Ammonium iron(III) citrate

Toxicity to reproduction

Administrative data

Endpoint:

reproductive toxicity, other

Remarks:

13 weeks subchronic toxicity study

Type of information:

experimental study

Adequacy of study:

weight of evidence

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

data from handbook or collection of data

Justification for type of information:

Data from peer reviewed journal

Data source

Reference

Reference Type:

publication

Title:

A 13-week subchronic toxicity study of test chemical in F344 rats

Author:

Takeshi Toyoda et. al.

Year:

2014

Bibliographic source:

Food and Chemical Toxicology,2014

Materials and methods

Test guideline

Qualifier:

equivalent or similar to guideline

Guideline:

other: As mentioned below

Principles of method if other than guideline:

13 weeks subchronic toxicity study was performed to determine the toxic nature of test chemical on male and female Fischer 344/DuCrj rats

GLP compliance:

not specified

Limit test:

no

Justification for study design:

No data available

Test material

Test material information

Constituent 1

Reference substance name:

2-hydroxypropane-1,2,3-tricarboxylate;iron(3+)

Cas Number:

2338-05-8

Molecular formula:

C6H8O7xFe

IUPAC Name:

2-hydroxypropane-1,2,3-tricarboxylate;iron(3+)

Details on test material:

- Name of test material: Ferric citrate
- Molecular formula: C6H8O7xFe
- Molecular weight: 244.9435 g/mol
- Substance type: Organic
- Physical state: Reddish brown color powder

Test animals

Species:

rat

Strain:

Fischer 344/DuCrj

Details on species / strain selection:

No data available

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan (Yokohama, Japan)
- Age at study initiation: 5 weeks old when purchased and used after oneweek acclimatization.
- Weight at study initiation: No data
- Fasting period before study:no data
- Housing: The animals were housed 3–4 rats per polycarbonate cage with soft chip bedding. The cages and chip bedding were exchanged twice a week.
- Diet (e.g. ad libitum): Basal diet ad libitum
- Water (e.g. ad libitum): Tap water ad libitum
- Acclimation period: one week

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 23 ± 2°C
- Humidity (%): 55 ± 5%
- Air changes (per hr): 18 times/hr
- Photoperiod (hrs dark / hrs light): 12 hr light dark cycle

IN-LIFE DATES: From: To: No data

Administration / exposure

Route of administration:

oral: feed

Vehicle:

other: feed

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: The test chemical was mixed with diet at dose level of 0%, 0.25%, 1.0%, and 4.0% (w/w) (0, 125, 500 or 2000 mg/Kg bw/day)

DIET PREPARATION
- Rate of preparation of diet (frequency): No data
- Mixing appropriate amounts with (Type of food): No data
- Storage temperature of food: No data

VEHICLE
- Justification for use and choice of vehicle (if other than water): Basal diet
- Concentration in vehicle: 0%, 0.25%, 1.0%, and 4.0% (w/w) (0, 125, 500 or 2000 mg/Kg bw/day)
- Amount of vehicle (if gavage): No data
- Lot/batch no. (if required): No data
- Purity: No data

Details on mating procedure:

No data available

Analytical verification of doses or concentrations:

not specified

Duration of treatment / exposure:

13 weeks

Frequency of treatment:

daily

Details on study schedule:

No data available

Doses / concentrations

Remarks:

0%, 0.25%, 1.0%, and 4.0% (w/w) (Males: 0, 143.9, 595.9 or 2834.7 mg/Kg bw/day; Females: 0, 147.7, 601.4 or 2845.6 mg/Kg bw/day)

No. of animals per sex per dose:

Total: 40 males and 40 females
0 mg/Kg bw/day: 10 males and 10 females
143.9 mg/Kg bw/day for males and 147.7 mg/Kg bw for females: 10 males and 10 females
595.9 mg/Kg bw/day for males and 601.4 mg/Kg bw for females: 10 males and 10 females
2834.7 mg/Kg bw/day for males and 2845.6 mg/Kg bw/day: 10 males and 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: In a preliminary 4-week palatability study of test chemical with administration at the highest dose of 5.0% (w/w), significant decrease of body weight gain (>20% compared to the controls) was observed in males and females (data not shown). From these results, doses of test chemical were decided as 0%, 0.25%, 1.0%, and 4.0% (w/w) in both sexes for the present 13-week toxicity study, with diet replacement every week.
- Rationale for animal assignment (if not random): At the beginning of the experiment, the animals were randomly allocated to four groups of 10 male and 10 female rats each, based on their body weights measured just before starting the test chemical treatment.
- Rationale for selecting satellite groups: No data
- Post-exposure recovery period in satellite groups: No data
- Section schedule rationale (if not random): No data

Positive control:

No data available

Examinations

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily
- Cage side observations checked in table [No.?] were included. General condition and mortality

DETAILED CLINICAL OBSERVATIONS: No data
- Time schedule: No data

BODY WEIGHT: Yes
- Time schedule for examinations: body weights were measured once a week

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes, The amounts of supplied and residual diet were weighed weekly in order to calculate the average daily food intake and test chemical intake through the entire treatment period.
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/kg body weight/day: Yes
- Compound intake calculated as time-weighted averages from the consumption and body weight gain data: Yes

FOOD EFFICIENCY: No data
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No data

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data
- Time schedule for examinations: No data

Oestrous cyclicity (parental animals):

No data available

Sperm parameters (parental animals):

No data available

Litter observations:

No data available

Postmortem examinations (parental animals):

GROSS PATHOLOGY: Yes, Complete necropsy was performed for all animals, and the brain, thymus, lungs, heart, spleen, liver, adrenal glands, kidneys, and gonads were weighed.

HISTOPATHOLOGY: Yes, the brain, thymus, lungs, heart, spleen, liver, adrenal glands, kidneys, and gonads, skin, mammary gland, sternum with marrow, femur with marrow, mandibular and mesenteric lymph nodes, salivary glands, aorta, trachea, tongue, esophagus, stomach, small and large intestines, pancreas, urinary bladder, epididymides, seminal vesicles, prostate gland, bulbourethral glands, uterus, vagina, pituitary gland, thyroid glands, parathyroid glands, spinal cord with vertebrae, trigeminal nerve, sciatic nerve, harderian glands, femoral skeletal muscle, and nasal cavity were stained with hematoxylin and eosin for histopathological examination. The testes and eyes were fixed in Bouin’s fixative and Davidson’s solution, respectively. Bony tissues including the nasal cavity, vertebrae, sternum, and femur were decalcified with a mixture of 10% formic acid and 10% buffered formalin for up to 2 weeks. Histopathological assessment was first performed on all tissues of the control and highest dose groups. If a chemical treatment-related change appeared at the highest dose, the relevant tissues from the lower dose groups then also underwent examination.

Postmortem examinations (offspring):

No data available

Statistics:

Variance in data for body and organ weights, as well as the results of hematology and serum biochemistry, was checked by Bartlett’s test for homogeneity. When the data were homogeneous, one-way analysis of variance (ANOVA) was conducted. In heterogeneous cases, the Kruskal–Wallis test was applied. When statistically significant differences were indicated, the Dunnett’s or non-parametric Dunnett’s multiple test were employed for comparisons between control and other groups. For incidences of histopathological findings, the Fisher’s exact probability test was applied. P values of < 0.05 were considered to be statistically significant.

Reproductive indices:

No data available

Offspring viability indices:

No data available

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:

no effects observed

Description (incidence and severity):

All animals were without any clinical signs

Dermal irritation (if dermal study):

not specified

Mortality:

no mortality observed

Description (incidence):

No deaths were noted

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Body weight gain of both sexes was significantly reduced with 4.0% (2834.7 mg/Kg bw/day for males and 2845.6 mg/Kg bw/day for females) test chemical treatment compared to the controls from the 1st week until the end of the experiment except in the 9th and 12th weeks in females. Comparing with the control groups, the average bodyweight gain during the experiment in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females were reduced by 13.4% and 5.9%, respectively.

Food consumption and compound intake (if feeding study):

no effects observed

Description (incidence and severity):

Daily food intake of 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females was slightly higher than in the other groups. Since there was no avoidance of the experimental diet, average intakes of test chemical per body weight was dose-dependent

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

Significant decreases of RBC and lymphocytes and increases of PLT and eosinophils were detected in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. In males, significant increases of MCV and MCH were observed in the 1.0% and 4.0% groups and with 4.0%, respectively

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

An increase of serum Fe levels and decreases of TP and transferrin were noted in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. Significant decrease of transferrin was also detected in 601.4 mg/Kg bw/day females. In males, increase of Na and decreases of AST and ALT were observed in the 2834.7 mg/Kg bw/day group. In females, significant increase of IP and decreases of Alb and Cl were found in the 2845.6 mg/Kg bw/day group. Increase of ALT in 595.9 mg/Kg bw/day males and decrease of K levels in 147.7 mg/Kg bw/day females were also detected, without any dose-dependence.

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

no effects observed

Description (incidence and severity):

Treatment related pathological findings were noted in the colon, mesenteric lymph node, spleen and bone marrow. There was moderate to severe colitis in all 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. In the affected colons, numbers of eosinophils as well as neutrophils and macrophages had infiltrated into the lamina propria and submucosa. Absorptive epithelial cells of colonic mucosa were replaced by hyperplastic mucous cells with frequent mitotic figures. Eosinophilic infiltration was also observed occasionally in the submucosa of stomach, duodenum and cecum in 4.0% males and females with a lower incidence than in the colon. Mesenteric lymph nodes of all animals of 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females were macroscopically enlarged and histologically showed dilated sinuses featuring aggregation of plasmacytes and hemosiderin deposits. Infiltration of eosinophils was noted not only in the sinuses of mesenteric lymph nodes but also in the surrounding adipose tissue. In spleens of 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females, the degree of hemosiderin deposition was higher than in control groups. Hematopoietic cells in the bone marrow were increased in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. Although several lesions in other organs were sporadically detected, no significant treatment dependent alteration in their incidences was apparent.

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Effect levels (P0)

1

Dose descriptor:

NOAEL

Effect level:

595.9 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

male

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

gross pathology

Remarks on result:

other: no toxic effects on reproductive organ

2

Dose descriptor:

NOAEL

Effect level:

601.4 mg/kg bw/day (nominal)

Based on:

test mat.

Sex:

female

Basis for effect level:

clinical signs

mortality

body weight and weight gain

food consumption and compound intake

haematology

clinical biochemistry

organ weights and organ / body weight ratios

Remarks on result:

other: No toxic effects on reproductive organ

Target system / organ toxicity (P0)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: P1 (second parental generation)

General toxicity (P1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Behaviour (functional findings):

not specified

Immunological findings:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Neuropathological findings:

not specified

Histopathological findings: non-neoplastic:

not specified

Histopathological findings: neoplastic:

not specified

Other effects:

not specified

Reproductive function / performance (P1)

Reproductive function: oestrous cycle:

not specified

Reproductive function: sperm measures:

not specified

Reproductive performance:

not specified

Results: F1 generation

General toxicity (F1)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F1)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F1)

Developmental immunotoxicity:

not specified

Effect levels (F1)

Dose descriptor:

other: not specified

Generation:

other: not specified

Based on:

not specified

Sex:

not specified

Remarks on result:

not measured/tested

Target system / organ toxicity (F1)

Critical effects observed:

not specified

System:

other: not specified

Organ:

not specified

Results: F2 generation

General toxicity (F2)

Clinical signs:

not specified

Dermal irritation (if dermal study):

not specified

Mortality / viability:

not specified

Body weight and weight changes:

not specified

Food consumption and compound intake (if feeding study):

not specified

Food efficiency:

not specified

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

not specified

Ophthalmological findings:

not specified

Haematological findings:

not specified

Clinical biochemistry findings:

not specified

Urinalysis findings:

not specified

Sexual maturation:

not specified

Anogenital distance (AGD):

not specified

Nipple retention in male pups:

not specified

Organ weight findings including organ / body weight ratios:

not specified

Gross pathological findings:

not specified

Histopathological findings:

not specified

Other effects:

not specified

Developmental neurotoxicity (F2)

Behaviour (functional findings):

not specified

Developmental immunotoxicity (F2)

Developmental immunotoxicity:

not specified

Overall reproductive toxicity

Reproductive effects observed:

not specified

Treatment related:

not specified

Any other information on results incl. tables

Table 1: Body weight and food intake data for F344 rats treated with test chemical for 13weeks

Group	Body weight (g)		Food intake (g/rat/day)	Chemical intake (mg/Kg bw/day)
	Initial	Final
Males
0	106.0 ± 5.2	305.0 ± 10.7	13.9	0
0.25	105.8 ± 4.1	301.5 ± 12.8	13.4	143.9
1.0	105.9 ± 4.4	304.5 ± 18.7	13.9	595.9
4.0	105.7 ± 4.1	266.6 ± 10.1**	14.5	2834.7
Females
0	94.1 ± 3.0	174.8 ± 5.8	9.2	0
0.25	93.3 ± 3.5	173.2 ± 9.5	8.8	147.7
1.0	93.7 ± 3.4	174.7 ± 6.1	9.0	601.4
4.0	93.4 ± 2.8	166.1 ± 6.0*	10.0	2845.6

Values are mean ± S.D. * and ** Significantly different from the controls at p < 0.05 and < 0.01, respectively

Table 2: Hematology data for F344 rats treated with test chemical for 13 weeks.

Dose Level (%)	0	0.25	1.0	4.0
Males
No. of animals examined	10	10	10	10
WBC × 102/μl	38.8 ± 11.6	33.9 ± 6.9	31.7 ± 4.4	45.0 ± 11.1
RBC × 104/µl	908.0 ± 57.2	889.3 ± 70.0	849.1 ± 47.9	838.3 ± 48.5*
HGB g/dl	16.0 ± 1.0	15.6 ± 1.0	15.2 ± 0.8	16.1 ± 0.7
HCT %	49.5 ± 2.7	49.1 ± 2.7	47.4 ± 2.3	49.3 ± 2.1
MCV fl	54.6 ± 1.1	55.4 ± 1.4	55.9 ± 0.9*	58.9 ± 1.3**
MCH pg	17.7 ± 0.3	17.6 ± 0.4	17.9 ± 0.2	19.2 ± 0.5**
MCHC g/dl	32.3 ± 0.7	31.8 ± 0.5	32.1 ± 0.5	32.6 ± 0.6
PLT × 104/μl	68.2 ± 11.1	64.2 ± 11.5	65.5 ± 5.3	79.5 ± 4.7*
Differential cell count
Band %	0.1 ± 0.2	0.3 ± 0.4	0.3 ± 0.3	0.3 ± 0.3
Seg %	28.2 ± 4.4	30.9 ± 8.4	31.7 ± 5.8	34.9 ± 3.8
Eosin %	1.3 ± 0.8	1.0 ± 0.5	1.3 ± 0.9	9.5 ± 7.0**
Baso %	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Lymph %	70.2 ± 4.6	67.6 ± 8.3	66.4 ± 5.8	55.1 ± 7.7**
Mono %	0.3 ± 0.4	0.3 ± 0.4	0.3 ± 0.4	0.3 ± 0.4
Ebl (per 200 WBC)	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.1 ± 0.3
Females
No. of animals examined	9^a	10	10	10
WBC × 102/μl	21.1 ± 3.2	17.9 ± 5.7	17.2 ± 2.2	24.5 ± 6.2
RBC × 104/µl	811.1 ± 33.9	770.5 ± 98.0	781.7 ± 48.2	749.9 ± 34.1*
HGB g/dl	15.3 ± 0.6	14.5 ± 1.9	15.0 ± 1.0	14.4 ± 0.7
HCT %	46.8 ± 1.7	45.3 ± 5.3	46.2 ± 2.7	44.4 ± 1.6
MCV fl	57.7 ± 0.9	58.9 ± 1.7	59.1 ± 1.1	59.2 ± 1.3
MCH pg	18.8 ± 0.4	18.8 ± 0.7	19.2 ± 0.2	19.3 ± 0.3
MCHC g/dl	32.7 ± 0.6	31.9 ± 0.8	32.4 ± 0.6	32.5 ± 0.9
PLT × 104/μl	69.5 ± 8.3	69.7 ± 9.7	71.5 ± 7.1	80.6 ± 8.1*
Differential cell count
Band %	0.2 ± 0.3	0.2 ± 0.2	0.1 ± 0.2	0.4 ± 0.5
Seg %	29.1 ± 2.8	29.2 ± 5.4	27.1 ± 4.5	32.7 ± 6.3
Eosin %	1.7 ± 0.8	0.9 ± 0.8	1.8 ± 2.1	10.2 ± 3.0*
Baso %	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0	0.0 ± 0.0
Lymph %	68.2 ± 3.4	69.6 ± 5.6	70.8 ± 3.6	56.2 ± 8.4*
Mono %	0.8 ± 1.0	0.2 ± 0.3	0.3 ± 0.4	0.6 ± 0.5
Ebl (per 200 WBC)	0.0 ± 0.0	0.0 ± 0.0	0.2 ± 0.6	0.0 ± 0.0

Values are mean ± S.D. * and ** Significantly different from the controls at p < 0.05 and < 0.01, respectively.^aThe number of effective animals was reduced to nine due to incidental nephroblastoma.

Table 3: Serum biochemistry data for F344 rats treated with test chemical for 13 weeks.

Dose Level (%)	0	0.25	1.0	4.0
Males
No. of animals examined	10	10	10	10
TP (g/dl)	6.3 ± 0.2	6.3 ± 0.2	6.4 ± 0.1	6.1 ± 0.2**
Na (mEq/l)	141.0 ± 0.5	141.8 ± 0.6	141.6 ± 2.0	142.4 ± 1.1**
AST (IU/l)	81.3 ± 5.6	78.2 ± 8.3	85.6 ± 9.8	72.0 ± 3.5*
ALT (IU/l)	53.4 ± 5.2	56.1 ± 7.2	60.6 ± 6.9*	41.7 ± 4.8**
Transferrin (mg/dl)	148.0 ± 3.3	148.6 ± 5.1	150.5 ± 5.5	141.5 ± 4.7*
Transferrin (mg/dl)	148.0 ± 3.3	148.6 ± 5.1	150.5 ± 5.5	141.5 ± 4.7*
Females
No. of animals examined	9^a	10	10	10
TP (g/dl)	6.2 ± 0.1	6.3 ± 0.3	6.2 ± 0.3	5.6 ± 0.2**
Alb (g/dl)	4.2 ± 0.1	4.2 ± 0.2	4.1 ± 0.1	3.8 ± 0.1**
Cl (mEq/l)	103.1 ± 0.6	103.1 ± 1.2	103.1 ± 1.1	101.5 ± 1.1**
IP (mg/dl)	4.1 ± 0.3	4.3 ± 0.9	4.6 ± 1.0	6.6 ± 1.2**
Transferrin (mg/dl)	152.8 ± 5.6	149.7 ± 4.4	145.6 ± 4.9*	137.9 ± 6.8**
Fe (μg/dl)	249.4 ± 36.9	288.8 ± 37.1	272.1 ± 33.4	308.4 ± 57.3*

Values are mean ± S.D. * and ** Significantly different from the controls at p < 0.05 and < 0.01, respectively.^aThe number of effective animals was reduced to nine due to incidental nephroblastoma.

Table 4: Organ weight data for F344 rats treated with test chemical for 13 weeks.

Dose Level (%)	0	0.25	1.0	4.0
Males
No. of animals examined	10	10	10	10
Body weight (g)	298.1 ± 11.4	293.7 ± 10.5	298.5 ± 17.8	265.2 ± 11.2**
Absolute (g)
Heart	0.828 ± 0.043	0.837 ± 0.025	0.850 ± 0.049	0.760 ± 0.039**
Relative (%)
Brain	0.641 ± 0.032	0.659 ± 0.020	0.639 ± 0.037	0.713 ± 0.030**
Spleen	0.202 ± 0.009	0.206 ± 0.008	0.201 ± 0.008	0.240 ± 0.009**
Adrenals	0.011 ± 0.002	0.012 ± 0.001	0.013 ± 0.001	0.013 ± 0.001**
Kidneys	0.586 ± 0.024	0.588 ± 0.020	0.586 ± 0.029	0.633 ± 0.029**
Testes	0.993 ± 0.065	1.010 ± 0.041	0.983 ± 0.061	1.102 ± 0.050**
Females
No. of animals examined	9^a	10	10	10
Body weight (g)	165.4 ± 5.4	167.5 ± 8.3	168.0 ± 5.6	161.7 ± 5.6
Absolute (g)
Heart	0.562 ± 0.036	0.563 ± 0.031	0.544 ± 0.026	0.508 ± 0.025**
Spleen	0.378 ± 0.035	0.402 ± 0.042	0.388 ± 0.024	0.438 ± 0.038**
Relative (%)
Heart	0.340 ± 0.024	0.336 ± 0.011	0.324 ± 0.020	0.315 ± 0.013*
Spleen	0.229 ± 0.020	0.239 ± 0.018	0.231 ± 0.014	0.271 ± 0.025**
Liver	2.258 ± 0.092	2.339 ± 0.079	2.234 ± 0.112	2.399 ± 0.070**

Values are mean ± S.D. * and ** Significantly different from the controls at p < 0.05 and < 0.01, respectively. a and b The number of effective animals was reduced to nine due to incidental nephroblastoma and failure of tissue sampling, respectively.

Table 5: Histopathological findings for F344 rats treated with test chemical for 13 weeks.

	Dose level (%)	Males				Females
		0	0.25	1.0	4.0	0	0.25	1.0	4.0
Organs and findings	No. of animals	10	10	10	10	9a	10	10	10
Spleen	Increased hemosiderosis	0	0	0	10**	0	0	0	6*
Colon	Inflammation with eosinophilic infiltration	0	0	0	10**	0	0	0	10**
	Mucosal hyperplasia	0	0	0	10**	0	0	0	10**
Mesentric lymphnode	Inflitration of eosinophils	0	0	0	10**	0	0	0	10**
	Plasmacytosis	0	0	0	7**	0	0	0	7**
	Hemosiderosis	0	0	0	10**	0	0	0	9**
Bone marrow Increased	hematopoiesis	0	0	0	8**	0	0	0	9**

** Significantly different from the controls at p < 0.05 and < 0.01, respectively. a The number of effective animals was reduced to nine due to incidental nephroblastoma.

Applicant's summary and conclusion

Conclusions:

The No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is considered to be 1.0% (595.9 mg/kg bw/day for males and 601.4 mg/kg bw/day for females), When male and female Fischer 344/DuCrj rats were treated with test chemical orally in the 13 weeks.

Executive summary:

The reproductive toxicity was considered on the basis of 13 weeks sub chronic toxicity study of test chemical .The study was performed using 40 male and 40 female Fischer 344/DuCrj rats. The test chemical was mixed with basal diet and used at dose level of 0%, 0.25%, 1.0%, and 4.0% (w/w) (Males: 0, 143.9, 595.9 or 2834.7 mg/Kg bw/day; Females: 0, 147.7, 601.4 or 2845.6 mg/Kg bw/day) for 13 weeks. During the study period, the animals were observed for general condition, mortality, changes in body weight and food intake, haemotology, clinical chemistry and were subjected to gross and histopathology. All animals were without any clinical signs and no deaths were reported. Reduction of body weight gain was noted in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. On hematology assessment, decreases of red blood cells and lymphocytes and increases of platelets and eosinophils were noted in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. Serum biochemistry demonstrated increased iron and decreased total protein and transferrin in both sexes treated with 2834.7 mg/Kg bw/day and 2845.6 mg/Kg bw/day test chemical respectively. In addition, an increase of serum inorganic phosphorus levels was noted in 2845.6 mg/kg bw females. Regarding organ weights, In males, absolute heart weights were decreased in the 4.0% group (2834.7 mg/Kg bw/day) compared to the controls. In addition, relative weights of brain, spleen, adrenals, kidneys, and testes were significantly increased in 2834.7 mg/Kg bw/day males. In females, decrease of absolute and relative heart weights, increase of absolute and relative spleen weights and increase of relative liver weights were observed in the 2845.6 mg/Kg bw/day group. Absolute weights of liver were also increased in 147.7 mg/Kg bw/day females without dose-dependence. On histopathological assessment, colitis with infiltration of eosinophils and hyperplasia of mucosal epithelium, eosinophilic infiltration in mesenteric lymph nodes, and increased hemosiderosis in spleen were observed as treatment-related toxicological changes in 2834.7 mg/Kg bw/day males and 2845.6 mg/Kg bw/day females. Based on the observations made, the No Observed Adverse Effect Level (NOAEL) for reproductive toxicity is considered to be 1.0% (595.9 mg/kg bw/day for males and 601.4 mg/kg bw/day for females), When male and female Fischer 344/DuCrj rats were treated with test chemical orally in the 13 weeks.