standard deviation (RSD) was less than 10 %.
The unused back up samples was disposed as analysis results of the first set of samples were within the acceptable limits. The results of dose concentration analyses are provided in Appendix 25.

Duration of treatment / exposure:

Males: The dose formulation was administered to the rats of the specific groups once daily at approximately the same time each day (varying by ± 3 hours) for 2 weeks prior to mating and the treatment was continued during the mating and post mating period until sacrifice.
Females: The dose formulations were administered to the specific group of rats once daily at approximately the same time each day (varying by ± 3 hours) throughout the treatment period. Treatment was done 2 weeks prior to the mating period and continued through mating, pregnancy and up to LD 13, after which, pups were sacrificed on LD 13 and parental females (dams) were sacrificed on LD 14 after overnight fasting (water allowed).
The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).
The dose volume administered for each rat was 10 mL/kg Bwt throughout the study. The dose volume was adjusted based on the most recent body weight of individual rat.
Similarly, vehicle was administered to rats in the vehicle control and vehicle control recovery groups at 10 mL/kg Bwt.
The vehicle and the test item was not administered for vehicle control recovery and high dose recovery groups, respectively for 14 days following the treatment period.

Frequency of treatment:

The dose formulations were administered to the high dose recovery group of rats once daily at approximately the same time each day (varying by ± 3 hours).

Details on study schedule:

One female was placed with one male from the same group in a 1:1 ratio. Cohabitation was continued until there is evidence of sperms in the vaginal smear and /or vaginal plug or for a maximum period of 2 weeks. Subsequently, pregnant females were housed individually until LD 14. All the mated females were maintained till they litter. Not littered females were sacrificed after 25 days from the day they are found of sperm positive (by vaginal smear examination).
The day of confirmed mating was designated as Gestation Day ‘0’ (GD ‘0’). The pre-coital time was calculated for each female.

Doses / concentrations

Doses / concentrations 1

Dose / conc.:

10 mg/kg bw/day (nominal)

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.:

50 mg/kg bw/day (nominal)

Remarks:

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

No. of animals per sex per dose:

100 (50 male 50 female)

Control animals:

yes, concurrent vehicle

Details on study design:

Grouping was done by the method of body weight stratification and distribution. On the day of randomization, based on the given temporary animal identification number, all males and female with normal oestrous cyclicity (4-5-day cycle) were weighed and the corresponding body weight was recorded. The data was transferred to EDP (Electronic Data Processing) for data input (temporary identification number and body weight) into an excel spread sheet. The body weight recorded was stratified in ascending order.
EDP was performed statistical analysis and ensured that the weight variation was minimal and inter group variation did not exceed ± 20% of the mean body weight in each sex. Rats with extreme body weights were discarded. Grouping was done one day prior to initiation of the treatment.

Positive control:

Animals in the recovery groups were kept only for observations of
reversibility, persistence or delayed occurrence of systemic toxic effects
for 14 days post treatment and these animals were not mated and
consequently not used for assessment of reproduction/developmental
toxicity.

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

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).

Dermal irritation (if dermal study):

not examined

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

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):

effects observed, treatment-related

Description (incidence and severity):

The food consumption was not altered by the treatment in both the sexes at
low and mid doses. At 300 mg/kg/day dose, the food consumption was
significantly lower during Days 1-3 in both sexes, with reduction up to 77 %
when compared to vehicle control group. After reducing to 50 mg/kg
Bwt/day, the food consumption was significantly lower during Days 43-50
in main group males with concomitant apparent decrease in body weight
during the same period.

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Description (incidence and severity):

Blood smears were prepared from the hematology (K2EDTA tube)
samples and stained with Wright’s stain solution. As the study findings did not
warrant blood smear evaluation, the slides were discarded.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

The test item administration did not reveal any treatment related changes in
the hematology, coagulation and clinical chemistry parameters of both
males and females.

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

Histopathological findings: non-neoplastic:

no effects observed

Histopathological findings: neoplastic:

no effects observed

Other effects:

no effects observed

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

no effects observed

Details on results (P0)

Daily oral (gavage) administration of Ortho Tolyl Biguanide to male and female
Wistar rats at dose levels of 10, 25 and 50 mg/kg Bwt/day for 2 weeks prior to
mating, during mating, and 4 weeks post mating (males) or 2 weeks prior to
mating, during mating, and during pregnancy until 13 days after delivery did not
induce any adverse effects on fertility and reproductive performance. The No
Observed Adverse Effect Level (NOAEL) Ortho Tolyl Biguanide for
reproductive toxicity in parental rats is considered to be 50 mg/kg Bwt/day.
Considering the changes observed in the body weights,food consumption and
microscopic changes of kidneys at 50 mg/kg Bwt/day, the No Observed
Adverse Effect Level (NOAEL) of Ortho Tolyl Biguanide for systemic toxicity
(repeated dose toxicity) in males is considered to be 25 mg/kg Bwt/day. As
there were no changes observed in the parameters related to systemic toxicity in
females, the No Observed Adverse Effect Level (NOAEL) of Ortho Tolyl
Biguanide for systemic toxicity (repeated dose toxicity) in females is considered
to be 50 mg/kg Bwt/day.

Effect levels (P0)

Key result

Dose descriptor:

NOAEL

Effect level:

50 mg/kg bw/day

Based on:

test mat.

Sex:

male/female

Basis for effect level:

body weight and weight gain

food consumption and compound intake

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Neuropathological findings:

not examined

Histopathological findings: non-neoplastic:

not examined

Histopathological findings: neoplastic:

not examined

Other effects:

not examined

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not examined

Reproductive function: sperm measures:

not examined

Reproductive performance:

not examined

Results: F1 generation

General toxicity (F1)

Clinical signs:

no effects observed

Dermal irritation (if dermal study):

not examined

Mortality / viability:

no mortality observed

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

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

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

no effects observed

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

no effects observed

Details on results (F1)

No treatment-related abnormalities were observed in
any of the doses tested in both sexes.

Effect levels (F1)

Remarks on result:

not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Critical effects observed:

no

Results: F2 generation

General toxicity (F2)

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

not examined

Body weight and weight changes:

not examined

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Organ weight findings including organ / body weight ratios:

not examined

Gross pathological findings:

not examined

Histopathological findings:

not examined

Other effects:

not examined

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not examined

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not examined

Overall reproductive toxicity

Key result

Reproductive effects observed:

no

Lowest effective dose / conc.:

25 mg/kg bw/day

Treatment related:

no

Any other information on results incl. tables

TABLE 2. Summary of Detailed Clinical Examination, Clinical Signs and Mortality

Refer Appendices 1 and 2

Group No. G1 G2 G3 G4 G1R G4R

Parameters Dose (mg/kg Bwt/day) 0 10 50/25 300/50 0 300/50

Sex M F M F M F M F M F M F

No. of rats 10 10 10 10 10 10 10 10 5 5 5 5

1. GENERAL CONDITION

- Dehydration (slight) 0 0 0 0 0 0 10 10 0 0 5 5

- Emaciation

- Salivation (slight)

- Piloerection (slight)

00

0

00

0

00

0

00

0

0

10

0

0

10

0

10

10

10

10

10

10

00

0

00

0

55

5

55

5

2. SKIN AFFECTIONS 0 0 0 0 0 0 0 0 0 0 0 0

3. EYE AFFECTIONS 0 0 0 0 0 0 0 0 0 0 0 0

4. UROGENITAL AFFECTIONS 0 0 0 0 0 0 0 0 0 0 0 0

5. RESPIRATORY AFFECTIONS 0 0 0 0 0 0 0 0 0 0 0 0

6. PRE-TERMINAL DEATHS (Total)

Death during treatment

Death during gestation

Death during lactation

Dystocia deaths

Moribund sacrifice

Total mortality

0

NA

NA

NA

0

0

0

0

0

0

0

0

0

NA

NA

NA

0

0

0

0

0

0

0

0

0

NA

NA

NA

0

0

0

0

0

0

0

0

0

NA

NA

NA

0

0

0

0

0

0

0

0

0

NA

NA

NA

0

0

0

NA

NA

NA

0

0

0

NA

NA

NA

0

0

0

NA

NA

NA

0

0

NA: Not Applicable; M: Male; F: Female

G3 and G4 rats were treated with 50 and 300 mg/kg/day respectively during days 1 and 2, and then 25 and 50 mg/kg/day respectively from day 3 till

sacrifice

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.