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Toxicological information

Toxicity to reproduction

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Administrative data

Endpoint:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Considering the metabolism pathway of chlorine dioxyde which is likely to undergo rapid redox reactions within biological tissues rather than to be absorbed as parent compound, this study can be used for the assessment of ClO2.
Cross-reference
Reason / purpose for cross-reference:
reference to same study
Reference
Endpoint:
neurotoxicity: oral
Remarks:
two-generation reproductive toxicity
Type of information:
experimental study
Adequacy of study:
key study
Study period:
No data
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
comparable to guideline study with acceptable restrictions
Justification for type of information:
Considering the metabolism pathway of chlorine dioxyde which is likely to undergo rapid redox reactions within biological tissues rather than to be absorbed as parent compound, this study can be used for the assessment of ClO2.
Reason / purpose for cross-reference:
reference to same study
Reason / purpose for cross-reference:
reference to other study
Qualifier:
equivalent or similar to guideline
Guideline:
other: EPA Guideline OPPTS 870.3800 (similar to OECD 416)
Deviations:
no
Principles of method if other than guideline:
The first 20 litters in each group (F1 and F2 generation) comprising at least 7 pups after culling on day 4 were selected to have pups assessed for the neurotoxicological evaluations. Acoustic startle habituation was also conducted for the F2b generation.
GLP compliance:
yes
Remarks:
USEPA, United Kingdom and European Commission GLP standards and principles
Limit test:
no
Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Belgium.
- Age at study initiation: approximately 6 weeks old at the start of F0 prebreed exposure period.
- Weight at study initiation: (P) Males: 80-99 g; Females: 60-79 g
- Housing: - during mating: one male was housed with each female
- during pregnancy: each female was housed individually
- during period of lactation: each dam was housed with its litter, in solid-bottomed polypropylene cages with sawdust bedding.
- Diet: pelleted SQC Rat and Mouse No. 3 expanded diet, Special Diets Services Limited, Witham, Essex (UK) ad libitum.
- Water: purified water (with or without chemical) provided from polycarbonates bottles fitted with stainless-steel tops and sipper tubes ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: appropriate
- Humidity: appropriate
- Air changes: no data
- Photoperiod: appropriate
Route of administration:
other: parents exposed via drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: direct addition of the test material to purified water (UHP). Treated drinking wter were made fresh each week.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity, stability and concentration of sodium chlorite in the drinking water solutions were evalued analytically. The results of these analysis confirmed that the treated drinking water solutions were homogeneous and stable under the conditions of use. The concentration of sodium chlorite in the drinking water was adjusted to account fot the purity of the test substance.
Duration of treatment / exposure:
Pups received the test article during gestation and lactation only (until day 21 post-partum). On day 21 the pups were offered purified water which was not formulated with test article. None of the pups selected in any group for neurotoxicological assessments received test article after weaning.
Frequency of treatment:
Continuously via mother
Remarks:
Doses / Concentrations:
0, 35, 70 and 300 ppm
Basis:
nominal in water
No. of animals per sex per dose:
10 animals/sex/dose/assessment (motor activity/swim maze/neuropathology).
20 animals/sex/dose/asessment (functional operation battery of tests/acoustic startle habituation).
Control animals:
yes, concurrent vehicle
Details on study design:
Acoustic startle habituation was also conducted for the F2b generation because data generated previously in the study for acoustic startle were deemed to be unreliable due to equipment malfunction.
Whenever possible, an equal number of rats of each sex from each treatment group was evaluated each day to limit bias resulting from possible experimental variation. All observations were conducted without knowledge of treatment group by the testing facility personnel who performed the observations and/or recorded the data.
Observations and clinical examinations performed and frequency:
General observations ie. bodyweights, clinical observations and pre-weaning observations were as for the unselected F1 and F2 generation animals.
Specific biochemical examinations:
Not done
Neurobehavioural examinations performed and frequency:
FUNCTIONAL OBSERVATIONAL BATTERY: Yes
- Description of procedures: The battery involved observing each animals in an open field arena for spontaneous behaviours (e.g. gait, respiratory pattern, convulsions, tremors, etc.). Manipulative procedures also were carried out by handling each animal and assessing reactions to various stimuli (handling reactivity, righting reflexes, tactiles placing reflexes, etc.).
- Technicians were blind to treatment status of animals: Yes
- Number of animals: 20 pups per sex and dose
- Time schedule for examinations: on days 21 post-partum and 60 +/- 2 days post-partum

MOTOR ACTIVITY: Yes
- Type of equipment used: Figure-of-height mazes equipped with photobearn designed to elicit moderately high levels of spontaneous activity (San Diego Instruments).
- Length of session, number and length of subsessions: 20 minuts on days 17, 21 and 1 hour on day 60 +/- 2 days post-partum
Additional information for developmental neurotoxicity study:
- Number and age of offspring/sex/group examined: 10

AUDITORY STARTLE REFLEX HABITUATION: Yes
- Number of animals: 20 pups per sex and dose
- Days of testing: postnatal days 25 and 60 +/- 2
- Same offspring evaluated at each preweaning time point: yes
- Exact age: no data
- Type of equipment used: SR-Screening System (San Diego instruments)
- Parameters evaluated: maximum startle amplitude, time to maximum startle amplitude
- Environmental conditions: 60 dB background white noise
- Number of trials performed: 50
- Length (msec) and intensity (dB) of sound: 40 msec / 120 dB noise
- Length of interval between trials: 8 sec

LEARNING AND MEMORY TESTING: Yes
(1) Overall testing design
- Number of animals: 10 pups per sex and dose
- Days of testing: day 22 and 65 +/- 2 post partum
(2) Equipment used
- Type of equipment: swimming E-Maze. The dimensions of the maze were approximately 90 cm wide, 50.5 cm deep and 40.6 cm high and it was filled to a depth of 12 cm with water at a temperature of 18-20°C.
(3) Testing and training procedures
- Number of trials per day: 6
- Number of days of testing: 2
- Inter-trial intervals: 30 minuts
- Learning criteria: number of correct turns from the center arm of the maze, time taken to exit the maze, and the percent of correct first turn during the initial trial of the reversal phase
(4) Control procedures
No data
(5) Performance measures
- Number of errors or trials to criterion: yes
- Time or latency to reach goal: yes
- Performance on "probe trials": yes
Sacrifice and (histo)pathology:
OFFSPRING
- Time point of sacrifice of offspring selected for brain weight or neuropathological evaluation: postnatal day 11 or 60+/-2
- At postnatal day 11, ten pups/sex/group were sacrificed by intracardiac injection of sodium pentobarbitone solution. The brains were weighted and the brains and spinal cords were embedded in parrafin, sectioned at 5 µm and stained with hematoxilin and eosin. Sections taken from the telencephalon, diencephalon, mesencephalon and myelencephalon were examined microscopically for pathological and developmental changes, including neuronal degeneration and anomalies of cell migration.
- On postnatal day 60 +/- 2, 10 animals/sex/group were anesthetized with an intraperitoneal injection of sodium pentobarbitone plus heparin and were perfused in situ with phosphate-buffered paraformaldehyde followed by phosphate-buffered glutaraldehyde.
The following tissues were examined for 6 animals/sex in the high-dose and control groups:
Brain including forebrain, cerebrum, hippocampus, midbrain, cerebellum and pons
Pituitary
Eyes (including ocular muscles)
Sensory ganglia (including those of the V cranial nerve, at least from four from the cervical bulb region and four from the lumbar region)
Spinal cord (cervical, thoracic and lumbar)
Dorsal and ventral nerve roots
Sciatic nerve
Peroneal nerve
Tibial nerve
Sural nerve
Anterior tibial muscle
Gastrocnemius muscle
Biceps femoris muscle
The sensory ganglia, dorsal and ventral nerve roots and the nerves were post-fixed in osmium tetroxide, processed and embedded in methylmethacrylate, sectioned at ca. and were stained with Toluidine Blue. The remaining tissues were embedded, in paraffin, sectioned at 5 µm and stained with haematoxylin and eosin.
Other examinations:
Not applicable
Positive control:
No
Statistics:
The neurotoxicological data were subjected to a repeated measures analysis of variance adjusting for time point, group, sex and also sex by group interaction. All the within animal interations were also included in the model, but the model refitted excluding non-significant interations (these were tested using the Greenhouse-Geisser adjustment for non-sphericity using a 5% significance level). The time effect was statistically assessed again using a G-G adjustment and in the absence of any significant interations, the statistical significance of the treated groups against the control was made using Dunnetts multiple-comparison test.
The neurotoxicological data consisted of startle response, motor activity, E-maze and functional observation battery data.
The F2b startle response amplitude was summarised from the 50 repeated measurements into 5 time intervals of 10 trials. The day 21 and day 60 data were anlysed separately.
The motor activity number of beam crossings was anlysed at days 17, 21 and 60 separately. There were 5 tile intervals and so the repeated measures analysis of variance described above was used.
For the E-maze, both the time to successfully exit the maze and the proportion of correct firts turns were nalysed. There were 6 runs at each of 2 sessions at each assessment, 24 hours appart. The run times for the sessions and assessments were analysed separately using the repeated measures approach described above.
Also analysed were the proportion of correct firts turns per session (non-parametric methods) made, and also the proportion of animals turning the correct way on the first run of session 2. These were anlysed using the Fidhers exact method and was intended as an indication of 24 hour memory retention
Clinical signs:
not specified
Mortality:
not specified
Body weight and weight changes:
effects observed, treatment-related
Food consumption and compound intake (if feeding study):
not examined
Food efficiency:
not examined
Water consumption and compound intake (if drinking water study):
not examined
Ophthalmological findings:
not examined
Clinical biochemistry findings:
not examined
Behaviour (functional findings):
effects observed, treatment-related
Description (incidence and severity):
decreased maximum startle amplitude
Gross pathological findings:
not examined
Neuropathological findings:
effects observed, treatment-related
Description (incidence and severity):
minor decrease in absolute brain weight
Other effects:
effects observed, treatment-related
Description (incidence and severity):
Migrated information from 'Further observations for developmental neurotoxicity study'



Details on results (for developmental neurotoxicity):LITTER DEVELOPMENT OBSERVATIONS
There were no clear treatment-related changes in pup developmental indices, including ear and eye opening, righting reflex, auditory startle response and pupil response. There was an decrease in the percent of F2a pups with eye open on PND 15 in the 300 ppm treatment group when compared to the control (71 +/- 30.6 % for control (mean +/- SD) and 47.4 +/- 38.1 % for 300 ppm) but similar effects were not observed for F1 or F2b pups (80.3 +/- 34.4 % for control F1 pups and 67.9 +/- 35.6 % for 300 ppm F1 pups; 73.0 +/- 32.6 % for controls F2b pups and 71.9 +/- 38.0 % for 300 ppm F2b pups)

SEXUAL MATURATION (OFFSPRING)
No treatment-related changes in ano-genital distances. There was a small but statistically significant increases in the average time to preputial separation for F1 pups in the70 and 300 ppm groups and in the vaginal opening for F1 pups in the 300 ppm group. Similar changes were not observed for F2 generation pups. (migrated information)
Details on results:
CLINICAL SIGNS AND MORTALITY
Not reported

MOTOR ACTIVITY
There were no treatment-related differences in motor activity observed in F1 animals evaluated on PND 17, 21 or 60.

FUNCTIONAL OBSERVATION BATTERY
There were no treatment-related changes observed in the FOB for F1 animals evaluated on PND 21 or 60

LEARNING ABILITY AND MEMORY RETENTION
There were no treatment-related effects on learning in the swim maze for F1 animals evaluated on PND 22 and 65

AUDITORY FUNCTION
In the auditory startle test, the time to maximum startle response was similar in all groups throughout testing. In addition, the maximum startle response tended to decreased across trials within a test session for all sodium chlorite treatment groups, indicating that the animals habituated to the startle stimulus. Small, statistically significant decreases in maximum response amplitude were observed for animals in the 70 and 300 ppm groups compared to control during the latter testing trials on PND 24. Similar effects were not observed on PND 60.

NEUROPATHOLOGY
There were no gross or microscopic lesions noted in the brains or spinal cords of F1 PND 11 pups. In addition there was no evidence of developmental changes, or anomalies in cell migration for PND 11 pups. A minor, albeit statistically significant, decrease in absolute brain weight (-8 %) was observed for male pups in the 300 ppm group sacrificed on PND 11 compared to control. Decreased brain weight for these pups was associated with decreased pup weight at birth and a 14 % decrease in pup weight on PND 11 compared to the control. Accordingly, brain weight to body weight ratios on PND 11 were increased for male pups in the 300 ppm group, although this increase (+6 %) was not statistically significantly different from the control. Decreases in absolute brain weight were not observed for female PND 11 pups, for male pups in the 35 and 70 ppm groups or for male or female PND 25 pups. Microscopic examination of the central and peripheral nervous system tissues for male and female PND 60 animals did not reveal any treatment-related alterations or pathology.
Dose descriptor:
NOAEL
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: offspring (migrated information)

No other information

Conclusions:
Based on the results of this study, the NOAEL for neurotoxicity is 300 ppm, the highest dose tested corresponding to 200 mg/kg bw/d of ClO2.
Executive summary:

In a two-generation study (Gill et al., 2000), Sodium chlorite was administered to 25-30 Sprague-Dawley rat/sex/dose in water at dose levels of 0, 35, 70 or 300 ppm for 10 weeks prior to mating, then throughout the study according to EPA Guideline OPPTS 870.3800 (similar to OECD 416) and in compliance with GLP. Due to the increase in water consumption normally observed in lactating rats, females received drinking water at concentrations of 17.5, 35 or 150 ppm during the lactation period.


25 male and 25 female F1 generation pups were selected from each group for rearing to sexual maturity. In addition animals from each group were allocated for neurotoxicity assessment:


- 10 animals/sex/dose/assessment (motor activity/swim maze/neuropathology).


- 20 animals/sex/dose/asessment (functional operation battery of tests/acoustic startle habituation).


F1 animals were paired for mating within dose group. Due to a reduced number of litters observed in the group receiving 70 ppm, the F1 generation animals were re-paired following weaning of the F2a generation to produce a F2b generation. Animals of F2a and F2b generations were also allocated for neurotoxicity assessments.


 


There were no mortalities or premature sacrifices in the F1, F2a or F2b generation selected animals.


There was an increase in animals in the F2b generation which exhibited general pallor, piloerection and hairloss in the high-dose group compared with the controls. These transitory observations were recorded for only a short time during the immediate post-weaning period. There was no evidence of any other effect of the test material on the clinical condition of the animals.


Statistical reduction of bodyweight was observed during the immediate post-weaning period but this was due to the reduced weight at weaning. The rate of bodyweight gain of the F2a generation males in the high-dose group was lower than that of the controls during the post-weaning period and the absolute bodyweights achieved statistical significance. However, a similar response was not observed in the F2b generation and therefore the effect observed in the F2a cannot be positively attributed to maternal treatment.


There was no effect of treatment on the mean activity counts observed on days 17, 21 or 60 post-partum.


There was no effect of treatment on the incidence of observations in the home cage, on removal from the home cage or in the open field in any of the treated groups on either days 21 or 60 post-partum.


There was no difference in response in the air righting reflex, vision, hindlimb tactile placing or righting reflex on days 21 or 60 post-partum which was considered to be related to treatment.


There was considered to be no effect of maternal treatment on learning ability and memory retention upon on either day 22 or day 65.


 


On day 24 post-partum, the maximum startle amplitude tended to be decreased compared to control in most trial blocks for medium- and high-dose group. These differences achieved statistical significance when compared with the controls. There were no statistically significant changes observed in the day 60 post-partum assessments. No effects were observed on the time to maximum startle amplitude and on the habituation response. Although changes in peak amplitude may be indicative of a neurotoxic effect the test substance is considered to be of low level of concern based on reversibility achieved at day 60 post-partum and on the lack of other effects indicating a neurotoxic effect (see EPA/630/R-95/001F). Observed changes may be a result of the habituation response for control animals rather than an effect of treatment with sodium chlorite. Habituation to auditory startle is demonstrated by decreased mean startle response amplitude the course of repeated trials during the test session (-22%, -30%, -30% and -30% for the control, 30 , 70 and 300 ppm treatment groups, respectively).


 


A minor, but statistically significant, decrease in absolute brain weight was observed for the high-dose group male pups killed on day 11 post-partum compared to the controls. Decreased brain weight for these pups was associated with decreased pup weight at birth and a decrease in pup weight on day 11 post-partum, compared with the controls. Similar effects on the brain weight were not observed for female pups of for pups in low- and medium-dose group. The decrease in absolute brain weight for male pups on day 11 post-partum was considered to be a result of small birth size for these pups and was considered not to be of toxicological significance based on:


1/ the small magnitude of the differences from control,


2/ the reversible nature of the effect,


3/ the lack of microscopic findings indicative of test article related alterations in brain development for animals sacrificed on day 11 post-partum.


 


Based on the results of this study, the NOAEL for developmental neurotoxicity is 300 ppm, the highest dose tested.


 


Considering the metabolism pathway of chlorine dioxyde which is likely to undergo rapid redox reactions within biological tissues rather than to be absorbed as parent compound, this study can be used for the assessment of ClO2.


 


However correction of doses should be done using the metabolism percentage of ClO2 into ClO2- which is 11 %(Abdel Rhaman, 1980a).


Based on the results of this study, the NOAEL for neurotoxicity is 300 ppm, the highest dose tested corresponding to 330 mg/kg bw/d of ClO2.


 


Even if the results of the study showed that ClO2 is of low level of concern for neurotoxicity at doses above 88 mg/kg bw, it has corrosive properties at doses as low as 40 mg/kg bw (Tos, 1996) and so effects that will be observed will be linked to corrosive properties rather than neurotoxicity.

Data source

Reference
Reference Type:
publication
Title:
Two-generation reproduction and development neurotoxicity study with sodium chlorite in the rat
Author:
Gill MW, Swanson MS, Murphy SR and Bailey GP
Year:
2000
Bibliographic source:
Journal of Applied Toxicology, 20: 291-303

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to guideline
Guideline:
EPA OPPTS 870.3800 (Reproduction and Fertility Effects)
Deviations:
no
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 416 (Two-Generation Reproduction Toxicity Study)
Deviations:
no
Principles of method if other than guideline:
Not applicable
GLP compliance:
not specified
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Sodium chlorite
EC Number:
231-836-6
EC Name:
Sodium chlorite
Cas Number:
7758-19-2
Molecular formula:
ClHO2.Na
IUPAC Name:
sodium chlorite
Details on test material:
- Name of test material (as cited in study report): Sodium chlorite
- Analytical purity: 81.4 %
- Impurities (identity and concentrations): Sodium chloride and small amount of Sodium hydroxide and Sodium chlorate

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals or test system and environmental conditions:
TEST ANIMALS
- Source: Iffa Credo, Belgium.
- Age at study initiation: approximately 6 weeks old at the start of F0 prebreed exposure period.
- Weight at study initiation: (P) Males: 80-99 g; Females: 60-79 g
- Housing: - during mating: one male was housed with each female
- during pregnancy: each female was housed individually
- during period of lactation: each dam was housed with its litter, in solid-bottomed polypropylene cages with sawdust bedding.
- Diet: pelleted SQC Rat and Mouse No. 3 expanded diet, Special Diets Services Limited, Witham, Essex (UK) ad libitum.
- Water: purified water (with or without chemical) provided from polycarbonates bottles fitted with stainless-steel tops and sipper tubes ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: appropriate
- Humidity: appropriate
- Air changes: no data
- Photoperiod: appropriate

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
PREPARATION OF DOSING SOLUTIONS: direct addition of the test material to purified water (UHP). Treated drinking wter were made fresh each week.
Details on mating procedure:
- M/F ratio per cage: 1:1
- Length of cohabitation: no data
- Proof of pregnancy: no data
- After successful mating each pregnant female was caged individually
- Any other deviations from standard protocol: due to a reduced number of litters observed in the F1-F2a generation, the F1 generation animals were re-paired following weaning of the F2a generation to produce and F2b generation.
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
The homogeneity, stability and concentration of sodium chlorite in the drinking water solutions were evalued analytically. The results of these analysis confirmed that the treated drinking water solutions were homogeneous and stable under the conditions of use. The concentration of sodium chlorite in the drinking water was adjusted to account fot the purity of the test substance.
Duration of treatment / exposure:
The F0 and F1 animals received treated drinking water through a 10-week prebreed period as well as during mating, gestation, parturition and lactation.
Frequency of treatment:
Continuously
Details on study schedule:
- Selection of parents from F1 generation: at weaning
Doses / concentrations
Remarks:
Doses / Concentrations:
0, 35, 70 and 300 ppm
Basis:
nominal in water
Dose concentrations decreased by 50% to 0, 17.5, 35 and 150 ppm during lactation.
No. of animals per sex per dose:
25-30 animals/sex/group
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: based on the results of a 90-day oral (gavage) toxicity study in rats, an oral (drinking water) developmental toxicity study in rabbits and an oral (drinking water) dose range-finding study in rats. The highest concentration selected (3000 ppm) was expected to be high enough to cause some systemic toxicity and to satisfy the regulatory requirement for a maximum tolerated dose, but not so high as to jeopardize the health of the animals as a result of decreases in palatability and water consupmtion. The 70 and 35 ppm concentrations were expected to produce graded effects or no effects.
Positive control:
No

Examinations

Parental animals: Observations and examinations:
CLINICAL OBSERVATIONS: Yes

BODY WEIGHT: Yes

FOOD CONSUMPTION: Yes

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes

Oestrous cyclicity (parental animals):
Yes.
Sperm parameters (parental animals):
Sperm number, sperm motility and sperm morphology.
Sperm number and motility were measured using an automated computer-assisted sperm motility analysis (CASA) system (Hobson Tracker, Uk)
Litter observations:
STANDARDISATION OF LITTERS: No data

PARAMETERS EXAMINED
The following parameters were examined in F1 and F2 offspring: bodyweights and bodyweights changes , landmarks of pup development and sexual maturation.

OTHER EXAMINATIONS:
- Hematological and thyroid hormone evaluation from 1 pup/sex/dose from each F1 generation, followed by additional evaluations at 13 weeks for all F1 animals selected to rear the F2 generation.
Red blood cell count (RBC), Hemoglobin levels (Hb), Hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentrations (MCHC), total white blood cell count (WBC), methemoglobin concentration (MetHb) and total serum T3 and T4 concentrations were evaluated.
- Neurotoxicological assessments were made on F1 and F2 generation
Postmortem examinations (parental animals):
HISTOPATHOLOGY: microscopic evaluation of gross lesions and reproductive organs from animals of the high-dose and control groups or any animals with suspected reduced fertility.
Postmortem examinations (offspring):
SACRIFICE
- The F1 offspring not selected as parental animals and all F2 offspring were sacrificed at 25 days of age.

GROSS NECROPSY
- Gross external evaluation

HISTOPATHOLOGY / ORGAN WEIGTHS
- Tissues prepared for microscopic examination and weighed: brain, liver, adrenals, spleen, thymus, kidneys, testes/ovaries
Statistics:
Interval data were subjected to an analysis of variance (ANOVA) with the absolute residuals from this analysissubjected to Levene’s test to identify possible differences in variances between treatment groups. For data with equal variances (P > 0.01), pairwise tests of all treated groups versus control were performed using William’s test. For a comparison of the high dose versus the control group, a two-sided test was performed with statistical significance noted at the P< 0.05, 0.01 and 0.001 levels. If the comparison of the high dose with the control group was not significant, one-sided tests were perfomed comparing the lower groups to the control. For data with unequal variances the Kruskall-Wallis-non-parametric ANOVA and Shirley’s non-parametric equivalent of Williams’ test were performed.
For nominal data Fisher’s Exact test was used to compare each treated group with the control.
Reproductive indices:
Mating index (%), Fertility index (%), Gestation index (%)
Offspring viability indices:
Viability and survival indices

Results and discussion

Results: P0 (first parental generation)

General toxicity (P0)

Clinical signs:
no effects observed
Body weight and weight changes:
effects observed, treatment-related
Description (incidence and severity):
Reduced body weights, food and water consumption
Food consumption and compound intake (if feeding study):
effects observed, treatment-related
Description (incidence and severity):
Reduced body weights, food and water consumption
Organ weight findings including organ / body weight ratios:
not specified
Histopathological findings: non-neoplastic:
no effects observed
Other effects:
not specified

Reproductive function / performance (P0)

Reproductive function: oestrous cycle:
no effects observed
Reproductive function: sperm measures:
not specified
Reproductive performance:
no effects observed

Details on results (P0)

CLINICAL SIGNS AND MORTALITY (PARENTAL ANIMALS)
No treatment-related clinical signs of toxicity or mortality that were attributed to treatment for F0 and F1 parental animals.

BODY WEIGHT AND FOOD CONSUMPTION (PARENTAL ANIMALS) (See Table 7.8.1/1)
- F0 generation: No treatment-related changes in body weigh or food consumption that were attributed to treatment
- F1 generation: body weight and food consumption were significantly decreased at all measurement intervals for F1 males in the 300 ppm group. Additionally, very small but statistically significant decreases in body weights were noted during the first 3-6 weeks of the prebreed treatment period for F1 males in the 70 ppm group and F1 females in the 300 ppm group.

During the last 7 days of gestation at parturition and for varying lenghts of time during lactation, body weights for F0 and F1 females in the 300 ppm group were decreased compared to the control group. The magnitude of the change in body weight from the control for dams in the 300 ppm treatment group generally was -4 % to -6 %.

WATER CONSUMPTION (PARENTAL ANIMALS)
- F0 generation: dose-related decreases in water consumption in the 70 and 300 ppm groups (ca. 10-25 % decrease compared to control). Statistically descreased occasionally for F0 males in the 35 ppm group.
- F1 generation: dose-related decreases in water consumption were observed for males at all sodium chlorite treatment levels (ca. 10-25 % decreased) and for females in the 300 ppm group (ca. 20 % decreased) at moset measurements intervals.

TEST SUBSTANCE INTAKE (PARENTAL ANIMALS)
No data

REPRODUCTIVE FUNCTION: ESTROUS CYCLE (PARENTAL ANIMALS)
No treatment-related changes in estrous cyclicity

REPRODUCTIVE FUNCTION: SPERM MEASURES (PARENTAL ANIMALS)
No treatment-related changes in sperm motility or morphology

REPRODUCTIVE PERFORMANCE (PARENTAL ANIMALS)
No treatment-related changes in mating, fertility or gestational indices for the F0 and F1 generations.

ORGAN WEIGHTS (PARENTAL ANIMALS)
No data

GROSS PATHOLOGY (PARENTAL ANIMALS)
No treatment-related effect observed.

HISTOPATHOLOGY (PARENTAL ANIMALS)
No treatment-related microscopic changes in reproductive tissues for male and female parental animals.

Effect levels (P0)

open allclose all
Dose descriptor:
LOAEL
Effect level:
70 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: Haematological toxicity
Dose descriptor:
NOAEL
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Dose descriptor:
NOAEL
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: fertility (migrated information)
Dose descriptor:
NOAEL
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects
Remarks on result:
other: Generation: reproduction (migrated information)

Results: F1 generation

General toxicity (F1)

Clinical signs:
no effects observed
Mortality / viability:
no mortality observed
Body weight and weight changes:
effects observed, treatment-related
Sexual maturation:
effects observed, treatment-related
Organ weight findings including organ / body weight ratios:
not specified
Gross pathological findings:
no effects observed
Histopathological findings:
no effects observed

Details on results (F1)

NUMBER AND SEXES OF PUPS BORN
No treatment-related changes in the number of pups born, the pup gender ratio, live birth index or pup survival indices.

BODY WEIGHT (OFFSPRING)
Treatment-related decreases in body weight were observed for male and female pups in the 300 ppm treatment group from the F1, F2a and F2b generations. The magnitude of the changes in pup body weight from control increased with age and ranged from -6 % at birth to -10 % on PND 24. The decreases were statistically significant from birth to weaning for F1 pups and during the final 2-3 weeks of lactation for F2a and F2b pups.

LITTER DEVELOPMENT OBSERVATIONS
There were no clear treatment-related changes in pup developmental indices, including ear and eye opening, righting reflex, auditory startle response and pupil response. There was an decrease in the percent of F2a pups with eye open on PND 15 in the 300 ppm treatment group when compared to the control (71 +/- 30.6 % for control (mean +/- SD) and 47.4 +/- 38.1 % for 300 ppm) but similar effects were not observed for F1 or F2b pups (80.3 +/- 34.4 % for control F1 pups and 67.9 +/- 35.6 % for 300 ppm F1 pups; 73.0 +/- 32.6 % for controls F2b pups and 71.9 +/- 38.0 % for 300 ppm F2b pups)

SEXUAL MATURATION (OFFSPRING)
No treatment-related changes in ano-genital distances. There was a small but statistically significant increases in the average time to preputial separation for F1 pups in the70 and 300 ppm groups and in the vaginal opening for F1 pups in the 300 ppm group. Similar changes were not observed for F2 generation pups.

ORGAN WEIGHTS (OFFSPRING)
No data

GROSS PATHOLOGY (OFFSPRING)
No treatment-related changes in gross external alterations.

HAEMATHOLOGY AND THYROID HORMONES (F1 GENERATION): (See Tables 7.8.1/2-3)
For F1 PND 25 pups, statistically significant decreases in red blood cell parameters, including decreased red blood cell (RBC) count, Hb, HCT, hematocrit mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentrations (MCHC) were observed for males and females in the 300 ppm group. In addition decreased WBC count was observed for animals in the 70 and 300 ppm groups. For F1 adult animals (at week 13), small decreases in RBC count, Hb, HCT, MCV, MCH and WBC count and a small increase in MCHC was observed for male and/or female rats in the 300 ppm group. Very small but statistically significant changes in some of these endpoints also were observed for male and female animals in the 35 and 70 ppm groups. The MetHb concentrations for PND 25 pups were increased compared to the control for males in the 300 ppm group and females in all treatment groups, although the increase for females occurred in a non-dose-related manner. The maximum MetHb concentration attained in any of the treatment groups was 1.5 % of Hb. Finally there were no treatment-related changes in the total serum concentrations of the thyroid hormones T3 or T4 for F1 PND 25 or F1 13-week-old animals.

Effect levels (F1)

Dose descriptor:
NOAEL
Generation:
F1
Effect level:
300 ppm (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: overall effects

Overall reproductive toxicity

Reproductive effects observed:
not specified

Any other information on results incl. tables

Table 7.8.1/1: Gestational and lactational mean body weights for female rats

Study period

Concentration (ppm)

0

35

70

300

F0 Generation

Gestation day

0

316 ± 22

306 ± 24

314 ± 23

304 ± 24

7

339 ± 25

329 ± 25

339 ± 23

327 ± 24

14

368 ± 27

360 ± 27

369 ± 27

355 ± 24

20

452 ± 35

441 ± 35

449 ± 36

426 ± 230**

Lactation day

0

342 ± 26

328 ± 27

333 ± 36

309 ± 24***

7

361 ± 25

349 ± 25

359 ± 30

341 ± 20**

14

374 ± 26

367 ± 22

375 ± 30

359 ± 22*

21

362 ± 24

352 ± 21

361 ± 26

348 ± 21*

F1 → F2a Generation

Gestation day

0

316 ± 25

309 ± 28

302 ± 44

314 ± 36

7

347 ± 24

339 ± 31

335 ± 44

341 ± 36

14

378 ± 24

371 ± 33

369 ± 47

372 ± 38

20

464 ± 32

455 ± 40

453 ± 53

438 ± 49

Lactation day

0

357 ± 30

344 ± 39

345 ± 53

330 ± 36*

7

377 ± 26

371 ± 37

374 ± 49

366 ± 37

14

386 ± 25

378 ± 36

389 ± 44

377 ± 32

21

368 ± 24

369 ± 27

368 ± 40

370 ± 38

F1 → F2b Generation

Gestation day

0

367 ± 28

363 ± 36

351 ± 35

354 ± 30

7

395 ± 29

390 ± 38

382 ± 36

380 ± 35

14

425 ± 32

420 ± 40

413 ± 39

412 ± 38

20

517 ± 36

503 ± 48

494 ± 37

485 ± 46*

Lactation day

0

401 ± 37

397 ± 40

396 ± 47

371 ± 49*

7

425 ± 32

417 ± 40

412 ± 35

400 ± 39*

14

428 ± 33

421 ± 34

416 ± 34

408 ± 35

21

398 ± 24

400 ± 31

394 ± 34

390 ± 90

* P < 0.05

** P < 0.01

*** P < 0.001

Table 7.8.1/2 : Haematology – group mean values on day 25 post-partum

 

RBC (106/µL)

Hb (%)

PCV (%)

MCV (fL)

MCH (pg

MCHC (g %)

WBC (103/µL)

Met. Hb (%)

MALES

0

4.8

9.3

29.7

61.5

19.3

31.4

5.8

0.7

35

4.8

9.1

28.8

59.9

18.8

31.5

5.4

0.9

70

4.9

9.1

28.5

58.5b

18.7

32.0

5.8

0.8

300

4.7

7.4c

24.2c

51.9c

16.0c

30.0a

4.1c

1.0a

FEMALES

0

5.1

10.2

31.6

62.2

20.1

32.3

5.6

0.9

35

5.0

9.6a

30.0a

59.8a

19.1b

31.9

5.1

1.2b

70

5.2

9.6a

30.0a

58.1c

18.6c

32.0

4.6a

1.0a

300

4.7c

7.7c

24.6c

52.5c

16.4c

31.3c

4.0b

1.4b

a = significantly different from control, p < 0.05

b = significantly different from control, p < 0.01

c = significantly different from control, p < 0.001

 

Table 7.8.1/3 : Haematology – group mean values at 13 weeks of age

 

RBC (106/µL)

Hb (%)

PCV (%)

MCV (fL)

MCH (pg

MCHC (g %)

WBC (103/µL)

Met. Hb (%)

MALES

0

8.8

16.4

44.7

50.6

18.5

36.6

12.9

1.1

35

8.7

16.1

43.4b

50.1

18.6

37.1a

13.7

1.2

70

8.7

16.1

42.9c

49.1b

18.4

37.5a

12.5

1.1

300

9.0

15.2c

41.5c

46.1c

16.9c

36.6a

12.5

1.3

FEMALES

0

8.0

15.6

42.9

53.4

19.5

36.4

9.7

1.4

35

7.9

15.5

42.0a

53.4

19.7

36.9b

9.0

1.5

70

7.9

15.4

41.7b

53.1

19.6

36.8b

9.1

1.4

300

7.7b

14.9c

40.2c

52.7

19.5

37.1c

8.5a

1.3

a = significantly different from control, p < 0.05

b = significantly different from control, p < 0.01

c = significantly different from control, p < 0.001

Applicant's summary and conclusion

Conclusions:
Under the test conditions, there were no adverse effects on reproduction and fertility, therefore:
The NOAEL (Parental) = 88 mg ClO2/kg bw/d, based on the hematotoxicity observed at 330 mg ClO2/kg bw/d
The NOAEL (Developmental) = 330 mg ClO2/kg bw/d, the highest dose tested.
The NOAEL (Fertility) = 330 mg ClO2/kg bw/d, the highest dose tested.
Executive summary:

In a two-generation study (Gill et al., 2000), Sodium chlorite was administered to 25-30 Sprague-Dawley rat/sex/dose in water at dose levels of 0, 35, 70 or 300 ppm for 10 weeks prior to mating, then throughout the study according to EPA Guideline OPPTS 870.3800 (similar to OECD 416) and in compliance with GLP. Due to the increase in water consumption normally observed in lactating rats, females received drinking water at concentrations of 17.5, 35 or 150 ppm during the lactation period.

25 male and 25 female F1 generation pups were selected from each group for rearing to sexual maturity. In addition animals from each group were allocated for neurotoxicity assessment.

F1 animals were paired for mating within dose group. Due to a reduced number of litters observed in the group receiving 70 ppm, the F1 generation animals were re-paired following weaning of the F2a generation to produce a F2b generation.

Blood samples were collected for haematological examination from the F1 pups killed on day 25 and at approximately 13 weeks of age for the F1 generation retained for the rearing.

Clinical signs, bodyweights, food and water consumption, fertility and mating performance, organ weights, macroscopic abnormalities at necropsy and histopathological findings were recorded for all parental animals. Litter size, clinical condition, growth and development to weaning, developmental neurotoxicological parameters and macroscopic abnormalities at necropsy were recorded for the offspring.

There were no effects of treatment at any dose level on parental mortality, mating performance, fertility, macroscopic findings at necropsy, or on the duration of gestation, numbers of pups born, pup survival, ano-genital distance at birth sexual development or macroscopic findings of the F1 and F2 pups at necropsy.

Reductions in bodyweight gain and food consumption observed in all generations are possibly related to the decrease in water consumption due to the palatability of the formulated drinking water.

Delays in preputial separation and vaginal opening observed in F1 animals were considered to be a result of lower body weight observed for these animals rather than a direct effect of Sodium chlorite on sexual developments.

At 300 ppm, significant lower red blood cell parameter values observed on day 25 and week 13. These effects were also observed in the 70 ppm dose group but they are not considered to be of toxicological concern since the hematological values are within the historical range or are sporadic.

Based on the results of these study, the following NOAEL were determined:

- NOAEL (parental) = 70 ppm (ca. 8 mg/kg bw/d for males and 10 mg/kg bw/d for females)

- NOAEL (developmental) = 300 ppm (ca. 30 mg/kg bw/d for males and 39 mg/kg bw/d for females), the highest dose tested

- NOAEL (fertility) = 300 ppm (ca. 30 mg/kg bw/d for males and 39 mg/kg bw/d for males), the highest dose tested.

Considering the metabolism pathway of chlorine dioxyde which is likely to undergo rapid redox reactions within biological tissues rather than to be absorbed as parent compound, this study can be used for the assessment of ClO2.

However correction of doses should be done using the metabolism percentage of ClO2 into ClO2- which is 11 % (Abdel Rhaman, 1980a).

In conclusion:

Under the test conditions, there were no adverse effects on reproduction and fertility, therefore:

The NOAEL (Parental) = 88 mg ClO2/kg bw/d, based on the hemato toxicity observed at 330 mg ClO2/kg bw/d.

The NOAEL (Developmental) = 330 mg ClO2/kg bw/d, the highest dose tested.

The NOAEL (Fertility) = 330 mg ClO2/kg bw/d, the highest dose tested.

Even if the results of the study showed that parental toxicity is observed at 300 ppm, ClO2 has corrosive properties at doses as low as 40 mg/kg bw (Tos, 1996) and so effects that will be observed will be linked to corrosive properties rather than to repeated dose toxicity.

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