Reaction mass of calcium dihydroxide and calcium peroxide

Endpoint summary

Description of key information

Repeated dose toxicity - oral:

Hydrogen peroxide: Key study: OECD Guideline 408. GLP study: Reduced food and water consumption were seen at ≥ 300 ppm. Body weight was reduced in mice receiving 3000 ppm during most of treatment period in male animals. Further signs of treatment indicated the duodenum as target organ with local mucosal hyperplasia at ≥ 1000 ppm. Mucosal hyperplasia in the duodenum was not found in any dose group after recovery. The no observed adverse effect level (NOAEL) was 100 ppm (26 mg/kg bw/day in males and 37 mg/kg bw/day in females).

Calcium lactate: Key study: Similar to OECD Guideline 453. It was concluded that calcium lactate had neither toxic nor carcinogenic activity in F344 rats when it was given continuously in the drinking-water for 2 yr at a dose up to 5% drinking water. Therefore, the NOAEL for both chronic toxicity and carcinogenicity was determined to be 5% drinking water, i.e. 2150 and 2280 mg/kg bw/day to male and female rats respectively, corresponding to 279.5 and 296.4 mgCa/kg bw/day (860 and 570 mgCa/rat).

Repeated dose toxicity - inhalation:

Hydrogen peroxide: Key study: OECD Guideline 412. GLP study. Local effects appeared in the nose with necrosis and inflammation at ≥10 ppm followed by respiratory irritation and reduced body weight gain in higher exposure concentrations. The no observed adverse effect level (NOAEL) was 2.9 mg/m³ (2.03 ppm).

Key value for chemical safety assessment

Repeated dose toxicity: via oral route - systemic effects

Link to relevant study records

Reference

Reference 1

Endpoint:

sub-chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: see 'Remark'

Remarks:

The study was performed in essential accordance with the OECD Guideline for Testing of Chemicals No. 408 with restrictions. No FOB and motor activity measurements were performed as they were not requested by the guideline at the time the study was performed.

Qualifier:

according to guideline

Guideline:

OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity Study in Rodents)

Deviations:

yes

Remarks:

None relevant for the integrity and validity of the study: no FOB and motor activity measurements were performed as they were not requested by the guideline at the time the study was performed (there were no indications for neurotoxic effects); blood clot

GLP compliance:

yes

Limit test:

no

Species:

mouse

Strain:

other: C57BL/6NCrlBR

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories
- Age at study initiation: 5 weeks
- Weight at study initiation:
- Fasting period before study: no data
- Housing: individually in suspended, stainless steel cages with wire bottom
- Diet (e.g. ad libitum): Purina Rodent Chow 5002 (meal) ad libitum
- Water (e.g. ad libitum): ad libitum
- Acclimation period: 7 days


ENVIRONMENTAL CONDITIONS
- Temperature: 65 to 71 °F
- Humidity (%): 41 to 78
- Air changes (per hr): no data
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Route of administration:

oral: drinking water

Vehicle:

water

Details on oral exposure:

The treated (and control) water for this study was prepared twice weekly and administered to the animals on the day of preparation. The drinking water solutions were made by adding preweighed amounts of 35 % hydrogen peroxide to distilled water. The solutions were mixed in carboys for at least 15 minutes prior to dispending the animals. Following administration, unused portions of treated water were stored refrigerated. All equipment for water solution preparation and administration was passivated with nitric acid before use.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Samples of the 100-ppm hydrogen peroxide stock solution and the 3000-ppm hydrogen peroxide stock solution used to dose mice at the 100 ppm and 3000 ppm hydrogen peroxide levels were taken together with a blank sample and analysed for concentration and homogeneity. A colourimetric analytical method designated Test Method APG No. 332 was applied which uses a ferrus thiocyanate reagent. The colour absorbance was measured with a Perkin Elmer Lambda 18 Spectrophotometer. Subsequently, on each of four date one distilled water blank, one control sample of 100 ppm and 3000 ppm hydrogen peroxide and five samples of 100 ppm and 3000 ppm dose solutions were analysed by the same method. Additionally, the 35 % hydrogen peroxide solution was analysed by an iodometric titration at 30 day intervals up to 120 days to test the stability of the solution under the storage conditions (4 °C, vented closed container).

Duration of treatment / exposure:

Approximately 90 days

Frequency of treatment:

Mice received drinking water ad libitum

Remarks:

Doses / Concentrations:
0, 100, 300, 1000 or 3000 ppm
Basis:
nominal in water

No. of animals per sex per dose:

Number of animals per group: 15/sex per treatment group; 10/sex were killed after ceasing the exposure period, whereas 5/sex were submitted to a six week recovery period.

Control animals:

yes

Details on study design:

C57BL/6NCRlBR mice were chosen due to their particular sensitivity to hydrogen peroxide because of a deficient detoxification pathway. The strain can therefore be regarded as a very sensitive animal model for this particular substance.

Observations and examinations performed and frequency:

Clinical signs: daily
Mortality: twice daily
Body weight: weekly
Food consumption: weekly
Water consumption: twice weekly
Blood analysis, haematology, clinical chemistry analyses: blood samples were taken immediately before the scheduled necropsy
Ophthalmic examinations: the eyes of all animals were checked for lesions before the study and one week before the study termination and only animals showing no lesions were used in the study

Sacrifice and pathology:

Animals that died before the study termination underwent a complete necropsy upon discovery of death. Animals sacrificed at their scheduled termination (days 91-93 of treatment period, days 133-134 of recovery period) were anaesthetised, bled for haematology and clinical chemistry determinations, sacrificed via exsanguination then necropsied. Animals were not fasted prior to sacrifice. The weights were determined of brain, liver, kidneys, spleen, testes, adrenals and heart. Samples from various tissues were saved in 10 % buffered formalin. All slides of organs and tissues in the control and high dose groups as well as tissues from mice that died out of schedule were investigated by an experienced pathologist and histological examinations were performed on all gross lesions, the tongue, esophagus, stomach, duodenum, ileum, jejunum, caecum, colon and rectum of all animals from all groups.

Statistics:

Body weights, food consumption, water consumption, absolute organ weights, organ:brain weight ratios, haematology and clinical chemistry data were analysed using the Ebar-Squared trend test. The test compared data from the high-dose group to control and computed a p-value to indicate whether the measured parameter was significantly different (p < 0.05 for statistically significant difference). Subsequent analyses compared data from the next highest dosage group to control, in the direction of the overall trend, and generated another p-value. These analyses continued in a stepwise manner for successively lower groups until the p-value was greater 0.05. When the trend test returned a value greater than 0.05, no subsequent comparisons of the lower dosage groups were performed.

Clinical signs:

no effects observed

Mortality:

no mortality observed

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not specified

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

effects observed, treatment-related

Ophthalmological findings:

no effects observed

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not specified

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

No treatment-related deaths occurred and no treatment-related clinical signs were noted at any time of the study. Male and females exhibited significant reductions in body weight at 3000 ppm. Food and water consumption were significantly reduced at 3000 ppm and notably reduced at 1000 and 300 ppm. Males receiving 3000 ppm displayed significant reductions in total protein and globulin levels in the blood, possibly caused by mucosal hyperplasia occurring in the duodenum of these animals. Necropsy revealed no treatment-related gross lesions. Tissue slides indicated an increase in the cross sectional diameter and wall thickness of the duodenum. Subsequent microscopic evaluations revealed mild mucosal hyperplasia in eight of nine males receiving 3000 ppm and in seven of ten males receiving 1000 ppm. Minimal mucosal hyperplasia was noted in one of ten males receiving 300 ppm. Minimal to mild mucosal hyperplasia was also seen in ten of ten females receiving 3000 ppm and in eight of ten females receiving 1000 ppm. No other areas of the gastrointestinal tract were affected. No evidence of cellular atypia or architectural disruptions nor any other indications of neoplastic changes were observed; therefore, the treatment-related mucosal hyperplasia noted was not considered as a neoplastic lesion.

Key result

Dose descriptor:

NOEL

Effect level:

100 ppm

Sex:

male/female

Basis for effect level:

other: 26 and 37 mg/kg bw/day for males and females, respectively; dose-related reductions in food and water consumption were seen at the next higher doses level of 300 ppm; additionally, duodenal mucosal hyperplasia was observed at 300 ppm

Key result

Critical effects observed:

no

Table 1: Results of analysis of stock solution (35 % hydrogen peroxide), dose solutions (100, 300, 1000 and 3000 ppm), and mean hydrogen peroxide consumption.

Stability of refrigerated 35 % test material			Range of % of target concentration in dose solutions			Hydrogen peroxide consumption (mg/kg/day) based on water consumption and nominal conc.
Time point of sampling	Total % hydrogen peroxide	% change from initial analysis	Group	Target concentration (ppm)	Initial range, % of target	Males	Females
Initial analysis	35.1	NA	1	0	NA	NA	NA
30-day analysis	35.0	-0.3	2	100	94.8-102	26 ± 6.0	37 ± 10.0
60-day analysis	34.7	-1.1	3	300	98.0-105	76 ± 17.1	103 ± 25.5
90-day analysis	34.7	-1.1	4	1000	102-105	239 ± 56.4	328 ± 81.4
120-day analysis	34.7	-1.1	5	3000	101-104	547 ± 95.3	785 ± 194.3

NA: not applicable

Table 2: Development of body weights, food and water consumption throughout the study

sex	males					females
group	0	1	2	3	4	0	1	2	3	4
ppm	0	100	300	1000	3000	0	100	300	1000	3000
Body weight [g/animal]
Day 0	19.7	19.7	19.6	19.6	19.7	16.6	16.6	16.7	16.7	16.7
Day 21	23.2	22.5	22.3	22.7	21.6	19.6	20.0	19.9	19.6	19.1
Day 42	24.9	24.5	24.1	24.5	23.6¯	22.1	22.5	22.3	21.7	21.7
Day 63	26.3	25.5	25.5	25.7	24.9¯	23.2	21.7	22.7	22.5	22.7
Day 91	28.1	27.1	27.3	27.2	26.7¯	25.1	24.9	25.2	24.8	24.1
Weight gain	9.2	8.4	8.7	9.2	7.3¯	8.5	10.3	10.6	10.4	8.9
Mean food consumption [g/animal/week]
Day 7	31	33	31	32	24¯	27	31	26	27	27
Day 35	39	41	35	38	36	56	45	40¯	36¯	39¯
Day 63	40	40	38	40	34¯	68	69	68	62	56¯
Day 91	43	41	43	41	36¯	47	48	46	35¯	39¯
Mean water consumption [g/animal/week]
Day 7	39	41	38	33	25¯	38	42	44	33	26¯
Day 35	52	52	43	47	33¯	90	75	56¯	61¯	52¯
Day 63	39	35	35	32	26¯	46	42	42	42	33¯
Day 91	40	37	41	36	35¯	47	50	48	48	43

-- Indicates a decrease in comparison with controls

Table 3: Results of clinical chemistry (blood samples)

parameter changed		control	100 ppm	300 ppm	1000 ppm	3000 ppm
Males
Total protein	g/dL	4.7	4.6	4.8	4.5	4.2¯
Globulin	g/dL	2.0	2.1	1.9	1.9	1.5¯
Females
Total protein	g/dL	4.9	4.8	4.6	4.7	4.5
Globulin	g/dL	2.3	2.2	2.1	2.1	2.0

-- Indicates a decrease in comparison with controls

Table 4: Incidence of histopathological findings

Parameter	Control		100 ppm		300 ppm		1000 ppm		3000 ppm
	m	f	m	f	m	f	m	f	m	f
number of animals examined	9	10	10	10	10	10	10	10	9	10
duodenum - mucosal hyperplasia	0	0	0	0	1	0	7	8	8	10

Conclusions:

No treatment-related effects were observed at 100 ppm dose level and the LOEL, based on decreased food and water consumption and the observation of duodenal mucosal hyperplasia, was 300 ppm.

Executive summary:

A 90-day oral, subchronic toxicity study with a 35 % aqueous solution of hydrogen peroxide dissolved in drinking water to produce concentrations ranging from 100 to 3000 ppm was performed with C57BL/6NCrlBR mice under GLP conditions and in essential accordance with OECD Guideline No. 408. C57BL/6NCRlBR mice were chosen due to their particular sensitivity to hydrogen peroxide because of a deficient detoxification pathway. The strain can therefore be regarded as a very sensitive animal model for this particular substance. Groups of 15 males and 15 females received different doses of hydrogen peroxide dissolved in their drinking water. After the 90 -day exposure duration, 10 animals/sex of each dose group were sacrificed, while the remaining five animals/sex were submitted to a six week recovery period. No treatment-related mortality or clinical signs were noted throughout the study. No other treatment-related effects were observed at the 100 ppm dose level. At 300 ppm, the consumption of food and water was reduced. Tissue slides indicated an increase in the cross sectional diameter and wall thickness of the duodenum. Subsequent microscopic evaluations revealed mild mucosal hyperplasia in eight of nine males and ten of ten females receiving 3000 ppm and in seven of ten males and eight of ten females receiving 1000 ppm. Minimal mucosal hyperplasia was noted in one of ten males but in none of the females receiving 300 ppm. No other areas of the gastrointestinal tract were affected. Microscopically, no evidence of cellular atypia or architectural disruptions nor any other indications of neoplastic changes were observed; therefore, the treatment-related mucosal hyperplasia noted in the study was not considered as neoplastic lesion. Based on dose-related reductions in food and water consumption and the observation of duodenal mucosal hyperplasia the lowest observed effect level in the study was 300 ppm and the no observed effect level (NOEL) was 100 ppm (26 and 37 mg/kg/day for males and females, respectively). Clinical pathologic effects (decreased total protein and globulin blood levels) were limited to the 3000 ppm level. All effects noted during the treatment period were reversible; animals sacrificed following the recovery period were considered biologically normal.

Reference 2

Endpoint:

chronic toxicity: oral

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

2 (reliable with restrictions)

Rationale for reliability incl. deficiencies:

comparable to guideline study with acceptable restrictions

Qualifier:

equivalent or similar to guideline

Guideline:

OECD Guideline 453 (Combined Chronic Toxicity / Carcinogenicity Studies)

Deviations:

yes

Remarks:

experimental design is poorly described.

Principles of method if other than guideline:

The long-term toxicity/carcinogenicity of calcium lactate, a food additive, was examined in F344 rats. Calcium lactate was given ad lib. in the drinking water at levels of 0, 2.5 or 5 % to groups of 50 male and 50 female rats for two years.

GLP compliance:

not specified

Limit test:

no

Species:

rat

Strain:

Fischer 344

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Japan Inc. (Kanagawa, Japan).
- Females (if applicable) nulliparous and non-pregnant: [yes/no]
- Age at study initiation: 6 wk old
- Weight at study initiation:
- Fasting period before study:
- Housing: Three (or four) males or five females to a plastic cage
- Diet (e.g. ad libitum): Ad libitum, basal diet (CRF-1; Oriental Yeast Inc., Tokyo, Japan)
- Water (e.g. ad libitum): Ad libitum, tap-water
- Acclimation period:

ENVIRONMENTAL CONDITIONS
- Temperature (°C): 24 ± 1
- Humidity (%): 55 ± 5

Route of administration:

oral: drinking water

Details on route of administration:

The calcium lactate solutions were replaced with freshly prepared solutions three times a week, on which occasions the amount of solutions consumed was measured in order to calculate the intake of calcium lactate. Administration of the compound ended after 104 wk, and the rats were then given distilled water for a recovery period of 9wk.

Vehicle:

water

Details on oral exposure:

- PREPARATION OF DOSING SOLUTIONS:
Calcium lactate was dissolved in distilled water at levels of 0 (control), 2.5 or 5%.

Duration of treatment / exposure:

Treatment: 104 weeks.
Recovery period: 9 weeks.

Frequency of treatment:

Daily.

Dose / conc.:

5 other: % drinking water

Dose / conc.:

2.5 other: % drinking water

No. of animals per sex per dose:

50

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: These doses were selected after a 13-wk subchronic toxicity study done prior to the present study (Matsushima et al., 1989).

Observations and examinations performed and frequency:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule: Daily

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: Daily

BODY WEIGHT: Yes
- Time schedule for examinations: Once a week for the first 13 wk of the study, and every 4 wk thereafter.

WATER CONSUMPTION AND COMPOUND INTAKE (if drinking water study): Yes
- Time schedule for examinations: The calcium lactate solutions were replaced with freshly prepared solutions three times a week, on which occasions the amount of solutions consumed was measured in order to calculate the intake of calcium lactate.

OPHTHALMOSCOPIC EXAMINATION: Not specified

HAEMATOLOGY: Yes

CLINICAL CHEMISTRY: Yes

URINALYSIS: Not specified

NEUROBEHAVIOURAL EXAMINATION: Not specified

Sacrifice and pathology:

GROSS PATHOLOGY: Yes
The animals were then examined macro- and microscopically for the presence of non-neoplastic and neoplastic lesions.

HISTOPATHOLOGY: Yes
All organs and/or tissues were routinely fixed in 10% buffered formalin, sectioned and stained with haematoxylin and eosin.

Statistics:

Statistical analyses were performed using Fisher's exact probability test and/or the chi-square test, and also the age-adjusted statistical test recommended by Peto et al. (1980).

Clinical signs:

not specified

Mortality:

mortality observed, non-treatment-related

Description (incidence):

In females, the mortality rate in the 5% group was slightly higher than those in the other two groups. This difference, however, was not significant.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

Throughout the administration period, there was a dose-dependent inhibitory effect of calcium lactate on the growth of rats of both sexes. Compared with the controls, a 13% decrease in body-weight gain was observed in male and female rats of the high-dose group.

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

no effects observed

Description (incidence and severity):

Daily water consumption was almost constant in all groups of both sexes.

Haematological findings:

no effects observed

Description (incidence and severity):

No specific dose-related changes were observed in any of the haematological parameters.

Clinical biochemistry findings:

no effects observed

Description (incidence and severity):

No specific dose-related changes were observed in any of the biochemical parameters.

Organ weight findings including organ / body weight ratios:

effects observed, non-treatment-related

Description (incidence and severity):

A significant dose-dependent increase was observed in the relative brain weights of both male and female rats although no histological change was detected. Females in the 5% group exhibited slightly but significantly higher kidney weights compared with controls.

Gross pathological findings:

effects observed, non-treatment-related

Description (incidence and severity):

A number of nonneoplastic lesions (e.g. myocardial fibrosis, bile-duct proliferation, hepatic microgranulomas and chronic nephropathy) were observed in all groups, with no difference in their incidences and/or degrees.

Histopathological findings: non-neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

There was no difference in the severity of chronic nephropathy between different groups. No toxic lesions such as severe cortico-medullary nephrocalcinosis were observed in the kidney of females in the 5% group despite a slight increase in calcium deposition in the papilla compared with controls. There was no difference in histological findings of the parathyroid gland between different groups of both sexes; also no metastatic calcification was detected in the various organs/tissues.

Histopathological findings: neoplastic:

effects observed, non-treatment-related

Description (incidence and severity):

The incidences of total tumours were 100% in all the male groups. In females, they were 80-86%, and there was no significant difference in the incidences between different groups. Tumours were found in many organs and/or tissues in all groups including the controls. In males from all groups, tumours of the testis were the most frequent, followed by those of the adrenal gland, thyroid gland, pituitary gland, haematopoietic organs, mammary gland, lung and pancreas. In females, the commonest tumours were those of the uterus, pituitary gland, haematopoietic organs, mammary gland, thyroid gland, adrenal gland and pancreas. Tumours were also detected in other organs/tissues from rats of all groups, but at lower incidences. Histologically, all the tumours observed in this experiment were similar to those known to occur spontaneously in F344 rats. None of the experimental groups showed a significant increase in the incidence of any specific tumours compared with the corresponding control value (chisquare and/or Fisher's test), and also no positive trend was noted in the occurrence of any tumour. Male rats in the 5% group, however, showed a slightly higher incidence of pheochromocytomas compared with the present controls and also with our historical controls (12-28%). The incidence of adrenal medullary hyperplasias in the high-dose group (24%) was also higher than that in the lowdose
and control groups (12% and 10%, respectively). A positive trend was observed in the occurrence of the two types of lesions (combined hyperplasias and pheochromocytomas).

Key result

Dose descriptor:

NOAEL

Effect level:

2 150 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male

Basis for effect level:

other: No test item related adverse effects at the highest dose

Remarks on result:

other: corresponding to 279.5 mgCa/kg bw/day (860 mgCa/rat)

Key result

Dose descriptor:

NOAEL

Effect level:

2 280 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

female

Basis for effect level:

other: No test item related adverse effect observed at the highest dose tested

Remarks on result:

other: corresponding to 296.4 mgCa/kg bw/day (570 mgCa/rat)

Key result

Critical effects observed:

no

Conclusions:

It was concluded that calcium lactate had neither toxic nor carcinogenic activity in F344 rats when it was given continuously in the drinking-water for 2 yr at a dose up to 5% drinking water. Therefore, the NOAEL for both chronic toxicity and carcinogenicity was determined to be 5% drinking water, i.e. 2150 and 2280 mg/kg bw/day to male and female rats respectively, corresponding to 279.5 and 296.4 mgCa/kg bw/day (860 and 570 mgCa/rat).

Executive summary:

The aim of the present study was to examine the toxicity/carcinogenicity of calcium lactate ingested over a long period. 50 F344 rats per sex and dose were exposed to calcium lactate by drinking water. Based in a previous 13 weeks subchronic toxicity test, calcium lactate was dissolved in distilled water at levels of 0 (control), 2.5 or 5%. The calcium lactate solutions were replaced with freshly prepared solutions three times a week, on which occasions the amount of solutions consumed was measured in order to calculate the intake of calcium lactate. Administration of the compound ended after 104 weeks, and the rats were then given distilled water for a recovery period of 9 weeks. At week 113, all surviving animals were killed and autopsied. Haematological and biochemical examinations were also carried out in these rats. They were observed daily and clinical signs and deaths were recorded. Body weights were measured once a week for the first 13 weeks of the study, and every 4 week thereafter. An autopsy was immediately performed on rats that died (or were killed when moribund) during the study and those killed at the end of the study. The animals were then examined macro- and microscopically for the presence of non-neoplastic and neoplastic lesions. All organs and/or tissues were routinely fixed in 10% buffered formalin, sectioned and stained with haematoxylin and eosin. In females, the mortality rate in the 5% group was slightly higher than those in the other two groups. This difference, however, was not significant. Throughout the administration period, there was a dose-dependent inhibitory effect of calcium lactate on the growth of rats of both sexes. Compared with the controls, a 13% decrease in body-weight gain was observed in male and female rats of the high-dose group. No specific dose-related changes were observed in any of the haematological and biochemical parameters. The anatomical sites and histological characteristics of the tumours were similar to those of spontaneous tumours, which occur commonly in the strain of rats studied. There is an established link between serum calcium levels and adrenal medullary function. Rats are very susceptible to the development of hyperplastic and neoplastic changes in the adrenal medulla in response to high levels of serum calcium. However, the serum calcium levels recorded in treated rats of both sexes were not different from the corresponding control levels. There was also no difference in histological findings of the thyroid and parathyroid glands and kidney and bone between different groups. In addition, the incidence of pheochromocytomas observed in the present male controls was also higher than that in our historical controls. Furthermore, no significant increase or positive trend in the incidences of hyperplasias/pheochromocytomas was detected in females from all groups. Thus, we consider that the increase in hyperplasias/pheochromocytomas of male rats in the high-dose group was probably due to experimental variability and not related to treatment with calcium lactate. The results from this study also show that many kinds of non-neoplastic lesions were detected in male and female rats from all groups. No clear toxic lesions specifically caused by long-term administration of calcium lactate, except for the slight calcium deposition in the renal papilla, were detected in any organ. The type of lesion observed in the kidney of female rats in the 5% group was histologically different from the so-called nephrocalcinosis, which is characterized by an intraluminal deposition of calcium observed mainly in the cortico-medullary region. The pathogenesis of this lesion is unclear and might depend on the increase in the urinary calcium level. The observed increase in relative brain weights of males and females in the high-dose group may result from the decrease in body-weight gains and not to the toxic effect of calcium lactate. It was concluded that calcium lactate had neither toxic nor carcinogenic activity in F344 rats when it was given continuously in the drinking-water for 2 yr. Based on these results, the NOAEL for both chronic toxicity and carcinogenicity was determined to be 5% drinking water, i.e. 2150 and 2280 mg/kg bw/day to male and female rats respectively, corresponding to 279.5 and 296.4 mgCa/kg bw/day (860 and 570 mgCa/rat).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - systemic effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with the respective OECD Guidelines and under GLP conditions. No deviations were reported that were likely to negatively influence the outcomes of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:ApfSD (Wistar derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, UK
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: for groups 1-4, 242.5 +/- 9.6 g (males), 190.2 +/- 12.3 g (females); four groups five and six, 300.3 +/- 9.1 g (males), 233.8 +/- 11.0 g (females)
- Fasting period before study:
- Housing: five rats, sexes separately, in stainless steel cages
- Diet (e.g. ad libitum): CT1 supplied by Special Diet Services Ltd, Witham, UK ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

Test atmospheres were generated using a glass concentric-jet atomiser to generate fine aerosol directly into a 3-necked quick-fit round bottomed flask, heated by placing it in a waterbath at 80 °C. The test substance was pumped to the atomiser using a peristaltic pump, typically operating at a pump speed giving a flow rate of test material of approximately 1 mL/min. Clean, dry air was passed through the atomiser at nominal flow rates of 2, 10 or 15 L/minute for groups 2, 3 and 4 respectively, and (together with heated generation air at 25 L/minute) carried the atmosphere to the lng term exposure chamber. Diluting air was added directly to the exposure chambers at a flow rate of 500-600 L/min. Air flows were monitored continuously using variable area flowmeters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres were sampled by passing the atmosphere, at a fixed flow rate for a known time period, through a known volume of de-ionised water in a midget impinger. The resulting solutions were analysed by flow injection analysis using a LC Module 1 (Waters) separations module at a flow rate of 5 L/minute, a dilute Cobalt-bicarbonate reagent mobile phase, and a 486 series UV detector (Waters) at 260 nm. The limit of detection of the method was assessed to be approximately 0.1 mg/mL corresponding to an atmosphere concentration of 0.1 ppm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours daily, 5 days per week

Remarks:

Doses / Concentrations:
2.03, 10.3, 23.3, 58.1/27.3 ppm (2.88, 14.6, 33, 82.4/38.7 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

five males, five females

Control animals:

yes

Details on study design:

Another group (group 4 of the main test) of animals was exposed to 60 ppm at day 1, 4, 5 and 6. Thereafter, the exposure level was reduced to 30 ppm at day 11 and 12. The treatment was terminated on day 13 and animals were sacrificed due to toxicity. The group treated with a target concentration of 25 ppm was introduced later in the test after termination of the test group 4 (60/30 ppm).

Observations and examinations performed and frequency:

Clinical observations: prior to the start of the study, frequently during exposure and at the end of the 6 hour exposure duration, daily before exposure
Body weight: before study initiation, weekly during exposure study
Food consumption: continuously throughout the study
Clinical pathology (haematology, blood clinical chemistry): at termination of study

Sacrifice and pathology:

Animals were killed by an overdose of halothane Ph. Eur. vapour followed by exsanguination. Weights of adrenal glands, kidneys, liver, lungs and testes were measured. All animals were subjected to full examination post mortem including external and careful internal examination of all organs and structures. Slides were prepared from various organs and tissues. All submitted tissues from control and high exposure animals together with the lungs, liver, kidney, trachea, nasal passages and abnormal tissues from the low and mid exposure groups were routinely processed, embedded in paraffin wax, sectioned at 5 micrometre and stained with haematoxylin and eosin. Examination by light microscopy was performed for these tissues.

Statistics:

Body weights were considered by analysis of covariance on initial body weight, separately for males and females. Haematology and blood clinical chemistry were considered by analysis of variance. Male and female data were analysed together. Organ weights were considered by analysis of variance and of covariance on final body weight, separately for males and females. Analyses were carried separately for main study and additional group 5 and 6 animals. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least-squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs were seen in animals exposed to 10.3 ppm and greater and in general the number and severity of these clinical signs increased with repeated exposure at low doses, whereas the onset of clinical signs was earlier at higher doses but also a certain degree of recovery from symptoms was seen at higher doses. Signs included reddening of the nose, stains around the snout, stains around the mouth, signs of salivation, signs of respiratory tract irritation, irregular breathing, signs of urinary incontinence, piloerection, chomodacryorrhoea, hunched posture, increased response to touch, thin appearance. Some evidence of recovery from these symptoms was seen during periods of non-exposure. Body weights gradually decreased in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Food consumption was affected in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Minor effects on haematology were seen at exposure levels of 23.3 ppm, which were considered as not biologically and toxicologically significant. In both sexes there was a minimal decrease in albumin and total protein levels at 23.3 ppm exposure. Kidney weight was increased in females exposed to 23.3 ppm and lung/body weight ratio in males and kidney/body weight ratios in females exposed to 23.3 ppm was increased. Treatment-related findings were seen in the nasal and oral cavities of rats at the necropsy following termination of the study. Staining of the nares was seen at 10 ppm and above and mouth staining was at 25 ppm. In both instances, no dose-response could be found. Increased incidences of findings in exposed animals over controls during the microscopic examinations were seen in the nasal cavity, larynx and lung including necrosis, inflammation and perivascular neutrophil infiltration.

Key result

Dose descriptor:

NOAEL

Effect level:

2.9 mg/m³ air

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

14.6 mg/m³ air

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Results of the clinical chemistry haematology

Parameter changed	Control group 1 (0 ppm)	2 ppm	10 ppm	Control group 2 (0 ppm)^A	25 ppm^A
Males
Mean cell volume (fl)	56.1	56.2	56.0	55.3	55.3*
Mean cell haemoglobin (pg)	19.4	19.6	19.4	19.0	18.8*
Albumin (g/L)	33.6	32.8	33.1	32.7	31.3*
Total protein (g/L)	66.	64.9	65.1	63.3	57.8**
Females
Red blood cell count (10e12/L)	7.56	7.72	7.42	7.77	7.43*
Albumin (g/L)	33.2	33.0	32.8	32.6	31.3*
Total protein (g/L)	61.1	61.3	60.2	61.4	57.0*
Glucose (mmol/L)	12.4	13.7	12.9	12.8	9.6**

A) 25 ppm group is compared to control group 2 since animals were treated simultaneously. *p <0.05, **p <0.01 (student's t-test, two-sided)

Table 2: Microscopic findings

Target organs	Control (0 ppm)	2 ppm	10 ppm	25 ppm
Nasal cavity	No finding	No finding	Necrosis and inflammation (sqamous epithelium, anterior region of nasal cavity) 3/5 males, 2/5 females	Rhinitis 1/5 males Necrosis and inflammation (sqamous epithelium, anterior regions of nasal cavity) 4/5 males, 4/5 females
Larynx	No finding	Inflammation 1/5 females	No finding	Mononuclear cell infiltration 2/5 females Epithelia erosion 1/5 males
Lung	No finding	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males, 1/5 females	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males	Increase in perivascular neurophil infiltration 1/5 males, 2/5 females

Conclusions:

Whole body exposure to hydrogen peroxide vapour for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm resulted in signs of general toxicity in males exposed to 23.3 ppm and were consistent with the material being a respiratory tract irritant. Treatment-related microscopic changes were seen in the nasal cavity in animals exposed to 10.3 ppm or above. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide.

Executive summary:

A repeated dose inhalation toxicity study was performed with male and female Alpk:APfSD (Wistar-derived) rats exposed to hydrogen peroxide vapours for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm. The study was carried out under GLP conditions and in accordance with OECD Guideline No. 412. Treatment of a group exposed initially to 58.1 ppm and subsequently to 27.3 ppm was terminated before schedule due to the toxicity of the test material. Clinical observations were consistent with the material being a respiratory tract irritant (reddened noses, stains around the nose, abnormal respiratory noise) and in general the time to onset, incidence and severity of clinical signs increased with exposure concentration and repeated exposure. Males exposed to 23.3 ppm hydrogen peroxide showed lower food consumption and body weight gain compared to controls. Minimal changes in albumin and total protein blood levels were found in males and females exposed to 23.3 ppm. Histopathological, treatment-related changes were seen in the anterior-most regions of the nasal cavity lined with squamous epithelium, where minimal to slight necrosis (with associated inflammation) and rhinitis were seen in animals exposed to 10.3 and 23.3 ppm hydrogen peroxide. Inflammation and epithelial erosion in the larynx and increased perivascular neutrophil infiltration in the lungs were considered unlikely to be related to treatment in the absence of a clear dose response relationship. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide (corresponding to 2.9 mg/m3).

Endpoint conclusion

Endpoint conclusion:

no adverse effect observed

Repeated dose toxicity: inhalation - local effects

Link to relevant study records

Reference

Endpoint:

short-term repeated dose toxicity: inhalation

Type of information:

experimental study

Adequacy of study:

key study

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

other: The study was carried out in accordance with the respective OECD Guidelines and under GLP conditions. No deviations were reported that were likely to negatively influence the outcomes of the study.

Qualifier:

according to guideline

Guideline:

OECD Guideline 412 (Subacute Inhalation Toxicity: 28-Day Study)

Deviations:

no

GLP compliance:

yes

Limit test:

no

Species:

rat

Strain:

other: Alpk:ApfSD (Wistar derived)

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Rodent Breeding Unit, Alderley Park, Macclesfield, UK
- Age at study initiation: 6 to 7 weeks
- Weight at study initiation: for groups 1-4, 242.5 +/- 9.6 g (males), 190.2 +/- 12.3 g (females); four groups five and six, 300.3 +/- 9.1 g (males), 233.8 +/- 11.0 g (females)
- Fasting period before study:
- Housing: five rats, sexes separately, in stainless steel cages
- Diet (e.g. ad libitum): CT1 supplied by Special Diet Services Ltd, Witham, UK ad libitum
- Water (e.g. ad libitum): mains water ad libitum
- Acclimation period: 5 days


ENVIRONMENTAL CONDITIONS
- Temperature (°C): 22 +/- 3
- Humidity (%): 30-70
- Air changes (per hr): at least 15
- Photoperiod (hrs dark / hrs light): 12 hours light/12 hours darkness

Route of administration:

inhalation

Type of inhalation exposure:

whole body

Vehicle:

other: unchanged (no vehicle)

Remarks on MMAD:

MMAD / GSD: not applicable

Details on inhalation exposure:

Test atmospheres were generated using a glass concentric-jet atomiser to generate fine aerosol directly into a 3-necked quick-fit round bottomed flask, heated by placing it in a waterbath at 80 °C. The test substance was pumped to the atomiser using a peristaltic pump, typically operating at a pump speed giving a flow rate of test material of approximately 1 mL/min. Clean, dry air was passed through the atomiser at nominal flow rates of 2, 10 or 15 L/minute for groups 2, 3 and 4 respectively, and (together with heated generation air at 25 L/minute) carried the atmosphere to the lng term exposure chamber. Diluting air was added directly to the exposure chambers at a flow rate of 500-600 L/min. Air flows were monitored continuously using variable area flowmeters.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

Test atmospheres were sampled by passing the atmosphere, at a fixed flow rate for a known time period, through a known volume of de-ionised water in a midget impinger. The resulting solutions were analysed by flow injection analysis using a LC Module 1 (Waters) separations module at a flow rate of 5 L/minute, a dilute Cobalt-bicarbonate reagent mobile phase, and a 486 series UV detector (Waters) at 260 nm. The limit of detection of the method was assessed to be approximately 0.1 mg/mL corresponding to an atmosphere concentration of 0.1 ppm.

Duration of treatment / exposure:

28 days

Frequency of treatment:

6 hours daily, 5 days per week

Remarks:

Doses / Concentrations:
2.03, 10.3, 23.3, 58.1/27.3 ppm (2.88, 14.6, 33, 82.4/38.7 mg/m3)
Basis:
analytical conc.

No. of animals per sex per dose:

five males, five females

Control animals:

yes

Details on study design:

Another group (group 4 of the main test) of animals was exposed to 60 ppm at day 1, 4, 5 and 6. Thereafter, the exposure level was reduced to 30 ppm at day 11 and 12. The treatment was terminated on day 13 and animals were sacrificed due to toxicity. The group treated with a target concentration of 25 ppm was introduced later in the test after termination of the test group 4 (60/30 ppm).

Observations and examinations performed and frequency:

Clinical observations: prior to the start of the study, frequently during exposure and at the end of the 6 hour exposure duration, daily before exposure
Body weight: before study initiation, weekly during exposure study
Food consumption: continuously throughout the study
Clinical pathology (haematology, blood clinical chemistry): at termination of study

Sacrifice and pathology:

Animals were killed by an overdose of halothane Ph. Eur. vapour followed by exsanguination. Weights of adrenal glands, kidneys, liver, lungs and testes were measured. All animals were subjected to full examination post mortem including external and careful internal examination of all organs and structures. Slides were prepared from various organs and tissues. All submitted tissues from control and high exposure animals together with the lungs, liver, kidney, trachea, nasal passages and abnormal tissues from the low and mid exposure groups were routinely processed, embedded in paraffin wax, sectioned at 5 micrometre and stained with haematoxylin and eosin. Examination by light microscopy was performed for these tissues.

Statistics:

Body weights were considered by analysis of covariance on initial body weight, separately for males and females. Haematology and blood clinical chemistry were considered by analysis of variance. Male and female data were analysed together. Organ weights were considered by analysis of variance and of covariance on final body weight, separately for males and females. Analyses were carried separately for main study and additional group 5 and 6 animals. Unbiased estimates of differences from control were provided by the difference between each treatment group least-squares mean and the control group least-squares mean. Differences from control were tested statistically by comparing each treatment group least-squares mean with the control group least squares mean using a two-sided Student's t-test, based on the error mean square in the analysis.

Clinical signs:

effects observed, treatment-related

Mortality:

mortality observed, treatment-related

Body weight and weight changes:

effects observed, treatment-related

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Food efficiency:

not examined

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

not examined

Ophthalmological findings:

not examined

Haematological findings:

no effects observed

Clinical biochemistry findings:

effects observed, treatment-related

Urinalysis findings:

not examined

Behaviour (functional findings):

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Gross pathological findings:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Histopathological findings: neoplastic:

no effects observed

Details on results:

Clinical signs were seen in animals exposed to 10.3 ppm and greater and in general the number and severity of these clinical signs increased with repeated exposure at low doses, whereas the onset of clinical signs was earlier at higher doses but also a certain degree of recovery from symptoms was seen at higher doses. Signs included reddening of the nose, stains around the snout, stains around the mouth, signs of salivation, signs of respiratory tract irritation, irregular breathing, signs of urinary incontinence, piloerection, chomodacryorrhoea, hunched posture, increased response to touch, thin appearance. Some evidence of recovery from these symptoms was seen during periods of non-exposure. Body weights gradually decreased in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Food consumption was affected in males exposed to 23.3 ppm and in males and females exposed to 58.1/27.3 ppm. Minor effects on haematology were seen at exposure levels of 23.3 ppm, which were considered as not biologically and toxicologically significant. In both sexes there was a minimal decrease in albumin and total protein levels at 23.3 ppm exposure. Kidney weight was increased in females exposed to 23.3 ppm and lung/body weight ratio in males and kidney/body weight ratios in females exposed to 23.3 ppm was increased. Treatment-related findings were seen in the nasal and oral cavities of rats at the necropsy following termination of the study. Staining of the nares was seen at 10 ppm and above and mouth staining was at 25 ppm. In both instances, no dose-response could be found. Increased incidences of findings in exposed animals over controls during the microscopic examinations were seen in the nasal cavity, larynx and lung including necrosis, inflammation and perivascular neutrophil infiltration.

Key result

Dose descriptor:

NOAEL

Effect level:

2.9 mg/m³ air

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Dose descriptor:

LOAEL

Effect level:

14.6 mg/m³ air

Sex:

male/female

Basis for effect level:

histopathology: non-neoplastic

Key result

Critical effects observed:

no

Table 1: Results of the clinical chemistry haematology

Parameter changed	Control group 1 (0 ppm)	2 ppm	10 ppm	Control group 2 (0 ppm)^A	25 ppm^A
Males
Mean cell volume (fl)	56.1	56.2	56.0	55.3	55.3*
Mean cell haemoglobin (pg)	19.4	19.6	19.4	19.0	18.8*
Albumin (g/L)	33.6	32.8	33.1	32.7	31.3*
Total protein (g/L)	66.	64.9	65.1	63.3	57.8**
Females
Red blood cell count (10e12/L)	7.56	7.72	7.42	7.77	7.43*
Albumin (g/L)	33.2	33.0	32.8	32.6	31.3*
Total protein (g/L)	61.1	61.3	60.2	61.4	57.0*
Glucose (mmol/L)	12.4	13.7	12.9	12.8	9.6**

A) 25 ppm group is compared to control group 2 since animals were treated simultaneously. *p <0.05, **p <0.01 (student's t-test, two-sided)

Table 2: Microscopic findings

Target organs	Control (0 ppm)	2 ppm	10 ppm	25 ppm
Nasal cavity	No finding	No finding	Necrosis and inflammation (sqamous epithelium, anterior region of nasal cavity) 3/5 males, 2/5 females	Rhinitis 1/5 males Necrosis and inflammation (sqamous epithelium, anterior regions of nasal cavity) 4/5 males, 4/5 females
Larynx	No finding	Inflammation 1/5 females	No finding	Mononuclear cell infiltration 2/5 females Epithelia erosion 1/5 males
Lung	No finding	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males, 1/5 females	Increase in perivascular neurophil infiltration 1/5 males Haemorrhage 2/5 males	Increase in perivascular neurophil infiltration 1/5 males, 2/5 females

Conclusions:

Whole body exposure to hydrogen peroxide vapour for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm resulted in signs of general toxicity in males exposed to 23.3 ppm and were consistent with the material being a respiratory tract irritant. Treatment-related microscopic changes were seen in the nasal cavity in animals exposed to 10.3 ppm or above. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide.

Executive summary:

A repeated dose inhalation toxicity study was performed with male and female Alpk:APfSD (Wistar-derived) rats exposed to hydrogen peroxide vapours for 6 hours per day, 5 days per week for a period of 28 days at concentrations of 2.03, 10.3 or 23.3 ppm. The study was carried out under GLP conditions and in accordance with OECD Guideline No. 412. Treatment of a group exposed initially to 58.1 ppm and subsequently to 27.3 ppm was terminated before schedule due to the toxicity of the test material. Clinical observations were consistent with the material being a respiratory tract irritant (reddened noses, stains around the nose, abnormal respiratory noise) and in general the time to onset, incidence and severity of clinical signs increased with exposure concentration and repeated exposure. Males exposed to 23.3 ppm hydrogen peroxide showed lower food consumption and body weight gain compared to controls. Minimal changes in albumin and total protein blood levels were found in males and females exposed to 23.3 ppm. Histopathological, treatment-related changes were seen in the anterior-most regions of the nasal cavity lined with squamous epithelium, where minimal to slight necrosis (with associated inflammation) and rhinitis were seen in animals exposed to 10.3 and 23.3 ppm hydrogen peroxide. Inflammation and epithelial erosion in the larynx and increased perivascular neutrophil infiltration in the lungs were considered unlikely to be related to treatment in the absence of a clear dose response relationship. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide (corresponding to 2.9 mg/m3).

Endpoint conclusion

Endpoint conclusion:

adverse effect observed

Dose descriptor:

NOAEC

2.9 mg/m³

Study duration:

subacute

Species:

rat

Quality of whole database:

One key studies available of Klimish score = 1.

Repeated dose toxicity: dermal - systemic effects

Endpoint conclusion

Endpoint conclusion:

no study available

Repeated dose toxicity: dermal - local effects

Endpoint conclusion

Endpoint conclusion:

no study available

Mode of Action Analysis / Human Relevance Framework

Calcium peroxide when in contact with water, on the one hand hydrolyses into hydrogen peroxide, and on the other hand results in a loss of active oxygen.

CaO₂  + H₂O → CaO + H₂O₂

2CaO₂→ 2CaO + O₂

2CaO₂+ 2H₂O → 2Ca²⁺+ 4OH^- + O₂

 

The toxicity of the reaction mass is due to the hydrolysis product hydrogen peroxide (H2O2) and calcium hydroxide (Ca(OH)2). Under physiological conditions, calcium hydroxide ultimately dissociates into calcium cations (Ca2 +) and hydroxyl anions (OH-). Calcium, is an essential and abundantly available mineral nutrient. Hydroxyl anion is neutralised in body fluids.

Additional information

Repeated dose toxicity - oral:

Hydrogen peroxide:

A reliable, well-conducted 90-day study with a catalase deficient strain of mice found a decrease in body weight at doses of 3000 ppm in drinking water (Freeman 1997). The study indicated that the NOAEL of hydrogen peroxide in drinking water was 100 ppm implying a daily dose of 26 mg/kg bw for males and 37 mg/kg bw for females. The LOAEL was 300 ppm (76 mg/kg bw for males, 103 mg/kg bw for females) based on dose-related reductions in food and water consumption and on the observation of duodenal mucosal hyperplasia in one male. Hyperplasia was a consistent finding at the higher levels of 1,000 and 3,000 ppm both in males and females (corresponding daily doses were 239 mg/kg for males, 328 mg/kg for females and 547 mg/kg for males, 785 mg/kg for females, respectively), and it was completely reversible in the recovery period. At the top dose (3,000 ppm) plasma total protein and globulin concentrations were reduced.

Calcium lactate:

A reliable, well-conducted 2 years combined chronic toxicity/carcinogenicity study, concluded that calcium lactate had neither toxic nor carcinogenic activity in F344 rats when it was given continuously in the drinking-water. In females, the mortality rate in the 5% group was slightly higher than those in the other two groups. This difference, however, was not significant. Throughout the administration period, there was a dose-dependent inhibitory effect of calcium lactate on the growth of rats of both sexes. Compared with the controls, a 13% decrease in body-weight gain was observed in male and female rats of the high-dose group. No specific dose-related changes were observed in any of the haematological and biochemical parameters. Increase in hyperplasias/pheochromocytomas of male rats in the high-dose group was probably due to experimental variability and not related to treatment with calcium lactate. No clear toxic lesions specifically caused by long-term administration of calcium lactate, except for the slight calcium deposition in the renal papilla, were detected in any organ. The type of lesion observed in the kidney of female rats in the 5% group was histologically different from the so-called nephrocalcinosis, which is characterized by an intraluminal deposition of calcium observed mainly in the cortico-medullary region. The pathogenesis of this lesion is unclear and might depend on the increase in the urinary calcium level. The observed increase in relative brain weights of males and females in the high-dose group may result from the decrease in body-weight gains and not to the toxic effect of calcium lactate. Based on these results, the NOAEL for both chronic toxicity and carcinogenicity was determined to be 5% drinking water, i.e. 2150 and 2280 mg/kg bw/day to male and female rats respectively, corresponding to 279.5 and 296.4 mgCa/kg bw/day (860 and 570 mgCa/rat).

Repeated dose toxicity - inhalation:

Hydrogen peroxide:

A reliable 28-day repeated dose inhalation toxicity study was performed with male and female Alpk:APfSD (Wistar-derived) rats exposed to hydrogen peroxide vapours for 6 hours per day, 5 days per week at concentrations of 2.03, 10.3 or 23.3 ppm (Kilgour 2002). The study was carried out under GLP conditions and in accordance with OECD Guideline No. 412. Treatment of a group exposed initially to 58.1 ppm and subsequently to 27.3 ppm was terminated before schedule due to the toxicity of the test material. Clinical observations were consistent with the material being a respiratory tract irritant (reddened noses, stains around the nose, abnormal respiratory noise) and in general the time to onset, incidence and severity of clinical signs increased with exposure concentration and repeated exposure. Males exposed to 23.3 ppm hydrogen peroxide showed lower food consumption and body weight gain compared to controls. Minimal changes in albumin and total protein blood levels were found in males and females exposed to 23.3 ppm. Histopathological, treatment-related changes were seen in the anterior-most regions of the nasal cavity lined with squamous epithelium, where minimal to slight necrosis (with associated inflammation) and rhinitis were seen in animals exposed to 10.3 and 23.3 ppm hydrogen peroxide. Inflammation and epithelial erosion in the larynx and increased perivascular neutrophil infiltration in the lungs were considered unlikely to be related to treatment in the absence of a clear dose response relationship. The no observed effect level (NOEL) for the study was considered to be 2.03 ppm hydrogen peroxide (corresponding to 2.9 mg/m3).

Justification for classification or non-classification

Based on available data, the substance is not classified for repeated dose toxicity according to the CLP Regulation (EC) no. 1272/2008. Hydrogen peroxide should not be classified for systemic effects as it is not systemically available and as the substance exhibits only local irritancy.