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Repeated dose toxicity: oral

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Administrative data

Endpoint:
sub-chronic toxicity: oral
Type of information:
experimental study
Adequacy of study:
key study
Study period:
Experimental completion date: 7 August 2002
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: GLP Guideline study

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2003
Report Date:
2003
Reference Type:
other company data
Title:
Unnamed
Year:
2004
Report Date:
2004

Materials and methods

Test guidelineopen allclose all
Qualifier:
according to
Guideline:
EU Method B.26 (Sub-Chronic Oral Toxicity Test: Repeated Dose 90-Day Oral Toxicity Study in Rodents)
Version / remarks:
(30 May 1988)
Deviations:
yes
Remarks:
(haematocrit not determined, but not of relevance)
Qualifier:
according to
Guideline:
OECD Guideline 408 (Repeated Dose 90-Day Oral Toxicity in Rodents)
Version / remarks:
(21 September 1998)
GLP compliance:
yes (incl. certificate)
Limit test:
no

Test material

Reference
Name:
Unnamed
Type:
Constituent

Test animals

Species:
rat
Strain:
Sprague-Dawley
Sex:
male/female
Details on test animals and environmental conditions:
- Strain: Sprague-Dawley: Crl CD (SD) IGS BR.
- Source: Charles River, l’Arbresle, France
- Age/weight at study initiation: On the first day of treatment, the animals were approximately 6 weeks old and had a mean body weight of 190 g (range: 175 g to 208 g) for the males and 160 g (range: 145 g to179 g) for the females.

Administration / exposure

Route of administration:
oral: gavage
Vehicle:
water
Details on oral exposure:
- Concentration in vehicle: 10, 33.3, 100 mg/mL from day 1 to 10; 10, 33.3, 66.7 mg/mL from day 11 to 22; 3.3, 10, 33.3 mg/mL from day 23 to 92
- Total volume applied: 1.5mL/kg bw/day
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
An aliquot of each dosage form was sampled then diluted appropriately for analysis by pHmetry. The acetic acid in the test item was titrated by a solution of sodium hydroxide and the equivalence point was determined from the curve pH versus volume of sodium hydroxide added (mL). Hereafter the concentration PAA was calculated using the measured concentration acetic acid.

Throughout the study, a satisfactory agreement was observed between the nominal and actual concentrations of the test item in the administered dosage forms since the deviations from nominal concentrations were in acceptable range of +/- 10 %.
Duration of treatment / exposure:
13 weeks ( 92 - 93 days) [exception: 82 days for the substitute surviving animals (two males and one female in high-dose group) which were sacrificed at the same time as the other animals of the study].
Frequency of treatment:
daily via the drinking water (ad libitum).
Doses / concentrationsopen allclose all
Remarks:
Doses / Concentrations:
0, 15, 50, 150 mg Acide peracétique 5%/kg bw/day from day 1 to 10; 0, 15, 50, 100 mg/kg bw/day from day 11 to 22; 0, 5, 15, 50 mg/kg bw/day from day 23 to 92
Basis:
nominal in water
Remarks:
Doses / Concentrations:
0, 0.75, 2.5, 7.5 mg peracetic acid/kg bw/day from day 1 to 10; 0, 0.75, 2.5, and 5.0 mg/kg bw/day from day 11 to 22; 0, 0.25, 0.75, and 2.5 mg/kg bw/day from day 23 to 92
Basis:
nominal in water
Remarks:
Doses / Concentrations:
0, 7.4, 23.4, 67.4 mg Acide peracétique 5%/kg bw/day corresponding to 0, 0.37, 1.17, 3.37 mg peracetic acid/kg bw/day
Basis:
other: TWA dose levels (nominal in water)
No. of animals per sex per dose:
4 groups of 10 males and 10 females (Due to mortality 2 males and 2 females were added in the highest dose group resulting in a total number of 84 animals.)
Control animals:
yes, concurrent vehicle
Details on study design:
- Dose selection rationale: The dose-levels were selected on the basis of the results of two preliminary 2-week toxicity studies performed in the same species at the dose-levels of 150, 450 and 1000 mg/kg/day (product) (with a constant concentration of 100 mg/mL) and 15, 50, 150 and 300 mg/kg/day (at the concentrations of 10, 33.3, 100 and 300 mg/mL, respectively). Mortality was observed at 300, 450 and 1000 mg/kg/day. A slightly lower body weight gain was noted in animals given 150 mg/kg/day and females given 50 mg/kg/day. No signs of overt toxicity were noted in animals given 15 mg/kg/day. Therefore, the dose-levels chosen for the present study were 15, 50 and 150 mg/kg/day.
However, high mortality was observed in the first weeks. Thus, the dose-levels were modified as specified in "Doses/concentrations". The probable reason behind this phenomenon is discussed in "Details on results".

- No post-exposure (recovery) period was included in this study.
Positive control:
not applicable

Examinations

Observations and examinations performed and frequency:
- Clinical signs and mortality: daily (Detailed clinical observations were made on all animals outside the cage, once before the beginning of the treatment period and once a week thereafter.)

- Functional observation battery (FOB): All animals were evaluated once in week 12. This included a detailed clinical examination, measurement of reactivity to manipulation or to different stimuli and motor activity.

- Body weight: on the first day of treatment, and once a week until the end of the study

- Food consumption: weekly recorded and calculated per day per animal

- Ophthalmoscopy: Before treatment on all animals, on day 87 on animals of the control group and the high-dose group

- Haematology: All animals at the end of the study; parameters: erythrocytes, haemoglobin, mean cell volume, packed cell volume, mean cell haemoglobin, mean cell haemoglobin, thrombocytes, leukocytes, differential white cell, neutrophils, eosinophils, basophils, lymphocytes, monocytes and prothrombin time.
Prior to blood sampling, the animals were deprived of food for an overnight period of at least 14 hours.

- Clinical chemistry: All animals at the end of study; parameters: sodium, potassium, chloride, calcium, ortho-cresolphthalein, inorganic phosphorus, glucose, urea, urease, creatinine, total bilirubin, total proteins, albumin, albumin/globulin, cholesterol, triglycerides, alkaline phosphatase, aspartate aminotransferase and alanine aminotransferase.

Sacrifice and pathology:
- Organ weights:
Adrenals, brain, epididymides, heart, kidneys, liver, ovaries, spleen, testes, thymus, thyroids with parathyroids and uterus.

- Gross and histopathology:
Macroscopic lesions, trachea and lungs of all animals. Organs of animals of the control group and high-dose group killed at the end of the treatment period and for all animals that died or were killed prematurely: adrenals, aorta, brain (including medulla/pons cerebellar and cerebral cortex), cecum, colon, duodenum, epididymides, oesophagus, eyes with Harderian glands, femoral bone with articulation, heart, ileum (with Peyers patches), jejunum, kidneys ,liver, lungs with bronchi, lymph nodes (mandibular and mesenteric), mammary gland area, ovaries (with oviducts), pancreas, pituitary gland, prostate (dorso-lateral and ventral), rectum, salivary glands (sublingual and submandibular), sciatic nerve ,seminal vesicles ,skeletal muscle, skin, spinal cord (cervical, thoracic and lumbar), spleen, sternum with bone marrow, stomach with forestomach, testes, thymus, thyroids with parathyroids, tongue, trachea, urinary bladder, uterus (horns and cervix), vagina
Other examinations:
None
Statistics:
Differentiated statistical analyses were performed on the basis of the following tests: Kolmogorov-Lillefors test, Bartlett test, Fisher test, Dunn test, Dunnett test, Mann-Whitney/Wilcoxon test.

Results and discussion

Results of examinations

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:
no effects observed
Haematological findings:
no effects observed
Clinical biochemistry findings:
effects observed, treatment-related
Description (incidence and severity):
significant effects with not toxicological relevance
Urinalysis findings:
not examined
Behaviour (functional findings):
no effects observed
Organ weight findings including organ / body weight ratios:
no effects observed
Gross pathological findings:
effects observed, treatment-related
Histopathological findings: non-neoplastic:
no effects observed
Histopathological findings: neoplastic:
no effects observed
Details on results:
The details are also summarised in Table 1 below.

- Clinical signs and mortality:
Ptyalism (in one male and eight females), loud breathing and/or dyspnea (in four males and seven females) and/or piloerection (in two males and five females) were observed in animals of the high-dose group.
Transient or intermittent loud breathing was also noted in two females in the mid-dose group. The onset of the symptoms was observed in both animals in the study period in which the 50 mg/kg/day dose-level was administered. The few other signs (i.e. swollen ear, scattered hair or area of hair loss, scabs and abnormal growth of teeth) were noted with the same incidence in control and treated groups. Thus, they were considered to be without relationship to the treatment with the test item. There were no treatment-related changes in autonomic, physiological or neurotoxicological parameters. Motor activity was not affected by the treatment with the test item.

There were no mortalities in the control and low-dose (15/5 mg/kg bw/day) group. One female was found dead on day 20 in the mid-dose group (50 mg/kg bw/day). There were no mortalities when the dose was reduced to 15 mg/kg bw/day.
In the high-dose group (150 mg/kg bw/day) two males and two females were found dead or killed prematurely on day 8, 9 or 10. Prior to death or sacrifice, signs of poor clinical condition (such as piloerection, emaciated appearance, tiptoe walking, dyspnea, loud breathing, swollen abdomen) were observed in 2/4 animals.
In the high-dose group (100 mg/kg bw/day) four females were found dead or killed prematurely on day 17, 19 or 20. Prior to death, signs of poor clinical condition (such as emaciated appearance, round back, piloerection, dyspnea, loud breathing) were noted in 2/4 females.
In the high-dose group (50 mg/kg bw/day) three females and one male were found dead or killed prematurely on day 26, 35, 60 or 71. Prior to death, the main clinical signs were loud and/or abdominal breathing and swollen abdomen. Signs of poor clinical condition were noted in one female.

- Body weight gain:
No relevant differences from controls were noted in the body weight gain in the low- and mid-dose groups. A moderately lower mean body weight gain was noted in males of the high-dose group over the first period of the study (150 mg/kg/day), and in females of the high-dose group throughout the study.

- Food consumption and food conversion rate:
During the first week of treatment animals of the high-dose group (150 mg/kg bw/day) showed a slightly lower mean food consumption when compared to control animals. No relevant differences were observed in food consumption between control and treated animals in all other groups and at all other treatment periods.

- Ophthalmoscopy:
There were no treatment-related ophthalmologic findings.

- Haematology:
No treatment related haematological findings were observed. Minor differences between treated and control animals were slight, not dose-related and within the historical background data and therefore considered to be incidental and not treatment related.

- Clinical chemistry:
Differences between control and high-dose animals included slightly decreased mean total protein level (65 g/L vs. 72 g/L), albumin level (36 g/L vs. 40 g/L) and alkaline phosphatase activity (143 IU/L vs. 195 IU/L) in males and slightly lower mean potassium (3.55 mmol/L vs. 4.18 mmol/L) and inorganic phosphorus levels. All these were within the range of historical control data and therefore considered to be without toxicological significance. Some differences in sodium and chloride levels were also noted between control and treated animals. These showed no dose-response relationship and were considered to be not treatment related.

- Organ weights:
Absolute and relative organ weights were slightly different in treated and control animals. However, these differences were minimal, not dose-related and/or of opposing trend in the two sexes and different groups. They were considered to be of no toxicological importance.

- Gross and histopathology:
In animals of the high-dose group found dead or killed prematurely several segments of the gastro-intestinal tract were distended with gas. Moreover, reddish coloured or dilated lungs, sometimes associated with foamy contents, were observed in one female of the mid-dose group and one male and four females of the high-dose group. These animals also showed marked pulmonary congestion, moderate emphysema and moderate to marked alveolar oedema, respectively. Animals of the high-dose group also had moderate to marked necrotising inflammation of the trachea (two males, six females) and moderate to severe acute bronchitis at the tracheobronchial bifurcation (one male, four females). One male and three females of the high-dose group showed a slight to marked lymphoid depletion. All of these animals were found dead or killed prematurely. Hence, this finding was considered to be due to stress and without direct relationship to the treatment with the test item. The few findings in animals of other groups were those commonly seen in rats of this strain and age and therefore considered to be of no toxicological relevance.

A microscopic examination of the sex organs of the animals was performed during the test but no effects of the test substance were observed. Furthermore the organ weight of the sex organs (testes, ovaries and uterus) was determined at the end of the treatment period. No statistically significant weight differences were noted between treated groups and controls.

- FOB and motor activity:
Detailed clinical observations and functional observation battery revealed no treatment-related changes in autonomic, physiological or neurotoxicological parameters. Motor activity was unaffected by treatment.

Effect levels

open allclose all
Dose descriptor:
NOAEL
Effect level:
23.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the TWA dose of the product. Relevant findings: mortality, clinical signs and histopathological changes due to local irritative effects on trachea and lungs caused by a physical effect (reflux from the stomach due to formation of oxygen gas)
Dose descriptor:
NOAEL
Effect level:
1.17 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on the TWA dose of PAA (5.5% in product)
Dose descriptor:
NOEL
Effect level:
7.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the TWA dose of the product
Dose descriptor:
NOEL
Effect level:
0.37 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: based on the TWA dose of PAA (5.5% in product)
Dose descriptor:
LOAEL
Effect level:
67.4 mg/kg bw/day (nominal)
Based on:
test mat.
Sex:
male/female
Basis for effect level:
other: based on the TWA dose of the product
Dose descriptor:
LOAEL
Effect level:
3.37 mg/kg bw/day (nominal)
Based on:
act. ingr.
Sex:
male/female
Basis for effect level:
other: Based on the TWA dose of PAA (5.5% in product)

Target system / organ toxicity

Critical effects observed:
not specified

Any other information on results incl. tables

Table 1: 13-Week Gavage Study in Rats: Results

Dose [mg/kg bw/day]a

0

15/5

50/15

150/100/50

dose-response

+/-

m

f

m

f

m

f

m

f

m

f

number of animals in group

10

10

10

10

10

10

12b

12b

mortality

0

0

0

0

0

1/10

3/12

9/12

+

+

clinical signs

ptyalism

0

0

0

0

0

0

1/12

8/12

+

+

loud breathing / dyspnea

0

0

0

0

0

2/10c

4/12

7/12

+

+

piloerection

0

0

0

0

0

0

2/12

5/12

+

+

FOB

ne

ne

ne

ne

ne

ne

ne

ne

body weight gain

week 1 to 2 in g (% control)

+63

+24

+67

(+6)

+26

(+8)

+63

(±0)

+27

(+13)

+50

(-21)

+18

(-25)

+

+

week 2 to 4 in g (% control)

+104

+40

+117

(+13)

+39

(-3)

+102

(-2)

+37

(-5)

+107

(+3)

+32

(-20)

-

+

week 4 to 14 in g (% control)

+173

+95

+182

(+5)

+93

(-2)

+163

(-6)

+87

(-8)

+184

(+6)

+75

(-20)

-

+

mean food consumption
 week 1 – 13 [g/animal/day]

28.4

20.6

26.7

20.4

27.2

20.4

27.8

20.6

-

-d

clinical chemistry

ne

ne

ne

ne

ne

ne

ne

ne

haematology

ne

ne

ne

ne

ne

ne

ne

ne

ophthalmology

ne

ne

ne

ne

ne

ne

ne

ne

pathology, histo-pathology

organ weights

ne

ne

ne

ne

ne

ne

ne

ne

stomach / GIT (distended with gas, reddish coloured)

ne

ne

ne

ne

ne

ne

3/12

9/12

+

+

lungs dilated

ne

ne

ne

ne

ne

ne

3/12

9/12

+

+

bronchitis/trachea inflammation

ne

ne

ne

ne

ne

ne

3/12

9/12

+

+

a Over the designated period the dose-levels for the low-, mid- and high-dose groups, respectively, were:

15, 50 and 150 mg/kg bw/day (day 1-10)

15, 50 and 100 mg/kg bw/day (day 11-22)

5, 15 and 50 mg/kg bw/day (from day 23 on).

b Animals in the high-dose group found dead or killed prematurely between day 8 and day 10, were replaced from day 11.

c Onset of symptoms observed during period in which animals received 50 mg/kg bw/day.

d During the first week of treatment female animals of the high-dose group (150 mg/kg bw/day) showed a slightly lower mean food consumption when compared to control animals.

ne no treatment related effects

Applicant's summary and conclusion

Conclusions:
The overall results demonstrated that following oral administration of peracetic acid by gavage for a period of a total of 92 days, no systemic effects were evident. Mortality, clinical signs and histopathological changes observed were due to local reactions in trachea and lungs caused by a physical effect (reflux from the stomach due to formation of oxygen gas). Apart from these local reactions no other observations were made which would be indicative for a systemic distribution resulting in systemic effects. The fact that no increase of toxicity was observed with time correlates with the hypothesis of a local effect.
Taking into account that dose levels were reduced during the study, it is justified to deduce an NOAEL of 23.4 mg product/kg bw/day (= 1.17 mg peracetic acid/kg bw/day ) based on the time-weighted-average (TWA).
Executive summary:

A 90-day gavage study was performed in Sprague-Dawley rats (10 animals/sex/dose group) with 0, 15, 50, 150 mg/kg bw/d from day 1 to 10, 0, 15, 50, 100 mg/kg bw/d from day 11 to 22, and 0, 5, 15, 50 mg/kg bw/d from day 23 to 92 (based on time-weighted average (TWA) dose levels: 0, 7.4, 23.4 and 67.4 mg/kg bw/day).

One female of the mid-dose group died showing reddened lungs with foamy content, lung congestion and alveolar oedema. Onset of transient or intermittent loud breathing was observed in two females during that time. No relevant laboratory or histopathological findings were observed in the other animals. In the high-dose group (150/100/50 mg/kg bw/d), a total of three males and nine females died or were killed prematurely (between day 8 and day 71). In most of these animals symptoms of loud breathing, dyspnea, abdominal swelling or ptyalism were observed. Some surviving animals of this dose group (three males and two females) also showed ptyalism, loud breathing and/or piloerection. In males (during the first week) and in females (throughout the study) a moderately lower body weight gain correlating in part with lower food consumption was noted. No relevant laboratory or histopathological findings were observed in the surviving animals. The stomach and several parts of the gastrointestinal tract (GIT) of animals found dead or killed prematurely were distended with gas and reddish coloured, the lungs were dilated. Treatment-related microscopic changes were found in the trachea (narcotising inflammation) and lungs (acute bronchitis at the tracheobronchial bifurcation) of these animals. Although trachea and lungs are not directly in contact with the test material when administered by gavage, it is reasonable that rapid degradation of hydrogen peroxide and peracetic acid and hence the formation of oxygen gas in the rat stomach and intestinal tract resulted in reflux of the test material together with the acid stomach fluid into trachea and lung leading to the local irritative and inflammatory changes. The higher sensitivity observed in females could be explained by their smaller size and the shorter distance between the stomach and the trachea and lungs leading probably to a higher exposure of these organs. Thus, a local effect on trachea and lungs is more likely to characterise the more acute and immediate toxicity of the test material than a systemic effect.

The overall results demonstrated that following oral administration of peracetic acid by gavage for a period of a total of 92 days, no systemic effects were evident. Apart from local reactions no other observations were made which would be indicative for a systemic distribution resulting in systemic effects.

Furthermore, investigations in this subchronic study included also a functional observation battery (FOB) and motor activity (MA) assessment. Results of FOB and MA examinations were not different from concurrent control animals which confirm the absence of a neurotoxicity potential of peracetic acid. Taking into account that dose levels were changed several times during the study, it is justified to apply the time-weighted-average approach for the deduction of the NOAEL. The NOAEL is derived at the mid dose level which is calculated to be 23.4 mg/kg bw/d (TWA).