Registration Dossier

Diss Factsheets

Administrative data

Key value for chemical safety assessment

Toxic effect type:
dose-dependent

Effects on fertility

Description of key information

In cases where data for potassium chlorate were not available or to support data that were found for potassium chlorate, tests performed with sodium chlorate were used in this dossier. Read across is possible for these two substances, because the toxicity of the test substance is expected to be related to the chlorate ion and not by the sodium or potassium ion.


 


From the two-generation reproduction study, the NOAEL for parental toxicity is 70 mg/kg bw/day for the females, based on signs of hyperactivity of the thyroid glands (grade 4) at the next highest dose level. The NOAEL for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny is 500 mg/kg/day (Gaoua 2004b).

Link to relevant study records

Referenceopen allclose all

Endpoint:
two-generation reproductive toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the RAAF document.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Description, severity, time of onset and duration of clinical signs: Parents: No effects. However, at 500 mg/kg/day, ptyalism was recorded in 1 male and a few other clinical signs were randomly distributed among the treated groups (1 or 2 males per group). According to females there were no relevant signs of toxicity in any group. In conclusion: All these signs were not considered to be of toxicological significance.
Mortality P: There was no death in any group during the whole study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
BW Parents: No effects.
Food Parents: No effects.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
-

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Changes in estrous cycles: The estrous cycle stages evaluated during the last three weeks of the premating period were not affected by the treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Effects on sperm in P and F1: The seminology parameters evaluated in both P and F1 males, namely epididymal sperm count, epididymal sperm motility and morphology, testicular sperm head count and daily sperm production were not affected by the treatment at any dose-level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In P and F1:
- Reproductive parameters in females with evidence of mating:
P
Dose mg/kg/day 0 10 70 500
Paired animals 25M+25F 25M+25F 25M+25F 25M+25F
evidence of mating 24(25) 24(25) 22 (24)25
Mating index (%) 96(100) 96(100) 88(96) 100
Pre-coital interval(d) 2.96 2.88 3.09 2.44
Fertility index (%) 96(96) 100(100) 95(96) 96
Females which delivered 23 24 21 24
Gestation index (%) 100 100 100 100
Gestation length (days) 21.5 21.4 21.3 21.4
Post-implantation + 6.5 7.4 7.9 5.8
neo-natal losses (%)
M: male, F: female, (): pregnant females with no evidence of mating are taken into account.

- Reproductive parameters in females with evidence of mating:
F1
Dose mg/kg/day 0 10 70 500
Paired animals 25M+25F 25M+25F 25M+25F 24M+24F
evidence of mating 25 24 22(23) 23(24)
Mating index (%) 100 96 88(92) 96(100)
Pre-coital interval (d) 3.28 3.29 2.23 2.70
Fertility index (%) 92 96 91(91) 96(96)
Pregnant mated females 23 23 20(21) 22(23)
Females which delivered 23 23 20 22
Gestation index (%) 100 100 100 100
Gestation length (days) 21.6 21.6 21.5 21.6
Post-implantation + 3.7 10.7 5.7 9.2
neo-natal losses (%)
M: male, F: female, (): pregnant females with no evidence of mating are taken into account.

- Ovarian primordial follicle counts and growing follicles:
P:
Dose mg/kg/day 0 500
Primordial follicle
Total number 477 755
Mean Value 19.08 30.20
Range 0-44 5-94
Growing Follicles
Total number 66 39
Mean Value 2.64 1.56
Range 0-9 0-6
F1:
Primordial follicle
Total number 457 538
Mean Value 18.28 21.52
Range 7-49 5-47
Growing Follicles
Total number 43 27
Mean Value 1.72 1.08
Range 0-5 0-5

A relationship to the treatment was considered to be unlikely. Considering the great variability described in published literature for such quantitative analysis and when the quantitative parameters were evaluated, as per defined intervals of values it appeared more evident that the differences observed were due, at least in part, to the great inter-individual variability.
ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weight changes P, F1 and F2:
An increased spleen weight was observed in 500 mg/kg day P and F1 males. It was considered that the differences in spleen weight were most probably secondary to the anaemia noted among the animals and consequently of no toxicological importance. Other differences in organ weights in P, F1 and F2 were not dose-related and/or without the same trend in both sexes. As the differences in thyroid and thymus weight were minimal and not dose-related, they were considered to be of no toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross pathology incidence and severity:
The only macroscopic findings observed were those which have been found to occur spontaneously in the untreated laboratory rat of this strain and age. They were therefore considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Histopathology incidence and severity, P, F1 and F2:
Parents:
At 500 mg/kg/day minimal to moderate follicular thyroidal hyperplasia and moderate hyperactivity of the thyroids in both parent males and females. At 70 mg/kg/day moderate follicular hyperactivity of the thyroids in the parents.

P:
Follicular thyroidal hyperplasia:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 0/25 0/25 0/24 20/24
Grade 1 - - - 0
Grade 2 - - - 18
Grade 3 - - - 2
Mean grading - - - 2.1

Females Incidence 0/25 0/25 0/25 18/25
Grade 1 - - - 7
Grade 2 - - - 8
Grade 3 - - - 3
Mean grading - - - 1.8

Follicular thyroidal hyperactivity:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 25/25 25/25 24/24 24/24
Grade 2 11 3 3 2
Grade 3 12 20 15 15
Grade 4 2 2 6 7
Mean grading 2.6 3.0 3.1 3.2

Females Incidence 24/25 25/25 25/25 25/25
Grade 2 16 8 8 3
Grade 3 8 16 17 16
Grade 4 0 1 0 6
Mean grading 2.3 2.7 2.7 3.1

F1:
Follicular thyroidal hyperplasia:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 0/25 0/25 0/24 21/22
Grade 1 - - - 3
Grade 2 - - - 12
Grade 3 - - - 6
Mean grading - - - 2.1

Females Incidence 0/25 0/25 0/25 20/25
Grade 1 - - - 6
Grade 2 - - - 10
Grade 3 - - - 4
Mean grading - - - 1.9

Follicular thyroidal hyperactivity:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 25/25 25/25 24/24 22/22
Grade 1 2 0 0 0
Grade 2 6 4 6 3
Grade 3 17 21 15 16
Grade 4 0 0 3 3
Mean grading 2.6 2.8 2.9 3.0

Females Incidence 25/25 25/25 24/25 24/25
Grade 1 2 1 1 0
Grade 2 12 13 12 5
Grade 3 11 11 11 16
Grade 4 0 0 0 3
Mean grading 2.4 2.4 2.4 2.9

F2:
Dose mg/kg/day Microscopic findings 0 10 70 500
Males Follicular cell hypertrophy 91 97 100 100
1.3 1.3 1.2 1.2
Small follicles 91 97 100 100
1.5 2.1 1.8 2.4
Pale or decreased colloid contents 91 95 100 100
1.2 2.0 1.7 2.3
Cords of epithelial cells 66 84 98 90
1.4 1.5 1.3 1.7
Females Follicular cell hypertrophy 100 98 98 100
1.0 1.0 1.1 1.0
Small follicles 100 98 98 100
1.9 2.0 1.9 2.4
Pale or decreased colloid contents 100 98 98 100
1.8 2.0 1.8 2.4
Cords of epithelial cells 88 95 95 95
1.3 1.7 1.7 1.8

There was an absence of characterized thyroidal hypertrophy at all dose-levels and in both sexes. The cords of epithelial cells with small follicles and decreased or pale colloid contents are morphological characteristics of an infant thyroidal tissue. As they were recorded without evidence of a dose or treatment relationship, it was concluded that the small morphological differences observed between treated and control F2 pups were without relationship to treatment with the test item and correlated with the morphological characteristics of an infant thyroidal tissue.

The only other microscopic findings were those which are commonly encountered in the untreated laboratory rat of this strain and age and were thus considered to be without relationship to treatment with the test item.

OTHER FINDINGS (PARENTAL ANIMALS)
- LACTATION:
Changes in lactation:
P
Dose mg/kg/day 0 10 70 500
lactation index (%) 100.0 98.9 99.4 97.4
CIT Historical Control Data for lactation index on day 21 pp: minimum = 96.4, maximum = 100%
The lactation index was in the range of historical control data.

- HEMATOLOGICAL FINDINGS
Hematological findings incidence and severity: Parents: At 500 mg/kg/day significant decrease in red blood cell count and mean hemoglobin concentration in both parental males and females.
Dose-level (mg/kg/day) 0 10 70 500
Males
RBC (T/L) 8.76 8.73 8.61 8.02**
HB (g/dL) 15.7 15.8 15.3 14.5**
PCV (L/L) 0.45 0.46 0.44 0.43**
MCHC (g/dL) 34.7 34.5 34.7 33.9**
Females
RBC (T/L) 7.62 7.57 7.29* 6.98**
HB (g/dL) 15.6 15.5 15.1* 14.7**
PCV (L/L) 0.45 0.45 0.44 0.43*
MCHC (g/dL) 34.7 34.5 34.5 34.1**
Statistically significant: * p <0.05, ** p <0.01

CIT historical control data:
Males
RBC (T/L) : mean = 8.64, minimum = 7.56, maximum = 9.56
HB (g/dL) : mean = 15.3, minimum = 13.8, maximum = 16.6
PCV (L/L) : mean = 0.45, minimum = 0.40, maximum = 0.49
MCHC (g/dL) : mean = 33.9, minimum = 32.4, maximum = 34.9
Females
RBC (T/L) : mean = 7.60, minimum = 6.53, maximum = 8.33
HB (g/dL) : mean = 14.3, minimum = 13.1, maximum = 15.5
PCV (L/L) : mean = 0.42, minimum = 0.37, maximum = 0.46
MCHC (g/dL) : mean = 34.3, minimum = 32.8, maximum = 35.7
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day
Sex:
male/female
Basis for effect level:
other: increase in spleen follicular cell hyperplasia and grade 4 follicular hyperactivity of the thyroids in males and females at 500 mg/kg bw/day in F0 and F1 parents
Dose descriptor:
NOEL
Remarks:
Parental tox M
Effect level:
10 mg/kg bw/day
Sex:
male
Basis for effect level:
other: increase in spleen follicular cell hyperplasia and grade 4 follicular hyperactivity of the thyroids in males and females at 500 mg/kg bw/day in F0 and F1 parents
Dose descriptor:
NOEL
Remarks:
Parental tox F
Effect level:
70 mg/kg bw/day
Sex:
female
Basis for effect level:
other: increase in spleen follicular cell hyperplasia and grade 4 follicular hyperactivity of the thyroids in males and females at 500 mg/kg bw/day in F0 and F1 parents
Dose descriptor:
NOEL
Remarks:
for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny
Effect level:
500 mg/kg bw/day
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
VIABILITY (OFFSPRING)
Mortality F1:
. There was no death in the control or the 10 mg/kg/day group during the whole treatment period.

. In the 70 mg/kg bw/day group 2 males died, on day 95 and day 98. In the first male a prematurely sacrificed on day 98 of the treatment period for humane reasons. Post-mortem macroscopic examination, noted a perforation in the cervical region of esophagus with a pouch, and found in adjacent tissue, measuring 2.5 cm in diameter and showing thick whitish contents at cut surface. At microscopic examination, there was a para-esophageal abscess and marked granulation tissue formation including the cervical skeletal muscles. In the second male no signs for cause of death could be determined.

. In the 500 mg/kg bw/day group 3 males died, on day 69, 96 and day 98.
- For the male found dead on day 98 of the treatment period:
At macroscopic post-mortem examination, perforation was noted in the cervical region of esophagus with a pouch, found in adjacent tissue, measuring 2.5 cm in diameter and showing thick whitish contents at cut surface. At microscopic examination, there was a para-esophageal abscess and marked granulation tissue formation including the cervical skeletal muscles. The major factor contributing to the death was esophagus perforation.
- For the male found dead on day 96 of the treatment period:
At macroscopic post-mortem examination, foamy contents were seen in the trachea and bronchi and the lungs and heart were blackish in color. Except for congestion, most probably passive, due to the absence of bleeding, no relevant microscopic finding was noted.
- For the male found dead on day 69 of the treatment period:
At macroscopic post-mortem examination, liquid whitish contents were noted in the thoracic cavity. In addition, moderate cortical cell hypertrophy was noted in the adrenals, associated with moderate lipid depletion. This was most probably related to the poor physical condition of this animal before death. No other relevant microscopic finding was noted.

During the premating period, one female given 500 mg/kg/day was found dead after dosing on day 41, no clinical signs were noted prior to death. At necropsy, foamy contents were seen in the trachea and the lungs which were reddish in color. Apart moderate congestion of the lungs with minimal alveolar edema, no relevant microscopic finding was noted.

There was no other death in any group during the premating, pregnancy or the lactation periods.

From the above clinical history, necropsy and microscopic findings, the above mentioned sporadic deaths were considered to be accidental and without relationship to the treatment with the test item.

Viability index:
P:
Dose-level (mg/kg/day) 0 10 70 500
Viability index (%) 98.1 97.3 98.0 96.6
CIT Historical Control Data for viability index on day 4 pp: minimum = 95.9, maximum = 100%

CLINICAL SIGNS (OFFSPRING)
Description, severity, time of onset and duration of clinical signs: F1: no other effects.

BODY WEIGHT + FOOD CONSUMPTION (OFFSPRING)
BW F1: at 500 mg/kg/day for the males, significantly lower body weight gain (days 1 to 15, -8%, p<0.05)
Food intake F1: at 500 mg/kg/day for the males, significantly lower food consumption (days 1 to 15, -11%, p<0.01) during the first weeks of the premating.

SEXUAL MATURATION (OFFSPRING)
- Offspring toxicity F1 and F2:
F1:
Litter data
Dose-level (mg/kg/day) 0 10 70 500
Pups delivered (mean number/F) 13.6 14.0 14.4 14.7
Sex-ratio (% of males) 51.8 53.7 50.8 53.0
Pup weight at birth (day 1 pp)(g) 6.7 6.6 6.5 6.5
. Day 1 pp 6.7 6.6(-1%) 6.5(-3%) 6.5(-3%)
. Day 4 pp (pre-culling) 9.6 9.5(-1%) 9.1(-5%) 9.1(-5%)
. Day 7 pp 16.3 15.9(-2%) 15.6(-4%) 15.1(-7%)
. Days 14 pp 34.5 34.7(+1%) 34.0(-1%) 33.5(-3%)
. Day 21 pp 53.8 54.9(+2%) 53.0(-1%) 52.0(-3%)
Body weight change (g)
. Days 1-4 pp 2.9 2.9(0%) 2.7(-7%) 2.6(-10%)
. Days 4-21 pp 44.1 45.4(+3%) 43.7(-1%) 42.9(-3%)
(): difference from controls
CIT Historical Control Data for body weight on day 4 pp: mean = 9.7, mini = 9.5, maxi = 10.0
CIT Historical Control Data for body weight on day 7 pp: mean = 14.5, mini = 13.9, maxi = 16.3
CIT Historical Control Data for body weight on day 21pp: mean = 43.3, mini = 40.6, maxi = 52.0

The litter size at birth showed normal fluctuations among treated and control groups, being slightly higher in the treated groups, compared to the control group; this finding was considered to be of fortuitous occurrence. The sex ratio was similar in all groups. Very few deaths within control range, not related to treatment. No macroscopic post-mortem changes. Anouria (absence of tail) was observed in one pup from dam given 10 mg/kg/day or 500 mg/kg/day. Since this finding was low in incidence and not dose-related, it was considered to have occurred by chance.
The other observations (necrosis on hindlimb, cold to the touch and scattered hair) were low in incidence and randomly distributed within the groups, thus, they were considered not to be related to the treatment. There were no effects on reflex development. No effects on auditory function, visual function and no significant effect on spontaneous locomoter activity.

F2:
Litter data
Dose-level (mg/kg/day) 0 10 70 500
Litter size at birth(mean/F) 13.6 13.4 14.9 14.3
Sex-ratio (% of males) 52.7 49.2 50.2 52.2
Pup weight at birth (day 1 pp) (g) 6.6 6.7 6.4 6.7
Body weight (g)
. Day 1 pp 6.6 6.7(+1%) 6.4(-3%) 6.7(+1%)
. Day 4 pp (pre-culling) 9.1 9.3(+2%) 9.3(+2%) 9.0(-1%)
. Day 7 pp 15.4 15.4(0%) 15.4(0%) 14.6(-5%)
. Day 14 pp 33.0 33.2(0%) 33.1(0%) 31.2(-5%)
. Day 21 pp 52.0 51.9(0%) 51.7(-1%) 48.8(-6%)
Body weight change (g)
. Days 1-4 pp 2.5 2.6(+4%) 2.8(+12%) 2.3(-8%)
. Days 4-7 pp 6.1 6.1(0%) 6.3(+3%) 5.6(-8%)
. Days 4-21 pp 42.6 42.6(0%) 42.6(0%) 39.9(-6%)
(): difference from controls
CIT Historical Control Data for body weight on day 4 pp: mean=9.7, mini=9.5, maxi=10.0
CIT Historical Control Data for body weight on day 7 pp: mean=14.5, mini=13.9, maxi=16.3
CIT Historical Control Data for body weight on day 21pp: mean= 43.3, mini=40.6, maxi= 52.0

No effect was recorded on pup survival over the lactation period. The slightly lower viability index recorded in the control group was the consequence of one entirely decedent litter. There were no notable clinical signs among the pups.
The pups body weight and body weight gain at 10 and 70 mg/kg/day were not affected by the treatment during the whole lactation period. At 500 mg/kg/day, the body weight was similar to the control group value while slightly non-significant lower body weight and body weight gain were recorded after one week of birth until the end of the lactation period. These differences were clearly not related to the treatment period since the control value on day 21 post-partum was particularly higher and within the highest body weight values recorded in CIT historical control data. No effects on the sex ratio, reflex development or sperm parameters.

ORGAN WEIGHTS (OFFSPRING)
Organ weight changes P, F1 and F2:
An increased spleen weight was observed in 500 mg/kg day P and F1 males. It was considered that the differences in spleen weight were most probably secondary to the anaemia noted among the animals and consequently of no toxicological importance. Other differences in organ weights in P, F1 and F2 were not dose-related and/or without the same trend in both sexes. As the differences in thyroid and thymus weight were minimal and not dose-related, they were considered to be of no toxicological importance.

GROSS PATHOLOGY (OFFSPRING)
Gross pathology incidence and severity:
The only macroscopic findings observed were those which have been found to occur spontaneously in the untreated laboratory rat of this strain and age. They were therefore considered to be of no toxicological importance.

HISTOPATHOLOGY (OFFSPRING)
Histopathology incidence and severity, P, F1 and F2:
See above (parental animals).

OTHER FINDINGS (OFFSPRING)
Dose descriptor:
NOEL
Remarks:
for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny
Generation:
F1
Effect level:
500 mg/kg bw/day
Sex:
male/female
Remarks on result:
not determinable due to absence of adverse toxic effects
Reproductive effects observed:
not specified
Conclusions:
No effect was observed on mating, fertility, gestation, fecundity or delivery at any dose-level for either generation.
No effect was recorded on litter parameters and on pre and post-natal development of either generation at any dose-level.
Therefore, the No Observed Effect Level (NOEL) for parental toxicity is 10 mg/kg/day for the male and 70 mg/kg/day for the female.
The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny is 500 mg/kg/day.
Executive summary:

The study was designed to investigate the effects of the test material, sodium chlorate, on the growth and reproductive performance of the Sprague-Dawley rat and complies with OECD Guideline 416. A former one-generation dose-range finding study was performed to select dose-levels for this two-generation study. The study followed the principles of GLP. The test material was administered orally, by gavage. In the P generation three groups of 25 male and 25 female rats received the test material 10 weeks before mating and during mating, pregnancy and lactation. In the F1 generation three groups of 25 male and 25 female rats received the test material at weaning, on day 22 post-partum, until weaning of the F2 generation. The dose levels were 10, 70 and 500 mg/kg/day. The control group (25 males and 25 females in both generations) received vehicle (purified water) alone.


 


For parent of the F0 generation, sign of toxicity was substantiated by a decrease in red blood cells and hemoglobin concentration levels at 500 mg/kg/day. For parent of the F1 generation, significantly lower body weight gain and food consumption were observed during the first week of treatment at 500 mg/kg/day. For both parent males and females of both generations given 500 mg/kg/day, signs of slight to moderate follicular hyperplasia and signs of slight to moderate hyperactivity of the thyroid glands were recorded. Signs of hyperactivity were also observed for males of both F0 and F1 generation at 70 mg/kg/day.


No effect was observed on mating, fertility, gestation, fecundity or delivery at any dose-level for either generation.


No effect was recorded on litter parameters and on pre and post-natal development of either generation at any dose-level.


 


Therefore, the No Observed Effect Level (NOEL) for parental toxicity is 10 mg/kg/day for the male and 70 mg/kg/day for the female. Due to an increase in spleen follicular cell hyperplasia and grade 4 follicular hyperactivity of the thyroids in males and females at 500 mg/kg bw/day in F0 and F1 parents, which is a non-adverse effects, the parental NOAEL was 70 mg/kg/day in males and females.


The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny is 500 mg/kg/day.

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
October 2002 - June 2003
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol.
Qualifier:
according to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Laboratories, L'Arbresle, France
- Age at study initiation: (P) 6 wks; (F1) 3 wks
- Weight at study initiation: (P) Males: 189 g (176-205g); Females: 153 g (138-170g); (F1) only detailed info
- Fasting period before study: no because of repeated exposure
- Housing: P animals were housed individually in wire mesh cages; prior to delivery and during lactation P and F1 females were housed individually in polycarbonate cages; after weaning the F1 animals were housed individually in wire-mesh cages
- Use of restrainers for preventing ingestion (if dermal): not applicable
- Diet (e.g. ad libitum): free access to A04 C pelleted maintenance diet
- Water (e.g. ad libitum): free access to tap water
- Acclimation period: 6-day acclimation period


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 ± 2
- Humidity (%): 50 ± 20
- Air changes (per hr): about 12 cycles/hour of filtered, non-recycled air
- Photoperiod (hrs dark / hrs light): 12h/12h (7:00 - 19:00)


IN-LIFE DATES: From: October 28, 2002 To: June 30, 2003
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: The test item was administered as a solution in the vehicle (concentrations of 2, 14 and 100 mg/mL). The test item dosage forms were prepared by the Pharmacy at weekly intervals and stored at +4°C, away from light prior to use.

VEHICLE
- Justification for use and choice of vehicle (if other than water): purified water
- Concentration in vehicle: 2, 14 and 100 mg/mL
- Amount of vehicle (if gavage): (The quantity of the dosage form administered to each animal was adjusted according to the most recently recorded body weight). A constant dosage-volume of 5 mL/kg/day was used.
- Lot/batch no. (if required): not required
- Purity: purified water
Details on mating procedure:
- M/F ratio per cage: 1
- Length of cohabitation: during mating: each female was placed with the same male until mating occurred or 14 days had elapsed
- Proof of pregnancy: vaginal plug and sperm in vaginal smear; referred to as day 0 of pregnancy
- After ... days of unsuccessful pairing replacement of first male by another male with proven fertility. No info.
- Further matings after two unsuccessful attempts: no info
- After successful mating each pregnant female was caged (how): Each pair was separated and by the end of pregnancy, the females were individually placed in appropiate cages with nesting material.
- Any other deviations from standard protocol: no
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Stability and Concentration are examined, Homogenicity isn't determined. For stability and concentration no acceptability criteria are given in the method section. In the result section criteria are given (±10%), however 2 of the samples differ more than 10%, so that's remarkable.
- Stability: A previous study using the same batch (CIT/ Study No. 22823RSR) demonstrated the satisfactory stability of dosage forms (range 8-200 mg/mL) over a 9-day period at, room temperature, protected from light. In the present study, the stability of the low concentration was checked.
A dosage form at 2 mg/mL was prepared, protected from light then sampled after 0 (just after preparation) and 9 days storage at +4°C. All samples were analyzed as soon as possible after sampling. The mean result of two determinations (day 0) was taken as the initial value for the stability test.
- Concentration: The concentration of samples taken from each dosage form (including the control) prepared for use in weeks 1, 6, 12, 19, 24, 30 and 35 was determined.
Duration of treatment / exposure:
Exposure period: see "details on study schedule"
Premating exposure period (males): 10 weeks
Premating exposure period (females): 10 weeks
Duration of test: 2 generations
Frequency of treatment:
daily
Details on study schedule:
- Number of generation studies: 2
- Detailed description of duration treatment / exposure:
F0 generation
. 10 weeks before mating, during the mating period until sacrifice (weaning of pups) for the males.
. 10 weeks before mating, during the mating, pregnancy and lactation periods (until day 21 post-partum) for the females.
F1 generation
At weaning of the F1 generation, on day 22 post-partum, three groups of 25 male and 25 female Sprague-Dawley rats received the test item under the same experimental conditions as above, during their growth, adulthood, mating, pregnancy and lactation, until weaning of the F2 generation.

- F1 parental animals not mated until they are 12 to 14 weeks old (9-11 weeks after selected from the F1 litters).
- Selection of parents from F1 generation when pups were 22 days post-partum of age.
- Age at mating of the mated animals in the study: 12 to 14 weeks
Remarks:
Doses / Concentrations:
10, 70 or 500 mg/kg/day
Basis:
nominal conc.
No. of animals per sex per dose:
25
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: the dose-levels were specified by the Sponsor and were selected on the basis of a previous 1-generation dose-range finding study in rats (CIT/Study No. 22823 RSR; dose-levels of 40, 200 and 1000 mg/kg/day).
- Rationale for animal assignment (if not random): animals were allocated to the groups (by sex), according to a computerized stratification procedure, so that the average body weight of each group was similar.
Positive control:
Not required.
Parental animals: Observations and examinations:
CAGE SIDE OBSERVATIONS + DETAILED CLINICAL OBSERVATIONS + BODY WEIGHT + FOOD CONSUMPTION AND COMPOUND INTAKE + WATER CONSUMPTION AND COMPOUND INTAKE
Clinical signs and mortality were checked daily. Food consumption and body weight were recorded at designated intervals. During lactation, the pups (F1 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded. On day 22 post-partum, one male and one female pup per litter were selected to constitute the F1 generation, which comprised 25 males and 25 females per group. The F1 animals were observed daily for clinical signs and mortality. Body weight and food consumption were recorded once a week. Sexual development of both males and females was assessed. Neurobehavioural tests were conducted at designated intervals to assess auditory and visual functions. Spontaneous locomotor activity was also evaluated over a 10-min interval when the animals were between 7 and 8 weeks. During lactation, the pups (F2 generation) were observed daily for survival and clinical signs; body weight was recorded at designated intervals; the sex-ratio was recorded.
Oestrous cyclicity (parental animals):
- Estrous cycle: The estrous cycle stage was determined was evaluated in both F0 and F1 females from a fresh vaginal lavage (stained with methylene blue), each morning as follows:
. during the last three weeks of the pre-mating period,
. during the mating period, until the females were mated.
Sperm parameters (parental animals):
- Sperm examination: Epididymal and testicular sperm parameters were evaluated in both F0 and F1 males. Before sacrifice, each male was kept under isoflurane anesthesia, the left epididymide was sampled, weighed and sperm was collected from the cauda for investigation.
Litter observations:
STANDARDISATION OF LITTERS
On day 4 post-partum, the size of each F1 litter was adjusted to obtain eight pups per litter (four males and four females).


PARAMETERS EXAMINED
See above 'Parental animals: observations and examinations'
Postmortem examinations (parental animals):
SACRIFICE + GROSS NECROPSY + HISTOPATHOLOGY / ORGAN WEIGHTS
A macroscopic post-mortem examination was performed on all F0 and F1 parent males and females and on three pups per sex and per litter of each F0 and F1 females killed at weaning. Any pups which died during the lactation period or were not selected were also submitted to macroscopic post-mortem examination. Macroscopic lesions, reproductive organs, adrenals, thyroids and pituitary glands were sampled in all parent animals. In all pups, the macroscopic lesions, the thyroid and pituitary glands were also preserved. A microscopic examination was performed on macroscopic lesions, reproductive organs, adrenals, and pituitary glands of all F0 and F1 parents of the control and high dose groups and on the thyroid gland for all F0 and F1 parents. Particular detailed histopathological examinations were performed for the ovaries and the testes. A microscopic examination of the thyroid gland was also performed for all pups of the F2 generation
- Organ weights P and F1: Designated organs were weighed for F0 and F1 parents as well as brain, spleen, thymus and thyroids of one pup per sex per litter of each generation.
- Histopathology P and F1: see above
- Histopathology F1 not selected for mating, F2: see above

OTHER EXAMINATIONS
After weaning of their respective progeny, F0 and F1 parent males and females were sacrificed. Blood was sampled for determination of hematology parameters in F0 males and F0 females only.
Postmortem examinations (offspring):
See above 'Postmortem examinations (Parental animals)'
Statistics:
Data other than organ weights: Mean values were compared by one-way analysis of variance and Dunnett test (mean values being considered as normally distributed and variances being considered as homogeneous). Percentage values were compared by the Fisher exact probability test.

For statistical methods used for organ weights: see appended table.
Clinical signs:
no effects observed
Body weight and weight changes:
no effects observed
Food consumption and compound intake (if feeding study):
no effects observed
Organ weight findings including organ / body weight ratios:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
effects observed, treatment-related
Other effects:
not examined
Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
no effects observed
Reproductive performance:
effects observed, treatment-related
CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
Description, severity, time of onset and duration of clinical signs: Parents: No effects. However, at 500 mg/kg/day, ptyalism was recorded in 1 male and a few other clinical signs were randomly distributed among the treated groups (1 or 2 males per group). According to females there were no relevant signs of toxicity in any group. In conclusion: All these signs were not considered to be of toxicological significance.
Mortality P: There was no death in any group during the whole study period.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS)
BW Parents: No effects.
Food Parents: No effects.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
-

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
Changes in estrous cycles: The estrous cycle stages evaluated during the last three weeks of the premating period were not affected by the treatment.

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
Effects on sperm in P and F1: The seminology parameters evaluated in both P and F1 males, namely epididymal sperm count, epididymal sperm motility and morphology, testicular sperm head count and daily sperm production were not affected by the treatment at any dose-level.

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
In P and F1:
- Reproductive parameters in females with evidence of mating:
P
Dose mg/kg/day 0 10 70 500
Paired animals 25M+25F 25M+25F 25M+25F 25M+25F
evidence of mating 24(25) 24(25) 22 (24)25
Mating index (%) 96(100) 96(100) 88(96) 100
Pre-coital interval(d) 2.96 2.88 3.09 2.44
Fertility index (%) 96(96) 100(100) 95(96) 96
Females which delivered 23 24 21 24
Gestation index (%) 100 100 100 100
Gestation length (days) 21.5 21.4 21.3 21.4
Post-implantation + 6.5 7.4 7.9 5.8
neo-natal losses (%)
M: male, F: female, (): pregnant females with no evidence of mating are taken into account.

- Reproductive parameters in females with evidence of mating:
F1
Dose mg/kg/day 0 10 70 500
Paired animals 25M+25F 25M+25F 25M+25F 24M+24F
evidence of mating 25 24 22(23) 23(24)
Mating index (%) 100 96 88(92) 96(100)
Pre-coital interval (d) 3.28 3.29 2.23 2.70
Fertility index (%) 92 96 91(91) 96(96)
Pregnant mated females 23 23 20(21) 22(23)
Females which delivered 23 23 20 22
Gestation index (%) 100 100 100 100
Gestation length (days) 21.6 21.6 21.5 21.6
Post-implantation + 3.7 10.7 5.7 9.2
neo-natal losses (%)
M: male, F: female, (): pregnant females with no evidence of mating are taken into account.

- Ovarian primordial follicle counts and growing follicles:
P:
Dose mg/kg/day 0 500
Primordial follicle
Total number 477 755
Mean Value 19.08 30.20
Range 0-44 5-94
Growing Follicles
Total number 66 39
Mean Value 2.64 1.56
Range 0-9 0-6
F1:
Primordial follicle
Total number 457 538
Mean Value 18.28 21.52
Range 7-49 5-47
Growing Follicles
Total number 43 27
Mean Value 1.72 1.08
Range 0-5 0-5

A relationship to the treatment was considered to be unlikely. Considering the great variability described in published literature for such quantitative analysis and when the quantitative parameters were evaluated, as per defined intervals of values it appeared more evident that the differences observed were due, at least in part, to the great inter-individual variability.
ORGAN WEIGHTS (PARENTAL ANIMALS)
Organ weight changes P, F1 and F2:
An increased spleen weight was observed in 500 mg/kg day P and F1 males. It was considered that the differences in spleen weight were most probably secondary to the anaemia noted among the animals and consequently of no toxicological importance. Other differences in organ weights in P, F1 and F2 were not dose-related and/or without the same trend in both sexes. As the differences in thyroid and thymus weight were minimal and not dose-related, they were considered to be of no toxicological importance.

GROSS PATHOLOGY (PARENTAL ANIMALS)
Gross pathology incidence and severity:
The only macroscopic findings observed were those which have been found to occur spontaneously in the untreated laboratory rat of this strain and age. They were therefore considered to be of no toxicological importance.

HISTOPATHOLOGY (PARENTAL ANIMALS)
- Histopathology incidence and severity, P, F1 and F2:
Parents:
At 500 mg/kg/day minimal to moderate follicular thyroidal hyperplasia and moderate hyperactivity of the thyroids in both parent males and females. At 70 mg/kg/day moderate follicular hyperactivity of the thyroids in the parents.

P:
Follicular thyroidal hyperplasia:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 0/25 0/25 0/24 20/24
Grade 1 - - - 0
Grade 2 - - - 18
Grade 3 - - - 2
Mean grading - - - 2.1

Females Incidence 0/25 0/25 0/25 18/25
Grade 1 - - - 7
Grade 2 - - - 8
Grade 3 - - - 3
Mean grading - - - 1.8

Follicular thyroidal hyperactivity:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 25/25 25/25 24/24 24/24
Grade 2 11 3 3 2
Grade 3 12 20 15 15
Grade 4 2 2 6 7
Mean grading 2.6 3.0 3.1 3.2

Females Incidence 24/25 25/25 25/25 25/25
Grade 2 16 8 8 3
Grade 3 8 16 17 16
Grade 4 0 1 0 6
Mean grading 2.3 2.7 2.7 3.1

F1:
Follicular thyroidal hyperplasia:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 0/25 0/25 0/24 21/22
Grade 1 - - - 3
Grade 2 - - - 12
Grade 3 - - - 6
Mean grading - - - 2.1

Females Incidence 0/25 0/25 0/25 20/25
Grade 1 - - - 6
Grade 2 - - - 10
Grade 3 - - - 4
Mean grading - - - 1.9

Follicular thyroidal hyperactivity:
Dose-level (mg/kg/day) 0 10 70 500
Males Incidence 25/25 25/25 24/24 22/22
Grade 1 2 0 0 0
Grade 2 6 4 6 3
Grade 3 17 21 15 16
Grade 4 0 0 3 3
Mean grading 2.6 2.8 2.9 3.0

Females Incidence 25/25 25/25 24/25 24/25
Grade 1 2 1 1 0
Grade 2 12 13 12 5
Grade 3 11 11 11 16
Grade 4 0 0 0 3
Mean grading 2.4 2.4 2.4 2.9

F2:
Dose mg/kg/day Microscopic findings 0 10 70 500
Males Follicular cell hypertrophy 91 97 100 100
1.3 1.3 1.2 1.2
Small follicles 91 97 100 100
1.5 2.1 1.8 2.4
Pale or decreased colloid contents 91 95 100 100
1.2 2.0 1.7 2.3
Cords of epithelial cells 66 84 98 90
1.4 1.5 1.3 1.7
Females Follicular cell hypertrophy 100 98 98 100
1.0 1.0 1.1 1.0
Small follicles 100 98 98 100
1.9 2.0 1.9 2.4
Pale or decreased colloid contents 100 98 98 100
1.8 2.0 1.8 2.4
Cords of epithelial cells 88 95 95 95
1.3 1.7 1.7 1.8

There was an absence of characterized thyroidal hypertrophy at all dose-levels and in both sexes. The cords of epithelial cells with small follicles and decreased or pale colloid contents are morphological characteristics of an infant thyroidal tissue. As they were recorded without evidence of a dose or treatment relationship, it was concluded that the small morphological differences observed between treated and control F2 pups were without relationship to treatment with the test item and correlated with the morphological characteristics of an infant thyroidal tissue.

The only other microscopic findings were those which are commonly encountered in the untreated laboratory rat of this strain and age and were thus considered to be without relationship to treatment with the test item.

OTHER FINDINGS (PARENTAL ANIMALS)
- LACTATION:
Changes in lactation:
P
Dose mg/kg/day 0 10 70 500
lactation index (%) 100.0 98.9 99.4 97.4
CIT Historical Control Data for lactation index on day 21 pp: minimum = 96.4, maximum = 100%
The lactation index was in the range of historical control data.

- HEMATOLOGICAL FINDINGS
Hematological findings incidence and severity: Parents: At 500 mg/kg/day significant decrease in red blood cell count and mean hemoglobin concentration in both parental males and females.
Dose-level (mg/kg/day) 0 10 70 500
Males
RBC (T/L) 8.76 8.73 8.61 8.02**
HB (g/dL) 15.7 15.8 15.3 14.5**
PCV (L/L) 0.45 0.46 0.44 0.43**
MCHC (g/dL) 34.7 34.5 34.7 33.9**
Females
RBC (T/L) 7.62 7.57 7.29* 6.98**
HB (g/dL) 15.6 15.5 15.1* 14.7**
PCV (L/L) 0.45 0.45 0.44 0.43*
MCHC (g/dL) 34.7 34.5 34.5 34.1**
Statistically significant: * p <0.05, ** p <0.01

CIT historical control data:
Males
RBC (T/L) : mean = 8.64, minimum = 7.56, maximum = 9.56
HB (g/dL) : mean = 15.3, minimum = 13.8, maximum = 16.6
PCV (L/L) : mean = 0.45, minimum = 0.40, maximum = 0.49
MCHC (g/dL) : mean = 33.9, minimum = 32.4, maximum = 34.9
Females
RBC (T/L) : mean = 7.60, minimum = 6.53, maximum = 8.33
HB (g/dL) : mean = 14.3, minimum = 13.1, maximum = 15.5
PCV (L/L) : mean = 0.42, minimum = 0.37, maximum = 0.46
MCHC (g/dL) : mean = 34.3, minimum = 32.8, maximum = 35.7
Dose descriptor:
NOAEL
Effect level:
70 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
haematology
histopathology: non-neoplastic
Dose descriptor:
NOEL
Effect level:
500 mg/kg bw/day
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny
Clinical signs:
no effects observed
Mortality / viability:
mortality observed, treatment-related
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
no effects observed
Histopathological findings:
effects observed, treatment-related
VIABILITY (OFFSPRING)
Mortality F1:
. There was no death in the control or the 10 mg/kg/day group during the whole treatment period.

. In the 70 mg/kg bw/day group 2 males died, on day 95 and day 98. In the first male a prematurely sacrificed on day 98 of the treatment period for humane reasons. Post-mortem macroscopic examination, noted a perforation in the cervical region of esophagus with a pouch, and found in adjacent tissue, measuring 2.5 cm in diameter and showing thick whitish contents at cut surface. At microscopic examination, there was a para-esophageal abscess and marked granulation tissue formation including the cervical skeletal muscles. In the second male no signs for cause of death could be determined.

. In the 500 mg/kg bw/day group 3 males died, on day 69, 96 and day 98.
- For the male found dead on day 98 of the treatment period:
At macroscopic post-mortem examination, perforation was noted in the cervical region of esophagus with a pouch, found in adjacent tissue, measuring 2.5 cm in diameter and showing thick whitish contents at cut surface. At microscopic examination, there was a para-esophageal abscess and marked granulation tissue formation including the cervical skeletal muscles. The major factor contributing to the death was esophagus perforation.
- For the male found dead on day 96 of the treatment period:
At macroscopic post-mortem examination, foamy contents were seen in the trachea and bronchi and the lungs and heart were blackish in color. Except for congestion, most probably passive, due to the absence of bleeding, no relevant microscopic finding was noted.
- For the male found dead on day 69 of the treatment period:
At macroscopic post-mortem examination, liquid whitish contents were noted in the thoracic cavity. In addition, moderate cortical cell hypertrophy was noted in the adrenals, associated with moderate lipid depletion. This was most probably related to the poor physical condition of this animal before death. No other relevant microscopic finding was noted.

During the premating period, one female given 500 mg/kg/day was found dead after dosing on day 41, no clinical signs were noted prior to death. At necropsy, foamy contents were seen in the trachea and the lungs which were reddish in color. Apart moderate congestion of the lungs with minimal alveolar edema, no relevant microscopic finding was noted.

There was no other death in any group during the premating, pregnancy or the lactation periods.

From the above clinical history, necropsy and microscopic findings, the above mentioned sporadic deaths were considered to be accidental and without relationship to the treatment with the test item.

Viability index:
P:
Dose-level (mg/kg/day) 0 10 70 500
Viability index (%) 98.1 97.3 98.0 96.6
CIT Historical Control Data for viability index on day 4 pp: minimum = 95.9, maximum = 100%

CLINICAL SIGNS (OFFSPRING)
Description, severity, time of onset and duration of clinical signs: F1: no other effects.

BODY WEIGHT + FOOD CONSUMPTION (OFFSPRING)
BW F1: at 500 mg/kg/day for the males, significantly lower body weight gain (days 1 to 15, -8%, p<0.05)
Food intake F1: at 500 mg/kg/day for the males, significantly lower food consumption (days 1 to 15, -11%, p<0.01) during the first weeks of the premating.

SEXUAL MATURATION (OFFSPRING)
- Offspring toxicity F1 and F2:
F1:
Litter data
Dose-level (mg/kg/day) 0 10 70 500
Pups delivered (mean number/F) 13.6 14.0 14.4 14.7
Sex-ratio (% of males) 51.8 53.7 50.8 53.0
Pup weight at birth (day 1 pp)(g) 6.7 6.6 6.5 6.5
. Day 1 pp 6.7 6.6(-1%) 6.5(-3%) 6.5(-3%)
. Day 4 pp (pre-culling) 9.6 9.5(-1%) 9.1(-5%) 9.1(-5%)
. Day 7 pp 16.3 15.9(-2%) 15.6(-4%) 15.1(-7%)
. Days 14 pp 34.5 34.7(+1%) 34.0(-1%) 33.5(-3%)
. Day 21 pp 53.8 54.9(+2%) 53.0(-1%) 52.0(-3%)
Body weight change (g)
. Days 1-4 pp 2.9 2.9(0%) 2.7(-7%) 2.6(-10%)
. Days 4-21 pp 44.1 45.4(+3%) 43.7(-1%) 42.9(-3%)
(): difference from controls
CIT Historical Control Data for body weight on day 4 pp: mean = 9.7, mini = 9.5, maxi = 10.0
CIT Historical Control Data for body weight on day 7 pp: mean = 14.5, mini = 13.9, maxi = 16.3
CIT Historical Control Data for body weight on day 21pp: mean = 43.3, mini = 40.6, maxi = 52.0

The litter size at birth showed normal fluctuations among treated and control groups, being slightly higher in the treated groups, compared to the control group; this finding was considered to be of fortuitous occurrence. The sex ratio was similar in all groups. Very few deaths within control range, not related to treatment. No macroscopic post-mortem changes. Anouria (absence of tail) was observed in one pup from dam given 10 mg/kg/day or 500 mg/kg/day. Since this finding was low in incidence and not dose-related, it was considered to have occurred by chance.
The other observations (necrosis on hindlimb, cold to the touch and scattered hair) were low in incidence and randomly distributed within the groups, thus, they were considered not to be related to the treatment. There were no effects on reflex development. No effects on auditory function, visual function and no significant effect on spontaneous locomoter activity.

F2:
Litter data
Dose-level (mg/kg/day) 0 10 70 500
Litter size at birth(mean/F) 13.6 13.4 14.9 14.3
Sex-ratio (% of males) 52.7 49.2 50.2 52.2
Pup weight at birth (day 1 pp) (g) 6.6 6.7 6.4 6.7
Body weight (g)
. Day 1 pp 6.6 6.7(+1%) 6.4(-3%) 6.7(+1%)
. Day 4 pp (pre-culling) 9.1 9.3(+2%) 9.3(+2%) 9.0(-1%)
. Day 7 pp 15.4 15.4(0%) 15.4(0%) 14.6(-5%)
. Day 14 pp 33.0 33.2(0%) 33.1(0%) 31.2(-5%)
. Day 21 pp 52.0 51.9(0%) 51.7(-1%) 48.8(-6%)
Body weight change (g)
. Days 1-4 pp 2.5 2.6(+4%) 2.8(+12%) 2.3(-8%)
. Days 4-7 pp 6.1 6.1(0%) 6.3(+3%) 5.6(-8%)
. Days 4-21 pp 42.6 42.6(0%) 42.6(0%) 39.9(-6%)
(): difference from controls
CIT Historical Control Data for body weight on day 4 pp: mean=9.7, mini=9.5, maxi=10.0
CIT Historical Control Data for body weight on day 7 pp: mean=14.5, mini=13.9, maxi=16.3
CIT Historical Control Data for body weight on day 21pp: mean= 43.3, mini=40.6, maxi= 52.0

No effect was recorded on pup survival over the lactation period. The slightly lower viability index recorded in the control group was the consequence of one entirely decedent litter. There were no notable clinical signs among the pups.
The pups body weight and body weight gain at 10 and 70 mg/kg/day were not affected by the treatment during the whole lactation period. At 500 mg/kg/day, the body weight was similar to the control group value while slightly non-significant lower body weight and body weight gain were recorded after one week of birth until the end of the lactation period. These differences were clearly not related to the treatment period since the control value on day 21 post-partum was particularly higher and within the highest body weight values recorded in CIT historical control data. No effects on the sex ratio, reflex development or sperm parameters.

ORGAN WEIGHTS (OFFSPRING)
Organ weight changes P, F1 and F2:
An increased spleen weight was observed in 500 mg/kg day P and F1 males. It was considered that the differences in spleen weight were most probably secondary to the anaemia noted among the animals and consequently of no toxicological importance. Other differences in organ weights in P, F1 and F2 were not dose-related and/or without the same trend in both sexes. As the differences in thyroid and thymus weight were minimal and not dose-related, they were considered to be of no toxicological importance.

GROSS PATHOLOGY (OFFSPRING)
Gross pathology incidence and severity:
The only macroscopic findings observed were those which have been found to occur spontaneously in the untreated laboratory rat of this strain and age. They were therefore considered to be of no toxicological importance.

HISTOPATHOLOGY (OFFSPRING)
Histopathology incidence and severity, P, F1 and F2:
See above (parental animals).

OTHER FINDINGS (OFFSPRING)
Dose descriptor:
NOAEL
Generation:
F1
Effect level:
500 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny
Reproductive effects observed:
not specified
Conclusions:
For parent of the F0 generation, sign of toxicity was substantiated by a decrease in red blood cells and hemoglobin concentration levels at 500 mg/kg/day. For parent of the F1 generation, significantly lower body weight gain and food consumption were observed during the first week of treatment at 500 mg/kg/day. For both parent males and females of both generations given 500 mg/kg/day, signs of slight to moderate follicular hyperplasia and signs of slight to moderate hyperactivity of the thyroid glands were recorded. Signs of hyperactivity were also observed for males of both F0 and F1 generation at 70 mg/kg/day. No effect was observed on mating, fertility, gestation, fecundity or delivery at any dose-level for either generation.
No effect was recorded on litter parameters and on pre and post-natal development of either generation at any dose-level.
Therefore, the No Observed Effect Level (NOEL) for parental toxicity is 10 mg/kg/day for the male and 70 mg/kg/day for the female.
The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny is 500 mg/kg/day.
Executive summary:

The study was designed to investigate the effects of the test material, sodium chlorate, on the growth and reproductive performance of the Sprague-Dawley rat and complies with OECD Guideline 416. A former one-generation dose-range finding study was performed to select dose-levels for this two-generation study. The study followed the principles of GLP. The test material was administered orally, by gavage. In the P generation three groups of 25 male and 25 female rats received the test material 10 weeks before mating and during mating, pregnancy and lactation. In the F1 generation three groups of 25 male and 25 female rats received the test material at weaning, on day 22 post-partum, until weaning of the F2 generation. The dose levels were 10, 70 and 500 mg/kg/day. The control group (25 makes and 25 females in both generations) received vehicle (purified water) alone.


For parent of the F0 generation, sign of toxicity was substantiated by a decrease in red blood cells and hemoglobin concentration levels at 500 mg/kg/day. For parent of the F1 generation, significantly lower body weight gain and food consumption were observed during the first week of treatment at 500 mg/kg/day. For both parent males and females of both generations given 500 mg/kg/day, signs of slight to moderate follicular hyperplasia and signs of slight to moderate hyperactivity of the thyroid glands were recorded. Signs of hyperactivity were also observed for males of both F0 and F1 generation at 70 mg/kg/day.


No effect was observed on mating, fertility, gestation, fecundity or delivery at any dose-level for either generation.


No effect was recorded on litter parameters and on pre and post-natal development of either generation at any dose-level.


Therefore, the No Observed Effect Level (NOEL) for parental toxicity is 10 mg/kg/day for the male and 70 mg/kg/day for the female. Due to an increase in spleen follicular cell hyperplasia and grade 4 follicular hyperactivity of the thyroids in males and females at 500 mg/kg bw/day in F0 and F1 parents, which is a non-adverse effects, the parental NOAEL was 70 mg/kg/day in males and females.


The No Observed Effect Level (NOEL) for mating behaviour, fertility and gestation, of each generation and for development, growth and survival of each progeny is 500 mg/kg/day.

Effect on fertility: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
500 mg/kg bw/day
Study duration:
subchronic
Species:
rat
Effect on fertility: via inhalation route
Endpoint conclusion:
no study available
Effect on fertility: via dermal route
Endpoint conclusion:
no study available

Effects on developmental toxicity

Description of key information

In cases where data for potassium chlorate were not available or to support data that were found for potassium chlorate tests performed with sodium chlorate were used in this dossier. Read across is possible for these two substances, because the toxicity of the test substance is expected to be related to the chlorate ion and not by the sodium or potassium ion.


 


In the rabbit teratology study, the maternal NOAEL was determined at 475 mg/kg bw/day, the highest dose level tested and the developmental NOAEL was established to be higher than 475 mg/kg/day. It was concluded that sodium chlorate was not teratogenic in rabbits (George and Price 2002). These findings are supported by a developmental toxicity study in rats (Schroeder 1987).

Link to relevant study records

Referenceopen allclose all

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
August 23, 1998 - November 9, 1998
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol.
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Method: other: US EPA guidline (1997)
GLP compliance:
yes
Limit test:
no
Species:
rabbit
Strain:
New Zealand White
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Covance Research Products, Inc., Denver, PA
- Age at study initiation: no info
- Weight at study initiation: (P) Females: 3017 g - 3976 g (replicate I), 2747 g - 4061 g (replicate II)
- Fasting period before study: no info
- Housing: individually housed in stainless steel cages with mesh flooring
- Diet (e.g. ad libitum): Purina Certified Rabbit chow. From gestational day (gd) 3: food ad lib, (on gd 1: 65 g and on gd 2: 125 g)
- Water (e.g. ad libitum): tap water, ad lib
- Acclimation period: no info


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 20 ºC (replicate I and II)
- Humidity (%): 46.9 - 62.2% (replicate I), 49.0 - 60.7% (replicate II)
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12:12 light: dark cycle


IN-LIFE DATES: From: August 23, 1998 To: November 9, 1998
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 0, 100, 250 and 475 mg/kg bw in deionized/distilled water
Sodium chlorate was dissolved in deionized/distilled water. Each concentration of sodium chlorate was formulated independently in a quantity sufficient to last the entire dosing period. Dose formulations were stored in sealed plastic botlles at room temperature, and mixed well prior to dosing. During the study, two sets of dose formulations were prepared (one set per replicate).

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable (deionized/distilled water)
- Concentration in vehicle: 33.3, 83.3, 158.3 mg/ml
- Amount of vehicle (if gavage): total volume applied: 3 ml/kg
- Lot/batch no. (if required): not required
- Purity: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Formulations were administered within the period of proven stability (44 days).
- Aliquots were submitted for verification of concentration before each period of use. Measured concentrations were within 98.2%-102.1% of the theoretical concentration. (BLOQ = below the limit of quantitation. Limit of Quantitation = 0.11348 µg/ml).
- Nothing mentioned about homogenicity.
Details on mating procedure:
- Impregnation procedure: purchased timed pregnant (animals were naturally mated at vendor’s facility prior to shipment).
- Verification of same strain and source of both sexes: [yes / no (explain)]
- Proof of pregnancy: no info, however, gd 0 = date of mating and pregnancy status was confirmed by uterine examination.
Duration of treatment / exposure:
gestation day 6 through 29
Frequency of treatment:
daily dose (1 oral dose per day. From day 6 through day 29 of gestation).
Duration of test:
Necropsy on gestation day 30.
Remarks:
Doses / Concentrations:
100, 250 and 475 mg/kg bw d
Basis:
nominal conc.
No. of animals per sex per dose:
96 females, 24 per group, 12 per replicate
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: day 6 through day 30 of gestation
- Dose selection rationale: Doses were selected on the basis of a screening study in which New Zealand White Rabbits (8/group) were given 100, 250, 500, 750 or 1000 mg/kg bw via gavage from gd 6-29. At the two highest dose morbidity and mortality was seen (and these groups were terminated at gd 24). In the 500 mg/kg group one female died on gd 26 and lethargy and respiratory distress were seen in the other animals on gd 28-29. Clinical signs were minimal at dose <250 mg/kg d. Above 250 mg/kg d significant reduction of food uptake was seen in the exposed animals. Until the 500 mg/kg d dose overal weight changes during treatment and during gestation (corrected for gravid uterine weights) were not affected. There were no statistically significant differences among groups for gravid uterine weight, absolute or relative maternal liver weight. Pregnancy rates were 83-100% per group. A slight (not significant) decrease in number of corpora lutea, implantation sites and live fetuses per litter were seen >500 mg/kg d. Above 500 mg/kg d there was no definitive evidence of developmental toxicity. Therefore 475 mg/kg bw was chosen as the highest dose in the main study.
- Rationale for animal assignment (if not random): no info
Maternal examinations:
CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Females were observed for clinical condition at least once each day from 1 or 2 (date of animal arrival) through day 5 (prior to dosing). From day 6 through 29, females were observed for clinical condition and signs of toxicity at dosing, and approximately 1-2 hours after each dose. On day 30, females were observed for clinical condition at weighing and at scheduled termination.


DETAILED CLINICAL OBSERVATIONS: No data


BODY WEIGHT: Yes
- Time schedule for examinations: Bodyweight were recorded on day 0 (vendor), 3, 6 through 30 and after sacrifice on day 30.


FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption for each animal determined and mean daily diet consumption calculated as g food/day: Yes
- Time schedule: Feed consumption was monitored during the study, with measurements on the mornings of day 3, 6, 9, 12, 15, 18, 21, 24, 27, 29, and 30.


WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No data


POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 30
- Organs examined: The body, liver, and gravid uterus of each female was weighed. Thoracic and abdominal cavities were examined. Sections of all livers, as well as any maternal organs which displayed gross pathology, were saved. Ovarian corpora lutea were counted. Pregnancy status was confirmed by uterine examination. Uterine contents were examined to determine the number of implantation sites, resorptions, dead fetuses, and live fetuses. Dead fetuses were counted, weighed, and discarded. Uteri which presented no visible implantation sites were stained in order to visualize any implantation sites which might have undergone very early resorption.


OTHER: At termination blood samples were collected from 2 females per group per replicate as well as from all animals killed before the end of the study. This blood was tested for pathogens.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
Fetuses: All live fetuses were counted, weighed, and examined for external morphological abnormalities, including cleft palate. All fetal carcasses were sexed and examined for visceral morphological abnormalities. Approximately 50% of the fetal carcasses were decapitated prior to dissection.
All fetal skeletons were examined for skeletal morphological abnormalities.

- External examinations: Yes: all per litter
- Soft tissue examinations: Yes: all per litter
- Skeletal examinations: Yes: all per litter
- Head examinations: Yes: half per litter
Statistics:
Unit for statistical measurement: pregnant female or the litter. Quantitative continuous data (e.g., maternal/fetal body weights, etc.) were evaluated for homogeneity of variance using Bartlett's test (Winer, 1962). (Reported when Bartlett's test indicated lack of homogeneity (p<0.001)).

Parametric statistical procedures were applied to selected measures from this developmental study. GLM procedures were applied to the ANOVA and the Tests for Linear Trend. Prior to GLM analysis, an arcsine-square root transformation was performed on all litter derived percentage data. For litter-derived perc. data, the ANOVA was weighted according to litter size. When a significant (p<0.05) main effect for dose or replicate occurred, Dunnett's Multiple Comparison Test (Dunnett,1955;1964) was used to compare each treatment group to the control group for that measure. A one-tailed test (Dunnett's Test) was used for all pairwise comparisons to the control group, except that a two-tailed test was used for maternal body and organ weight parameters, maternal feed consumption, fetal body weight, and percent males per litter. Data for any measure which showed a significant (p<0.05) dose X replicate interaction in a two-way (dose X replicate) ANOVA was presented as Mean±SEM for each cell in the ANOVAdesign. Dose effects within each replicate were further evaluated using a one-way ANOVA, Test for Linear Trend, and Dunnett's Test. Nominal scale measures were analyzed by Chi-Square Test for Independence for differences among treatment groups and by the Cochran-Armitage Test for Linear Trend on Proportions. When Chi-Square revealed significant (p<0.05) differences among groups, a one-tailed Fisher's Exact Probability Test (with adjustments for multiple comparisons) was used for pairwise comparisons between each treatment group and control group. Alpha level for each statist. comparison was 0.05, and significance levels for trend tests and pairwise comparisons were reported as p<0.05 or p<0.01.
Indices:
Particular focus on embryo/fetal growth, viability, and morphological development after implantation and prior to birth.
Historical control data:
Included in the report.
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Parental data and F1:
- Body weight: No significant treatment related effect was observed.
- Food/water consumption: No significant treatment related effect was observed on food consumption.
- Description, severity, time of onset and duration of clinical signs: urinary changes (orange or brown urine; little or no urine). The incidence of females exhibiting these signs was dose-related (i.e., 0, 0, 2 and 8 for color changes; 0, 1, 6 and 8 for reduced urine output). Urinary changes in individual females (one or both signs) were noted on one to seven days, but never for more than four consecutive days. Only two females (in the mid- and high dose group) showed both signs.
- Fertility index: -
- Precoital interval: -
- Duration of gestation: terminated at day 30.
- Gestation index: At termination of the study the pregnancy rates ranged from 90-100%.
- Changes in lactation: -
- Changes in estrus cycles: -
- Effects on sperm: -
- Hematological findings incidence and severity:
- Clinical biochemistry findings incidence and severity:
- Mortality: 1 animals died in each treatment group.
- Gross pathology incidence and severity:
Parents: Necropsy findings included primarily changes in lung appearance, which may have been secondary to gavage administration of the sodium chlorate. Gross necropsy findings did not occur in a dose-related manner.
- Number of implantations:
Dose 0 100 250 475
Sites 8.11+-/0.35 8.88+/-0.45 8.17+/-0.51 7.80+/-0.50
per litter
Not affected by treatment with sodium chlorate.
- Number of corpora lutea:
Dose 0 100 250 475
per dam 9.16+/-0.30 9.41+/-0.44 9.22+/-0.38 9.00+/-0.52
Not affected by treatment with sodium chlorate.
- Ovarian primordial follicle counts: -
- Organ weight changes: Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were similar among exposed and control animals.
- Histopathology incidence and severity: -
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.
Dose descriptor:
NOAEL
Effect level:
>= 475 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Offspring toxicity F1 and F2:
- Litter size and weights: Not affected by treatment with sodium chlorate.
- Sex and sex ratios: -
- Viability index: Not affected by treatment with sodium chlorate.
- Post natal survival until weaning: -
- Effects on offspring: -
- Postnatal growth, growth rate: -
- Vaginal opening (F) or preputial separation (M): -
- Gross pathology incidence and severity:
Fetuses: There were no effects of treatment with sodium chlorate on the incidence of external, visceral, or skeletal malformations at any dose.
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.
Dose descriptor:
NOAEL
Effect level:
>= 475 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In summary, transient changes in maternal food intake, urinary color and/or output were noted at 100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998). Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were 475 mg/kg/day.
Executive summary:

The purpose of this study was to assess the effects of sodium chlorate on the pregnant female and on embryonic and fetal development when administered orally by gavage once daily to mated female New Zealand White rabbits from gestational days (gd) 6 through 29. Each group consisted of 24 mated female rabbits (12 per replicate). Sodium chlorate was administered once daily at dose levels of 0, 100, 250 and 475 mg/kg body weight/day. A standard dose volume of 3 mL/kg body weight was used. Control animals were dosed with the vehicle alone (deionized/distilled water). All surviving females were sacrificed on gestational day 30. Examination of dams and fetuses was performed in accordance with international recommendations. The study complies with OECD Guideline 414. Dose selection was based on a screening study in which New Zealand White rabbits were treated by gavage with 0, 100, 250, 500, 750, or 1000 mg sodium chlorate/kg body weight/day on gd 6 through 29. The study followed the principles of GLP.


 


Maternal toxicity:


Confirmed pregnancy rates were 90-100% per group. One maternal death occurred in each dose group. Clinical signs associated with sodium chlorate exposure included urinary changes (orange or brown urine; and/or little or no urine) on one or more days of treatment. The number of animals affected showed dose-response patterns across the control through high dose groups (i.e., 0, 0, 2, and 8 for color changes and 0, 1, 6 and 8 for urine output), respectively. Nevertheless, these changes in individual animals were transient and not clearly indicative of toxicity.


No significant treatment-related effects were observed for maternal body weight, body weight gain, or corrected weight gain. Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were equivalent among groups.


Relative maternal food intake (g/kg/day) exhibited an increasing trend prior to initiation of treatment, but was reduced to 82-83% of control intake in the mid and high dose groups during early treatment (gd 6 to 9). Thereafter, no significant differences were noted among treatment groups for maternal relative feed consumption, suggesting that the decrease on gd 6 to 9 was related to initiation of treatment. A decreasing dose-related trend for the periods of gd 15 to 18 and gd 18 to 21 was observed, although no significant differences between the control group and treated groups were observed for these periods. These effects were not noted later in the treatment period.


 


Fetal toxicity:


Sodium chlorate exposure did not significantly affect any endpoints related to prenatal viability. Average live litter size in sodium chlorate-treated groups was between 100-112% of the control mean, with no statistically significant difference among groups. There were no treatment-related effects on fetal body weight. There were no effects of treatment on the incidence of external, visceral, or skeletal malformations. In summary, transient changes in maternal food intake, urinary color and/or output were noted at ³100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998).


 


Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were ³475 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the RAAF document.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Details on maternal toxic effects:
Maternal toxic effects:no effects

Details on maternal toxic effects:
- Parental data and F1:
- Body weight: No significant treatment related effect was observed.
- Food/water consumption: No significant treatment related effect was observed on food consumption.
- Description, severity, time of onset and duration of clinical signs: urinary changes (orange or brown urine; little or no urine). The incidence of females exhibiting these signs was dose-related (i.e., 0, 0, 2 and 8 for color changes; 0, 1, 6 and 8 for reduced urine output). Urinary changes in individual females (one or both signs) were noted on one to seven days, but never for more than four consecutive days. Only two females (in the mid- and high dose group) showed both signs.
- Fertility index: -
- Precoital interval: -
- Duration of gestation: terminated at day 30.
- Gestation index: At termination of the study the pregnancy rates ranged from 90-100%.
- Changes in lactation: -
- Changes in estrus cycles: -
- Effects on sperm: -
- Hematological findings incidence and severity:
- Clinical biochemistry findings incidence and severity:
- Mortality: 1 animals died in each treatment group.
- Gross pathology incidence and severity:
Parents: Necropsy findings included primarily changes in lung appearance, which may have been secondary to gavage administration of the sodium chlorate. Gross necropsy findings did not occur in a dose-related manner.
- Number of implantations:
Dose 0 100 250 475
Sites 8.11+-/0.35 8.88+/-0.45 8.17+/-0.51 7.80+/-0.50
per litter
Not affected by treatment with sodium chlorate.
- Number of corpora lutea:
Dose 0 100 250 475
per dam 9.16+/-0.30 9.41+/-0.44 9.22+/-0.38 9.00+/-0.52
Not affected by treatment with sodium chlorate.
- Ovarian primordial follicle counts: -
- Organ weight changes: Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were similar among exposed and control animals.
- Histopathology incidence and severity: -
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.
Dose descriptor:
NOAEL
Effect level:
>= 475 mg/kg bw/day
Based on:
test mat.
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
- Offspring toxicity F1 and F2:
- Litter size and weights: Not affected by treatment with sodium chlorate.
- Sex and sex ratios: -
- Viability index: Not affected by treatment with sodium chlorate.
- Post natal survival until weaning: -
- Effects on offspring: -
- Postnatal growth, growth rate: -
- Vaginal opening (F) or preputial separation (M): -
- Gross pathology incidence and severity:
Fetuses: There were no effects of treatment with sodium chlorate on the incidence of external, visceral, or skeletal malformations at any dose.
- Other observations: blood samples were positive for ROTA and negative for all other test. This is normal for the specified vendor.
Dose descriptor:
NOAEL
Effect level:
>= 475 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: developmental toxicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
In summary, transient changes in maternal food intake, urinary color and/or output were noted at 100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998). Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were 475 mg/kg/day.
Executive summary:

The purpose of this study was to assess the effects of sodium chlorate on the pregnant female and on embryonic and fetal development when administered orally by gavage once daily to mated female New Zealand White rabbits from gestational days (gd) 6 through 29. Each group consisted of 24 mated female rabbits (12 per replicate). Sodium chlorate was administered once daily at dose levels of 0, 100, 250 and 475 mg/kg body weight/day. A standard dose volume of 3 mL/kg body weight was used. Control animals were dosed with the vehicle alone (deionized/distilled water). All surviving females were sacrificed on gestational day 30. Examination of dams and fetuses was performed in accordance with international recommendations. The study complies with OECD Guideline 414. Dose selection was based on a screening study in which New Zealand White rabbits were treated by gavage with 0, 100, 250, 500, 750, or 1000 mg sodium chlorate/kg body weight/day on gd 6 through 29. The study followed the principles of GLP.


 


Maternal toxicity:


Confirmed pregnancy rates were 90-100% per group. One maternal death occurred in each dose group. Clinical signs associated with sodium chlorate exposure included urinary changes (orange or brown urine; and/or little or no urine) on one or more days of treatment. The number of animals affected showed dose-response patterns across the control through high dose groups (i.e., 0, 0, 2, and 8 for color changes and 0, 1, 6 and 8 for urine output), respectively. Nevertheless, these changes in individual animals were transient and not clearly indicative of toxicity.


No significant treatment-related effects were observed for maternal body weight, body weight gain, or corrected weight gain. Maternal liver weight (absolute and relative to body weight) and gravid uterine weight were equivalent among groups.


Relative maternal food intake (g/kg/day) exhibited an increasing trend prior to initiation of treatment, but was reduced to 82-83% of control intake in the mid and high dose groups during early treatment (gd 6 to 9). Thereafter, no significant differences were noted among treatment groups for maternal relative feed consumption, suggesting that the decrease on gd 6 to 9 was related to initiation of treatment. A decreasing dose-related trend for the periods of gd 15 to 18 and gd 18 to 21 was observed, although no significant differences between the control group and treated groups were observed for these periods. These effects were not noted later in the treatment period.


 


Fetal toxicity:


Sodium chlorate exposure did not significantly affect any endpoints related to prenatal viability. Average live litter size in sodium chlorate-treated groups was between 100-112% of the control mean, with no statistically significant difference among groups. There were no treatment-related effects on fetal body weight. There were no effects of treatment on the incidence of external, visceral, or skeletal malformations. In summary, transient changes in maternal food intake, urinary color and/or output were noted at ³100 mg/kg/day in this study, but clear evidence of maternal toxicity was observed only at doses greater than 475 mg/kg/day in the screening study (NTP, 1998).


 


Sodium chlorate did not cause any significant treatment-related developmental toxicity under the conditions of this study. Thus, the maternal and developmental toxicity NOAELs were ³475 mg/kg/day.

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
January 1987 - March 1987
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study performed under GLP and according to standard protocol
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Principles of method if other than guideline:
Method: other: US EPA Assessment guidelines (1982)
GLP compliance:
yes
Species:
rat
Strain:
Sprague-Dawley
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Charles River Breeding Laboratories, Inc. Portage, Michigan 49081
- Age at study initiation: 9 weeks (age at initiation of mating) (age at receipt: 7 weeks, males + females)
- Weight at study initiation: 174 - 247 g (weight of mated females used on test, Day 0)
- Fasting period before study: no info
- Housing: individually, except during the first week of the acclimation period (two females/cage) and mating (one male and one female per cage) in stainless steel suspended cages with wire mesh floors.
- Diet (e.g. ad libitum): Purina Certified Rodent Chow, ad lib
- Water (e.g. ad libitum): tap water, ad lib
- Acclimation period: 2 weeks: 27 January - 9 February 1987 (females)


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 18 - 24ºC
- Humidity (%): 33 - 67%
- Air changes (per hr): no info
- Photoperiod (hrs dark / hrs light): 12 hour light/dark cycle (7 am to 7 pm)


IN-LIFE DATES: From: January 27, 1987 To: March 24, 1987
Route of administration:
oral: gavage
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: 0, 10, 100 and 1000 mg/kg/d

VEHICLE
- Justification for use and choice of vehicle (if other than water): not applicable; distilled water
- Concentration in vehicle: 0, 2, 20 and 200 mg/ml
- Amount of vehicle (if gavage): 5 ml/kg/d
- Lot/batch no. (if required): not required
- Purity: not applicable
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
- Homogeniety Analyses: Homogeneity of dosing solutions was determined prior to the initiation of the study. Mock batches o f dosing solutions at the low- and high-dose levels were made in a volume equivalent to a weekly mixture and six samples were taken at each dose level from the storage beaker. Two samples were taken from each level of the beaker (top, middle and bottom portions).
- Stability Analyses: Stability of dosing solutions was determined prior to the initiation of the study. The stability of dosing solutions were determined at Days 1, 4 and 8 post-preparation. Analyses were conducted in duplicate at the low- and high-dose levels.
- Analyses o f Dosing Solutions: Samples (approximately 20 mls) were taken at each dose level at each preparation interval during the study. Samples were analyzed to verify that concentrations were within an acceptable range from nominal (+/ - 15% of nominal).
Details on mating procedure:
- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Females were placed with one male overnight (conducted on 13 nights). Early in the morning vaginal smears were taken and females were considered to have mated if sperm and/or a vaginal plug was observed. The day on which this was observed was defined as day 0 of gestation..
Duration of treatment / exposure:
day 6-15 of gestation
Frequency of treatment:
each day a single dose
Duration of test:
from day 6 through day 20 of gestation
Remarks:
Doses / Concentrations:
10, 100 and 1,000 mg/kg d
Basis:

No. of animals per sex per dose:
96 females, 24 per group
Control animals:
yes, concurrent vehicle
Details on study design:
Sex: female
Duration of test: from day 6 through day 20 of gestation
Maternal examinations:
CAGE SIDE OBSERVATIONS / DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Clinical observations: Animals were observed twice daily. On day 0, 6, 10, 15 and 20 a detailed physical examination was performed.
- Cage side observations: signs of pharmacologic or toxicologic effects and mortality.

BODY WEIGHT: Yes
- Time schedule for examinations: Body weights were recorded on day 0, 6, 10, 12, 15 and 20.

FOOD CONSUMPTION AND COMPOUND INTAKE (if feeding study): Yes
- Food consumption was recorded for the following intervals: day 0-6, 6-10, 10-15 and 15-20.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): No

POST-MORTEM EXAMINATIONS: Yes
- On all mated females a complete gross postmortem examination was performed. External surfaces, all orifices, the cranial cavity, carcass, the external surface of the spinal cord and sectioned surfaces of the brain, nasal cavity and paranasal sinuses, the thoracic, abdonminal and pelvic cavities and their viscera and the cervical tissues and organs were examined for all animals. The intact uterus was removed and the following was recorded: weight, live fetuses, dead fetuses (no evidence of tissue degeneration), late resorptions (recognizable dead fetus undergoing degeneration regardless of size), early resorptions (evidence of implantation but no recognizable fetus), implantation sites. Ovaries were investigated for corpora lutea. The uterus was stained when no impants were grossly apparent. When staining revealed nothing the animal was considered not pregnant.
Ovaries and uterine content:
The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Gravid uterus weight: Yes
- Number of corpora lutea: Yes
- Number of implantations: Yes
- Number of early resorptions: Yes
- Number of late resorptions: Yes
Fetal examinations:
- External examinations: Yes: all per litter
- All fetuses were grossly examinated for external malformations/variations including observation for palatel defects. Each fetus was weighed and the sex was noted. Fetuses with external malformations were saved. Abou 1/2 of the fetuses were evaluated for soft tissue malformations. The other half was examined for skeletal malformations.
Statistics:
data were analyzed between control and treated groups.

METHOD A:
Statistical evaluation of equality of means was made by the appropriate one way analysis of variance technique, followed by a multiple comparison procedure if needed. First, Bartlett' s test was performed to determine if groups had equal variance. If the variances were equal, parametric procedures were used; if not, nonparametric procedures were used. The parametric procedures were the standard one way ANOVA using the F distribution to assess significance. If significant differences among the means were indicated, Dunnett's test was used to determine which means were significantly different from the control. If a nonparametric procedure for testing equality of means was needed, the Kruskal -Wallis test was used, and if differences were indicated a summed rank test (Dunn) was used to determine which treatments differed from control. A statistical test for trend in the dose levels was also performed. In the parametric case (i .e., equal variance) standard regression techniques with a test for trend and lack off it were used. In the nonparametric case, Jonckheere's test for monotonic trend was used. The test for equal variance (Bartlett' s) was conducted at the 1%, two-sided risklevel. All other statisticaltests were conducted at the 5% and 1%, twosided risklevel. All ratios were transformed via the arcsine transformation prior to analysis. Data is presented untransformed.
METHOD B:
See below at 'Any other information'
Indices:
Maternal and fetal parameters.
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Parental data and F1:
- Body weight: There was no adverse effect of treatment on body weight.
- Food/water consumption: No adverse effect of treatment was evident from food consumption data during gestation.
- Description, severity, time of onset and duration of clinical signs: No adverse effect of treatment was evident from physical observation data.
- Fertility index: -
- Precoital interval: -
- Duration of gestation: Terminated at day 20.
- Gestation index: Pregnancy rates were 100% in the control and high-dose group, 91.7% in the low-dose group and 95.8% in the mid-dose group.
- Changes in lactation: -
- Changes in estrus cycles: -
- Effects on sperm: -
- Hematological findings incidence and severity: -
- Clinical biochemistry findings incidence and severity: -
- Mortality: 1 female in the control group died on day 9 due to a dosing related injury.
- Gross pathology incidence and severity:
Parents: A slightly higher incidence of discolored lungs was present in 4 animals in the 1000 mg/kg d groups; either red, tan or brown areas were seen. The toxicological significance of these findings in a few animals remains equivocal on the basis of a gross postmortem examination only. Other post-mortem findings observed occurred sporadically and were not considered to be related to the test article.
- Number of implantations: The mean numbers of implantations per pregnant female were comparable between the control and treated groups.
- Number of corpora lutea: The mean numbers of corpora lutea per pregnant female were comparable between the control and treated groups.
- Ovarian primordial follicle counts: -
- Organ weight changes: No difference between control and treated group gravid uterine weights.
- Histopathology incidence and severity: -
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Offspring toxicity F1 and F2: -
- Gross pathology incidence and severity:
Fetuses: The incidences of external malformations, skeletal malformations and ossification variation both on a per fetus and per litter basis, were comparable between the control and treated groups. No adverse effect of treatment was evident from external variation data. Visceral malformations did not differ significantly between control and treated groups. The most seen defects wer ocular.
- Litter size and weights: No adverse effect of treatment was evident from fetal weight data. The results were comparable between the exposed and the control group.
- Sex and sex ratios: No adverse effect of treatment was evident from fetal sex distribution data. The results were comparable between the exposed and the control group.
- Viability index: -
- Post natal survival until weaning: -
- Effects on offspring: -
- Postnatal growth, growth rate: -
- Vaginal opening (F) or preputial separation (M): -
- Other observations: The mean numbers of resorptions per pregnant female were comparable between the control and treated groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
A dose levels of sodium chlorate up to 1000 mg/kg bw/day did not induce any maternal toxicity, embryotoxicity or fetotoxicity.
Executive summary:

This study was conducted to evaluate the embryotoxic, fetotoxic and teratogenic potential of Sodium Chlorate in the pregnant rat. The study is performed according to GLP standards.


Test material was dissolved in distilled water and administered by gastric intubation during the Day 6-15 gestation interval; dosing solutions were prepared fresh weekly. Dose levels were 10, 100 and 1000 mg/kg/day. Each study group was comprised of 24 mated female CDâ rats. Included in the study was a control group comprised of 24 mated female rats that received distilled water.


Study animals were observed twice daily for mortality/morbidity and for obvious pharmacologic and/or toxicologic effects. All females were weighed and given detailed in-life physical evaluations at regular intervals during gestation (i.e., Days 0, 6, 10, 12, 15 and 20). Similarly, food consumption data were recorded for each female for Days 0-6, 6-10, 10-15 and 15-20 of gestation. All females were sacrificed on Day 20 of gestation and given a gross postmortem evaluation. The uterus of each female was removed at sacrifice, weighed and evaluated for the number of fetuses and resorption sites. The ovaries were also dissected free and the number of corpora lutea recorded. Fetuses recovered at this time were evaluated for external malformations/variations, sexed and weighed. Subsequently, one-half of the fetuses in each litter were processed for visceral evaluation (microdissection procedure) and the remaining fetuses from each litter were processed for staining of the ossified structures with Alizarin Red S and evaluated for skeletal malformations and/or ossification variations.


No mortality occurred in the treated groups; all females survived to scheduled sacrifice. In the control group, one female died on Day 9 of gestation. Pregnancy rates were 100% in the control and high-dose group, 91.7% in the low-dose group and 95.8% in the mid-dose group.


No adverse effects of treatment at the dose levels evaluated were evident from maternal parameters (body weight and weight change data, food consumption data, physical observations, uterine implantation data, or gross post-mortem observations) or fetal parameters (body weights and sex distribution).


Additionally, no adverse effects of treatment were evident from the external, visceral or skeletal evaluations performed on fetuses recovered from treated females.


 


In conclusion, a dose levels of sodium chlorate up to 1000 mg/kg bw/day did not induce any maternal toxicity, embryotoxicity or fetotoxicity.

Endpoint:
developmental toxicity
Type of information:
read-across from supporting substance (structural analogue or surrogate)
Adequacy of study:
key study
Justification for type of information:
See the RAAF document.
Reason / purpose for cross-reference:
read-across: supporting information
Reason / purpose for cross-reference:
read-across source
Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Parental data and F1:
- Body weight: There was no adverse effect of treatment on body weight.
- Food/water consumption: No adverse effect of treatment was evident from food consumption data during gestation.
- Description, severity, time of onset and duration of clinical signs: No adverse effect of treatment was evident from physical observation data.
- Fertility index: -
- Precoital interval: -
- Duration of gestation: Terminated at day 20.
- Gestation index: Pregnancy rates were 100% in the control and high-dose group, 91.7% in the low-dose group and 95.8% in the mid-dose group.
- Changes in lactation: -
- Changes in estrus cycles: -
- Effects on sperm: -
- Hematological findings incidence and severity: -
- Clinical biochemistry findings incidence and severity: -
- Mortality: 1 female in the control group died on day 9 due to a dosing related injury.
- Gross pathology incidence and severity:
Parents: A slightly higher incidence of discolored lungs was present in 4 animals in the 1000 mg/kg d groups; either red, tan or brown areas were seen. The toxicological significance of these findings in a few animals remains equivocal on the basis of a gross postmortem examination only. Other post-mortem findings observed occurred sporadically and were not considered to be related to the test article.
- Number of implantations: The mean numbers of implantations per pregnant female were comparable between the control and treated groups.
- Number of corpora lutea: The mean numbers of corpora lutea per pregnant female were comparable between the control and treated groups.
- Ovarian primordial follicle counts: -
- Organ weight changes: No difference between control and treated group gravid uterine weights.
- Histopathology incidence and severity: -
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: maternal toxicity
Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Offspring toxicity F1 and F2: -
- Gross pathology incidence and severity:
Fetuses: The incidences of external malformations, skeletal malformations and ossification variation both on a per fetus and per litter basis, were comparable between the control and treated groups. No adverse effect of treatment was evident from external variation data. Visceral malformations did not differ significantly between control and treated groups. The most seen defects wer ocular.
- Litter size and weights: No adverse effect of treatment was evident from fetal weight data. The results were comparable between the exposed and the control group.
- Sex and sex ratios: No adverse effect of treatment was evident from fetal sex distribution data. The results were comparable between the exposed and the control group.
- Viability index: -
- Post natal survival until weaning: -
- Effects on offspring: -
- Postnatal growth, growth rate: -
- Vaginal opening (F) or preputial separation (M): -
- Other observations: The mean numbers of resorptions per pregnant female were comparable between the control and treated groups.
Dose descriptor:
NOAEL
Effect level:
1 000 mg/kg bw/day
Basis for effect level:
other: teratogenicity
Abnormalities:
not specified
Developmental effects observed:
not specified
Conclusions:
A dose levels of sodium chlorate up to 1000 mg/kg bw/day did not induce any maternal toxicity, embryotoxicity or fetotoxicity.
Executive summary:

This study was conducted to evaluate the embryotoxic, fetotoxic and teratogenic potential of Sodium Chlorate in the pregnant rat. The study is performed according to GLP standards.


Test material was dissolved in distilled water and administered by gastric intubation during the Day 6-15 gestation interval; dosing solutions were prepared fresh weekly. Dose levels were 10, 100 and 1000 mg/kg/day. Each study group was comprised of 24 mated female CDâ rats. Included in the study was a control group comprised of 24 mated female rats that received distilled water.


Study animals were observed twice daily for mortality/morbidity and for obvious pharmacologic and/or toxicologic effects. All females were weighed and given detailed in-life physical evaluations at regular intervals during gestation (i.e., Days 0, 6, 10, 12, 15 and 20). Similarly, food consumption data were recorded for each female for Days 0-6, 6-10, 10-15 and 15-20 of gestation. All females were sacrificed on Day 20 of gestation and given a gross postmortem evaluation. The uterus of each female was removed at sacrifice, weighed and evaluated for the number of fetuses and resorption sites. The ovaries were also dissected free and the number of corpora lutea recorded. Fetuses recovered at this time were evaluated for external malformations/variations, sexed and weighed. Subsequently, one-half of the fetuses in each litter were processed for visceral evaluation (microdissection procedure) and the remaining fetuses from each litter were processed for staining of the ossified structures with Alizarin Red S and evaluated for skeletal malformations and/or ossification variations.


No mortality occurred in the treated groups; all females survived to scheduled sacrifice. In the control group, one female died on Day 9 of gestation. Pregnancy rates were 100% in the control and high-dose group, 91.7% in the low-dose group and 95.8% in the mid-dose group.


No adverse effects of treatment at the dose levels evaluated were evident from maternal parameters (body weight and weight change data, food consumption data, physical observations, uterine implantation data, or gross post-mortem observations) or fetal parameters (body weights and sex distribution).


Additionally, no adverse effects of treatment were evident from the external, visceral or skeletal evaluations performed on fetuses recovered from treated females.


 


In conclusion, a dose levels of sodium chlorate up to 1000 mg/kg bw/day did not induce any maternal toxicity, embryotoxicity or fetotoxicity.

Effect on developmental toxicity: via oral route
Endpoint conclusion:
no adverse effect observed
Dose descriptor:
NOAEL
475 mg/kg bw/day
Study duration:
subacute
Species:
rabbit
Effect on developmental toxicity: via inhalation route
Endpoint conclusion:
no study available
Effect on developmental toxicity: via dermal route
Endpoint conclusion:
no study available

Justification for classification or non-classification

From existing studies there is no indication for reproductive toxicity of sodium chlorate therefore potassium chlorate is not classified (cross reading sodium chlorate).

Additional information