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Toxicological information

Developmental toxicity / teratogenicity

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Administrative data

Endpoint:
developmental toxicity
Type of information:
experimental study
Adequacy of study:
key study
Reliability:
2 (reliable with restrictions)
Rationale for reliability incl. deficiencies:
guideline study with acceptable restrictions
Remarks:
Study was performed in full compliance with OECD 414 as of January 2001 and was done according to the principles of GLP. Deficiency of the study: Inappropriate methodology for fixation of fetal tissues for visceral examinations was employed.

Data source

Referenceopen allclose all

Reference Type:
study report
Title:
Unnamed
Year:
2005
Reference Type:
study report
Title:
Unnamed
Year:
2007

Materials and methods

Test guideline
Qualifier:
equivalent or similar to guideline
Guideline:
OECD Guideline 414 (Prenatal Developmental Toxicity Study)
Deviations:
yes
Remarks:
Inappropriate methodology for fixation of fetal tissues for visceral examinations was employed.
Principles of method if other than guideline:
Study was performed in full compliance with OECD 414 as of January 2001 and was done according to the principles of GLP. Deficiency of the study: Inappropriate methodology for fixation of fetal tissues for visceral examinations was employed.
GLP compliance:
yes
Limit test:
no

Test material

Constituent 1
Chemical structure
Reference substance name:
Peracetic acid
EC Number:
201-186-8
EC Name:
Peracetic acid
Cas Number:
79-21-0
Molecular formula:
C2H4O3
IUPAC Name:
Peracetic acid generated by perhydrolysis of N-acetylcaprolactam by hydrogen peroxide in alkaline conditions

Test animals

Species:
rat
Strain:
Wistar
Details on test animals or test system and environmental conditions:
source: Charles River Wiga GmbH, Sulzfeld, Germany
Age: 9 – 10 weeks at pairing.
Weight: 220.9 (mean)
Number of animals per group: 23-24

Administration / exposure

Route of administration:
oral: drinking water
Vehicle:
water
Details on exposure:
Dose selection:
The dose levels used in the teratology study in rats were based upon the results obtained in a 28-day oral (drinking water) study in the rat. In the 28-day study, dose levels of 13.1, 30.5 and 61.9 mg peracetic acid/kg bw/day were administered to the female animals. The dose levels used in this 28-day drinking water study were in turn based on results obtained in a 12-day dose-range-finding gavage study performed in 3 female rats each at dose levels of 100, 150 and 200 mg peracetic acid/kg bw/day. All animals with 150 and 200 mg/kg bw/day died during study days 2-6. One animal dosed with 100 mg/kg bw/day died on study day 8.On necropsy, findings were made which could be ascribed to the irritating nature of peracetic acid to the gastro-intestinal tract.

The test substance was given ad lib via the drinking water from day 5 p.c. until day 20 p.c..
Analytical verification of doses or concentrations:
yes
Details on analytical verification of doses or concentrations:
0, 100, 300 and 700 mg peracetic acid/L drinking water corresponding to 12.5, 30.4 and 48.1 mg peracetic acid/kg bw/day and 4.2, 10.1 and 16 mg hydrogen peroxide/kg bw/day, respectively.
Details on mating procedure:
Females were mated with proven males at a 2-1:1 ratio was performed overnight for a period of four weeks. If not vaginal plug was observed the following morning, a vaginal smear was taken. Female with vaginal plug or sperm-positive smear were considered as mated and caged individually thereafter. Pairing was performed from a pool of 100 females and 30 males.

The day of observation of vaginal plug or sperm-positive vaginal smear was defined as day 0 p.c. (post conception).
Duration of treatment / exposure:
Day 5 p.c. –day 20 p.c.
Frequency of treatment:
The test material was offered ad libitum in the drinking water from day 5 of gestation until one day prior to scheduled Cesarean section.
Duration of test:
about 15 days.
Doses / concentrationsopen allclose all
Dose / conc.:
48.1 mg/kg bw/day (actual dose received)
Remarks:
actual ingested peracetic acid
Dose / conc.:
30.4 mg/kg bw/day (actual dose received)
Remarks:
actual ingested peracetic acid
Dose / conc.:
700 other: mg/L
Remarks:
nominal in water
Dose / conc.:
300 other: mg/L
Remarks:
nominal in water
Dose / conc.:
100 other: mg/L
Remarks:
nominal in water
Dose / conc.:
12.5 mg/kg bw/day (actual dose received)
Remarks:
actual ingested peracetic acid
No. of animals per sex per dose:
23-24 pregnant females per dose group including controls.
Control animals:
yes, concurrent vehicle
Details on study design:
The developmental toxicity and teratogenicity of Wofasteril containing 32 – 38 % (w/w) peracetic acid and 10 – 14 % (w/w) hydrogen peroxide was investigated in pregnant rats. Groups of twenty-one (low and high dose level) and twenty (mid dose level) pregnant Wistar rats were administered dose levels of 100, 300 or 700 mg peracetic acid/L (corresponding to 12.5, 30.4 and 48.1 mg peracetic acid/kg bw/day and 4.2, 10.1 and 16 mg hydrogen peroxide/kg bw/day, respectively.) via the drinking water from day 5 p.c. through day 20 p.c.. A group of twenty-one control animals received untreated drinking water only.
Clinical signs and mortality of dams were daily observed. Food consumption was recorded on the days when body weights were measured. Drinking water consumptions was recorded daily. Body weights were determined on day 0 p.c., day 3 p.c., day 5 p.c. and every 3 days during the dosing period and on day 20. Body weight gains were determined on study days 0-2, 3-4, 5-7, 8-10, 11-14, 14-16, 17-19, respectively. On scheduled termination of the study on day 20 p.c., dams were sacrificed and dams received a macroscopic examination. Uterine contents were examined and reproduction parameters (number of corpora lutea, number or pre- and postimplantation losses including early and late resorptions) was determined. The number of dead and live fetuses, fetuses per dam, fetal weight, sex ratio and external appearance of the fetuses were recorded. Fetuses were subsequently processed for visceral and skeletal examinations.
Based on the analytical results obtained for both peracetic acid and hydrogen peroxide, fresh drinking water preparations were offered daily for the mid and high dose group while for the low dose group, fresh drinking water was offered twice daily.
The study was conducted following OECD 414 (January 2001).
The dose levels used in the teratology study in rats were based upon the results obtained in a 28-day oral (drinking water) study in the rat following administration of dose levels of 13.1, 30.5 and 61.9 mg peracetic acid/kg bw/day and on results obtained in a 12-day dose-range-finding gavage study performed in 3 female rats each at dose levels of 100, 150 and 200 mg peracetic acid/kg bw/day, respectively.

Examinations

Maternal examinations:
Clinical signs and mortality of dams was regularly observed throughout the entire study period. Body weight of dams was determined on day 0 p.c., day 3 p.c., day 5 p.c. (first day of dosing) every 3 days during the dosing period and on day 20 p.c. (i.e day of scheduled sacrifice). Body weight gains were determined on study days 0-2, 3-4, 5-7, 8-10, 11-14, 14-16, 17-19, respectively. The overall body weight gain and body weight gain corrected for uterine weight was calculated as well.
Food consumption of pregnant females was recorded on the days of body weight determination and the mean daily intake for each group was calculated. Food consumption data were presented for study days 0-2, 3-4, 5-7, 8-10, 11-14, 14-16, 17-19, respectively.
Drinking water consumption of pregnant females was recorded daily throughout the entire treatment period and the mean daily intake for each group was calculated for study days 0-4, 5-7, 8-10, 11-13, 14-16, 17-19, respectively.
Since fresh drinking water was offered to animals of the low dose group twice a day, water consumption data were added up.
On termination of treatment (day 20), dams were sacrificed by carbon dioxide asphyxiation and examined externally. The thoracic and abdominal cavities were opened and the major organs subjected to a macroscopic examination.
Ovaries and uterine content:
On termination of the study, the following examinations were performed:
- gravid uterine weight including cervix and both ovaries
- the number of corpora lutea
- the number of implantations in uterine horns including pre- and postimplantation losses (the latter differentiated in early and late resorptions), number of dead and live fetuses and
- uteri appearing non-gravid were examined by ammonium sulphide staining for confirmation of the absence of pregnancies
Fetal examinations:
Fetal examinations included:
- Litter size, number of live and dead foetuses, foetal weight, sex ratio

One half of the fetuses was fixed in ethanol and then processed (evisceration) to visualise the skeleton and to examine skeletal abnormalities
Skeletal examinations were carried out using a stereomicroscope. The remaining half of the fetuses was processed for examination of soft tissue anomalies using 5 frontal sections of the head and 10 – 12 horizontal sections of the trunk with particular emphasis on the reproduction tract.
Soft tissue examinations were carried out using a stereomicroscope.

Statistics:
Yes, the following statistical methods were used:
Welch t-test:
Drinking water consumption, body weights, food consumption and weights of gravid uteri of dams
Dunnett test:
Number of corpora lutea, number of implantations, number of live and dead fetuses, number of resorptions, sex ratio, number of skeletal/visceral abnormalities and number of skeletal/visceral variations.
Indices:
- corporal lutea
- pre- and postimplantation loss
- dead and live fetuses
Historical control data:
Since this was the first study of this type performed by the testing laboratory, no in-house historical background data for the strain of rats and the parameters examined were available.

Results and discussion

Results: maternal animals

Maternal developmental toxicity

Details on maternal toxic effects:
Maternal toxic effects:yes

Details on maternal toxic effects:
Clinical signs and mortality:
None of the dams died during the study period until Caesarean section on day 20. There was no effect on general behaviour. On termination of the study, no external findings and no alterations were evident in dams following macroscopic examination.

Drinking water consumption:
Treatment-related effects in dams were characterised by a statistically significant and dose-related reduction in drinking water consumption at all dose levels from day 5 p.c. until termination of the study. When compared to controls receiving untreated drinking water only, drinking water intake was 83-93 %, 64-74 % and 37-49 % of controls at the low, mid and high dose level, respectively. At the mid and high dose level, decreases in water intake were most pronounced during days 5-7 of the study and during study days 11-13 at the low dose level.

Food consumption:
At the low dose level, food consumption was significantly increased during the most part of the study while at the mid dose level, food intake was significantly lower than that of controls during study days 5-7 only and comparable to or not significantly different from controls thereafter. At the high dose level, food consumption was significantly decreased by 19-39 % during the entire study period when compared with control intake rates.

Body weight and body weight gain:
Absolute body weight, although not attaining statistical significance, was higher than that of concurrent controls in dams of the low dose group throughout the treatment period and comparable to control body weights in dams of the mid dose group. Starting with study day 8, absolute body weight of high dose dams was statistically significantly reduced by 9 – 18 % when compared to body weight of control dams.
On days 5-7 of the study, body weight gains were statistically significantly reduced at all dose levels in a dose-dependent manner. Thereafter, body weight gains measured on the different occasions as well as overall weight gain was not statistically significantly higher in low dose dams when compared to controls. In mid dose dams, individual body weight gains determined on the different occasions as well as overall body weight were slightly but not significantly lower than that of concurrent controls (91 – 94 % of controls) while on study days 8-10 only, body weight gain was 137 % of controls. In the high dose group, body weight gain on each occasion and overall weight gain was statistically significantly reduced throughout the entire study period and overall weight gain achieved 53 % of controls only.

Corrected body weight gain:
Body weight gain corrected for uterine weight was slightly higher in the low dose group (104 % of controls) while a dose-related and statistically significant reduction was noted at the mid (83 % of control) and high dose level. Most importantly, no weight gain at all was noted in high dose dams which lost 1.5 % of weight when compared with their initial body weight.
The statistically significant reduction in corrected body weight gain of dams of the mid dose group was considered to be not of toxicological relevance as there was no effect on absolute body weight, body weight gain as well as reproduction parameters and since dams of the mid dose level did not demonstrate any signs of toxicity.

Necropsy dams:
Necropsy did not reveal treatment-related macroscopic findings in any groups of treated dams.

Effect levels (maternal animals)

open allclose all
Key result
Dose descriptor:
NOAEL
Effect level:
30.4 other: mg/kg peracetic acid bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: maternal toxicity
Key result
Dose descriptor:
NOAEL
Effect level:
30 other: mg/kg peracetic acid bw/day (actual dose received)
Based on:
act. ingr.
Basis for effect level:
other: developmental toxicity

Results (fetuses)

Details on embryotoxic / teratogenic effects:
Embryotoxic / teratogenic effects:yes

Details on embryotoxic / teratogenic effects:
External examinations:
On external examination of fetuses, no abnormalities directly related to treatment with peracetic acid were evident at any dose level. There were a few macroscopic findings in all dose groups including controls which were accidental in nature e.g. umbilical/abdominal hernia, haematoma and which were caused by the handling procedures applied.
Skeletal examinations:
On skeletal examination, there were no findings in pups of the low dose level which could be directly attributed to treatment. At the mid and high dose levels increased incidence of skeletal changes (mainly poor or hypertrophic ossification) was observed. In detail, impairment of ossification in one or more bones (i.e. bones missing or poor/hypertrophic ossification), occurrence of an additional 14th costa or deformed costa were observable at the mid and high dose levels. At the high dose only, a hypertrophic trachea was noted in 8 fetuses of 2 dams. The incidences in the impairment of ossification were statistically significant at the high dose level only. At this dose level, severe maternal toxicity characterised by statistically significant reduced body weight, overall body weight gain, corrected body weight gain, water and food consumption was evident. Large fontanel was observable both in control and high dose pups the incidence/percentage of which was slightly lower in high dose pups and thus, not considered to be of any toxicological relevance.
Other findings such as bones with reduced length, one incomplete chondrification of the nasal septum (one fetus of the low dose group), one costa-like transverse process at the 7th vertebrae cervicales (one fetus of the mid dose group) and one malformation in the region of the viscerocranium (one fetus of the high dose group) were not considered to be related to treatment as these findings were made in one individual fetus of the low, mid and high dose group, respectively, only.
Please refer to Table A6.8.1/01-3.
Visceral (soft tissue) examinations:
According to the evaluation of the study pathologist, there were several findings at all dose levels on visceral examination of fetuses. The most prominent finding was a greyish brown to yellowish or yellow discoloration of fetal livers the severity of which was differing across groups including controls when the size (classified in 0 – 33 %, 34 – 66 % or 67 – 100 % of the liver area affected) of the liver discoloration was considered. The extent and incidence of discoloration in fetal livers was reported to be clearly dose-dependent and statistical significance was attained at the mid and high dose levels. The number and percentage of fetuses with each kind (i.e. greyish brown, yellowish and yellow discoloration) and each affected area were statistically significantly increased in high dose fetuses. At the mid dose level, the percentage of fetuses with greyish brown discoloration of the liver and affecting 34 – 66 % of the liver area was statistically significantly increased. No further statistical significances in the extent and size of liver discoloration was noted in the mid dose group. At the low dose level, incidences in the extent and size of liver discoloration were comparable to or not significantly different from incidences in control fetuses.
Please refer to Table A6.8.1/01-3.
Other observations made on visceral examination of fetuses such as different types of aplasia, microphthalmia, microrchidia, lung not completely developed, diaphragmatic hernia, yellowish discolored brain, poorly enlarged renal pelvis with thin parenchyma in the kidney, poorly dilated urinary bladder, were not considered to be directly related to treatment since these findings were made in single fetuses across groups and were not associated with a dose-response relationship. Furthermore, haematoma seen in different areas were present in all groups including controls and were associated with handling of the animals rather than related to treatment.
Histological examination of fetal livers:
Discolorations in fetal livers were histologically examined following staining.
According to the evaluation of the study pathologist, severe damage of the hepatic cells was seen to some extent. The degree of severity was reported to be clearly dose-dependent. Alterations in fetal livers comprised loosening or unrecognisable structure of the parenchyma, degeneration to necrosis of the nuclei, atypical mitosis (up to 6 binuclei in one cell), lysis of hepatic cells and partially large blood islands with cell detritus and pyknotic nuclei.
In fetuses of the low dose group, liver damage was reported to be of slight severity while in fetuses of the high dose group, only a few intact cells were observable. The degeneration or necrosis in fetal hepatocytes was interpreted by the study pathologist to be indicative for cell death.

Effect levels (fetuses)

Key result
Remarks on result:
other: see Remarks
Remarks:
see "Any other information"

Fetal abnormalities

Key result
Abnormalities:
not specified

Overall developmental toxicity

Key result
Developmental effects observed:
not specified

Any other information on results incl. tables

Pregnancy data


Uterine weights:


There was no treatment-related adverse effect on uterine weights which were comparable to control figures (107 %, 103 % and 102 % at the low, mid and high dose level, respectively).


 


Reproduction parameters:


There were no treatment-related effects on the number of corpora lutea, pre- and post-implantation losses as well as on the number of total resorption across groups of treated dams. The number of pre- and post-implantation losses was slightly higher in dams of the concurrent control group.


 


Fetal effects:


No dead fetuses were observed at any dose level. The number of live fetuses per dam was slightly, but not statistically significantly higher in treated groups with the largest litter size observable at the high dose level. There was no effect on the sex ratio.


 


Fetal body weights:


At the low and mid dose level, fetal body weights were slightly, but not statistically significantly higher when weight of male and female pups was considered separately or combined. At the high dose level, fetal body weight was statistically significantly reduced by 5 – 7 % (male and female fetuses separately and combined). However, the litter size in the high dose group was about 13 % higher than in controls which might have contributed to slightly smaller and thus, lighter fetuses at the high dose level.

Applicant's summary and conclusion

Conclusions:
In the developmental toxicity and teratogenicity study performed with Wofasteril, no teratogenic effect was evident up to and including the high dose level of 700 mg peracetic acid/L (48.1 mg peracetic acid/kg bw/day or 16 mg hydrogen peroxide/kg bw/day).
Treatment-related maternal toxicity was observed at 700 mg peracetic acid/L (48.1 mg peracetic acid/kg bw/day or 16 mg hydrogen peroxide/kg bw/day) and was characterised by reductions in drinking water and food consumption and in absolute body weight as well as by a drastic reduction in overall body weight gain and in body weight gain corrected for uterine weight. At the high dose level, fetal weight was significantly reduced but litter size at this dose level was about 13 % higher than in controls which might have contributed to reduced fetal weights. Furthermore, the incidence of poor, hypertrophic ossification was increased at the high dose level. Most importantly, a pathological/histopathological re-evaluation of fetal livers demonstrated no effect of treatment. The originally reported discolorations of the liver and also in other organs were a direct consequence of the fixation method used and in contrast to the evaluation in the original study report, there was no liver damage evident when fetal livers were histologically re-examined.
Overall, toxicity to the fetus was only noted at dose levels exerting severe maternal toxicity.
Executive summary:

The developmental toxicity and teratogenicity of Wofasteril containing 32 – 38 % (w/w) peracetic acid and 10 – 14 % (w/w) hydrogen peroxide was investigated in pregnant rats. Groups of twenty-one (low and high dose level) and twenty (mid dose level) pregnant Wistar rats were administered dose levels of 100, 300 or 700 mg peracetic acid/L (corresponding to 12.5, 30.4 and 48.1 mg peracetic acid/kg bw/day and 4.2, 10.1 and 16 mg hydrogen peroxide/kg bw/day, respectively.) via the drinking water from day 5 p.c. through day 20 p.c.. A group of twenty-one control animals received untreated drinking water only.


 


In the teratology study peformed, there were no treatment-related clinical signs of toxicity evident in dams at any dose level and no mortalities were observed. No effects on reproduction parameters as well as uterine weights and no macroscopic findings were noted across groups of treated dams.


 


Maternal effects


Treatment-related effects were basically restricted to dams of the high dose group and characterised by statistically significant reductions in water and food consumption throughout the entire treatment period in addition to a statistically significant reduction in terminal body weight, corrected body weight and body weight gain from day 5 to 20 of the study. Reductions in body weight gain of dams at the low and mid dose level were transient and shown to be reversible by the end of the study.


 


Fetal effects


No dead fetuses were observed at any dose level and the number of live fetuses per dam was slightly higher in treated groups. There was no effect on the sex ratio. No external abnormalities were evident in fetuses of any treatment group.


 


High dose fetuses demonstrated a significantly lower body weight and an increased incidence of poor and/or hypertrophic ossification. The latter finding is considered to be secondary to severe toxicity in high dose dams while in the absence of (severe) maternal toxicity, no statistically significant findings in fetuses were made. In addition, the toxicological relevance of reduced fetal weights at the high dose level is questioned as litter size in the high dose group was about 13 % higher than in controls which might have contributed to slightly smaller and thus, lighter fetuses at the high dose level.


 


The most prominent finding in this study was related to a dose-dependent discoloration (greyish brown, yellowish or yellow) of fetal livers the severity of which ranged from 5 – 100 % when the size of the discoloration or the affected area of the livers are considered. Furthermore, the incidence and severity of fetal liver discoloration was reported to be increased with dose according to the assessment of the study pathologist. In addition, a dose-related increase in the severity of fetal liver damage was observed and characterised by loosening or unrecognisable structure of liver parenchyma, degeneration to necrosis of the nuclei, atypical mitosis, lysis of hepatic cells, partly large blood islands with cell detritus and pyknotic nuclei.


 


 


Other findings made on visceral examination of fetuses were not considered to be directly related to treatment since these findings were made in single fetuses across groups, were not associated with a dose-response relationship or were considered to be a result of the handling procedures applied. Pathological/histopathological re-evaluation of fetal livers:


 


In view of the findings made in the fetal livers of the rat teratology study performed with an aqueous solution containing peracetic acid (32 – 38 % w/w) and hydrogen peroxide (10 – 14 % w/w), the PAR Task Force initiated a review of the fetal liver preparations of this study.


 


The re-examination of the fetal liver preparations was performed by an independent experienced veterinary pathologist and by the responsible pathologist of the laboratory which conducted the teratology study. The overall purpose of the review was to assess the distribution and incidences of a pale discoloration of fetal livers observed in all dose groups of the rat teratology study and to determine the toxicological relevance of this alteration. In addition, the re-examination served also to assess any histological changes in the fetal liver preparations.


 


A pale liver discoloration was recorded in almost all fetuses of all dose groups including the control group. The liver was affected focally to multifocally or diffuse. The discoloration ranged from grey to yellow or white and incidences seen in control and the low and middle treatment groups were comparable. The discoloration observed in the high dose treatment group was not significantly different from controls. Surrounding tissues in the body cavities and the carcass were discolored in the same manner as observed in the livers. Furthermore, the higher incidence of discolored livers among high dose fetuses could be due to the lower body weight of these fetuses resulting from maternal toxicity and, hence, delayed development.


 


The discoloration was evaluated as a typical alteration of improperly chemically-fixed organs. Such discoloration is known to progress with time (wet tissue storage). The exact cause for the discoloration is unknown but may be considered to be a consequence of the fixation method chosen. The fixative used in the study was 96% Ethanol: Formaldehyde: Glacial Acetic Acid as 12:6:1 proportions. This fixative is not routinely used today and probably contributed to the discoloration of the fetal livers and other organs across all groups. However, histopathological examination of fetal livers did not reveal lesions related to the dose of the test substance administered. Histological evaluation revealed a slight autolysed liver tissue in all cases examined but the status of the livers was considered sufficient to recognize all structures and did not lead to the conclusion of any liver pathological injury. All tissues examined did not demonstrate any abnormalities. This was supported by the absence of any shrinkage or swelling as well as the presence of sharp liver lobe margins. An inadequate fixation method was found to be the cause for a misinterpretation of the discoloration findings observed in the fetal livers.


 


The results of the re-evaluation of fetal livers demonstrate that a treatment-related liver discoloration in fetal livers as well as fetal liver damage could be excluded and an aqueous solution containing 35 % (w/w) peracetic acid and 12 % (w/w) hydrogen peroxide demonstrated that both substances are not teratogenic to the fetus.


 


Please refer to Table A6.8.1/01-4.

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