Registration Dossier

Administrative data

Endpoint:
screening for reproductive / developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
From 2011-07-20 to 2012-03-26
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 guidelineopen allclose all
Qualifier:
according to
Guideline:
OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)
Version / remarks:
OECD 422, Section 4: Health Effects, 1996
Deviations:
yes
Remarks:
No chemical analysis of the test solutions prepared was carried out
Qualifier:
according to
Guideline:
other: OPPTS 870.3650.2000
Deviations:
no
GLP compliance:
yes (incl. certificate)
Remarks:
The study followed the SOP-M 285 and accomplished in agreement with the OECD 422, 1996, Section 4, and OPPTS 870.3650.2000.
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent
Test material form:
liquid
Specific details on test material used for the study:
- Source and lot/batch No.of test material:
8684-90
- Storage of the test item: the samples were stored in a specific room, at room temperature, protected from humidity and light

Test animals

Species:
rat
Strain:
Wistar
Details on species / strain selection:
Currently, no acceptable non-animal alternative methodology is accepted by the relevant regulatory agencies. The rat was selected because this species has proven to be sensitive to chemicals; the rat is the preferred rodent species for prenatal developmental toxicity studies because of its small size, short gestation period, high fertility rate, large litter size and ease of maintenance. The Wistar Hannover rat was selected due to the large amount of background knowledge accumulated on this strain.
Sex:
male/female
Details on test animals and environmental conditions:
TEST ANIMALS
- Source: BIOAGRI Laboratorios
- Females (if applicable) nulliparous and non-pregnant: yes
- Age at study initiation: 11 - 12 weeks old
- Weight at study initiation: Males 352.3±20.7 - 359.5±19.9 grams; Females 216.2± 8.6 - 220.4 ± 9.6 grams
- Fasting period before study: no data
- Housing: Each animal was housed individually, except during cohabitation. After acclimation one male was placed into each female cage for pairing. After pairing, females that presented vaginal smears with the presence of sperm were considered mated and housed individually. The rats were housed in polypropylene cages (41x34x19 cm) with wire mesh tops and bedding material (wood shavings). Clean cages were provided twice weekly for all animals. The cages with the test animals were arranged on the racks in such a way that uniform experimental conditions (ventilation and light) were ensured.
During gestation period all females were housed individually in the cages. Females without copulation date were housed in individual cages at the end of the mating period. Females showing no evidence of copulation were euthanized 24-26 days after the last day of mating period.
- Diet (e.g. ad libitum): ad libitum, Nuvilab CR-1 diet for rats supplied by Nuvital Nutrientes Ltda.
- Water (e.g. ad libitum): ad libitum, supplied by CAESB (Compania de Saneamento Ambiental do Distrito Federal) in water bottles
- Acclimation period: 5 days prior to dosing


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 19.0 - 24.4° C
- Humidity (%): 40.8 - 70.0%
- Air changes (per hr): 10-20 air changes per hour
- Photoperiod (hrs dark / hrs light): 12 hours light and 12 hours dark

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
corn oil
Details on exposure:
- PREPARATION OF DOSING SOLUTIONS:
- For each dosage group, the appropriate amount of JEFFSOL® AG 1700 was weighed into a precalibrated beaker. The vehicle (corn oil) was added in sufficient quantity to achieve the desired concentration. Each solution was homogenized and dispensed into individual containers properly identified. A sufficient quantity of the vehicle was similarly dispensed for administration to control animals.
- the prepared dolutions were stored at room temperature.
- Test solutions were prepared daily at the testing facility and were administered within 2 hours after preparation. The test solutions were stirred continuously during the administration to maintain homogeneity.

- VEHICLE
- Justification for use and choice of vehicle (if other than water):
- Amount of vehicle (if gavage): 1 ml/250g body weight.
Details on mating procedure:
- M/F ratio per cage: 1 female: 1 male
- Length of cohabitation: until evidence of copulation is observed or 2 weeks had elapsed
- Proof of pregnancy: . Vaginal smears were collected daily during mating period and examined for the presence of sperm.
Day O of gestation was defined as the day a sperm was found in the vaginal smear.
Analytical verification of doses or concentrations:
no
Frequency of treatment:
daily administration, on a 7 day per week basis
Doses / concentrationsopen allclose all
Dose / conc.:
0 mg/kg bw/day (nominal)
Remarks:
Group 1, vehicle only
Dose / conc.:
250 mg/kg bw/day (nominal)
Remarks:
Group 2, the expected dose which causes no signs of toxicity
Dose / conc.:
500 mg/kg bw/day (nominal)
Remarks:
Group 3, the intermediate dose level
Dose / conc.:
1 000 mg/kg bw/day (nominal)
Remarks:
Group 4, the expected dose which causes signs of systemic toxicity, but not death or severe suffering
No. of animals per sex per dose:
12 animals/sex/group
5 animals/sex/satllite group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dosage levels (in mg/kg body weight/day) were selected in agreement with the Sponsor, based upon the results of a dose range-finding study (P.T. 3984.329.003.11).
- Rationale for animal assignment (if not random): randomized
Positive control:
No

Examinations

Parental animals: Observations and examinations:
DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: weekly
- All animals underwent a daily clinical observation for overt signs of ill health. These included, but were not limited to, changes in skin and fur, eye and mucous membranes, respiratory, circulatory, autonomic and central nervous system, motor activity and behavioral patterns.


BODY WEIGHT: Yes
- Time schedule for examinations: Males were weighed on the first day of dosing and weekly thereafter (including mating and post-mating periods). Females were weighed on first day of dosing and once a week during premating and mating periods, on days 0, 7, 14 and 20 of gestation, and during lactation on the same days as the weighing of litters (on days 0 and 4 postnatal).

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study):
Food consumption of females was determined on the same day of body weight determination during premating and lactation periods. During the gestation period, food consumption was determined on days 3, 6, 9, 12, 15, 18 and 20. After the mating period, food consumption of males was determined weekly. Food consumption was not determined during the mating period.

HAEMATOLOGY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy
- Anaesthetic used for blood collection: Yes, animals were anesthetized by CO2 prior to blood collection (cardiac puncture).
- Animals fasted: Yes , overnight
- How many animals: 5 parental animals/sex/group, randomly selected
- Parameters examined: Red Blood Cell Count (RBC), Hemoglobin (HB), Hematocrit (HCT), Platelets (PLT), Mean Corpuscular Volume (MCV), Mean Corpuscular Hemoglobin (MCH), Mean Corpuscular Hemoglobin Concentration (MCHC), Total White Blood Cell Count (WBC), Differential Leukocyte Count, Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT)

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood: on the day of scheduled necropsy.
- Animals fasted: Yes, overnight
- How many animals: 5 parental animals/sex/group, randomly selected
- Parameters examined: Aspartate Aminotransferase (AST), Alanine Aminotransferase (ALT), Alkaline Phosphatase (ALP), Total Protein (TP), Albumin (ALB), Glucose (GLU), Total Cholesterol (CHOL), Urea Nitrogen (BUN), Creatinine (CREA), Sodium (Na), Potassium (K), Calcium (Ca), Globulin (GLOB), Albumin/Globulin Ratio (A/G)

FUNCTIONAL OBSERVATIONS: yes
Sensory reactivity to stimuli and motor activity assessment were performed in 5 animals/sex/group. For males these evaluations were performed at the end of the dosing period before scheduled necropsy, and for females, these evaluations were performed during the lactation period (day 3). The following parameters were assessed:
A - Autonomic Functions: lacrimation, salivation, palpebral closure, prominence of the eye, piloerection and respiration.
B - Reactivity and Sensitivity: sensor motor responses to approach tactile and tail flick. C - Excitability: reactions to handling and behavior in an open field.
D - Gait and Sensor Motor Coordination: degree of mobility and gait pattern in an open field.
E - Abnormal Clinical Signs: including convulsions, tremors, unusual behavior and deposits around the eyes, nose or mouth.
Oestrous cyclicity (parental animals):
Not examined
Sperm parameters (parental animals):
Not examined
Litter observations:
Live pups were counted, sexed and weighed on days 0 and 4 postnatal.
The day when delivery is completed was designated day O of lactation (postnatal day 0). On day O of lactation the number of alive and dead pups/sex were recorded. Dams with offspring were euthanized on day 4 postnatal. All pups were euthanized at day 4 postnatal.
Postmortem examinations (parental animals):
GROSS PATHOLOGY: Yes
At termination, all parental animals were examined macroscopically for any structural abnormalities or pathological changes. The animals were euthanized in a carbon dioxide chamber. The numbers of implantation sites and corpora lutea were recorded. Animals found dead were necropsied, discarded in biological garbage and incinerated.

ORGAN WEIGHTS:
- At scheduled necropsy, testes and epididymides of all males were weighed.
- Organ weights were obtained for the following organs from 5 animals/sex/group: liver; kidneys; adrenals; thymus; spleen; brain; heart.

HISTOPATHOLOGY: Yes
- For all animals necropsied, tissues were preserved in 10% neutral buffered formalin (except for the testes, which were preserved in Davidson's fixative)
- At scheduled necropsy, the following organs of all animals were preserved: testes, epididymides, ovaries, prostate, seminal vesicle and coagulating gland , bulbourethral gland ,organs showing alterations
- The following organs and tissues of 5 animals/sex/group were preserved: adrenals (right and left); bone marrow (femur); brain (cerebrum, cerebellum and pons); heart; intestine (duodenum, jejunum, ileum - including Peyer's patches, colon, rectum/anus); kidneys (right and left); liver (3 lobes); lungs; lymph nodes (mesenteric and submaxillary); peripheral nerve (sciatic); spinal cord (cervical, midthoracic and lumbar sections); spleen; stomach (glandular and non-glandular); trachea; thymus; thyroid; urinary bladder; uterus; all gross lesions.
- Full histopathology of the preserved organs and tissues listed above were performed in high dose and control animals.
Postmortem examinations (offspring):
All pups were grosly examined for abnormalities of the oral, thoracic and abdominal cavities.
Statistics:
Quantitative variables such as body weights, food consumption and organs weights were analyzed by One Way Analysis of Variance (ANOVA), followed by Dunnett's test if significance is detected, or by the non-parametric test of Kruskal-Wallis, according to the results of tests for normality and homogeneity of variance. For qualitative or non-parametric data such as clinical findings, macroscopic and microscopic findings and fetal findings, comparison between means were carried out using Fisher's Exact Test or the Chi-Square Test.
The level of significance was set at 5%.
Reproductive indices:
Mating index index (%): (number of female mated x 100/ number of females paired)
Fertility index (%): (number of pregnant animals/number of presumed pregnant animals) x 100
Preimplantation loss (%): (no. of corpora lutea - number of implantations/number of corpora lutea) x 100
Offspring viability indices:
Postimplantation loss (%): (number of implantations-number of live fetuses/ number of implantations) x 100
Gestation index (%): (number of female with live pups at birth x 100 /number of pregnant animals) x 100

Results and discussion

Results: P0 (first parental animals)

General toxicity (P0)

Clinical signs:
no effects observed
Description (incidence and severity):
No clinical signs were observed.
Dermal irritation (if dermal study):
not examined
Mortality:
no mortality observed
Description (incidence):
No mortality occurred during the study.
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No alteration on body weight in treated males was observed when compared to the control group. Lower body weight gain was observed in males exposed to 250 mg/kg/day between days 7 to 14 (-11.3%) and days 28 to 35 (-16%). Similar diminution on body weight gain was observed in males at 500 mg/kg/day between days 14 to 21 (-55.8%) and at 1000 mg/kg/day between days 7 to 14 and days 14 to 21 (-6.5% and -97.7%, respectively). In all cases, these changes were not considered to be test item related, because they were isolated, not statistically significant and did not affect overall body weight gain.
In treated satellite males, at the end of the first observation interval no alteration on body weight was observed, but some variations on body weight gain occurred, however, these variations did not affect overall body weight gain. In the recovery period, lower body weight gain occurred (- 29.7%), but was not statistically significant and did not affect the body weight. This finding was not considered to be test item related.
No alteration on body weight was observed in treated females compared to the control group. Differences on body weight gain were observed at several observation periods, affecting overall body weight gain in the gestation period (-15.7% low, -11.9% mid and -10.6% high dose) and lactation period (-60.9% low, -18.6% mid and -51.9% high dose). These differences were not dose related and not statistically significant, and therefore were not considered to be test item related.
In treated satellite females, at the end of the first observation period no alteration on body weight was observed, but some variations on body weight gain, statistically significant between days 0 to 21 were observed, but did not affect total body weight gain. In the recovery period, although the body weight gain was decreased (-57.5%), this change did not affect overall body weight, therefore this finding was considered incidental.
Food consumption and compound intake (if feeding study):
effects observed, non-treatment-related
Description (incidence and severity):
Differences in food consumption were observed in treated males when compared to the control group.
Similar effects occurred in treated satellite males. Although these differences were sometimes statistically significant, they did not affect overall food consumption in either treated males or treated satellite males.
In treated females, differences in food consumption were observed, with statistical significance at some observation periods. In the lactation period, these differences were observed in high dose females, statistically significantly affecting overall food consumption (-24.46%) compared to the control group. This finding was not considered to be test item related since the body weight was not affected.
Statistically significant differences in food consumption were observed at several observation periods in treated satellite females, affecting total food consumption (-5%) in the first observation period and (-8.8%) in the recovery period compared to the control group. These findings were small and did not affect overall body weight, and, therefore are not considered to be test item related.
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):
In treated males at 500 mg/kg/day, statistically significant higher lymphocytes counts were observed (+7.8%) but considered incidental because they occurred at the mid dose group. No other alterations on hematological and clotting parameters were observed in treated males.
Mean hemoglobin in treated satellite males was statistically significant lower (-6.7%) compared to the control group, but this finding was not considered to be test item related, because the difference was very small, and within Bioagri's historical range (14.1-17.9). No other hematological parameters were affected.
Statistically significant higher hematocrit (+6.6%) was observed in females exposed to 250 mg/kg/day, but it was an isolated finding at low dose, and therefore not considered to be test item related.
Statistically significant higher mean corpuscular hemoglobin concentration was noted in treated satellite females (+2.8%), but the difference was very small and considered incidental, as a normal biological variation.
In treated satellite males, white blood cell counts (-30.1%) and total lymphocyte counts (-34%) were lower than control group. Total lymphocyte count is within Bioagri's historical range and total white blood cells count is slightly outside (-3.9%). Considering this difference very small in magnitude and no other white cells were affected, this finding was not considered to be test item related. No other changes in white blood cells were observed.
Changes in clotting parameters were observed in both prothrombin time in females at 1000 mg/kg/day (+11.8%) and in treated satellite females (+7.2%), statistically significant in both cases. These
values were small and are within Bioagri's baseline (13.5-23.1) and are not considered to be test item related.
For more detailed information, see section 'Any other information on results'
Clinical biochemistry findings:
effects observed, non-treatment-related
Description (incidence and severity):
In treated males, statistically significantly lower creatinine (-20%) at 500 mg/kg/day and albumin (-7.7% low and -15.4% mid dose) were observed compared to the control group, but not considered to be dose related.
No alteration in the clinical chemistry parameters were noted in treated female groups.
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Description (incidence and severity):
No alteration in the Functional Observation Battery was found in male or female treated groups compared to the control.
Immunological findings:
not examined
Organ weight findings including organ / body weight ratios:
effects observed, non-treatment-related
Description (incidence and severity):
Statistically significant lower right testis weight relative to body weight was observed in treated males at 1000 mg/kg/day (-9.7%) but not relative to brain weight. This unilateral difference was small in magnitude, and considered incidental. No other changes in organ weight were noted in the remaining groups.
For further information, see section 'Any other information on results'
Gross pathological findings:
effects observed, non-treatment-related
Description (incidence and severity):
In one male exposed to 1000 mg/kg/day erosions in the stomach were observed, but this was an isolated finding not considered to be test item related. No other macroscopic lesion was observed in rats from the remaining groups.
Neuropathological findings:
not examined
Histopathological findings: non-neoplastic:
effects observed, non-treatment-related
Description (incidence and severity):
Slight reduction of germ cells (bilateral) was the most common lesion noted. This finding was observed in three males exposed to 1000 mg/kg/day. This change should not be considered to be test item related, since this focal isolated reduction of germ cells unilateral in epididymis was also observed in two treated satellite males and two control satellites.
Other microscopic lesions were observed in two animals; one male, at 1000 mg/kg/day presented erosion in the stomach mucosa, and in one female presented focal chronic proliferative nephropathy in the left kidney. These lesions were considered normal background findings and were not considered to be test item related.
For more detailed information, see section 'Any other information on results'
Histopathological findings: neoplastic:
not examined
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
not examined
Reproductive function: sperm measures:
not examined
Reproductive performance:
effects observed, non-treatment-related
Description (incidence and severity):
Similar gestation index were observed when treated groups were compared to the controls and no differences were observed. In dams exposed to the high dose, the number of total pups and the number of live pups at day 0 were lower compared to the control group. These findings, while decreased compared to controls, were not statistically significant and are not likely to be test item related.
For further detailed information, see section 'Any other information on results'

Details on results (P0)

The test substance did not cause any mortality or clinical signs of toxicity during the study. No effects on body weight, body weight gain or food consumption were observed. No effects were observed in the functional observational battery, hematology and clotting parameters, clinical chemistry parameters or organ weights. No macroscopic findings related to the test item were observed.
In this study, the alterations occurred at high dose levels in male and female. These changes were observed on testes (slight reduction of germ cells) and reproductive parameters. Slight reduction of germ cells was observed in three males exposed to the highest dose level. This result was not considered test item related because this lesion appeared in equal proportions in both control and high dose satellites and the frequency of this lesion was not statistically significant. Despite that these males produced offspring from their respective females.

Effect levels (P0)

open allclose all
Key result
Dose descriptor:
NOAEL
Remarks:
parental
Effect level:
>= 1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Key result
Dose descriptor:
NOAEL
Remarks:
reproductive
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (P0)

Key result
Critical effects observed:
no

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Description (incidence and severity):
No specific findings in all animals.
Dermal irritation (if dermal study):
not examined
Mortality / viability:
mortality observed, non-treatment-related
Description (incidence and severity):
While the absolute number of dead pups in the high dose group was statistically significantly increased when compared to controls on day 0, the mean number of live and dead pups per litter was not significantly increased when compared to controls on day 4. No differences were observed when delivery day 1 was compared to day 4 postnatal.
For further information, see section 'Any other information on results'
Body weight and weight changes:
effects observed, non-treatment-related
Description (incidence and severity):
No difference in pup body weight was observed between the treated and control groups at day 0. The body weight of pups at day 4 postnatal were lower compared to the control group. This finding, while decreased compared to controls, was not statistically significant and is not likely to be test item related.
For further information, see section 'Any other information on results'
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:
no effects observed
Description (incidence and severity):
No macroscopic finding was observed in treated or control groups.
Histopathological findings:
no effects observed
Other effects:
not examined

Developmental neurotoxicity (F1)

Behaviour (functional findings):
not examined

Developmental immunotoxicity (F1)

Developmental immunotoxicity:
not examined

Details on results (F1)

In dams exposed to the high dose, the number of total pups, live pups at day 0, and body weight of pups at day 4 postnatal were lower compared to the control group. These findings, while decreased compared to controls, were not statistically significant and are not likely to be test item related. While the absolute number of dead pups in the high dose group was statistically significantly increased when compared to controls on day 0, the mean number of live and dead pups per litter was not significantly increased when compared to controls on day 4.

Effect levels (F1)

Key result
Dose descriptor:
NOAEL
Remarks:
developmental
Generation:
F1
Effect level:
1 000 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects

Target system / organ toxicity (F1)

Key result
Critical effects observed:
no

Overall reproductive toxicity

Key result
Reproductive effects observed:
no

Any other information on results incl. tables

Hematology: data is presented as mean+/-SD (No. animals)

Parameter

unit

group

0 mg/kg/day (Control)

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

WBC

/mm3

Males

5360+/-2304

(5)

6640+/-1569 (5)

5500+/-1158 (5)

5040+/-2556 (5)

WBC

/mm3

Satellite males

5180+/-832 (5)

 

 

3620+/-1246* (5)

WBC

/mm3

Females

3260+/-1316 (5)

3040+/-1234 (5)

3500+/-1030 (5)

4680+/-2989 (5)

WBC

/mm3

Satellite females

3180+/-853 (5)

 

 

3580+/-1743 (5)

Lymphocytes

/mm3

Males

4333+/-2148 (5)

5027+/-1550 (5)

4744+/-981 (5)

3917+/-1886 (5)

Lymphocytes

WBC%

Males

78.6+/-10.1 (5)

76.0+/-13.2 (5)

86.4+/-4.2* (5)

79.0+/-6.5 (5)

Lymphocytes

/mm3

Satellite males

4249+/-705 (5)

 

 

2801+/-876* (5)

Lymphocytes

WBC%

Satellite males

82.0+/-2.0 (5)

 

 

78.2+/-6.4 (5) (5)

Lymphocytes

/mm3

Females

2546+/-1140 (5)

2420+/-1028 (5)

2611+/-890 (5)

3867+/-2563 (5)

Lymphocytes

WBC%

Females

77.2+/-4.6 (5)

79.2+/-5.6 (5)

74.0+/-9.0 (5)

81.4+/-3.4 (5)

Lymphocytes

/mm3

Satellite females

3455+/-741 (5)

 

 

2735+/-1348 (5)

Lymphocytes

WBC%

Satellite females

76.8+/-7.4 (5)

 

 

76.6+/-5.0 (5)

PT

sec

Males

14.4+/-1.2 (5)

14.1 +/-1.0 (5)

14.8+/-0.8 (5)

14.2+/-1.3 (5)

PT

sec

Satellite males

19.9+/-0.6 (5)

 

 

19.5+/-0.9 (5)

PT

sec

Females

13.6+/-0.7 (5)

14.6+/-0.8 (5)

13.6+/-1.0 (5)

15.2+/-0.8* (4)

PT

sec

Satellite females

19.5+/-0.7 (5)

 

 

20.9+/-0.8* (4)

hemoglobin

g/dL

Males

15.66+/-3.11 (5)

16.36+/-0.61 (5)

15.96+/-1.00 (5)

16.32+/-0.76 (5)

hemoglobin

g/dL

Satellite males

16.18+/-0.44 (5)

 

 

15.10+/-0.75* (5)

hemoglobin

g/dL

Females

14.74+/-0.46 (5)

15.36+/-0.51 (5)

15.30+/-0.64 (5)

13.16+/- 3.97 (5)

hemoglobin

g/dL

Satellite females

15.18+/-0.87 (5)

 

 

15.38+/-0.41 (5)

Hematocrit

%

Males

48.64+/-10.08 (5)

50.32+/-2.09 (5)

49.52+/-3.38 (5)

50.56+/-2.71 (5)

Hematocrit

%

Satellite males

47.66+/-0.74 (5)

 

 

45.06+/-2.81 (5)

Hematocrit

%

Females

45.04+/-1.60 (5)

48.02+/-1.35* (5)

47.46+/-3.64 (5)

41.28+/-12.65 (5)

Hematocrit

%

Satellite females

45.54+/-2.49 (5)

 

 

44.92+/-1.47

MCHC

g/dL

Males

32.22+/-0.48 (5)

32.48+/-0.26 (5)

32.24+/-0.34 (5)

32.28 +/- 0.72 (5)

MCHC

g/dL

Satellite males

33.96+/-0.59 (5)

 

 

33.58+/-0.74 (5)

MCHC

g/dL

Females

32.74+/-0.99 (5)

31.94+/-0.76 (5)

32.34+/-1.22 (5)

31.98+/-0.45 (5)

MCHC

g/dL

Satellite females

33.34+/-0.56 (5)

 

 

34.26+/-0.43* (5)

* statistically significant p<0.05 difference between the means of the group treated and control group

Organ weight: data is presented as mean+/-SD (N° of animals)

Organ

 

group

0 mg/kg/d (control)

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

Right testis

Absolute weight

Males

2.027+/-0.153 (12)

2.038+/-0.180 (12)

1.978+/-0.158 (12)

1.879+/-0.186 (12)

Right testis

Relative to body weight

Males

0.527+/-0.043 (12)

0.509+/-0.048 (12)

0.492+/-0.048 (12)

0.476+/-0.044* (12)

Right testis

Relative to brain weight

Males

99.36+/-7.73

96.44+/-8.55

101.96+/-6.94

95.13+/-7.23

Left testis

Absolute weight

Males

2.061+/-0.209 (12)

2.048+/-0.198 (12)

2.030+/-0.162 (12)

1.937+/-0.194 (12)

Left testis

Relative to body weight

Males

0.535+/-0.047 (12)

0.511+/-0.054 (12)

0.506+/-0.053 (12)

0.491+/-0.051 (12)

Left testis

Relative to brain weight

Males

98.71+/-9.58 (12)

94.97+/-7.02 (12)

103.45+/-8.34 (12)

96.97+/-7.94 (12)

Right testis

Absolute weight

Satellite males

2.120+/-0.188 (5)

 

 

1.866+/-0.212 (5)

Right testis

Relative to body weight

Satellite males

0.505+/-0.058 (5)

 

 

0.450+/-0.034 (5)

Right testis

Relative to brain weight

Satellite males

102.18+/-11.32 (5)

 

 

95.19+/-10.67 (5)

Left testis

Absolute weight

Satellite males

2.033+/-0.201 (5)

 

 

1.990+/-0.185 (5)

Left testis

Relative to body weight

Satellite males

0.483+/-0.052 (5)

 

 

0.481+/-0.040 (5)

Left testis

Relative to brain weight

Satellite males

97.99+/-11.82 (5)

 

 

101.45+/-8.77 (5)

* statistically significant (p<0.05)

Histopathology: individual data (presented as x out of y animals)

Organ

observation

Group

0 mg/kg/day

250 mg/kg/day

500 mg/kg/day

1000 mg/kg/day

Testis (bilateral)

Slight reduction of germ cells

Males

0/12

-

-

3/12

Epididymis (right)

Focal isolated reduction of germ cells

Satellite males

2/5

-

-

2/5

 

Reproduction data of dams on day 0

Test group

Animal/litter number

 

Total pups

N° of live pups

N° of dead pups

0 mg/kg/day (control)

13

Not pregnant

-

-

-

 

14

 

11

11

0

 

15

 

11

11

0

 

16

No viable pups

0

0

0

 

17

 

11

11

0

 

18

 

6

6

0

 

19

Not pregnant

-

-

-

 

20

Not pregnant

-

-

-

 

21

 

8

8

0

 

22

 

13

13

0

 

23

No viable pups

0

0

0

 

24

 

11

11

0

 

Total

 

71

71

0

 

Mean+/-SD (N° animals)

 

7.9+/-4.9 (9)

7.9+/-4.9 (9)

0+/-0 (9)

250 mg/kg/day

47

Not pregnant

-

-

-

 

48

 

3

3

0

 

49

 

10

10

0

 

50

 

12

12

0

 

51

 

10

10

0

 

52

 

4

4

0

 

53

 

6

6

0

 

54

Not pregnant

-

-

-

 

55

Not pregnant

-

-

-

 

56

 

7

7

0

 

57

 

8

8

0

 

58

 

10

10

0

 

Total

 

70

70

0

 

Mean+/-SD (N° animals)

 

7.8+/-3.0 (9)

7.8+/-3.0 (9)

0+/-0 (9)

500 mg/kg/day

71

 

5

4

1

 

72

Not pregnant

-

-

-

 

73

 

1

1

0

 

74

 

7

7

0

 

75

 

9

9

0

 

76

 

2

2

0

 

77

 

11

11

0

 

78

 

12

11

1

 

79

 

11

11

0

 

80

 

9

9

0

 

81

 

10

10

0

 

82

Not pregnant

-

-

-

 

Total

 

77

75

2

 

Mean+/-SD (N° animals)

 

7.7+/-3.9 (10)

7.5+/-3.8 (10)

0.2+/-0.4 (10)

1000 mg/kg/day

95

No viable pups

0

0

0

 

96

 

7

5

2

 

97

 

7

0

7

 

98

 

3

0

3

 

99

 

12

12

0

 

100

 

10

10

0

 

101

 

10

10

0

 

102

 

4

4

0

 

103

 

5

5

0

 

104

 

3

3

0

 

105

Not pregnant

-

-

-

 

106

Not pregnant

-

-

-

 

Total

 

61

49

12

 

Mean+/-SD (N° animals)

 

6.1+/-3.8 (10)

4.9+/-4.5 (10)

1.2+/-2.3 (10)

  Individual body weights (g) of pups

Test group

Litter number

Day 0 postnatal

mean pup weight

Day 4 postnatal

mean pup weight

0 mg/kg/day (control)

14

6.4

9.9

 

15

6.6

11.2

 

17

6.7

10.6

 

18

5.3

8.1

 

21

6.7

13.0

 

22

6.4

9.7

 

24

5.7

9.3

 

Mean+/-SD (N° of animals)

6.3+/-0.5 (7)

10.3+/-1.6 (7)

250 mg/kg/day

48

7.6

12.3

 

49

6.7

9.7

 

50

5.7

8.8

 

51

6.0

9.4

 

52

7.3

12.2

 

53

7.0

11.4

 

56

6.5

9.1

 

57

6.1

9.1

 

58

6.2

10.1

 

Mean+/-SD (N° of animals)

8.6+/-0.6 (9)

10.2+/-1.4 (9)

500 mg/kg/day

71

8.5

10.2

 

73

4.6

-

 

74

6.5

11.6

 

75

6.7

11.8

 

76

7.1

9.1

 

77

5.6

10.1

 

78

5.9

10.7

 

79

7.3

12.7

 

80

6.4

10.3

 

81

6.1

10.5

 

Mean+/-SD (N° of animals)

6.3+/-0.8 (10)

10.8+/-1.1 (9)

1000 mg/kg/day

96

5.4

3.6

 

99

5.9

8.0

 

100

6.3

11.2

 

101

5.3

7.8

 

102

7.4

13.0

 

103

6.6

8.7

 

104

6.9

11.8

 

Mean+/-SD (N° of animals)

6.3+/-0.8 (7)

9.2+/-3.2 (7)

Applicant's summary and conclusion

Conclusions:
The No Observed Adverse Effect Level (NOAEL) of the test item in Wistar rats was 1000 mg/kg/day for males and females and 1000 mg/kg/day for embryo-fetal toxicity. The substance is not to be classified as reproductive toxicant.