Chlorine dioxide

Neurotoxicity

Description of key information

Chlorine dioxide (ClO2) is an unstable gas which must be stabilized with water. After skin contact, the ClO2 in aqueous solution is rapidly reduced in chlorite and chloride (see § 7.1). Therefore, it is considered as relevant to use the structurally close metabolite, sodium chlorite, to evaluate the potential developmental neurotoxicity of ClO2 according to a read-across approach.

 

Cross reading to valid animal studies with Sodium chlorite do not indicate adverse effects on developmental neurotoxicity as NOAEL developmental neurotoxicity = 300 ppm, corresponding to 330 mg/kg bw/d of ClO2 (i.e. highest dose tested): Neurotoxicity, eq. to OECD 416, 2000, K, RS, rel.2.

Key value for chemical safety assessment

Effect on neurotoxicity: via oral route

Link to relevant study records

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.

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

330 mg/kg bw/day

Species:

rat

Effect on neurotoxicity: via inhalation route

Endpoint conclusion

Endpoint conclusion:

no study available

Effect on neurotoxicity: via dermal route

Endpoint conclusion

Endpoint conclusion:

no study available

Additional information

A key study was identified (Gill et al., 2000). In this two-generation study conducted according the EPA Guideline OPP 870 -3800 (equivalent to OECD 416) and in compliance with GLP, sodium chlorite was administered to 25-30 Sprague-Dawley rat/sex/dose in water at dose levels of 0, 30, 70 ot 300 ppm chlorite for 10 weeks prior to mating, then throughout the study.

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/assessment (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 hair loss 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 dioxide 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 ClO₂into 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.

Justification for classification or non-classification

Based on the results of the study of Gill et al. (2000) no self-classification is proposed for neurotoxicity of Chlorine dioxide.