Chlorine dioxide

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

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 effects on reproduction and fertility of ClO2 according to a read-across approach.

Cross reading to valid animal studies with Sodium chlorite do not indicate adverse effects on reproduction and fertility (i.e. highest dose tested): Toxicity to reproduction, OECD 416, 2000, K, RS, rel.2.

Link to relevant study records

Reference

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.

Reason / purpose for cross-reference:

reference to same study

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

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:

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

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

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

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: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

not specified

Reproductive performance:

no effects observed

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.

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)

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

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.

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

300 ppm (nominal)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

other: overall effects

Reproductive effects observed:

not specified

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 (10^6/µL)	Hb (%)	PCV (%)	MCV (fL)	MCH (pg	MCHC (g %)	WBC (10^3/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 (10^6/µL)	Hb (%)	PCV (%)	MCV (fL)	MCH (pg	MCHC (g %)	WBC (10^3/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

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 ClO_2^- 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.

Effect on fertility: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

330 mg/kg bw/day

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

A key study was identified (Gill et al., 2000). In this two-generation study, 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.

 

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 haematological 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 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).

 

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 haemato 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.

Effects on developmental toxicity

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 effects on development of ClO2 according to a read-across approach.

Cross reading to valid animal studies with Sodium chlorite do not indicate adverse effects on development (i.e. highest dose tested): Developmental toxicity/teratogenicity, EPA OPP 83-3 guideline, 1995, K, RS, rel.2.

 

In addition, the effects on teratogenicity has been studied in a weight-of-evidence approach based on rats and mice studies on Chlorine dioxide.

Link to relevant study records

Reference

Reference 1

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The teratogenic potential of Alcide® gel was evaluated.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, New York
- Age at study initiation: mature
- Weight at study initiation: 225-250 g
- Housing: wire mesh bottom cages
- Diet: Purina laboratory rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: 25 °C
- Humidity: 50 %
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the dose administered to each animal was based on the individual animal's body weight on the first day of administration.
Group (1): High-dose gel, 2 g/kg bw of part A + part B, 1:1, respectively
Group (2): Low-dose gel, 1 g/kg bw of part A + part B, 1:1, respectively
Group (3): Placebo gel
Group (4): Control, untreated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not done

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: overnight
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy
Females that were sperm-positive were weighted and placed in their original cages (3 animals per cage).

Duration of treatment / exposure:

From day 6 to day 15 of gestation

Frequency of treatment:

Daily

Duration of test:

until day 20 of gestation (1 day prior the expected delivery date)

Remarks:

Doses / Concentrations:
not quantified
Basis:

No. of animals per sex per dose:

20 dams per dose

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Details on study design:

- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: gross appearance, mortality, behavior

BODY WEIGHT: Yes
- Time schedule for examinations: daily

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of live fetuses/dam: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Data were analyzed using analysis of variance with Duncan's multiple range test.

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No signs of maternal toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistically significant effects was observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

No other information

Executive summary:

In a developmental toxicity study Alcide® was administered to females Sprague-Dawley rats (20/dose) by dermal application from days 5 through 20 of gestation. Untreated animals and placebo animal served as controls.

Maternal body weight changes were observed, uterine content was examined and fetal examinations were performed.

There were no signs of maternal toxicity. There were no significant difference between mother weight gain, implantations, live foetuses, male:female ratios and fetal weights and lenghts of the Alcide® gel treated groups and the controls. The percentage of the total number of resorptions (low-dose group) and the percentage of the total number of dead foetuses (high-dose group) were slightly higher compared to controls but not statistically different.

The incidences of skeletal and visceral abnormalities were comparable to the control fetuses.

Reference 2

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

no data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

The teratogenic potential of Alcide® gel was evaluated.

GLP compliance:

not specified

Limit test:

no

Species:

mouse

Strain:

Swiss Webster

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Marland breeding farms, Hewitt, New Jersey
- Age at study initiation: mature
- Weight at study initiation: 30-40 g
- Housing: wire mesh bottom cages
- Diet: Purina laboratory rodent chow ad libitum
- Water: tap water ad libitum
- Acclimation period: no data

ENVIRONMENTAL CONDITIONS
- Temperature: 25 °C
- Humidity: 50 %
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

dermal

Vehicle:

unchanged (no vehicle)

Details on exposure:

TEST MATERIAL
- Amount(s) applied (volume or weight with unit): the dose administered to each animal was based on the individual animal's body weight on the first day of administration.
Group (1): High-dose gel, 2 g/kg bw of part A + part B, 1:1, respectively
Group (2): Low-dose gel, 1 g/kg bw of part A + part B, 1:1, respectively
Group (3): Placebo gel
Group (4): Control, untreated

Analytical verification of doses or concentrations:

no

Details on analytical verification of doses or concentrations:

not done

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1/3
- Length of cohabitation: overnight
- Proof of pregnancy: sperm plug referred to as day 0 of pregnancy
Females that were sperm-positive were weighted and placed in their original cages (3 animals per cage).

Duration of treatment / exposure:

From day 6 to day 15 of gestation

Frequency of treatment:

Daily

Duration of test:

until day 18 of gestation (1 day prior the expected delivery date)

Remarks:

Doses / Concentrations:
not quantified
Basis:

No. of animals per sex per dose:

20 dams/dose

Control animals:

yes, concurrent no treatment

yes, sham-exposed

Details on study design:

- Rationale for animal assignment: random

Maternal examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: daily
- Cage side observations: gross appearance, mortality, behavior

BODY WEIGHT: Yes
- Time schedule for examinations: daily

Ovaries and uterine content:

The ovaries and uterine content was examined after termination: Yes
Examinations included:
- Number of live fetuses/dam: Yes
- Number of implantations: Yes
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: all per litter
- Visceral examinations: Yes: half per litter
- Skeletal examinations: Yes: half per litter

Statistics:

Data were analyzed using analysis of variance with Duncan's multiple range test.

Indices:

no data

Historical control data:

no data

Details on maternal toxic effects:

Maternal toxic effects:no effects

Details on maternal toxic effects:
No signs of maternal toxicity

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:no effects

Details on embryotoxic / teratogenic effects:
No statistically significant effects was observed

Remarks on result:

not determinable due to absence of adverse toxic effects

Abnormalities:

not specified

Developmental effects observed:

not specified

No other information

Executive summary:

In a developmental toxicity study Alcide® was administered to female Swiss Webster mice (20/dose) by dermal application from days 5 through 20 of gestation. Untreated animals and placebo animal served as controls.

Maternal body weight changes were observed, uterine content was examined and fetal examinations were performed.

There were no signs of maternal toxicity. There were no significant difference between mother weight gain, implantations, live fetuses, male:female ratios and fetal weights and lenghts of the Alcide® gel treated groups and the controls. The percentage of the total number of resorptions (high-dose group) and the percentage of the total number of dead fetuses (low- and high-dose group) were slightly higher compared to controls but not statistically different.

The incidences of skeletal and visceral abnormalities were comparable to the control fetuses.

Reference 3

Endpoint:

developmental toxicity

Type of information:

experimental study

Adequacy of study:

weight of evidence

Study period:

No data

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

study well documented, meets generally accepted scientific principles, acceptable for assessment

Reason / purpose for cross-reference:

reference to same study

Reason / purpose for cross-reference:

reference to other study

Qualifier:

no guideline followed

Principles of method if other than guideline:

Determination of the teratogenic effect of ClO2 in rats, when administered prior to and throughout gestation.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Sprague-Dawley

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Taconic Farms, Germantown, New York, USA
- Age at study initiation: no data
- Weight at study initiation: 150 – 170 g
- Fasting period before study: no data
- Housing: throughout gestation mated females were placed in plastic cages.
- Diet: Purina Rodent Chow ad libitum
- Water: ad libitum with treatment
- Acclimation period: 1 week

ENVIRONMENTAL CONDITIONS
- Temperature: 25-25 °C
- Humidity: 50-55%
- Air changes (per hr): no data
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

No data

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

The concentrations of the ClO2 in water was determined daily by diethyl-p-phenylene diamine (DPD) method of Palin.

Details on mating procedure:

- Impregnation procedure: cohoused
- If cohoused:
- M/F ratio per cage: 1:1
- Length of cohabitation: 1 day
- Proof of pregnancy: sperm in vaginal smear referred to as day 0 of pregnancy

Duration of treatment / exposure:

2 and a half months prior to and throughout gestation

Frequency of treatment:

Continuous

Duration of test:

No data

Remarks:

Doses / Concentrations:
0.1, 10, 100 mg/L
Basis:
nominal in water

No. of animals per sex per dose:

Between 6 and 8

Control animals:

yes, concurrent vehicle

Details on study design:

On day 20 of gestation, animals were killed by an overdose of ether.

Maternal examinations:

Bodyweight

Ovaries and uterine content:

The uterine content was examined after termination: Yes
Examinations included:
- Number of live fetuses: Yes
- Number of dead fetuses: Yes
- Number of implantations: Yes / No / No data
- Number of resorptions: Yes

Fetal examinations:

- External examinations: Yes: live fetuses were weighted and measured lenght (crown-rump) individually, sexed externally and examined for external malformations.
- Soft tissue examinations: Yes:half per litter
- Skeletal examinations: Yes: half per litter
- Head examinations: Noa

Statistics:

Each parameter measured was compared between control and treated groups by one-way analysis of variance. In addition, the average numbers of implants and live fetuses per dam were analyzed by the Kruskall-Wallis test. Jonckheere's test was used to test the significance of dose-response relationship.

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
One dam of the 10 mg/L ClO2 group died during pregnancy. Slight decreases in weight gain during pregnancy were seen in the ClO2 administered group. The average number of implants and live foetuses per dam were decreased in the ClO2 treatment groups. A one way analysis of variance showed that the decreases were not significantly different, however the decrease showed a significant trend as the dose was increased (p < 0.01).
Slightly increased incidences of resorptions were observed in the treatment groups, one dead fetus was found in the 100 mg/L ClO2 group.

Dose descriptor:

NOAEL

Effect level:

100 mg/L drinking water

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
Fetal weights in the ClO2 treatment groups were increased compared with those in the control group, although foetuses in the 100 mg/L group were the only ones to show a significant increase. Fetal length was increased significantly in the 10 mg/L ClO2- group. The foetuses in all groups except the 1 mg/L ClO2 group showed slight increases in total skeletal anomalies compared with the control group. Increased incidences of the commonly occured skeletal defects such as incomplete or bipartite sternebrae and rudimentary ribs were observed at the high doses of ClO2. A few cases of hypoplastic kidney, hydronephrosis and dextrocardia were observed in the ClO2 treatments groups.

Dose descriptor:

LOAEL

Effect level:

100 mg/L drinking water

Basis for effect level:

other: embryotoxicity

Dose descriptor:

NOAEL

Effect level:

10 mg/L drinking water

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 7.8.2/1: Effects of ClO₂in drinking water on pregnant rats.

	ClO2(mg L^-1)
	Control	1	10	100
Dams bred	6	6	7	8
Dams pregnant	6	6	7	8
Dams died	0	0	1	0
Weight gain during pregnancy (g)	124.5 ± 3.1	132.2 ± 6.7	109.0 ± 2.9	109.5 ± 3.4

Table 7.8.2/2: Effects of ClO₂in drinking water on rat reproduction.

	Control	1 mg L^-1	10 mg L^-1	100 mg L^-1
Litters examined	6	6	6	8
Total implants	74	74	68	82
Implants per dam	12.3 ± 0.7	12.3 ± 0.4	11.3 ± 0.7	10.3 ± 0.3
Live fetuses per dam	12.0 ± 0.4	12.2 ± 0.4	10.8 ± 0.8	10.0 ± 0.3
Total resorptions	0 (0)	1 (1.4)	3 (4.4)	1 (1.2)
Total dead fetuses	0	0	0	1
Male:female live fetuses	38:34	44:29	33:32	45:34
Fetal body weight Total	3.98 ± 0.05	4.13 ± 0.04	4.10 ± 0.05	4.22 ± 0.04
Fetal body weight Male	4.08 ± 0.07	4.20 ± 0.05	4.25 ± 0.05	4.36 ± 0.04
Fetal body weight Female	3.86 ± 0.07	4.03 ± 0.06	3.95 ± 0.08	4.04 ± 0.05
Fetal length Total	3.76 ± 0.03	3.81 ± 0.02	3.74 ± 0.03	3.79 ± 0.02
Fetal length Male	3.78 ± 0.04	3.83 ± 0.03	3.81 ± 0.02	3.84 ± 0.02
Fetal length Female	3.74 ± 0.04	3.78 ± 0.02	3.67 ± 0.05	3.73 ± 0.02

 

Table 7.8.2/3: Effects of ClO₂in drinking water on skeletal anomaly in rat fetuses.

	Control	ClO2(mgL^-1)
		1	10	100
Fetuses examined	35	36	31	32
Fetuses anomaly	11	9	12	16
Litters containing anomalous fetuses	5	4	6	7
Average % anomalous fetuses	30.7 ± 9.8	24.5 ± 10.7	41.0 ± 9.5	49.3 ± 5.2

Table 7.8.2/4: Types of skeletal anomaly in rat fetuses after treatment with ClO_2.

	Control	1 mg L^-1	10 mg L^-1	100 mg L^-1
Incomplete or bipartite sternabrae	6 (17.1)*	8 (22.2)	8 (25.8)	8 (25.0)
Missing sternabrae	2 (5.7)	0	0	0
Extra ribs	0	0	0	0
Rudimantary ribs	3 (8.6)	1 (2.8)	2 (6.5)	4 (12.5)
Extra vertebrae	0	0	0	0
Missing vertabrae	0	0	0	0
Incomplete ossification:
Vertebrae	0	0	0	0
Perietal	0	1	1	0
Interparietal	3 (8.6)	2 (5.6)	5 (16.1)	6 (18.8)
Occipital	0	0	0	1

*Values in parentheses represent the percentage of total number of each skeletal anomaly versus number of fetuses examined.

 

Table 7.8.2/5: Table for effects of ClO₂in drinking water on visceral anomaly in rat fetuses.

	Control	1 mg L^-1	10 mg L^-1	100 mg L^-1
Foetuses examines	37	37	34	42
Foetuses anomaly	0	2	1	3
Litters containing anomalous foetuses	0	2	1	2
Hypoplastic kidney	0	1	1	0
Hydronephrosis	0	1	0	2
Dextrocardia	0	0	0	1

 

Conclusions:

NOAEL for maternal toxicity is 100 mg/L drinking water, LOAEL for embryotoxicity is 100 mg/L drinking water and NOAEL for embryotoxicity is 10 mg/L drinking water base on reduced implants/live fetuses.

Executive summary:

In a developmental toxicity study, Chlorine Dioxide was administered to female Sprague-Dawley rats in water drinking at dose levels of 0.1, 10, 100 mg/L, daily for 2 ½ months prior to mating and throughout gestation. Rat fetuses were exposed in utero.

Maternal body weight changes were observed, uterine content was examined (total implants, implants per dam, live fetuses per dam and total resorptions (%)) and fetal examinations were performed.

 

Maternal toxic effects were observed. One dam of the 10 mg/L ClO₂ group died during pregnancy.

The average number of implants and live fetuses per dam were decreased in the ClO₂ treatment groups. A one way analysis of variance showed that the decreases were not significantly different, however the decrease showed a significant trend as the dose was increased (p < 0.01). One dead fetus was found in the 100 mg/L group and foetal weights in this group were significantly increased.

A few cases of hypoplastic kidney, hydronephrosis and dextrocardia were observed.

 

The fetuses in all treatment groups except the 1 mg/L group showed slight increases in total skeletal anomalies compared with the control group. Increased incidences of skeletal defects, such as rudimentary ribs, were observed. Because of their frequency, even in the control animals, researchers classify them either as anomalities or as skeletal variants.

The occurrence of the incompletely ossified interparietal bone was increased at the 10 and 100 mg/L doses. However in rodents the calvarium is among the regions that ossify rapidly during late gestation. A variable ossification of the intraparietal bone is thus considered as normal.

Hence, the slight ossification retardation observed in this study is considered to be of low level of concern.

 

Under the conditions of this study, the NOAEL for maternal toxicity is 100 mg/L drinking water (c.a. 8 mg/kg bw/day). The LOAEL for embryotoxicity is 100 mg/L drinking water and the NOAEL for embryotoxicity is 10 mg/L (c.a. 0.8 mg/kg bw/day) drinking water based on reduced implants/live fetuses.

Reference 4

Endpoint:

developmental 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

Qualifier:

equivalent or similar to guideline

Guideline:

EPA OPP 83-3 (Prenatal Developmental Toxicity Study)

Deviations:

no

Principles of method if other than guideline:

Not applicable

GLP compliance:

not specified

Limit test:

no

Species:

rabbit

Strain:

New Zealand White

Details on test animals or test system and environmental conditions:

TEST ANIMALS: Time-mated females
- Source: Interfauna UK Ltd. (Huntington, Cambridge, UK)
- Age at study initiation: 4–5 months old
- Weight at study initiation: 3–4 kg (at mating)
- Housing: individually, in grid bottomed metal cages suspended over paper lined trays
- Diet: SQC Rabbit Standard (Special Diets Services Limited, Witham, Essex, U.K.) ad libitum
- Water: tap water (purified by reverse osmosis) ad libitum

ENVIRONMENTAL CONDITIONS
- Temperature: 18-22 °C
- Humidity: 42-59 %
- Air changes: no data
- Photoperiod: 12 hrs dark / 12 hrs light

Route of administration:

oral: drinking water

Vehicle:

water

Details on exposure:

PREPARATION OF DOSING SOLUTIONS: the test article was formulated for dosing by dissolution in the drinking water. Separate formulations were prepared daily for each dose level.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Analysis of samples of the formulations used in this study were assessed for concentration on the first and last days of dosing to determine accuracy of preparation.

Details on mating procedure:

- Impregnation procedure: purchased timed pregnant

Duration of treatment / exposure:

Duration of treatment: Days 7-19 (post mating)

Frequency of treatment:

Continuously

Duration of test:

Until day 28 of pregnancy

Remarks:

Doses / Concentrations:
0, 200, 600 and 1200 mg/L
Basis:
nominal in water

No. of animals per sex per dose:

16 females/dose. One additional female was allocated to dose groups 200 and 600 ppm on day 7 of pregnancy as there was one female in each of these group showing poor clinical condition which may have necessitated sacrifice.

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected following examination of results of a preliminary maximum tolerated dose study in non-mated female rabbits. The dose levels were selected to cover a range between one expected to elicit minimal maternal effects and a no observable effect level.
- Rationale for animal assignment: randomisation procedure based on bodyweight

Maternal examinations:

CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily from day 3 of pregnancy until necropsy.

BODY WEIGHT: Yes
- Time schedule for examinations: day 0, daily from day 3 to day 22 and on days 25 and 28 of pregnancy.

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- The amount of food was recorded every 2 days from day 3 to day 27 and over 1 day from day 27 to day 28 of pregnancy.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
Water consumption was measured daily from day 3 to day 22 of pregnancy.

POST-MORTEM EXAMINATIONS: Yes
- Sacrifice on gestation day 28
- Organs examined: major organs

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 live and dead implantations: Yes

Fetal examinations:

GENERAL
Number of live foetuses, number of dead foetuses, foetal weight, external abnormalities.

SKELETAL
The bones were identified and examined for normality with respect to shape, size and the extent of ossification.

SOFT TISSUE
Foetuses were briefly fixed in alcohol prior to being skinned and dissected. The brain, eyes, palate and major organs and blood vessels in the thorax and abdomen were examined. The sex of the foetuses, as assessed from the appearance of the internal genitalia were recorded.

Statistics:

Analysis of variance; Student's t test; Kruskal-Wallis test; Dunn's multiple comparison test.

Indices:

No data

Historical control data:

No data

Details on maternal toxic effects:

Maternal toxic effects:yes

Details on maternal toxic effects:
Two rabbits were sacrificed in extremis during the study because of a general deterioratio of clinical condition that was not considered to be related to treatment. One of the rabbits was from the control group and the other was from the 600 ppm group. There were no mortalities at the highest dose level of 1200 ppm. The only clinical observation was a dose-related increase in the incidence of rabbits with reduced fecal production during the dosing period, which coincided with the observed reduction in food consumption.
Because of palatability, there were significant decreases in water consumption. At 200 ppm, water consumption was significantly reduced only during days 16-20 of pregnancy. Intake of NaClO2 averaged 13.0 mg/kg bw/d (equivalent to 9.7 mg/kg bw/d ClO2-) during the treatment period. At 600 ppm, there was a significant decrease in water consumption for the entire dosing period. The mean NaClO2 intake at 600 ppm during the dosing period was 35.5 mg/kg bw/d (equivalent to 2.5 mg/kg bw/d ClO2-). At 1200 ppm, mean water consumption was reduced by > 50 % at the onset of dosing. Water consumption increased by remained significantly lower than that of the control group throughout the entire dosing period. The average NaClO2 intake at 1200 ppm was 53.1 mg/kg bw/d (equivalent to 39.6 mg/kg bw/d ClO2-).
There was no significant reduction of food consumption at 200 ppm. At 600 ppm, ther was a significant reduction in food consumption during days 7-11 of pregnancy (p < 0.001). For the remainder of pregnancy, food consumption was generally similar to that of the control group. At 1200 ppm, food consumption was significantly (p < 0.001) lower than that in the control group for the remainder of the dosing period; food consumption increased the levels greater than that for the control group.
There were no statistically significant changes in absolute body weights in any treatment group throughout the study. At 200 and 600 ppm, there were no significant differences from the controls in body weight gain. At 1200 ppm, animals lost a significant amount of weight at the onset of dosing (days 7-11). This was only a transient loss since these animals gained more weight during the remainder of the pregnancy that did the control group.
There were no treatment-related abnormalities observed at maternal necropsy. Pregnancy incidence was slightly lower than expected in the 200- and 600-ppm groups but was within expected limits in the control and the 1200-ppm groups.

Dose descriptor:

NOAEL

Effect level:

1 200 mg/L drinking water

Basis for effect level:

other: maternal toxicity

Details on embryotoxic / teratogenic effects:

Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
The mean numbers of corpora lutea, implantations, and live fetuses were similar in all groups. The sex distribution of the live fetuses was similar in all groups with near equal proportions of male and female fetuses.
Mean fetal weight was similar between the control group and the group treated at 200 ppm. Fetal weights were marginally lower than control values at 600 and 1200 ppm. Since fetal weights at 1200 ppm were greater that at 600 ppm, these slightly lower mean fetal weights may not have been related to treatment. Mean gravid uterus weight was similar in the control group and the group treated at 600 and 1200 ppm. It was slightly higher in the group treated at 200 ppm where there were slightly higher mean numbers of live fetuses in utero. Differences from the control group were not statistically significant.
At fetal examination, the major and minor external/visceral abnormalities observed were of a type and incidence that occur spontaneously in this strain of rabbit. There were no dose-related abnormalities. Most of the major and minor visceral abnormalities were associated with the aortic arch, a particularly labile region during organogenesis in this strain of rabbit. The minor aortic arch abnormalities were mainly either additional minor blood vessels or abnormal origin of the common carotid artery. The major aortic arch abnormalities involved severe dilations or constrictions of the great vessels.
There were no treatment-related skeletal abnormalities. The higher incidence of major skeletal abnormalities in the 600 ppm in comparison with the control was considered coincidental. The abnormalities were of a type that occur spontaneously in this strain of rabbit, and there were no major skeletal abnormalities at the highest dose tested, which achieved a higher NaClO2 intake throughout the exposure period. At 600 and 1200 ppm there was an increase in both minor skeletal abnormalities and skeletal variants related to incomplete ossification of certain bones such as the pubis and sternebrae. This was not unexpected since the fetuses in these groups were slightly lower in body weight. The incidences of minor skeletal abnormalities and skeletal variants at 200 ppm was similar to that of the controls.

Dose descriptor:

NOAEL

Effect level:

1 200 mg/L drinking water

Basis for effect level:

other: embryotoxicity

Abnormalities:

not specified

Developmental effects observed:

not specified

Table 7.8.1/1: Mean maternal body weight and body weight gain of rabbits exposed to NaClO2 during pregnancy

NaClO2 concentration (ppm)		Control	200	600	1200
n		13	13	12	14
Body weight (mg) ± SD on Day 0 of pregancy		3.59 ± 0.26	3.61 ± 0.24	3.53 ± 0.25	3.61 ± 0.21
Body weight gain (kg) ± SD on indicated day of pregnancy	0-7	0.10 ± 0.13	0.05 ± 0.11	0.06 ± 0.13	0.09 ± 0.11
	7-11	0.09 ± 0.10	0.05 ± 0.11	0.02 ± 0.08	-0.16 ± 0.11*
	11-15	0.14 ± 0.06	0.16 ± 0.04	0.15 ± 0.08	0.20 ± 0.11
	15-19	0.06 ± 0.09	0.04 ± 0.09	0.05 ± 0.05	0.08 ± 0.05
	19-22	0.05 ± 0.03	0.04 ± 0.05	0.03 ± 0.04	0.13 ± 0.07*
	22-26	0.14 ± 0.06	0.16 ± 0.08	0.16 ± 0.07	0.14 ± 0.05

* significantly different from control p< 0.01: Student's test  

Table 7.8.1/2: Reproductive parameters in rabbits exposed to NaClO₂during pregnancy

NaClO2 concentration (ppm)	Control	200	600	1200
No. in group on Day 28	15	17	16	16
No. pregnant on Day 28	13	13	12	14
Mean no. of corpora lutea ± SD	11.9 ± 2.3	11.9 ± 1.9	12.8 ± 2.3	12.1 ± 2.7
Mean no. of implantation ± SD	10.7 ± 2.2	10.8 ± 1.8	10.7 ± 2.4	10.1 ± 2.2
Mean no. of live fetuses ± SD	8.5 ± 2.9	9.6 ± 1.9	9.0 ± 2.6	8.9 ± 2.4
Mean preimplantation loss (%)	10.4	8.4	16.1	14.9
Mean postimplantation loss (%)	21.3	11.1	15.4	12.6
Sex ratio (M:F)	55:45	41:59	48:52	52:48

 

Table 7.8.1/3: Mean fetal and gravid uterus weights in rabbits exposed to NaClO2 during pregnancy

NaClO2 concentration (ppm)	Control	200	600	1200
n	13	13	12	14
Mean male fetuses weight (g)	35.2 ± 4.9	36.7 ± 4.7	32.2 ± 4.7	33.5 ± 3.4
Mean female fetuses weight (g)	35.1 ± 4.2	35.3 ± 3.6	33.1 ± 3.2*	33.4 ± 3.4
Mean fetuses weight (g)	35.0 ± 4.2	35.8 ± 3.7	33.1 ± 2.6	33.2 ± 3.1
Gravid uterus weight (g)	494.5 ± 132.5	553.7 ± 69.7	480.4 ± 112.1	471.2 ± 105.5

* n = 11 for this value

Table 7.8.1/4: Incidences of fetal abnormalities in rabbits exposed to NaClO2 during pregnancy

NaClO2 concentration (ppm)	Control	200	600	1200
Total no. of fetuses (litters) examined	111 (13)	125 (13)	108 (12)	124 (14)
External and visceral examination : - No. with minor abnormalities only - Mean % with minor abnormalities only - No. of major abnormalities - Mean % with major abnormalities	33 (9) 28.6 2 (1) 1.5	29 (9) 22.8 1 (1) 0.5	36 (11) 32.2 4 (3) 6.6	32 (11) 26.8 3 (3) 2.9
Skeletal examination : - No. with minor abnormalities only - Mean % with minor abnormalities only - No. of major abnormalities - Mean % with major abnormalities - No. with any major abnormalities - Mean % with any major abnormalities	10 (4) 7.7 0 (0) 0.0 2 (1) 1.5	9 (7) 6.3 1 (1) 0.8 2 (2) 1.3	12 (6) 14.2 4 (4) 5.4* 5 (4) 7.5	18 (10) 13.9 0 (0) 0.0 3 (3) 2.9

Major abnormalities:

- 0 ppm x aortic arch abnormalities

- 200 ppm x aortic arch abnormalities, 1 x scoliosis

- 600 ppm x spinal dysmorphogenesis, 1 x spina bifida, 1 + microphtalmia, 1 x fused ribs

- 1200 ppm 1 x cleft plate, 1 x aortic arch abnormalities, 1 x umbilical hernia

* Significantly different from the control: p < 0.01: Dunn’s multiple comparison test

Conclusions:

The NOAEL for both maternal and developmental effects in this study was determined to be 1200 mg/L, the highest dose tested corresponding to 44 mg chlorite/kg bw/day.

Executive summary:

In a developmental toxicity study conducted similarly to EPA OPP 83-3 guideline, Sodium chlorite (80.58 % pure) was administered to 16 or 17 females New Zealand White rabbits/dose in drinking water at dose levels of 0, 200, 600 and 1200 mg/L (0, 9.7, 26.5 and 39.6 mg chlorite/kg bw/day) from days 7 through 19 of gestation. Dams were sacrificed on gestation day 28.

Oral administration of sodium chlorite during organogenesis at 600 and 1200 mg/L elicited dose-related reductions in maternal water with consequent reductions in maternal food consumption, production of fecal pellets and body weight gain.

Despite the maternal effects there was no evidence of embryo-lethality or teratogenicity. Although the number and mean percentage of major external and visceral and skeletal abnormalities were increased in the 600 and 1200 mg/L groups (external/visceral: 6.6 and 2.9%, respectively, vs. 1.5% in controls; skeletal: 5.4 and 0%, respectively, vs. 0% in controls), the authors did not consider these to be treatment-related adverse effects. Mean fetal weights in the 600 and 1200 mg/L groups were slightly decreased (<9%, relative to controls). In the 600 and 1200 mg/L groups, the incidence of minor skeletal abnormalities (13.9 and 14.2% for the 600 and 1200 mg/L groups, respectively, vs. 7.7% in controls) and skeletal variants related to incomplete fetal bone ossification was higher than for controls. The authors state in their discussion that these alterations in fetal body weight and delayed ossification indicate embryonic growth retardation. Decreases in maternal food and water consumption and body weight gain may be responsible, at least in part, for some of the fetal effects.

Sodium chlorite was not considered to have any effect on maternal toxicity or reproductive development.

The NOAEL for both maternal and developmental effects in this study was determined to be 1200 mg/L, 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 ClO₂into ClO_{2 -}(Abdel Rhaman, 1980a). Using a percentage of 11%, adjusted doses are the following: 0; 88.1; 240; 360 mg/kg bw/d.

Therefore the NOAEL for both maternal and developmental toxicity of ClO₂ in this study is 360 mg/kg bw/d. As ClO₂has corrosive properties at doses as low as 40 mg/kg bw (Tos, 1996), effects that will be observed will be linked to corrosive properties.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

no adverse effect observed

Dose descriptor:

NOAEL

360 mg/kg bw/day

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

Additional information

A key study was identified (Harrington, 1995). In this developmental toxicity study conducted similarly to EPA OPP 83-3 guideline, Sodium chlorite (80.58 % pure) was administered to 16 or 17 females New Zealand White rabbits/dose in drinking water at dose levels of 0, 200, 600 and 1200 ppm (0, 9.7, 26.5 and 39.6 mg chlorite/kg bw/day) from days 7 through 19 of gestation. Dams were sacrificed on gestation day 28.

Oral administration of sodium chlorite during organogenesis at 600 and 1200 mg/L elicited dose-related reductions in maternal water with consequent reductions in maternal food consumption, production of fecal pellets and body weight gain.

Despite the maternal effects there was no evidence of embryo-lethality or teratogenicity. Although the number and mean percentage of major external and visceral and skeletal abnormalities were increased in the 600 and 1200 mg/L groups (external/visceral: 6.6 and 2.9%, respectively, vs. 1.5% in controls; skeletal: 5.4 and 0%, respectively, vs. 0% in controls), the authors did not consider these to be treatment-related adverse effects. Mean foetal weights in the 600 and 1200 mg/L groups were slightly decreased (<9%, relative to controls). In the 600 and 1200 ppm groups, the incidence of minor skeletal abnormalities (13.9 and 14.2% for the 600 and 1200 ppm groups, respectively, vs. 7.7% in controls) and skeletal variants related to incomplete foetal bone ossification was higher than for controls. The authors state in their discussion that these alterations in foetal body weight and delayed ossification indicate embryonic growth retardation. Decreases in maternal food and water consumption and body weight gain may be responsible, at least in part, for some of the foetal effects.

Sodium chlorite was not considered to have any effect on maternal toxicity or reproductive development.

The NOAEL for both maternal and developmental effects in this study was determined to be 1200 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 ClO₂ into ClO_2^- (Abdel Rhaman, 1980a). Using a percentage of 11%, adjusted doses are the following: 0; 88.2; 240; 360 mg/kg bw/d.

Therefore, the NOAEL for both maternal and developmental toxicity of ClO₂ in this study is 360 mg/kg bw/d. As ClO₂ has corrosive properties at doses as low as 40 mg/kg bw (Tos, 1996), effects that will be observed will be linked to corrosive properties.

 

Justification for classification or non-classification

Harmonised classification:

The classification entered in the Annex VI to the CLP Regulation for Chlorine dioxide is not harmonised for the "toxicity to the reproduction" category.

 

 

Self-classification:

Considering the whole data available on reproduction and developmental endpoints, no self-classification is proposed for chlorine dioxide.